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Consistent high concentration of the viral microRNA BART17 in plasma samples from nasopharyngeal carcinoma patients - evidence of non-exosomal transport

Claire Gourzones1, François-Régis Ferrand12, Corinne Amiel3, Benjamin Vérillaud14, Ana Barat1, Maryse Guérin5, Charles-Henry Gattolliat1, Aurore Gelin1, Jihène Klibi1, Arij Ben Chaaben6, Véronique Schneider3, Fethi Guemira6, Joël Guigay7, Philippe Lang8, Anne-Sophie Jimenez-Pailhes1 and Pierre Busson1*

Author Affiliations

1 Université Paris-Sud 11, CNRS-UMR 8126 and Institut de Cancérologie Gustave Roussy, 39 rue Camille Desmoulins, Villejuif 94805, France

2 Ecole du Val de Grâce, 1 place Alphonse Laveran, Paris 75005, France

3 Virology Department, Hôpital Tenon, 4 rue de la Chine, Paris 75020, France

4 Head and Neck Department, Hôpital Lariboisière, 2 rue Ambroise Paré, Paris 75010, France

5 INSERM UMRS 939 - Université Pierre et Marie Curie - Paris6, Hôpital de la Pitié, 83 boulevard de l’Hôpital, Paris 75013, France

6 Clinical Biology Department, Institut Salah Azaiz, Tunis, Tunisia

7 Head and Neck Tumors Department, Institut de Cancérologie Gustave Roussy, 39 rue Camille Desmoulins, Villejuif 94805, France

8 Radiotherapy Department, Hôpital Pitié-Salpétrière, 47 Boulevard de l’hôpital, Paris 75013, France

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Virology Journal 2013, 10:119  doi:10.1186/1743-422X-10-119

Published: 16 April 2013

Abstract

Background

Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes.

Patients and methods

Plasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation.

Results

The miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes.

Conclusions

Detection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes.

Keywords:
Biomarker; DNA load; Epstein-Barr virus; Exosomes; Head and Neck carcinomas; Lipoproteins; miR-BART; microRNA; Nasopharyngeal carcinoma; Plasma