Assessing p16 Status of Oropharyngeal Squamous Cell Carcinoma by Combined Assessment of the Number of Cells Stained and the Confluence of p16 Staining

Abstract

Human papilloma virus (HPV) related oropharyngeal squamous cell carcinoma (OPSCC) has favorable prognosis relative to other head and neck squamous cell carcinomas. Criteria for predicting HPV status based upon p16 staining, including difficult cases with partial staining patterns, have been developed; however, clinical validation of these criteria and the clinical significance of partial p16 staining have not been reported. 81 archival OPSCC cases were initially stained for p16 by immunohistochemistry with clone G175-405. The percentage of p16+ cells and percentage of confluence of p16+ cells were categorized as 25%, 26-75% or >75%. Of all cases, 16 (20%) had partial p16 expression, with 26-75% p16+ cells. Applying previously developed criteria of >75% p16+ cells or >50% positive cells with >25% confluence, 48 (59%) patients were categorized p16+ and demonstrated expected clinical characteristics and superior disease-free survival (DFS) and overall survival (p < 0.001) compared to p16- patients. By themselves, the partial staining patients had intermediate outcomes however, separating the partial staining cases by degree of confluence showed those with >75% confluence had superior DFS (p = 0.042). When the 16 original partial staining cases were re-stained with the alternative anti-p16 E6H4 clone, p16 status remained concordant for all cases, but only 3 of the 16 were interpreted as demonstrating partial staining. This report shows the prevalence of partial p16 staining varies with the antibody utilized and clinically validates the application of a graded evaluation of both the number as well as confluence of positive cells for risk-stratification of patients with OPSCC.

Current status of clinical testing for human papillomavirus in oropharyngeal squamous cell carcinoma

“…Thus, establishing an accurate and reliable method of HPV detection in cytologic samples is critical [31]. Table 2 summarizes the many of the studies conducted in recent years comparing commercially available tests for their performance in HNSCC specimens [28,[32][33][34][35][36][37][38][39][40][41][42][43][44].…”

Application of radiomics for the prediction of HPV status for patients with head and neck cancers

“…In particular, oropharyngeal cancer patients with HPV+ status have been found to represent a subgroup with markedly better prognosis and improved response to conventional radiotherapy . Typically, HPV status testing is performed using immunohistochemistry staining to determine the expression of presence of p16, a protein‐based biomarker for the detection of viral deoxyribonucleic acid . The application of quantitative image analysis methods, such as radiomics, with reliable power in detection of the HPV status may serve as attractive alternative approaches toward determining the HPV status …”

“…[4][5][6] Typically, HPV status testing is performed using immunohistochemistry staining to determine the expression of presence of p16, a proteinbased biomarker for the detection of viral deoxyribonucleic acid. [7][8][9][10] The application of quantitative image analysis methods, such as radiomics, with reliable power in detection of the HPV status may serve as attractive alternative approaches toward determining the HPV status. [11][12][13][14] Computed tomography (CT) imaging plays a central role in early detection and prognosis for different types of cancers.…”

p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: a comparison of antibody clones using patient outcomes and high-risk human papillomavirus RNA status

“…1,6 This partial staining is a realistic problem occasionally encountered in clinical practice. [12][13][14] As p16 testing is becoming a recommended standard for oropharyngeal squamous cell carcinoma, more technical and method-based studies are needed to identify the best and most reproducible test for use in clinical practice, and as groundwork for anticipated laboratory proficiency testing. There are a number of commercially available immunohistochemical assays (antibody clones) for p16, and, although they largely have performed well at prognostication and patient risk stratification in large studies, there are some suggestions of variable performance characteristics.…”

“…There are a number of commercially available immunohistochemical assays (antibody clones) for p16, and, although they largely have performed well at prognostication and patient risk stratification in large studies, there are some suggestions of variable performance characteristics. 12,14 As there is, to our knowledge, no study directly comparing performance of different p16 antibodies in oropharyngeal squamous cell carcinoma, and because patient outcomes are the standard by which they should be evaluated, we performed a comparative study of three different commercially available p16 antibodies in a large tissue microarray cohort of patients with robust clinical follow-up information.…”