Non‑invasive prostate cancer detection by measuring miRNA variants (isomiRs) in urine extracellular vesicles
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Danijela Koppers-Lalic1,2,3, Michael Hackenberg4, Renee de Menezes5, Branislav Misovic6, Magda Wachalska7, Albert Geldof8, Nicoletta Zini8,9, Theo de Reijke10, Thomas Wurdinger2,11, Andre Vis7, Jeroen van Moorselaar7, Michiel Pegtel1,*, Irene Bijnsdorp7,*
1Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
2Department of Neurosurgery, Neuro-Oncology Research Group, VU University Medical Center, Amsterdam, The Netherlands
3Exbiome B.V., 1016 PL Amsterdam, The Netherlands
4Department of Genetics, Computational Genomics and Bioinformatics Group, University of Granada, Granada Spain
5Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
6Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
7Department of Urology, VU University Medical Center, Amsterdam, The Netherlands
8CNR-National Research Council of Italy, IGM, Bologna, Italy
9SC Laboratory of Musculoskeletal Cell Biology, IOR-IRCCS, Bologna, Italy
10Department of Urology, Academic Medical Centre, Amsterdam, The Netherlands
11Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA
*These authors contributed equally to this work
Irene Bijnsdorp, e-mail: [email protected]
Danijela Koppers-Lalic, e-mail: [email protected]
Keywords: liquid biopsy, isomiRs, miRNA, RNA sequencing, extracellular vesicles
Received: December 16, 2015 Accepted: February 24, 2016 Published: March 16, 2016
In many cancer types, the expression and function of ~22 nucleotide-long microRNAs (miRNA) is deregulated. Mature miRNAs can be stably detected in extracellular vesicles (EVs) in biofluids, therefore they are considered to have great potential as biomarkers. In the present study, we investigated whether miRNAs have a distinct expression pattern in urine-EVs of prostate cancer (PCa) patients compared to control males. By next generation sequencing, we determined the miRNA expression in a discovery cohort of 4 control men and 9 PCa patients. miRNAs were validated by using a stemloop RT-PCR in an independent cohort of 74 patients (26 control and 48 PCa-patients). Whereas standard mapping protocols identified > 10 PCa associated miRNAs in urinary EVs, miR-21, miR-375 and miR-204 failed to robustly discriminate for disease in a validation study with RT-PCR-detection of mature miRNA sequences. In contrast, we observed that miRNA isoforms (isomiRs) with 3′ end modifications were highly discriminatory between samples from control men and PCa patients. Highly differentially expressed isomiRs of miR-21, miR-204 and miR-375 were subsequently validated in an independent group of 74 patients. Receiver-operating characteristic analysis was performed to evaluate the diagnostic performance of three isomiRs, resulting in a 72.9% sensitivity with a high (88%) specificity and an area under the curve (AUC) of 0.866. In comparison, prostate specific antigen had an AUC of 0.707 and measuring the mature form of these miRNAs yielded a lower 70.8% sensitivity and 72% specificity (AUC 0.766). We propose that isomiRs may carry discriminatory information which is useful to generate stronger biomarkers.
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