A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis

Abstract

Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).

Figure 1

The expression profiles of HPV E6/E7 and functionally-related human genes in 150 oropharyngeal tumors. (A) The expression profiles of HPV E6 and E7 transcripts were determined by real-time RT-PCR. (B–F) The average expression of E6 and E7 transcripts was used to represent the expression of HPV in each tumor. Each data point in the graph represents one tumor sample, with the x-axis representing normalized HPV expression and the y-axis representing normalized expression of a human gene.

Figure 2

HPV and p16 have consistent expression profiles in oropharyngeal cancer. (A) Correlation of HPV RT-qPCR profile and p16 RT-qPCR profile. A normalized expression value of 4 or higher was used as the threshold to define high p16 mRNA expression. (B) In situ hybridization for high-risk HPV DNA and p16 immunohistochemistry. A tumor that is strongly positive for HPV DNA by in situ hybridization showing granular, blue staining in tumor cell nuclei; a tumor that is strongly and diffusely positive for p16 with strong nuclear and cytoplasmic staining in the tumor cells. (C) Correlation of HPV RT-qPCR profile, HPV DNA ISH profile and p16 IHC profile. (D) Correlation of p16 RT-qPCR profile, HPV DNA ISH profile and p16 IHC profile.

Figure 3

Kaplan-Meier survival analysis to evaluate the prognostic value of HPV and p16 expression status in 150 tumors as revealed by RT-qPCR. (A) HPV expression and overall survival. (B) HPV expression and disease-free survival. (C) p16 expression and overall survival. A normalized expression value of 4 or higher was used as the threshold to define high p16 mRNA expression. (D) p16 expression and disease-free survival. The p-values were calculated with the log-rank test.