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UK Study Finds DNA qPCR-Immuno Combo Assay Superior for Diagnosing Cancer-Causing HPV Strain


Clinical researchers from the University of Liverpool have determined that a combination of DNA-based quantitative PCR and immunohistochemistry is more specific and sensitive than several other single and combination methods — including DNA qPCR alone — for diagnosing a form of head and neck cancer caused by a subtype of human papillomavirus.

The group also determined that the combination DNA qPCR/IHC test was the best discriminator of favorable outcomes in patients with the cancer, called oropharyngeal squamous cell carcinoma, or OPSCC, and could serve as an important tool in facilitating patient recruitment for clinical trials.

The study also represents the first incidence data for the HPV strain, HPV16, in OPSCC with outcome data in a UK cohort, indicating that the number of HPV-mediated OPSCC cases has increased from 14 percent to 57 percent in the last decade or so.

The researchers, led by corresponding author Andrew Schache, a research fellow and surgeon at the University of Liverpool, presented their findings this week in a Clinical Cancer Research paper.

In the absence of a true gold standard for diagnosing HPV16-mediated OPSCC, Schache and colleagues hypothesized that the most reliable way to do so would be detecting viral mRNA expression using qPCR on fresh-frozen-derived samples – specifically, with primers and TaqMan-based probes from Life Technologies' Applied Biosystems unit designed to amplify the HPV16 E6 gene and run on an ABI 7500 Fast system.

"Although this gold standard may prove valuable in a research setting, it would be logistically difficult to introduce into a routine pathology service where diagnostic algorithms are based on the assessment of formalin-fixed, paraffin-embedded tissue," the researchers wrote.

Thus, using the mRNA qPCR test as a benchmark, the researchers compared the prognostic ability and sensitivity and specificity of seven other detection methods or combinations thereof, including qPCR of HPV16 E6 DNA alone; p16 immunohistochemistry alone; and a DNA qPCR/p16 IHC combo assay.

Other tested methods included high-risk HPV in situ hybridization; an HPV ISH/p16 IHC combo; DNA/RNA qPCR; and a p16 IHC/RNA qPCR combo.

Like the RNA qPCR assay, the DNA qPCR assay featured primers and a FAM-MGB-labeled TaqMan probe designed by Applied Biosystems and optimized to specifically amplify the HPV16 E6 region using a 7500 Fast platform.

Using the above methods, Schache and colleagues tested 108 cases of HPV16-derived OPSCC. Tissue samples were collected between 1998 and 2009 and included both fresh-frozen tumor specimens and FFPE tissue. The samples were matched prior to the experiments to various demographic details including age at diagnosis, gender, subsite of tumor and, most importantly, clinical outcomes including disease-specific survival and overall survival.

The group found that of all the tests and test combos, DNA qPCR and p16 immunohistochemistry delivered the best sensitivity and specificity of 97 percent and 94 percent, respectively. The test also was the best discriminator of favorable outcome.

In addition, they observed an "acceptable" specificity of 90 percent for the HPV ISH/p16 IHC combo, but coupled with a subpar sensitivity of about 88 percent; and insufficient specificity for the other assays.

"Our data suggest that HPV16 status determination with the p16 IHC/DNA qPCR combination test offers a valuable alternative to RNA analysis, with excellent prognostic value" and sensitivity/specificity, the researchers wrote.

"As a potential alternative with [fewer] logistical constraints in routine pathology practice, the combination of p16 IHC/high-risk HPV ISH is worthy of consideration," which is consistent with the previous findings of other groups, the researchers noted.

Despite underscoring the fact that implementing qPCR along with immunohistochemistry may be somewhat more labor-intensive for labs recruiting for clinical trials, the researchers concluded that when faced with the potential "loss" of about 10 percent of eligible patients using the IHC/ISH method, "the benefits to sensitivity of using DNA or RNA qPCR assays seem to easily balance the additional logistical costs."

Both the DNA qPCR assay and immunohistochemistry assays are commercially available in proprietary and generic forms, and thus could be administered to patients routinely from now on.

"This has immediate clinical applications as we consider recruitment to clinical trials designed to de-escalate the intensity of therapy based on HPV status" Schache said in a statement.

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