RNA chromogenic in situ hybridization assay with clinical automated platform is a sensitive method in detecting high-risk human papillomavirus in squamous cell carcinoma

Summary Detection of active human papillomavirus (HPV) is clinically important because its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas. Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 squamous cell carcinomas. Importantly, a clinical automated platform gives (1) consistent and reproducible results for HPV ISH and (2) better standardization across clinical laboratories. Compared with polymerase chain reaction results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%) and more specific (87% versus 74%). When only accounting for 2+-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity, whereas specificity was only 55%. In 3 cases, both RNA and DNA ISH were positive, whereas polymerase chain reaction results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed, paraffin-embedded tissues and has the potential of being a standalone clinical test.