Polycomb preferentially targets stalled promoters of coding and noncoding transcripts.

The Polycomb group (PcG) and Trithorax group (TrxG) of proteins are required for stable and heritable maintenance of repressed and active gene expression states. Their antagonistic function on gene control, repression for PcG and activity for TrxG, is mediated by binding to chromatin and subsequent epigenetic modification of target loci. Despite our broad knowledge about composition and enzymatic activities of the protein complexes involved, our understanding still lacks important mechanistic detail and a comprehensive view on target genes. In this study we use an extensive data set of ChIP-seq, RNA-seq, and genome-wide detection of transcription start sites (TSSs) to identify and analyze thousands of binding sites for the PcG proteins and Trithorax from a Drosophila S2 cell line. In addition of finding a preference for stalled promoter regions of annotated genes, we uncover many intergenic PcG binding sites coinciding with nonannotated TSSs. Interestingly, this set includes previously unknown promoters for primary transcripts of microRNA genes, thereby expanding the scope of Polycomb control to noncoding RNAs essential for development, apoptosis, and growth.

A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain.

Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.

Validation of a CE-IVD, urine exosomal RNA expression assay for risk assessment of prostate cancer prior to biopsy.

Improved risk stratification of patients suspected of prostate cancer prior to biopsy continues to be an unmet clinical need. ExoDx Prostate (IntelliScore) "EPI" is a non-invasive urine test utilizing RNA from exosomes to provide a risk score that correlates with the likelihood of finding high grade prostate cancer at biopsy. Here, we present the results from a prospective clinical validation study of EPI-CE, a CE-marked in-vitro diagnostic (IVD) assay, specifically developed for use in European clinical laboratories. The study (NCT04720599) enrolled patients with ≥ 50 years, PSA 2-10 ng/mL, prior to MRI, who were scheduled for initial biopsy. First catch urine samples were collected from participants without prior digital rectal examination or prostate massage. Exosomal RNA was isolated and expression levels of three biomarkers ERG, PCA3 and SPDEF were analyzed according to the EPI-CE Instructions For Use. In the study cohort of N = 109 patients, EPI-CE was validated to have a Negative Predictive Value of 89%, a Sensitivity of 92% and a superior performance to two commonly used multiparametric risk calculators (PCPT and ERSPC) in both Receiver Operating Characteristics with a higher Area Under the Curve for EPI-CE 0.67 (95% CI 0.56-0.77) versus PCPT 0.59 (95% CI 0.47-0.71) and ERSPC 0.60 (95% CI 0.49-0.72) and higher Net Benefits analysis across a wide range of risk acceptance levels. This is the first clinical study reporting on the performance of EPI-CE. We demonstrate that EPI-CE provides information beyond standard clinical parameters and provides a better risk assessment prior to MRI, of patients suspected of prostate cancer, than the commonly used multiparametric risk calculators.

Proteomic Exploration of Plasma Exosomes and Other Small Extracellular Vesicles in Pediatric Hodgkin Lymphoma: A Potential Source of Biomarkers for Relapse Occurrence.

Exosomes and other small extracellular vesicles (EVs) are potential sources of cancer biomarkers. Plasma-derived EVs have not yet been studied in pediatric Hodgkin lymphoma (HL), for which predictive biomarkers of relapse are greatly needed. In this two-part proteomic study, we used two-dimensional difference gel electrophoresis (2D-DIGE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze EV proteins of plasma collected at diagnosis from children with nodular sclerosis HL, relapsed or not. EVs isolated using membrane affinity had radii ranging from 20 to 130 nm and contained the programmed cell death 6-interacting (ALIX) and the tumor susceptibility gene 101 (TSG101) proteins, whereas calnexin (CANX) was not detected. 2D-DIGE identified 16 spots as differentially abundant between non-relapsed and relapsed HL (|fold change| ≥ 1.5, p < 0.05). LC-MS/MS identified these spots as 11 unique proteins, including five more abundant in non-relapsed HL (e.g., complement C4b, C4B; fibrinogen γ chain, FGG) and six more abundant in relapsed HL (e.g., transthyretin, TTR). Shotgun LC-MS/MS on pooled EV proteins from non-relapsed HL identified 161 proteins, including 127 already identified in human exosomes (ExoCarta data). This EV cargo included 89 proteins not yet identified in exosomes from healthy plasma. Functional interrogation by the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that the EV proteins participate in platelet degranulation and serine-type endopeptidase activity as the most significant Gene Ontology (GO) biological process and molecular function (p < 0.01).

Extracellular vesicles from plasma have higher tumour RNA fraction than platelets.

In addition to Circulating Tumour Cells (CTCs), cell-free DNA (cfDNA) and Extracellular Vesicles (EVs), the notion of "Tumour-Educated Platelets" (TEP) has recently emerged as a potential source of tumour-derived biomarkers accessible through blood liquid biopsies. Here we sought to confirm the suitability of the platelet blood fraction for biomarker detection in comparison to their corresponding EV fraction. As publications have claimed that tumour RNA and other tumour-derived material are transferred from tumour cells to the platelets and that tumour-derived transcripts can be detected in platelets, we chose to focus on RNA carrying a mutation as being of bona fide tumour origin. After informed consent, we collected prospective blood samples from a cohort of 12 melanoma patients with tissue-confirmed BRAF V600E mutation. Each blood specimen was processed immediately post collection applying two published standard protocols in parallel selecting for EVs and platelets, respectively. The RNA of each fraction was analysed by a highly sensitive ARMS RT-qPCR enabling the quantification of the mutant allele fraction (%MAF) of BRAF V600E down to 0.01%. In a direct comparative analysis, the EV fraction contained detectable BRAF V600E in 10 out of 12 patients, whereas none of the patient platelet fractions resulted in a mutant allele signal. The platelet fraction of all 12 patients contained high amounts of wild-type BRAF signal, but no mutation signal above background was detectable in any of the samples. Our observations suggest that the phenomenon of tumour RNA transfer to platelets occurs below detection limit since even a very sensitive qPCR assay did not allow for a reliable detection of BRAF V600E in the platelet fraction. In contrast, EV fractions derived from the same patients allowed for detection of BRAF V600E in 10 of 12 blood specimens.

Liquid Biopsies Using Plasma Exosomal Nucleic Acids and Plasma Cell-Free DNA Compared with Clinical Outcomes of Patients with Advanced Cancers.

Purpose: Blood-based liquid biopsies offer easy access to genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. Exosomal nucleic acids (exoNAs) originate from living cells, which can better reflect underlying cancer biology.Experimental Design: Next-generation sequencing (NGS) was used to test exoNA, and droplet digital PCR (ddPCR) and BEAMing PCR were used to test cfDNA for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations in 43 patients with progressing advanced cancers. Results were compared with clinical testing of archival tumor tissue and clinical outcomes.Results: Forty-one patients had BRAF, KRAS, or EGFR mutations in tumor tissue. These mutations were detected by NGS in 95% of plasma exoNA samples, by ddPCR in 92% of cfDNA samples, and by BEAMing in 97% cfDNA samples. NGS of exoNA did not detect any mutations not present in tumor, whereas ddPCR and BEAMing detected one and two such mutations, respectively. Compared with patients with high exoNA mutation allelic frequency (MAF), patients with low MAF had longer median survival (11.8 vs. 5.9 months; P = 0.006) and time to treatment failure (7.4 vs. 2.3 months; P = 0.009). A low amount of exoNA was associated with partial response and stable disease ≥6 months (P = 0.006).Conclusions: NGS of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared with clinical testing of archival tumor and testing of plasma cfDNA. Low exoNA MAF is an independent prognostic factor for longer survival. Clin Cancer Res; 24(1); 181-8. ©2017 AACR.

Implementing liquid biopsies into clinical decision making for cancer immunotherapy.

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies.Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients' outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.

Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as "exoRNeasy Serum/Plasma Maxi Kit".

Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium.

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.