Evaluation of Diagnostic Utility of a High-Risk Human Papillomavirus PCR Test on Formalin-Fixed, Paraffin-Embedded Head and Neck Tumor Tissues

Abstract

The increasing prevalence of high-risk human papillomavirus (HR-HPV)-associated head and neck squamous cell carcinoma (HNSCC) has prompted strong clinical demands for detecting HR-HPV directly in the tumor. Although p16 immunohistochemistry (IHC) has been the standard testing method, it has limitations including false positivity, lack of sensitivity in low tumor cell samples such as fine-needle aspirate (FNA), and its subjectivity. We developed a modified method based on a commercial automated HR-HPV PCR assay and evaluated the performance characteristics and the diagnostic utility of this assay for direct HR-HPV detection in the HNSCC samples. HNSCC formalin-fixed, paraffin-embedded blocks were retrieved from archives including 44 excisions, 63 biopsies, and 16 FNAs. Tissue slices were trimmed from the blocks, deparaffinized, lysed, and loaded on the commercial automated platform for HR-HPV PCR. All specimens had a concurrent p16 IHC performed. The PCR assay showed high concordance with the p16 IHC (96%; 99/103) and excellent positive agreement (91.5%) and negative agreement (100%). In addition, the PCR assay provided more conclusive results in samples with equivocal p16 IHC results. The modified commercial automated HR-HPV PCR test is a labor-efficient, quick, reliable, sensitive, and specific method for detecting HR-HPV in formalin-fixed, paraffin-embedded samples. This assay also showed excellent diagnostic utility in samples with equivocal p16 IHC results, including FNA cell blocks.

The Role of Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma

“…2 Recently, several studies have suggested that the Roche cobas 4800 (Roche Diagnostics USA, Indianapolis, Indiana) platform be used to determine HPV-positivity in formalin-fixed, paraffin-embedded tissue, although that would require further validation. [16][17][18] PREVALENCE AND TYPES OF HPV IN OROPHARYNGEAL CARCINOMAS The authors of a large international study stringently defined the criteria for HPV-positivity in head and neck cancer as a combination of positivity for HPV-DNA and either HPV E6 mRNA or p16 as biomarkers. 12 Using those criteria, they found that 22% of oropharyngeal carcinomas (anatomic site not further defined) were HPV þ .…”

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].…”

HPV Detection in Head and Neck Squamous Cell Carcinomas: What Is the Issue?

“…These techniques are known to be stable and reproducible, and a recent meta-analysis found the pooled sensitivity and specificity of HPV DNA PCR to be respectively 98 and 84% for HPV detection in OPSCCs ( 49 ). However, FFPE samples are often the only material available for molecular testing after pathological examination in the OPSCC context and only few studies have evaluated different commercial molecular assays on head and neck FFPE biopsies ( 50 – 55 ). Regarding the frequent proportion of degraded DNA in FFPE samples, some authors such as Steinau et al suggest to pretreat FFPE tissues using specific protocols to enhance DNA extraction yields before PCR assay ( 56 ).…”