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Australian Team Developing Saliva-Based Assay for HPV-Driven Oropharyngeal Cancer


NEW YORK ─ An Australian research team is developing a DNA-based saliva test that it believes could one day become a screening tool to detect hidden oropharyngeal cancers in patients that are asymptomatic.

Reporting their findings recently in Frontiers in Oncology, the researchers said that they used an internally developed molecular assay to conduct serial measurements of HPV-16 DNA in the saliva of 650 study participants.

The investigators ─ involving researchers at the University of Queensland, Royal Brisbane and Women's Hospital, and Queensland University of Technology ─ observed that the HPV-16 biomarker fell to undetectable levels in saliva after surgeons removed cancerous tumors from a high-risk study participant that they had been tracking for three years.

Though the report does not provide enough evidence that the test can be used as a screening tool, it demonstrates that a screened individual can receive "significantly less morbid treatment than would be required for the standard presentation at a more advanced stage," the researchers said.

Further, the study points to a need to conduct a broad, long-term study to evaluate the clinical utility of the biomarker and assay for throat cancer screening, said Chamindie Punyadeera, head of the saliva and liquid biopsy translational laboratory at Brisbane, Australia-based Queensland University of Technology (QUT), and one of the study's lead investigators.

"HPV-driven oropharyngeal cancers are on the rise, but we have no screening tests or programs like there are for cervical cancer," Punyadeera said.

The detection of HPV-16 levels in cervical tissue is frequently used as an indicator of high-risk HPV-related cervical cancer.

People have said that it may not be possible to noninvasively screen for oropharyngeal cancers because patients are generally asymptomatic and the cancer is hidden, but the results of the study suggest otherwise, Punyadeera said.

Their study, initiated to evaluate the prevalence of high-risk HPV in the general population and funded by Johnson & Johnson's Janssen Pharmaceuticals, involved testing of healthy study participants in Queensland for a year and a half.

The investigators developed and used a molecular assay to detect HPV-16 DNA in saliva samples. Punyadeera and her colleagues used Qiagen's QIAmp DNA Mini Kit for DNA extraction from a salivary oral rinse and tonsillar tissue samples, and Thermo Fisher's QuantStudio 7 Flex Real-Time PCR System for detection of HPV-16 DNA.

Though the study was not intended to be interventional, the investigators found three asymptomatic people that had a persistent oral HPV-16 infection. One person continues to be monitored by the clinicians while a second has had abnormal mucosal lesions removed. The third person ─ a man in his mid-60s ─ exhibited a steadily rising HPV-16 viral load in his salivary oral rinse samples over three years of intermittent testing.

Following a consultation with clinicians, the patient agreed to have both tonsils removed. Afterward, the HPV-16 DNA levels in his saliva dropped to where they were undetectable, and the diagnosis of oropharyngeal cancer was confirmed by histology, Punyadeera said.

Despite the promising result for this patient, the Australian researchers still have much work ahead before they could initiate routine screening.

The group would need to "demonstrate that screening reduces mortality from the cancer for it to be implemented as public policy," said Maura Gillison, a professor of thoracic/head and neck medical oncology at the University of Texas MD Anderson Cancer Center, who is not affiliated with the QUT research. "Finding one case such as this is very, very far from the high bar one needs to leap to establish policy," Gillison said.

Further, the perception of the clinical need for screening needs to reflect competing priorities, such as the need for screening for other cancers, Gillison added.

The estimate for new US cases of oral cavity and pharynx cancer from all causes in 2020 is 53,260, 2.9 percent of new cancer cases, according to the National Institutes of Health National Cancer Institute (NCI). By comparison, this year, the estimate for new US cases of cervical cancer, which is caused by an HPV infection, is 13,800, and accounts for 0.8 percent of all new cancer cases.

Diagnostic screening programs and vaccination have played an important role in cervical cancer prevention and driving down the rate of new diagnoses, according to NCI.

Punyadeera and her colleagues, with the belief that similar screening is needed for oropharyngeal cancers, is seeking funding for a four-year 1,000-patient study to validate the clinical utility of her team's molecular assay, a project that could cost around AU$1 million ($656,455).

In a 2019 study published in Cancers, Punyadeera and colleagues ─ including Ian Fraser, a developer of technology that became the basis of vaccines against HPV to prevent cervical cancer ─ reported that the same saliva-based DNA assay achieved 80 percent sensitivity and 91 percent specificity, using the expression of p16 in tissue as a comparison test. 

According to William Coman, a trustee and foundation chair of the Melbourne-based Garnett Passe & Rodney Williams Memorial Foundation, there is enough evidence to launch a broader study to further evaluate the potential for this type of test for screening. Though the foundation did not fund the current study, it has funded some of Punyadeera's diagnostics research in the past, and Coman ─ an emeritus professor at the University of Queensland ─ continues to work with her as an advisor.

The test, which shows a positive predictive value of about 96 percent, is the kind of screening tool that people working to advance the field of head-and-neck cancer diagnostics and treatment have been waiting for, Coman said.

"It is very difficult to find the primary cancer site in the head and neck," said Coman, who is also an otolaryngology, head, and neck surgeon. "When a noninvasive test you trust tells you that a patient has a tumor, you can consider a more aggressive treatment, such as in this case removing the tonsils."

Apart from its associated health benefits, the screening test used for early detection could also contribute significant cost savings, Coman said. An inexpensive DNA laboratory test completed after a patient provides a saliva sample by mail or at a clinic can lead to an early intervention that costs a few thousand dollars, he noted. By comparison, treating a patient with oropharyngeal cancer that has spread to lymph nodes can cost AU$250,000, he said.

Oropharyngeal cancers also have a high rate of recurrence after treatment, and the test used in the recent study could be deployed to continuously monitor patients, Coman said.

If the clinical utility of the assay can be demonstrated in the long-term study, the tool could easily be deployed for screening by labs that specialize in laboratory-developed tests, Punyadeera said. That will take at least four years because patients must be tracked for a long time during the study, she said.