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).

Clinical characteristics and outcomes of oropharyngeal carcinoma related to high‐risk non–human papillomavirus16 viral subtypes

“…One Canadian study suggests that high-risk non-HPV16 genotypes were associated with less cervical metastasis and a trend toward better disease-free survival compared with HPV16 types; however, these conclusions were based on only 5 patients with high-risk non-HPV16. 17 In both this article and the study by Gao et al, 16 only a very small number of patients with high-risk non-HPV types were identified, making it difficult to draw conclusions about relative prevalence or clinical behavior.…”

Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice

“…In their study, the RNAscope HPV-test had a sensitivity of 97% and a specificity of 93% against the reference test, with positive and negative predictive values of 91% and 98%, respectively. Recently, Gao et al 21 have developed a novel qRT-PCR method for quantification of HPV transcripts in archived tissues. They have noted that qRT-PCR and RNAscope HPV-test were 100% concordant in determining HPV status in a series of 45 OPSCC.…”

Human Papillomavirus (HPV) Biomarkers in Head and Neck Squamous Cell Carcinoma (HNSCC)

“…A similar study employing a PCR-based approach found that 57 % of tonsillar tumors were positive for HPV16 which represented a significant increase from 1993-1999 to 2006-2011, indicating an increase in total HPV-positive cancers from 25 % to 62 % (P < 0.002) (Nichols et al 2013). RT-qPCR analyses conducted on 150 oral squamous cell carcinoma tissues revealed the presence of HPV in 81.3 % of the samples (Gao et al 2013). Multiplex human papillomavirus genotyping (MPG) is a PCR assay that detects HPV DNA by utilizing a broad-spectrum general primer.…”