p16: a protein of significance

In some persistent high-risk HPV (hrHPV) infections, certain hrHPV oncogenes trigger the disruption of cellular mechanisms, altering normal control of the cell cycle. This leads to deregulated cell reproduction, with stimulation of cell division and replication.1

This is the molecular process that can drive the transformation from persistent HPV infection to pre-cancer and, ultimately, cancer.

HPV infection: a closer look

The key to the utility of p16 diffuse immunostaining is that it is only overexpressed as a consequence of transforming HPV infection.

The cell-cycle protein p16INK4a (also referred to as p16) is a key marker of this process. As such, it has been identified and clinically validated as a biomarker of HPV-mediated transforming disease.1-3

p16 in HPV transforming disease

In cervical dysplasia, HPV oncoproteins interfere with cell-cycle control. This disruption may lead to cell-cycle deregulation and oncogenic transformation, resulting in the overexpression of p16.1

Click on the figure to explore this process.

Watch the video on transforming HPV infection

Transforming HPV infection: a closer look
transforming_HPV_infectionUpon infection with HPV, viral DNA is introduced into cervical basal cells. The virus replicates in the differentiated cell layers and new infectious particles are produced.4
transforming_HPV_infectionIn transforming HPV infections the viral oncoprotein E7 starts to interfere with the cell-cycle control mechanism.1
transforming_HPV_infectionViral oncoprotein E7 binds to pRB preventing E2F from binding. As a result, the cell cycle becomes permanently stuck in the “on position,” and p16 is overexpressed.3-5This marks the onset of the transforming process.

Clinical utility

p16 overexpression has been found to be associated with HPV-mediated transforming infections that may potentially lead to development of cervical pre-cancer, and if untreated, cervical cancer.

p16 overexpression is detectable by immunohistochemical staining within cervical biopsy tissues. Therefore, p16 immunohistochemisty may help identify transforming lesions with oncogenic potential.6A positive p16 immunostain result is demonstrated by a “diffuse immunostain pattern.” Negative p16 immunostain results are demonstrated as either a “focal immunostain pattern” or no immunostaining (see figure).

p16 immunohistology: staining patterns in cervical tissue

Diffuse PatternFocal PatternNegative Pattern

p16 positive
A continuous staining of cells of the basal and parabasal cell layers of the cervical squamous epithelium, with or without staining of cells of intermediate and superficial cell layers.3
p16 negative
Non-continuous staining of isolated cells or small cell clusters, particularly not of the basal and parabasal cells.7
p16 negative

Download a collection of cervical histology images

Download a guide to interpreting cervical stains

Histologic analysis is important in confirming the presence or absence of disease.

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p16 cytology versus dual staining

Conversely from histology, there are limitations to using p16 as a single biomarker in cytology. Specifically, when cells are collected for cytologic examination, the context of the tissue morphology is lost, and it is not known from where each cell originated. It is critical to distinguish between proliferating parabasal cells from dysplastic cells, and thus determine which cells would actually contribute to the development of pre-cancerous dysplasia.9

Researchers have addressed this issue by devising a dual biomarker approach that incorporates both p16 and Ki-67, the latter of which is a common indicator of cell proliferation. The co-detection of p16 and Ki-67 in the same cell serves as an indicator of cell-cycle de-regulation that occurs during hrHPV-mediated oncogenic transformation.9

Learn more about p16/Ki-67 co-expression