A Diagnostic to Drool Over?

New diagnostic methods continually arise – something that is driven by our determination to detect disease as early as possible, and thus give patients the best chance of successful treatment. Although liquid biopsy testing has primarily focused on blood, an innovative approach seeks to use an even less invasive bodily fluid – saliva.

Originally trained as a dentist, David Wong has spent the last 12 years working on the use of saliva for health surveillance – a goal that he says is compelling and achievable. Despite humans producing around one liter of it every day, the diagnostic potential of saliva has not been evaluated with as much vigor as other less accessible fluids, such as plasma and spinal fluid. Wong believes that existing social and cultural stigmas surrounding saliva need to be uprooted to harness its potential for the early assessment of disease.

Within spitting distance of success

“It seems strange that, as such an abundant fluid, saliva has never been used in the same way as blood and urine in terms of liquid biopsy. There could be a whole host of reasons for this. Perhaps it is the negative social and behavioral associations with the fluid that discourage scientists from appreciating the clinical value of saliva,” says Wong. “This needs to change, and we are taking baby steps to achieve these changes in perception.” Given that the ultimate goal for early disease detection is noninvasive diagnostic testing, the fact that every one of us produces enough saliva to fill three soda cans per day makes it an appealing target. Its collection is about as noninvasive and painless for the patient as any sample could be. But Wong recognizes that accessibility is just one parameter: “Obviously, the most important aspect of any screening tool or diagnostic is performance. Although noninvasive fluids are easy to obtain, that’s irrelevant if their diagnostic ability is marginal or less than current practice.”

“Twelve years ago, the National Institutes of Health directed investment into this landscape in an attempt to give it scientific credibility and prove that saliva has clear clinical utility. And that’s how the journey first began – the effort was bolstered by the finding that mixed constituents, such as genetic material, proteins, microRNA, and microbial content, are equally as endowed in this fluid as others,” says Wong.

Wong and his team are currently focusing on the use of saliva for lung cancer detection. Shed by tumor cells and released into the blood, circulating tumor DNA (ctDNA) fragments are known to be fingerprints of human tumors. “In our first application – using saliva to detect circulating tumor targets in non-small cell lung carcinoma – the performance of saliva was as good as that of blood, and in one case even better,” (1) (2). As the ability and accuracy of liquid biopsy testing has advanced in recent years, it has set a high bar for saliva to achieve the same early detection credibility as other methods, including the traditional tissue biopsy.

Wong believes that saliva could be used for screening and risk assessment. “In cases where a computed tomography (CT) scan of someone’s chest shows that there is a mass present in the lung, the majority turn out not to be a tumor. There’s no way an image can determine whether it is, in fact, a tumor, so it is routine practice for the patient to return six months down the line for another scan. If we could obtain a drop of saliva in tandem with the radiological image and evaluate whether or not a tumor is present, it would be a game-changer in assisting clinical decisions.” The use of saliva also extends to measuring stress hormones, enzyme levels, and developmental disease biomarkers. In addition, salivary testing is highly relevant for head and neck cancer, which is rising in prevalence with nearly 690,000 new cases annually (3). Future efforts to use saliva to detect this disease are boosted by the finding that about 70 percent of head and neck cancer squamous cell carcinomas show detectable circulating tumor DNA mutant fragments (3).

In addition to the collection of saliva being truly noninvasive, only a single drop is required, which Wong considers far more practical for liquid biopsy than the use of blood. “Repeatedly drawing 10 mL of blood is not trivial, so having the ability to access a bodily fluid that i) has these discriminatory contents and ii) can be harnessed noninvasively is truly empowering. Notably, oncologists are also able to more easily access a diagnostic fluid in small children, because you can’t simply perform repeated blood draws on infants whenever you need a new sample.”

Another benefit of the technique is the speed with which it can be completed. The entire assay – from loading the sample onto a microarray detection platform to obtaining the results – can be performed within 20 minutes. Currently, saliva samples must be processed within an hour of collection from the patient, and factors such as the time of collection – due to diurnal variation – and variable consistency between patients also need to be accounted for (4). Despite these considerations, the future still looks good for salivary testing. Recent research found that an absorbent that can be inserted into the mouth, along with a filtered nozzle to remove cellular debris and salivary mucins, will facilitate easier collection, storage, and transportation of saliva (5).

“I think the next steps for us will be to continue the scientific credentialing of salivary testing by seeking clinical context that will have a big impact,” says Wong. “The most important outcome, in our opinion, is advancing toward using saliva in a specific clinical context. For instance, we could use it to monitor patients at risk of lung cancer to detect malignancy, which would then facilitate regulatory approval. That’s the ultimate goal.”

Within the next three to five years, Wong hopes that saliva will become a widely used sample type for liquid biopsy testing. He offers one potential “killer app” in the form of codon 12 mutation testing for pancreatic cancer – a disease that, at the moment, is generally diagnosed late in its course, often only after it has metastasized. Although not diagnostic, 90 percent of pancreatic cancer patients have a mutation on codon 12; early screening through saliva samples could help physicians to detect high-risk individuals.

Looking ahead, Wong hopes that the negative social and cultural connotations surrounding saliva won’t impede its development as a diagnostic tool. “Even with regulatory approval, there will inevitably be reluctance within clinics to say, ‘Now I’m going to stop drawing blood from my patients and start working on spit.’ This process won’t happen quickly, but if there’s demonstration of superior performance, clinical utility, and credible scientific data, I think people will begin to view the prospect differently. It may take another decade or two, but if saliva continues to sustain itself in terms of its performance, I think it has huge potential in years to come.”

David Wong is a Professor in the Division of Oral Biology and Medicine, and Associate Dean of Research and Director of the UCLA Center for Oral/Head and Neck Oncology Research.