Squamous cell carcinomas represent the most common histologic entity of all anal cancers (ASCC). Human papillomavirus (HPV) DNA has been detected in up to 90% of ASCC in previous studies. The frequent accompanying overexpression of the cell cycle protein p16INK4A has indicated an etiological association with HPV in a large proportion of ASCC. However, the gold standard to prove etiological relevance of HPV in carcinogenesis is the detection of HPV E6/E7 oncogene transcripts. The aim of this study was to identify the proportion of ASCC that are etiologically driven by HPV in a German cohort of 82 ASCC. Furthermore, the diagnostic value of HPV DNA detection and p16INK4A immunohistochemistry (IHC) alone and in combination to indicate HPV-induced ASCC was analyzed.
82 patients diagnosed with ASCC at the University Hospital Heidelberg, Germany between 2000 and 2011 were included in the study. Previously, the samples had been tested for HPV DNA using Luminex technology and p16INK4A immunohistochemical expression. ASCC containing ≥50% p16INK4A-overexpressing tumor cells were considered p16INK4A-positive. A transforming relevance of HPV in the ASCC was analyzed by E6*I mRNA detection using quantitative real-time-PCR. E6*I mRNA is a splice variant product of the E6 full-length transcript which is generally highly expressed in HPV-transformed cells. Sensitivity and specificity of HPV DNA PCR and p16INK4A IHC alone or in combination to detect a transforming HPV-infection (using E6*I identification as gold standard) were analyzed.
Eighty-four percent (69/82) of the samples were tested positive for HPV 16/18 DNA. Based on IHC, 82% (67/82) of the ASCC demonstrated overexpression of p16INK4A. E6*I mRNA was identified in 79.3% (65/82) of samples. Single application of the surrogate markers p16INK4A IHC or HPV DNA showed high sensitivity (98.5% and 100.0%, respectively) and moderate specificity (82.3% and 76.5%, respectively.). Co-testing for p16INK4A IHC and HPV DNA PCR demonstrated high sensitivity (98.4%) and excellent specificity (100.0%) for a transforming High Risk-HPV-infection (HR-HPV) in ASCC.
About 80% of ASCC of the analyzed cohort were shown to be etiologically driven by HR-HPV. The exclusive detection of HR-HPV DNA or p16INK4A overexpression demonstrates moderate specificity for a transforming HPV-infection. However, the combination of the surrogate markers identifies a transforming HPV-infection in ASCC with a high sensitivity and excellent specificity. In conclusion, co-testing of p16INK4A IHC and HPV DNA PCR is suggested as a reliable test combination to identify HPV-driven ASCC with high diagnostic accuracy.