Stratification of HPV-Induced Cervical Pathology using the Virally-Encoded Molecular Marker E4 in Combination with p16 or MCM

High-risk HPV types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16INK4a and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble non-productive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated haematoxylin & eosin image. Conventional grading of neoplasia was established by review panel, and compared directly to the composite molecular pathology visualised on the same tissue section. The detection of E4 coincided with the onset of vacuolation