Modern Pathology



Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM

Heather Griffin, Yasmina Soneji, Romy Van Baars, Rupali Arora, David Jenkins, Miekel van de Sandt, Zhonglin Wu, Wim Quint, Robert Jach, Krzysztof Okon, Hubert Huras, Albert Singer and John Doorbar


Figure 7.

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Molecular principles underlying the use of p16, MCM, and E4 as HPV-associated disease biomarkers. (a) In uninfected epithelium, the cellular MCM protein (red) is usually detectable at low levels only in the basal and parabasal cell layers as a result of cell cycle stimulation by growth factors. This facilitates the phosphorylation of pRb by cyclin-dependent kinases, the release of the E2F transcription factor, and the regulated expression of MCM. During normal metaplasia or wound healing, MCM may also be detected in the upper epithelial layers. The cellular p16INK4a protein is also stimulated by E2F, but does not usually accumulate to detectable levels in uninfected epithelium. It provides feedback regulation on the activity of cyclin-dependent kinases. p16INK4a is sometimes visualized as a weak cytoplasmic stain in cells undergoing senescence (pale brown). The HPV-encoded E4 protein is never expressed in uninfected epithelium and E4 antibodies show no reactivity with cellular proteins. (bi) In HPV-infected epithelial tissue, the high-risk E6 and E7 genes (red) are expressed together from the viral early promoter (PE), and function to drive cell-cycle entry in order to allow cell proliferation and genome amplification. The high-risk E4 gene (green) is expressed from a spliced mRNA, and becomes abundant following the activation of the viral late promoter (PL) as the infected cell exits the cell cycle and commits to true differentiation. (ii) E6 and E7 are expressed at low level in the cell, but the consequences of their presence can be visualized by alterations in the presence of p16INK4a and MCM. The association of E7 with pRb leads to E2F release irrespective of growth factor stimulation. This allows MCM and also p16INK4a to accumulate to higher levels than are typically seen in uninfected epithelium where expression is dependent on cyclin-dependent kinase activation. The E7 protein also acts to increase the transcription of p16INK4a as a result of epigenetic modification of the p16INK4a promoter. In this context, p16INK4a and MCM can be used with caution as surrogate markers of E6/E7 deregulation. The viral E4 protein becomes abundant in the upper layers of HPV-infected epithelium as a result of viral late promoter activation and the cleavage of the full-length E4 protein by calpain. Calpain-cleavage exposes a C-terminal multimerization motif in E4 that allows its assembly into amyloid-like fibers. The high-level accumulation of E4 amyloid is thought to coincide with progression of the infected cell through the G2 phase of the cell cycle and eventually to cell cycle exit, explaining its appearance as MCM levels decline.