High-risk human papillomavirus detection in oropharyngeal, nasopharyngeal, and oral cavity cancers: comparison of multiple methods

Abstract

Importance: Human papillomaviruses are now recognized as an etiologic factor in a growing subset of head and neck cancers and have critical prognostic importance that affects therapeutic decision making. There is no universally accepted gold standard for high-risk HPV (hrHPV) assessment in formalin-fixed, paraffin-embedded (FFPE) tissue specimens, nor is there a clear understanding of the frequency or role of hrHPV in sites other than oropharynx.

Objective: To determine the optimal assessment of hrHPV in FFPE head and neck tumor tissue specimens.

Design, setting, participants: In the setting of a large Midwestern referral center, assessment of hrHPV by p16 immunohistochemical staining, in situ hybridization, and polymerase chain reaction (PCR)-MassArray (PCR-MA), with L1 PGMY-PCR and sequencing to resolve method discordance, was conducted for 338 FFPE oropharyngeal, nasopharyngeal, and oral cavity tumor tissue specimens. Relative sensitivity and specificity were compared to develop a standard optimal test protocol. Tissue specimens were collected from 338 patients with head and neck cancer treated during the period 2001 through 2011 in the departments of Otolaryngology, Radiation Oncology, and Medical Oncology.

Intervention: Patients received standard therapy.

Main outcomes and measures: Optimal hrHPV identification, detection, and activity in head and neck cancers.

Results: Using combined PCR-MA with L1 PGMY-PCR and sequencing for conclusive results, we found PCR-MA to have 99.5% sensitivity and 100% specificity, p16 to have 94.2% sensitivity and 85.5% specificity, and in situ hybridization to have 82.9% sensitivity and 81.0% specificity. Among HPV-positive tumors, HPV16 was most frequently detected, but 10 non-HPV16 types accounted for 6% to 50% of tumors, depending on the site. Overall, 86% of oropharynx, 50% of nasopharynx, and 26% of oral cavity tumors were positive for hrHPV.

Conclusions and relevance: PCR-MA has a low DNA (5 ng) requirement effective for testing small tissue samples; high throughput; and rapid identification of HPV types, with high sensitivity and specificity. PCR-MA together with p16INK4a provided accurate assessment of HPV presence, type, and activity and was determined to be the best approach for HPV testing in FFPE head and neck tumor tissue specimens.

Figure 1. HPV PCR-MA Mass Spectrum of HPV16 and HPV33 from a single oropharynx tumor

PCR-MassArray detection of hrHPV, illustrating an expanded area of the mass spectrum region from 5900 to 7100 Daltons, showing HPV16 (at 6071) and HPV33 (at 6757) peaks in this oropharyngeal cancer sample.

Figure 2. Venn diagrams showing concordance of all three (panel A) or two (panel B) HPV detection assays

Assay agreement among detection methods tested. Panel A: Assay agreement among 167 samples tested with all 3 methods. Panel B: Assay agreement among 171 samples tested with 2/3 methods. Circled numbers represent samples retested by L1 HPV consensus PGMY-PCR.