Searching Saliva for Signs of Disease

The recent and premature death of ESPN anchor Stuart Scott at just 49 years old focused the media spotlight on a lesser-known form of cancer with a deadly track record: Gastric (stomach) cancer kills more than 800,000 people worldwide each year. Unfortunately, most patients do not notice symptoms until the disease has advanced too far for current treatments to be effective.

What if clinicians could diagnose and treat a wide range of diseases, including stomach cancer, just by understanding the “conversations” between cells and then eavesdropping on the human body? Extracellular RNA (exRNA) — RNA that is outside the wall of a cell — could enable just that. Scientists have discovered that exRNAs help cells talk to each other. But before exRNA can fulfill its potential, researchers need to know more about how it works in the body.

RNA helps translate genes into the proteins that organisms need to function. Recent research has shown that cells can release RNAs in the form of exRNAs to travel via fluids such as blood and saliva. Acting as a signaling molecule, exRNAs communicate with other cells and carry information from cell to cell throughout the body. Sampling and sequencing these exRNAs may provide a snapshot of what is going on in the body at that time and provide scientists with insights into disease.

Through the NIH Common Fund’s Extracellular RNA Communication program, NCATS and other NIH Institutes and Centers support research on how exRNA could be used to improve diagnosis and treatment for diseases such as cancer, bone marrow disorders, heart disease, Alzheimer’s disease and multiple sclerosis. This trans-NIH effort includes leadership and participation by NCATS, the National Cancer Institute, the National Heart, Lung, and Blood Institute, and the National Institute on Drug Abuse. Specifically, NCATS oversees the part of the program that uses exRNA for biomarker and therapy development.

Potential Biomarkers for Cancer

Researchers at the University of California, Los Angeles (UCLA) are exploring how exRNA in saliva could be used as biomarkers to detect gastric cancer. The team announced findings published in the January 2015 issue of Clinical Chemistry. While previous studies described only some of the exRNAs found in saliva, this study was the first to catalog all small noncoding exRNAs in saliva.

The UCLA team found that saliva contains many of the same molecules found in blood. The molecular profiles of different people varied in the same way that molecular profiles of blood do. This finding suggests that saliva represents a person’s health just as well as blood can. The researchers are using these findings to develop a new, noninvasive diagnostic test for stomach cancer.

“Had it not been for the NIH Common Fund initiative, we would not have been able to see what we are seeing now,” said David T.W. Wong, D.M.D., D.M.Sc., a senior author of the study and director of the UCLA Center for Oral/Head & Neck Oncology Research. Wong has been studying saliva for more than a decade. Working with the other senior author of the study, RNA bioinformatics expert Xinshu (Grace) Xiao, Ph.D., was key. “One of the benefits of this program is cross-collaboration with a brilliant RNA biologist,” Wong noted. “Dr. Xiao’s expertise enabled us to see the classes of RNA in the saliva. For this, I am scientifically grateful.”

Describing the Contents of Saliva

Wong and Xiao began with saliva samples from healthy volunteers. They removed the cells from the sample and worked only with the fluid. Using high-throughput RNA sequencing techniques, they analyzed the small noncoding RNA in the saliva.

One type of RNA that they found was microRNA (miRNA), which help regulate gene expression inside a cell’s nucleus. The scientists found that miRNAs were abundant in saliva and that they were present at similar levels in different people. They also found that the miRNA profiles of saliva are similar to those of other body fluids.

Circular RNAs (circRNAs) were also common in the samples; Wong and Xiao identified more than 400 circRNAs in saliva. Unlike linear RNA, which break down easily, circRNAs are stable. “Circular RNAs in saliva may be protecting other RNA,” Xiao said. They may keep the vulnerable miRNAs from degrading during travel to another part of the body. Other research funded by the same NIH initiative suggests that circRNAs may pick up other types of RNA and carry them around the body to deliver messages, similar to the function of hormones.

Of special note, the team also found piwi-interacting RNAs (piRNAs), which are involved in silencing DNA. Until now, scientists commonly found this kind of RNA in stem cells and the cells that become eggs and sperm, but rarely in bodily fluids. Wong and Xiao’s discovery of high levels of piRNA in saliva was unexpected and indicates a need for further scientific exploration.

Is the Mouth a Window to the Body?

Although Wong and Xiao do not know what biological role the noncoding RNA play in saliva, the results show that this fluid could have huge value as the basis for diagnostic tests because it reflects what is going on in other parts of the body.

RNA arrive in the mouth by two routes: (1) from leaking out of blood vessels into the crevices between the teeth and gums and (2) from the salivary glands, which are fed by blood vessels and can actively pull in molecules from the blood. “That information from the bloodstream is downloading into the saliva,” Wong explained.

“No part of our body is in isolation,” Wong said. “Our opportunity is to identify how the parts cross-talk.” The information in saliva could provide a snapshot of the biological processes going on throughout the body. That means sequencing the right molecules could tell you whether a person has a disease.

“Noninvasive tests are the holy grail of diagnostics,” Wong said. Saliva is a particularly good candidate for diagnostic tests because it is abundant — we make about a liter to a liter and a half every day — and easy to sample. Providing a saliva sample is more pleasant than having blood taken and less embarrassing than providing some of the other fluids used in diagnostic tests.

Translating Discovery into Action

Wong and Xiao are working to develop a saliva test to detect gastric cancer by collecting saliva samples from 200 people, half with and half without cancer. Now they are sequencing the exRNAs in the samples. When they have found possible cancer markers, they will recruit 1,500 more people to test the markers in a more powerful study. Wong expects to know definitively whether the test works in about four years.

“This research not only shows how exRNAs could be useful for detecting stomach cancer, but it opens the door for scientists to use saliva and other biofluids as a way to detect other diseases,” said NCATS Associate Director for Special Initiatives Dan A. Tagle, Ph.D., M.S. “Here at NCATS, we look for truly innovative approaches in what is common across diseases in an effort to accelerate discovery before sharing this information broadly to the scientific community.”

Collaboration among exRNA consortia investigators has meant rapid development of new thinking about what exRNAs can do. Not only could this research advance the field of exRNA communication, it also could lead to real applications in the clinic. A more thorough understanding of exRNA could even lead to the development of new devices, such as wearable chips that could detect new diseases and monitor the status of chronic ones.

These kinds of ideas, made possible by exRNA, could help change a diagnosis of stomach cancer — and many other cancers and unrelated conditions — from a grim identification of advanced disease to early detection and effective treatment. It could help prevent the untimely deaths of people like Stuart Scott. It could even point the way toward the future of precision medicine.


Posted February 2015