Month: December 2019

Thanks to Eileen Leahy from Elsevier and Chhavi Chauhan, Director of Scientific Outreach for the Journal of Molecular Diagnostics, for sharing this post here.

A novel non-invasive technique may detect human papilloma virus-16, the strain associated with oropharyngeal cancer, in saliva samples, reports The Journal of Molecular Diagnostics.

Philadelphia, December 13, 2019 – Unfortunately, cancers that occur in the back of the mouth and upper throat are often not diagnosed until they become advanced, partly because their location makes them difficult to see during routine clinical exams. A report in The Journal of Molecular Diagnostics, published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of co published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of confirmed OPC patients.

“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,” explained co-lead investigator Tony Jun Huang, PhD, Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA.

“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,” added Dr. Huang. This technique may also help physicians predict which patients will respond well to radiation therapy or achieve longer progression-free survival.

Exosomes are tiny microvesicles originating within cells that are secreted into body fluids. They are believed to play a role in intercellular communication and their numbers are elevated in association with several types of cancers. Acoustofluidics is an advanced technology that fuses acoustics and microfluidics. Fluid samples are analyzed using a tiny acoustofluidic chip developed to isolate salivary exosomes by removing unwanted particles based on size, leaving exosome-rich concentrated samples that make it easier to detect tumor-specific biomarkers.

Acoustofluidic exosome isolation chip
Acoustofluidic exosome isolation chip for salivary exosome isolation. The microfluidic channels are shown by red dye, and the coin demonstrates the size of the chip. Two pairs of gold interdigital transducers are deposited along the channel, which separates particles according to size.

In this study investigators analyzed saliva samples from 10 patients diagnosed with HPV-OPC using traditional methods. They found that the technique identified the tumor biomarker HPV-16 DNA in 80 percent of the cases when coupled with droplet digital PCR. Since this method is independent of sample variability arising from changes in saliva viscosity and collection method, it may prove ideal for use in clinical settings.

Dr. Huang highlighted some of the technique’s features, including automated and fast exosome isolation (less than five minutes of processing time compared to approximately eight hours of processing time using benchmark technologies). Analyses can be performed at relatively low cost and at points of care. Also, it is suitable for repeated and continuous monitoring of tumor progression and treatment, unlike traditional biopsy.

“With these features, the acoustofluidic technology has the potential to significantly exceed current industry standards, address unmet needs in the field, help expedite exosome-related biomedical research, and aid in the discovery of new exosomal biomarkers,” commented Dr. Huang.

“The saliva exosome liquid biopsy is an effective early detection and risk assessment approach for OPC,” said co-lead investigator David T.W. Wong, DMD, DMSc, of the Center for Oral/Head and Neck Oncology Research, School of Dentistry at the University of California Los Angeles, CA, USA. “The acoustofluidic separation technique provides a fast, biocompatible, high-yield, high-purity, label-free method for exosome isolation from saliva.” According to the researchers, this technology can also be used to analyze other biofluids such as blood, urine, and plasma.

The study was an international collaboration between Duke University, UCLA, and University of Birmingham (UK). According to Prof Hisham Mehanna, Director of the Institute of Head and Neck Studies and Education, University of Birmingham, Birmingham, UK, “The results are a testament to the power of interdisciplinary research and international collaboration.”

Reference
Wang Z et al. Acoustofluidic salivary exosome isolation: A liquid biopsy compatible approach for human papillomavirus—associated oropharyngeal cancer detection. Journal of Molecular Diagnostics v22, January 2020. doi: 10.1016/j.jmoldx.2019.08.004.

This work was supported by the National Institutes of Health (D.T.W.W.: UG3/UH3 TR002978, UH3 TR000923, U01 CA233370, UH2 CA206126), (T.J.H.: R01GM132603, R01 HD086325), (D.TW.W. and F.L.: R21 CA239052) and Canadian Institute of Health (CIHR) Doctoral Foreign Student Award (J.C.), Tobacco Related Disease Research Program (TRDRP) Predoctoral Fellowship (J.C.). Funding was also provided by the Queen Elizabeth Hospital Birmingham (QEHB) Charity UK and the Get-A-Head charity UK.

Malignant gliomas are highly aggressive brain tumors. Surgical removal and chemoradiation of the tumor are the standard of care. Recently, the U.S. Food and Drug Administration (FDA) approved a compound called 5-aminolevulinic acid (5-ALA) as an imaging agent to aid in differentiating tumor from normal tissue during surgery. 5-ALA is a precursor in the heme biosynthesis pathway, which is inefficient in glioma cells because their strongly rewired metabolism does not rely on heme. When patients with malignant glioma ingest 5-ALA prior to surgery, the glioma cells fluoresce pink under a blue light due to their preferential uptake and conversion of 5-ALA to the final precursor in heme biosynthesis, the fluorescent molecule protoporphyrin IX (PpIX). We sought to investigate whether extracellular vesicles (EVs) released from PpIX-enriched glioma cells would fluoresce and be detectable in the blood of these patients.

We employed Amnis® Imaging Flow, which combines flow cytometry and microscopy to detect PpIX-positive EVs. We first determined the optimal 5-ALA dose to maximize fluorescence and minimize cell death. We used a combination of beads of different size (100-500nm) and liposomes with different emission spectra to ensure that the signal emitted in Channel 11 (~640nm) of the Amnis® output was indeed from PpIX, and that all other channels reported no signal. Controls also included lysis with Triton-X of liposomes and EVs.

Importantly, we showed that glioma cells released a significantly higher number of PpIX-positive EVs (247-fold increase) than normal endothelial cells (6-fold increase) after 5-ALA ingestion. We also used xenograft mouse models to show that the presence of PpIX-positive EVs in circulating plasma after 5-ALA ingestion correlated strongly with the presence of a primary brain tumor, while the signal from the plasma of normal control mice remained below background both before and after 5-ALA ingestion.

Finally, we tested the optimized assay in the plasma of patients with gliomas undergoing 5-ALA fluorescence guided surgery at the Massachusetts General Hospital. Samples were collected prior to 5-ALA intake as well as at the time of surgery, prior to tumor removal. Pre- and post-5-ALA plasma samples were kept in the dark to avoid bleaching of the PpIX signal, as were the patients for 24 hours post 5-ALA. We collected samples from 4 patients whose tumors were avidly fluorescent during surgery and 2 patients whose tumors showed minimal fluorescence. Interestingly, we detected PpIX-positive EVs only in the plasma samples from patients whose tumors were avidly fluorescent. Finally, when we compared the fold increase (pre/post-5-ALA) in PpIX-positive signal to the size of the tumor, we found a clear correlation, suggesting that the detected events are likely coming directly from the tumor. This is the first time intracranially derived EVs have been quantified in circulating plasma, and this development opens the door for many exciting studies that can shed light on brain-derived EV dynamics and half-life. For example, we detected between 3,000 and 8,000 PpIX-positive events per mL of plasma. Assuming each 1 mL of plasma contains roughly 1010 EV/mL, we can deduct that only 0.00008% of EVs in blood are of glioma tumor origin. Furthermore, this assay allows us to study EV dynamics in tumor patients undergoing therapy as well as determine the effects of medications such as dexamethasone on the release of EVs into the bloodstream.

Clinically, there is a major need for minimally invasive diagnosis of brain cancer, and characterizing circulating tumor-specific fluorescent EVs provides a window into the primary tumor’s presence and status. Detecting and characterizing fluorescent EVs after administering 5-ALA allows for diagnosis and potentially monitoring of malignant gliomas over time.

Reference

Jones PS, et al. Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma. (2019) eBioMedicine 48:23-35. doi: 10.1016/j.ebiom.2019.09.025. PMID: 31628025.