A Rapid Method for Isolating Viral DNA-enriched Salivary Exosomes for Diagnosis and Monitoring of Oropharyngeal Cancer

A novel method, based on the combination of microfluidics and acoustics, for concentrating exosomes containing viral DNA from the saliva of oropharyngeal cancer patients was found to be 15 times more sensitive than the current gold standard differential centrifugation method.

Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) did not demonstrate sensitivity sufficient for routine clinical use. In this regard, investigators at Duke University (Durham, NC, USA) hypothesized that salivary exosomes were packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation would enhance diagnostic and prognostic performance for HPV-OPC.

Exosomes are cell-derived vesicles that are present in many and perhaps all biological fluids, including blood, urine, and cultured medium of cell cultures. The reported diameter of exosomes is between 30 and 100 nanometers, which is larger than low-density lipoproteins but much smaller than red blood cells. Exosomes, which contain RNA, proteins, lipids, and metabolites that are reflective of the cell type of origin, are either released from the cell when multivesicular bodies (MVBs) fuse with the plasma membrane, or they are released directly from the plasma membrane. Exosomes have specialized functions and play a key role in coagulation, intercellular signaling, and waste management.

To generate samples enriched with salivary exosomes, the investigators developed an acoustofluidic (the fusion of acoustics and microfluidics) platform. The minute acoustofluidic chip removed unwanted particles based on size, leaving exosome-rich concentrated samples that facilitated detection of tumor-specific biomarkers. The automated and fast exosome isolation (less than five minutes of processing time compared to approximately eight hours of processing time using benchmark technologies) could be performed at relatively low cost and was suitable for repeated and continuous monitoring of tumor progression and treatment.

The investigators used the acoustofluidic system to analyze saliva samples from 10 patients diagnosed with HPV-OPC using traditional methods. They found that the platform was capable of consistently isolating exosomes from saliva samples, regardless of viscosity variation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform was 15 times higher. With this high-yield exosome isolation platform, they showed that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16.

"OPC has an approximate incidence of 115,000 cases per year worldwide and is one of the fastest-rising cancers in Western countries due to increasing HPV-related incidence, especially in younger patients. It is paramount that surveillance methods are developed to improve early detection and outcomes," said senior author Dr. Tony Jun Huang, professor of mechanical engineering and materials science at Duke University. "Considering these factors, the successful detection of HPV from salivary exosomes isolated by our acoustofluidic platform offers distinct advantages, including early detection, risk assessment, and screening. This technique may also help physicians predict which patients will respond well to radiation therapy or achieve longer progression-free survival."

The acoustofluidic method was described in the December 13, 2019, online edition of the Journal of Molecular Diagnostics.

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