Molecular characterization of p16-immunopositive but HPV DNA-negative oropharyngeal carcinomas

Abstract

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...

Comparison of Polymerase Chain Reaction and Immunohistochemistry Assays for Analysing Human Papillomavirus Infection in Oral Squamous Cell Carcinoma

“…Another study stated that HPV DNA-negative but p16-immunopositive cases are actually HPV-negative, based on genetic profiling. It was revealed that the survival rates of these patients was the same as that of HPV-negative patients, which indicated the significance of testing for HPV DNA along with immunostaining to check for HPV positive oral cancers [29]. Similarly, one more study showed p16-IHC/DNA qPCR to be more sensitive and specific (97% and 94% respectively) as compared to RNA qPCR, proving to be the best contradistinguishing test for a suitable outcome.…”

Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice

“…Owing to its numerous advantages, p16 immunohistochemistry (Table 1) has emerged as a key test to define HPV status and prognosis in a clinical setting. 3,8 However, it is clearly acknowledged that ∼ 8-20% of p16-positive oropharyngeal squamous cell carcinomas are HPV-negatives [10][11][12] and this is even more problematic in other upper aerodigestive anatomical subsites, where p16's positive predictive value drops significantly. 25 This highlights the absence of a direct and exclusive link between HPV infection, p16 over-expression, and carcinogenesis and suggests that non-viral mechanisms may have a role in p16 over-expression.…”

“…3 Several authors have reported that ∼ 8-20% of p16-positive oropharyngeal squamous cell carcinomas do not contain HPV-DNA and have a molecular profile that is closer to HPV-negative cancers. [10][11][12] In this context, determining the presence and activity of HPV with a single test is very attractive. The RNAscope HPV-test (Advanced Cell Diagnostics, Hayward, CA, USA) is a recent in situ hybridization technique that detects E6/E7 mRNA from up to 18 high-risk HPVs (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82).…”

Quantitative Diffusion-Weighted MRI Parameters and Human Papillomavirus Status in Oropharyngeal Squamous Cell Carcinoma

“…However, p16 immunohistochemistry is not an ideal surrogate marker for an HPV infection because a considerable percentage of patients are p16-positive but HPV-negative. 17,28 The application of p16 protein overexpression as a surrogate marker for HPV infection could have affected their results. In our institution, we use a validated algorithm for HPV detection: p16 immunostaining, followed by a general primer 5ϩ/6 polymerase chain reaction on the p16-immunopositive cases.…”