Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (p16 IHC) but lack HPV DNA. The objective of this study was to characterize this group at the molecular level by performing sensitive HPV DNA-and RNA-based PCR methods and genetic profiling. All patients diagnosed with an OPSCC in the period 2000-2006 in two Dutch university medical centers were included (n 5 841). The presence of HPV in a tumor sample was tested by p16 IHC followed by an HPV DNA GP51/61 PCR. p16 IHC scored positive in 195 samples, of which 161 were HPV DNA-positive and 34 (17%) HPV DNA-negative. In the latter group, a SPF 10 -LiPA25 assay, an HPV16 type-specific E7 PCR and an E6 mRNA RT-PCR were performed. Next, ten of these cases were further analyzed for loss of heterozygosity (LOH) of 15 microsatellite markers at chromosome arms 3p, 9p and 17p. Of the 34 p16-positive but PCR-negative OPSCCs, two samples tested positive by SPF 10 assay, HPV16 E7 PCR and HPV16 E6 mRNA RT-PCR. Three samples tested positive by SPF 10 assay but negative by the HPV16-specific assays. Nine of ten cases that were tested for LOH showed a genetic pattern comparable to that of HPV-negative tumors. This study categorizes p16-positive but HPV DNA-negative OPSCCs as HPV-negative tumors based on genetic profiling. This study highlights the importance of performing HPV testing in addition to p16 IHC for proper identification of HPV-associated OPSCCs.Head and neck squamous cell carcinoma (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx and is the sixth most common cancer worldwide, accounting for 4% of all tumors.1 Besides tobacco and alcohol consumption, high-risk human papillomavirus (HPV) is also etiologically linked to the development of HNSCCs, particularly those carcinomas that arise in the oropharyngeal region. It is widely acknowledged that HPV-attributable oropharyngeal squamous cell carcinomas (OPSCCs) have a unique biology and also have improved treatment responses and patient outcomes.2,3 Knowledge of HPV status does provide prognostic information, and more importantly, it may guide specific treatment decisions. At this moment, clinical trials are being conducted to investigate de-escalation of therapy for patients with an HPV-positive OPSCC. The hazards to patient safety of inaccurately assigning HPV-negative tumors to a HPVpositive category are clear. Therefore, a reliable HPV detection method is of major importance.The most commonly applied HPV detection methods are based on PCR amplification of HPV DNA. The problem with most of these assays is their extremely high sensitivity, which may easily identify transient infections or contaminations yielding false-positive results rather than identifying biologically relevant oncogenic infections. 4 Another approach is the use of surrogate biomarkers of t...