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Q&A: UNCs Webster-Cyriaque Discusses qPCR Assay for Common HPV Types in Genital, Oral Tissue


NAME: Jennifer Webster-Cyriaque

TITLE: Associate professor of dental ecology, School of Dentistry, University of North Carolina at Chapel Hill

BACKGROUND: PhD in microbiology and immunology and general practice residency, University of North Carolina at Chapel Hill; DDS, dentistry, State University of New York

Clinical researchers from the University of North Carolina-Chapel Hill School have developed a nested, quantitative PCR assay to specifically detect four of the most common types of disease-associated human papillomavirus and generally detect any HPV type from both genital and oral tissues.

In a paper published last month in Virology Journal, the researchers demonstrated that their assay, which targets gene sequences in HPV 6, 11, 16, and 18, as well as the highly conserved HPV E1 region, was able to detect 35 oncogenic and low-risk HPV types in benign and malignant anogenital, cutaneous, or oral specimens.

Compared to HPV anogenital and oral cancer specimens that had been previously typed using standard PCR with consensus primers, the new test had an overall sensitivity of 89 percent and specificity of 90 percent, according to the paper.

The assay, which currently comprises two separate PCR reactions, may eventually be combined into a single-tube PCR assay that can provide insight into the relationships between various HPV types and their associated diseases — not just cervical cancer, but also potentially head and neck cancers and HPV-associated genital and oral warts.

In addition, the research group is already exploring potential avenues for commercializing the test.

Corresponding author Jennifer Webster-Cyriaque, who specializes in dental medicine and microbiology and immunology, took a few moments this week to discuss the development of the test with PCR Insider. Following is an edited version of the conversation.

What was your motivation for conducting this study and developing this test?

We are a translational group doing a number of clinical trials through the oral HIV-AIDS research alliance, which is part of the AIDS Clinical Trial Group. An oral component was added to that a few years ago. And part of our scientific agenda was to characterize oral HPV infection, and to gain an understanding of the natural history and the potential for local and systemic therapeutics.

That was really the impetus, because we've seen a significant increase in HPV-associated oral disease over time. With the advent of highly active anti-retroviral drugs for HIV, we've seen an increase in the incidence of oral warts. And now we're seeing more head and neck cancers that are HPV-associated.

Why did you develop this detection method for both oncogenic and wart-causing HPV types?

We're looking a lot at oral fluids. The HPV types present there are not the same as those that have been targeted in other assay systems. Those systems are largely looking at type-specific to the anal-genital region.

We picked the two most common types associated with malignancy, 16 and 18; and two most common benign types, 6 and 11; and then we did an E1-based degenerate assay that should pick up any other types of HPV. That will allow us not to miss anything as we're looking at these different biological specimens.

Is this why it is important for your test to be able to distinguish between the more oncogenic types of HPV and the more common types that are not necessarily associated with cervical and other cancers?

And also to look between the compartments and understand the differences between the oral and genital compartment. For instance, in our clinical cohort, we saw couples who came in within a year of each other that had the exact same HPV type, which says that this can clearly be transmitted orally or sexually.

And a significant amount of literature, mostly epidemiologic, shows that people who have oral sex have a higher incidence of oral warts and oral cancer, and these may be HPV-associated. Having these kinds of tools we can actually answer these kinds of questions beyond the epidemiology, moving to more of a molecular level … to look at exactly what is happening there.

Do other commercial HPV molecular tests only assess the types most frequently associated with anal-genital tumors?

Right, those commercially available tests focus on anal-genital subtypes, and those that are highest risk. But in terms of the warts, we see a different spectrum of HPVs that come up, and that's why we did the E1-based assay.

The four HPV types you chose for this are the same four types represented in Merck's Gardasil vaccine.

Those four types are associated with the majority of HPV-associated disease, both benign and malignant. Also, we're doing a couple of clinical trials right now looking at the effectiveness of the vaccine and how it influences oral HPV shedding. There are two studies ongoing right now, one in men and one in women.

What types of PCR probes and methodologies are you using to develop these tests?

One is SYBR Green-based, the E1. The other one is TaqMan based with specific probes that target each of the four types.

Are you getting adequate sensitivity and specificity with your test? Are there any changes you could make to increase that?

One of the co-authors, Joel Palefsky from [the University of California, San Francisco], we sent him blinded specimens, and he sent us a bunch of blinded specimens. For his, the way he previously tested them was to use, I believe, nested PCR and hybridization. And we had 100 percent concordance with him. So I felt very good about our test.

Do you believe this assay will be useful in a diagnostic setting in addition to a research setting?

I think it will, and that was one reason we elected to use a qPCR assay. This technology is broadly available in many clinical laboratory settings. We know with many viruses that you see an increase in viral load prior to disease development. And because this test is quantitative, rather than a plus-minus like many other tests, this can tell us about therapeutic benefit; or whether over time we see over time if a person is at risk of development of the disease. I think that's of significant value, the quantitative nature of the test.

What is next for this research?

It's already being implemented in a number of clinical trial settings, and we're also working hard to merge these so that we have a single assay.

Meaning a single-tube assay? What are the challenges there?

Melting point. Just making sure that we are able to omtpimize it. We've been able to optimize quite well the four HPV types. It's just optimizing the E1 assay. We're working on developing probes that will work at the optimal temperature.

Do you think there is commercial potential for this, and are you exploring that?

I really hope so. I think that a test that picks up other types quickly and in a quantitative manner can be very useful. We've touched base with our [tech-transfer] office here at the university. Now we need to start talking with companies to see if they're interested.

Also, using this test as a basis, we'd really like to develop an oral-specific test. It would use all of the same elements, plus all of the types we are seeing more frequently in the oral cavity. This would be for diagnosing potential HPV-associated head and neck cancers as well as oral warts.

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