Background Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and PCR amplification - a challenging technique that is restricted to the research laboratory. The development of RNA in-situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Methods Tissue microarrays (TMAs) containing 282 head and neck squamous cell carcinomas (HNSCCs) from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and a RNA ISH assay (RNAscope®) targeting high risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas where HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (i.e. p16 positive, DNA ISH negative). Results By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. P16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV positive tumors exhibited p16 expression, whereas only 22 of 233 HPV negative tumors were p16 positive (94% versus 9%, p < .0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16+/HPV DNA-), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. Conclusions The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.

Basaloid squamous cell carcinoma (BSCC) of the head and neck is set apart as a distinct subtype of squamous cell carcinoma on the basis of its basaloid appearance and aggressive behavior. The purpose of this study was to determine whether BSCC could be further subdivided on the basis of human papillomavirus 16 (HPV16) status. HPV16 in situ hybridization was performed on 53 BSCCs of the head and neck. Of the 53 BSCCs, 21 (40%) arose in the oropharynx and 32 (60%) arose in nonoropharyngeal sites. HPV16 was detected in 34% of BSCCs overall, but the frequency varied by site. HPV16 was detected in 16 of 21 (76%) BSCCs of the oropharynx, but in only 2 of 32 (6%) BSCCs from nonoropharyngeal sites (P<0.0001, Fisher exact). The absence of HPV16 was significantly associated with decreased overall survival (Hazard ratio=17.1; 95% confidence interval=7.2-40.3, log-rank P=0.0001), even though patients with HPV16-positive carcinomas were more likely to present with lymph nodes metastases (P=0.01, Fisher exact). Morphologic similarities aside, BSCCs are composed of a mixed group of tumors that can be separated on the basis of HPV16 status. The distinction is important. HPV16-positivity in squamous cell carcinomas of the head and neck is now recognized as a powerful indicator of improved patient survival. HPV16 detection thus permits resolution of a less aggressive component within a high-grade subtype of head and neck carcinoma.