Comparison of molecular assays for HPV testing in oropharyngeal squamous cell carcinomas: a population-based study in Northern Ireland.
Background Determination of HPV status has become clinically relevant for patient stratification under UICC TNM8 staging. Within the UK, a combination of p16 IHC and HPV DNA-ISH is recommended for classifying HPV status. This study will assess a series of clinically applicable second-line molecular tests to run in combination with p16 IHC to optimally determine HPV status. Methods The ability of HPV RNA-ISH, HPV DNA-ISH and HPV DNA-PCR to identify p16-positive/HPV-positive patients was investigated in a population-based OPSCC cohort of patients diagnosed in Northern Ireland from 2000-2011. Results Only 41% of the Northern Irish OPSCC patient population was associated with HPV-driven carcinogenesis. Both ISH assays were more specific than the DNA-PCR assay (100% and 95% vs 67%) and were less likely to be affected by pre-analytical factors such as increasing block age. A pooled HPV genotype probe for RNA-ISH was found to be the most accurate molecular assay assessed (95% accuracy) when compared to p16 positivity. Conclusions Our study demonstrates the advantage of tissue-based molecular assays when determining HPV status in retrospective samples. Specifically, we demonstrate the enhanced sensitivity and specificity of ISH techniques compared to PCR based methodology when working with FFPE tissue and found HPV RNA-ISH to be the most effective assay for determining HPV status. Impact As p16 IHC is a relatively inexpensive, accessible and sensitive test for stratifying patients by HPV status, this study finds more patients would benefit from first-line p16 IHC followed by specific HPV testing using HPV RNA-ISH to confirm HPV status.