High-Risk HPV E6/E7 mRNA Detection by a Novel In-Situ Hybridization Assay Strongly Correlates with p16 Expression and Patient Outcomes in Oropharyngeal Squamous Cell Carcinoma.
Odey C Ukpo, John Flanagan, Yuling Luo, Xiao-Jun Ma, James S Lewis. Washington University in St. Louis, MO; Advanced Cell Diagnostics, Hayward, CA
Background: The association of human papillomavirus (HPV) with oropharyngeal squamous cell carcinomas (OSCC) has been well established. HPV has been detected in 60-80% of these tumors by DNA-based in-situ hybridization (DNA ISH) and/or PCR. Though these methods detect the presence of HPV, they do not provide information on active viral transcription. HPV E6/E7 mRNA is considered by many to be the best indicator of tumor HPV status, but its detection in situ has not been demonstrated. We utilized a novel RNA in-situ hybridization (RNA ISH) assay called RNAscope to detect HPV E6/E7 mRNA in OSCC and correlated the results with DNA ISH, p16 expression, and patient outcomes.
Design: We constructed tissue microarrays (TMAs) of OSCC tumors for which p16 immunohistochemistry and HPV DNA ISH were previously performed. These TMAs were used to detect E6/E7 mRNA of HPV-16 and other high risk (18,31,33,35,52, and 58) HPV types in tumor cells by a chromogenic RNAscope assay. Staining was blindly reviewed and scored for the presence of HPV E6/E7 mRNA as positive or negative. P16 immunohistochemistry was classified as positive or negative: 95% of the cases were either strongly or diffusely positive or were completely negative. HPV DNA was considered positive for any definitive nuclear staining in tumor cells.
Results: There were 192 cases. HPV E6/E7 mRNA was detected in 154 patients by RNA ISH (80.2%) and p16 was positive in 146 (76.0%). Concordance of p16 and HPV E6/E7 mRNA, p16 and HPV DNA, and HPV E6/E7 mRNA and HPV DNA was 95%, 82%, and 81%, respectively. Only one case was p16-positive, HPV E6/E7 mRNA-negative, and only 9 cases were p16-negative, HPV E6/E7-positive. Overall survival strongly correlated with HPV E6/E7 mRNA (HR= 0.24, p= 9.59E-08), HPV DNA (HR= 0.36, p=2.23E-04), and p16 (HR= 0.24, p=1.93E-07). Disease-specific survival also strongly correlated with HPV E6/E7 mRNA expression (HR= 0.25, p=3.76E-04), HPV DNA (HR= 0.25, p=5.29E-04), and p16 (HR= 0.20, p= 1.39E-05). The clinical significance of HPV E6/E7 mRNA-positive, p16-negative cases could not be defined due to the small number of cases.
Conclusions: RNA ISH is significantly more sensitive than DNA ISH in detecting HPV in OSCC. There is a very tight correlation between p16 and HPV E6/E7 mRNA, such that both were comparable in stratifying patient outcomes and both doing so more strongly than DNA ISH.
Category: Head & Neck
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 154, Monday Morning