In the late 1950s, dentist and U.S. Navy Captain Kirk C. Hoerman, then a young man in his 30s, attempted to answer a bold question: Might the saliva of prostate cancer patients have different characteristics from that of healthy people? Could it contain traces of a disease that’s so far away from the mouth?

Without wasting more of their own saliva on elaborate discussion, Hoerman and his colleagues from the department of dental research at the Naval Training Center in Great Lakes, Illinois, got down to work. They analyzed samples from more than 200 patients and healthy controls, and found that the saliva of patients with untreated prostate cancer showed a significant increase in the levels of enzymes called acid phosphatases.

Writing in 1959 in the journal Cancer, the researchers then made a prescient reflection: that it may be valuable to observe discrete biochemical changes in tissues distant from the site of tumor origin.

More than 60 years later, the idea that saliva analysis can be used to detect different types of cancer is gaining traction in the scientific community. In the specialized literature, papers containing the keywords “diagnosis,” “cancer” and “saliva” grew more than tenfold over the past two decades, from 26 in 2001 to 117 in 2011, 183 in 2016 and 319 in 2021, according to the PubMed database, a search engine for biomedical research articles.

The appeal of this approach is obvious. Although cancer can be diagnosed through tissue biopsy, that requires trained physicians wielding long needles, scalpels, endoscopes or other tools to pry into the body to take samples. Liquid biopsy, which looks for traces of tumor components in fluids such as blood, urine, cerebrospinal fluid, semen or saliva, is a less invasive alternative. Of these, the simplest sample to collect is undoubtedly saliva.

The approach has already paid off: In 2021, the U.S. Food and Drug Administration gave an innovative device designation to a saliva-based oral and throat cancer prediagnostic tool developed by the U.S. company Viome. (Such designations are granted to novel medical devices that have the potential to provide more effective treatment or diagnosis of life-threatening diseases.) Based on artificial intelligence and machine learning, the tool analyzes a saliva sample for the activity of genes (in particular, messenger RNA) belonging to the bacterial community housed in the mouth. For unknown reasons, this community is modified when a tumor develops on the lips, tongue, throat or surrounding areas.

“For decades, saliva was considered a stepchild of blood,” says chemist Chamindie Punyadeera, who spent a decade working on Viome’s saliva diagnostic test. Now at Griffith University in Australia, she is lead author of a 2021 study describing the test’s development in NPJ Genomic Medicine. But that view of saliva as an afterthought could begin to change in the coming years as techniques to analyze it advance and a better understanding develops of what information it can hold. “Because saliva can be collected noninvasively, an empowered patient could take multiple samples and become a steward of his or her own diagnostic tests,” Punyadeera predicts.

The treasure contained in saliva

Every day, the salivary glands of an average adult produce between 500 and 1,500 milliliters of saliva to aid digestion and preserve oral health. In addition to enzymes, hormones, antibodies, inflammatory mediators, food debris and microorganisms, saliva has been found to contain traces of DNA and RNA or proteins from tumors.

“The aim of saliva diagnostics is … to develop a rapid and noninvasive detection of oral and systemic diseases,” write dental scientists Taichiro Nonaka of Louisiana State University and David T.W. Wong of the University of California, Los Angeles, in an article on saliva diagnostics published in the 2022 Annual Review of Analytical Chemistry. The field is developing rapidly due to the progress of “omics sciences” that analyze large collections of molecules involved in the functioning of an organism—such as genomics (genomes), proteomics (proteins) or metabolomics (metabolites)—as well as methods for analyzing large quantities of data. For example, the proteome of saliva—an exhaustive catalog of the proteins present in this fluid—is already available, and it is known that between 20 percent and 30 percent of the saliva proteome overlaps with that of blood.

But “the study of diagnostics through saliva is a relatively new field,” says Nonaka. It wasn’t until the last decade, he says, that it became known that salivary glands—parotid, submandibular, and sublingual, as well as other minor glands, in close proximity to blood vessels—transfer molecular information.

Today, in saliva—and in blood—scientists are beginning to look for and find circulating tumor DNA, which is DNA that is shed from cancer cells when a tumor is present in the body. Multiple studies have identified biomarkers—such as proteins that are produced in higher quantities in cancer cells or genetic changes that occur in tumor cells—that could be used to detect tumors of the head and neck, breast, esophagus, lung, pancreas and ovary, as well as to monitor the patient’s response to therapies.

Is Saliva the Next Frontier in Cancer Detection?
Scientists have found that saliva may contain biochemical signals (biomarkers) that provide an alert that a person has non-small cell lung cancer, the most common type of lung cancer. These signals are circulating tumor DNA (ctDNA) and tumor-derived exosomes that enter the circulation and reach the salivary glands. Both are taken up by secretory (acinar) cells of the salivary glands and are included in the saliva the glands produce. The presence of certain ctDNA mutations, combined with information provided by the exosomes, allow early detection and offer insight into the treatment of this type of cancer.

For example, in 2015 Chinese researchers published that the identification of two fragments of an RNA strand (microRNA) in saliva allowed the detection of malignant pancreatic cancer in seven out of ten patients with the disease. A more recent review of 14 studies involving more than 8,000 participants estimated that breast cancer patients were 2.58 times more likely to have certain saliva-detectable biomarkers—although 39 percent of the negative test results were in patients who actually had breast cancer. The research in the field is promising, but it will require further prospective studies to determine its clinical applicability, Nonaka says.

“A great advantage of liquid biopsies is that they can sweep for up to 50 types of cancers in early stages at once, when they can be surgically treated or are candidates for short, targeted treatments,” says biologist Marina Simian, a researcher at Argentina’s National Scientific and Technical Research Council at the Nanosystems Institute of the National University of San Martín, in Buenos Aires. Simian is also a co-founder of the company Oncoliq, which aims for the early detection of breast, prostate and other tumors from a blood sample.

“With today’s tools, very few organs are screened for cancer,” says Simian. Common screens include ones for cancer of the prostate, breast, cervix, colon after the age of 50, and the lungs for those who have smoked heavily. And globally, she says, only half of these people undergo these tests; in many countries, not even 10 percent do. The hope is to add many more tests that can be done on a single blood or saliva sample.

It is possible that in the future, testing of both blood and saliva will be the norm. Although there is still a long way to go, Nonaka believes that, except for oral cancers, saliva testing should most likely be supplemented with liquid biopsies in blood or urine, plus other parameters to increase sensitivity and practical utility.

In pursuit of exosomes

One particularly promising type of component to look for in saliva is the exosome. Exosomes are tiny lipid-wrapped vesicles that are present in almost all types of body fluids. They are transporters or messengers that travel from one cell to another—even to those in very distant organs. They carry a cargo of genetic material and proteins, which is taken up by a recipient cell in an organ and plays important roles in cell-to-cell signaling. But exosomes also have an important role in cancer. “They are key players,” says Punyadeera. Released by cancer cells, they pass into the blood and, from there, can reach the salivary glands. The exosomes are thus dumped into the saliva, from which they can be collected.

Is Saliva the Next Frontier in Cancer Detection?
Exosomes in saliva contain diverse cargoes. Shown are a variety of nucleic acids — messenger RNA (mRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) — as well as classes of proteins that serve different functions. They include, among others, enzymes that catalyze biochemical reactions, proteins involved in the immune system, ones that regulate water transport across membranes and ones that modulate calcium activity. The exosome’s cargo may also include “messages” from nearby or remote tumor cells, known as ctDNA or circulating tumor DNA.

Exosomes from tumor cells have a specific composition and are suspected of contributing to the spread of cancer to other organs or tissues. But from a diagnostic perspective, one of their main advantages is that they package and protect the cargo—in other words, they do not mix with the other components of saliva. In this way, they provide “more stable and accurate clinically relevant information for disease detection,” Nonaka explains.

For example, for squamous cell esophageal cancer, scientists have found two signatures or signals in salivary exosomes that allow detection of this disease with a sensitivity and specificity of more than 90 percent, in addition to providing guidance on prognosis and treatment, as reported in January 2022 in Molecular Cancer.

Factors such as the concentration or appearance of exosomes under the microscope can also be revealing. Patients with oral cancer, for example, have exosomes with different shapes and sizes than those found in healthy individuals.

However, the techniques available so far to isolate and study the exosome content of saliva are expensive and laborious. In response to this challenge, a new method known as electric field-induced release and measurement has emerged; it integrates electrochemical sensors and magnetic fields to elegantly capture minute amounts of circulating tumor DNA and other molecules—biomarkers—that indicate the presence of cancer. This technique has already shown encouraging results in the early detection of non-small cell lung cancer and could also be used to assess response to treatment.

Is Saliva the Next Frontier in Cancer Detection?
Although often described as “little bubbles,” exosomes are not necessarily perfect, solitary spheres. In the first image, taken with an atomic force microscope, lobes or waves are observed. In the second, taken with a field emission scanning electron microscope, connections between exosomes are visualized. The third, an electron microscopy image, shows the external “roughness” of the exosome due to the presence of proteins on its surface.

The U.S. company Liquid Diagnostics, in which Wong has a stake, already offers this technology, having christened it Amperial and promising “the highest specificity and sensitivity for early stage cancers” at “much lower cost.” Those most enthusiastic about the technology propose a world where a routine visit to the dentist saves lives and it is not necessary to draw blood to check if someone is ill. But experts agree that for that dream to become a reality on a large scale, more studies are still needed.

“To achieve the translation of salivary biomarkers to the clinic, it is necessary, on the one hand, to develop standardized protocols and, on the other, to carry out large multicenter studies in which the influence of different confounding variables such as age, sex or lifestyle is analyzed,” says dental scientist Óscar Rapado-González, of the Health Research Institute of Santiago de Compostela, in Spain, where he is investigating the use of saliva samples for the detection of head and neck cancers, as well as colorectal tumors.

The identification in saliva or other fluids of molecules directly or indirectly related to tumors has potential apart from early detection, says Rapado-González. It might make it possible to assess individual risk of developing cancer, predict how a tumor will evolve or monitor the therapeutic response in a noninvasive way, allowing the development of personalized medicine.

“Undoubtedly,” Rapado-González says, “more research in this field will drive progress toward the applicability of saliva in precision oncology in the coming years.”

Knowable Magazine is an independent journalistic endeavor from Annual Reviews.

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