Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer

Genome-wide association analysis of more than 120,000 individuals identifies 15 new susceptibility loci for breast cancer.

Genome-wide association studies (GWAS) and large-scale replication studies have identified common variants in 79 loci associated with breast cancer, explaining ∼14% of the familial risk of the disease. To identify new susceptibility loci, we performed a meta-analysis of 11 GWAS, comprising 15,748 breast cancer cases and 18,084 controls together with 46,785 cases and 42,892 controls from 41 studies genotyped on a 211,155-marker custom array (iCOGS). Analyses were restricted to women of European ancestry. We generated genotypes for more than 11 million SNPs by imputation using the 1000 Genomes Project reference panel, and we identified 15 new loci associated with breast cancer at P < 5 × 10(-8). Combining association analysis with ChIP-seq chromatin binding data in mammary cell lines and ChIA-PET chromatin interaction data from ENCODE, we identified likely target genes in two regions: SETBP1 at 18q12.3 and RNF115 and PDZK1 at 1q21.1. One association appears to be driven by an amino acid substitution encoded in EXO1.BCAC is funded by Cancer Research UK (C1287/A10118, C1287/A12014) and by the European Community's Seventh Framework Programme under grant agreement 223175 (HEALTH-F2-2009-223175) (COGS). Meetings of the BCAC have been funded by the European Union COST programme (BM0606). Genotyping on the iCOGS array was funded by the European Union (HEALTH-F2-2009-223175), Cancer Research UK (C1287/A10710, C8197/A16565), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer program and the Ministry of Economic Development, Innovation and Export Trade of Quebec, grant PSR-SIIRI-701. Combination of the GWAS data was supported in part by the US National Institutes of Health (NIH) Cancer Post-Cancer GWAS initiative, grant 1 U19 CA148065-01 (DRIVE, part of the GAME-ON initiative). For a full description of funding and acknowledgments, see the Supplementary Note.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.324

Circulating MicroRNAs in cancer

It is believed that microRNAs have potential as circulating biomarkers of disease, however successful clinical implementation remains a challenge. This chapter highlights broad variations in approaches to microRNA analysis where whole blood, serum and plasma have each been employed as viable sources. Further discrepancies in approaches are seen in endogenous controls and extraction methods utilised. This has resulted in contradictory publications, even when the same microRNA is targeted in the same disease setting. Analysis of blood samples highlighted the impact of both collection method and storage, on the microRNA profile. Analysis of a panel of microRNAs across whole blood, serum and plasma originating from the same individual emphasised the impact of starting material on microRNA profile. This is a highly topical field of research with immense potential for translation into the clinical setting. Standardisation of sample harvesting, processing and analysis will be key to this translation. Methods of sample harvesting, preservation and analysis are outlined, with important mitigating factors highlighted.This material is based upon works supported by the Irish Cancer Society collaborative cancer research centre BREAST-PREDICT Grant CCRC13GAL and funding agency Breast Cancer Research.Peer reviewe

Circulating MicroRNAs in cancer

It is believed that microRNAs have potential as circulating biomarkers of disease, however successful clinical implementation remains a challenge. This chapter highlights broad variations in approaches to microRNA analysis where whole blood, serum and plasma have each been employed as viable sources. Further discrepancies in approaches are seen in endogenous controls and extraction methods utilised. This has resulted in contradictory publications, even when the same microRNA is targeted in the same disease setting. Analysis of blood samples highlighted the impact of both collection method and storage, on the microRNA profile. Analysis of a panel of microRNAs across whole blood, serum and plasma originating from the same individual emphasised the impact of starting material on microRNA profile. This is a highly topical field of research with immense potential for translation into the clinical setting. Standardisation of sample harvesting, processing and analysis will be key to this translation. Methods of sample harvesting, preservation and analysis are outlined, with important mitigating factors highlighted.This material is based upon works supported by the Irish Cancer Society collaborative cancer research centre BREAST-PREDICT Grant CCRC13GAL and funding agency Breast Cancer Research.Peer reviewe

Colonic perforation in Behçet’s syndrome

A 17-year-old gentleman was admitted to our hospital for headache, the differential diagnosis of which included Behçet’s syndrome (BS). He developed an acute abdomen and was found to have air under the diaphragm on erect chest X-ray. Subsequent laparotomy revealed multiple perforations throughout the colon. This report describes an unusual complication of Behcets syndrome occurring at the time of presentation and a review of the current literature of reported cases