Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 
and E7 expression in infected cells. Oncogene expression analysis is, however, 
not part of the routine diagnostic evaluation of HPV-associated oropharyngeal 
squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We 
compared the diagnostic accuracy of several methods commonly employed for HPV 
characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA 
in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, 
in order to establish if the latter should be introduced in the diagnostic 
routine to increase accuracy when fresh tissue is not available. p16 
immunostain, DNA ISH for high-risk HPV genotypes, SPF LiPA amplification and 
genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC 
samples. Twenty (48.7%) cases were positive by mRNA ISH; sensitivity and 
specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for 
SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering 
p16 immunostain as first step followed by either high-risk HPV DNA ISH or HPV16 
E6 amplification in p16-positive cases correctly characterized 90% of 
mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly 
identified all cases. While no stand-alone test was sufficiently accurate for 
classifying HPV-associated OPSCC, the high sensitivity and specificity of the 
established combination of p16 immunostain, DNA ISH, and HPV16 DNA amplification 
suggests that the introduction of labour- and cost-intensive mRNA ISH, is not 
necessary in the diagnostic routine of oropharyngeal tumors.