p16 is a marker primary used as a surrogate marker for high risk HPV infection. The physiologic role of p16 when it is expressed leads to cell cycle arrest. Normal levels are below the threshold for detection by immunohistochemical methods (IHC). Sometimes occasional non-proliferating epithelial cells may express p16 by IHC (these cells are usually in the upper aspects of the stratified epithelium.
In cervical dysplasia high risk HPV E7 leads to inactivation of pRB, and normal expression of p16 is by-passed because of the inactivation by pRB. p16 in the setting of cervical dysplasia is highly sensitive (not necessarily specific) for high grade dysplasia.
IHC Staining Patterns
- Diffuse Staining – Basal and parabasal cell layers show continuous staining.
- Focal Staining – Isolated cells or in small clusters (non-continuous)
- No Staining – Self Explanatory
Positive staining is defined as a DIFFUSE STAINING PATTERN.
Negative staining is defined as either focal staining or no staining.
Staining patterns in cervical lesions (Klaes).
|
Diagnosis
|
Negative
|
Focal
|
Diffuse
|
|
Normal
|
100%
|
–
|
–
|
|
Inflammation
|
73%
|
27%
|
–
|
|
CIN-1
|
14%
|
25%
|
61%
|
|
CIN-2
|
–
|
–
|
100%
|
|
CIN-3
|
–
|
–
|
100%
|
|
Cx. Ca.
|
3%
|
–
|
97%
|
The take home message is that p16 is highly sensitive for high grade dysplasia, but low grade dysplasia may also show positive (diffuse) staining. The grade of dysplasia is still an H&E diagnosis, but p16 is very helpful in differentiating benign mimics from dysplasia (e.g. atypical squamous metaplasia).
Other Pearls
p16 may show patchy positivity in completely normal squamous epithelium, and such staining patterns should not be used to justify a diagnosis of low grade dysplasia. p16 may randomly (and strongly) stain endocervical cells, and should not be mistaken for dysplasia. High grade mullein tumors arising in the endometrium (that may contaminate cervical specimens) are often strongly positive for p16.
Head & Neck
Tumors of the oralpharynx may be associated with high-risk HPV infection, which is driven by HPV proteins E6 and E7. This results in decreased levels of p53 and functional RN tumor-suppressor protein, and increased expression of p16INK4a (cell cycle protein).
P16 is therefore a surrogate for high-risk HPV, and is required by the AJCC 8th edition for the following head and neck tumors:
- Oropharyngeal squamous cell carcinoma
- Cervical (neck) squamous cell carcinomas of unknown primary
What is POSITIVE (for Head & Neck)
- Positivity of p16 is (commonly) defined as >70% nuclear and cytoplasmic expression
- Differences exist between p16 clones, but E6H4 appears to have the best staining characteristics. Shelton et. al showed that E6H4 performed better than JC8, which performed better than G175-405.
- 8th Ed. AJCC Staging manual uses >75% as a cutoff for positive
- 50-80% of patients with partial p16 expression have a HPV driven tumor
- Lowering the cutoff threshold results in significant false-positive cases
- Many use 50% as a cutoff for positive (particularly if using a different clone)
- Important to have close communication with clinicians, and understanding of the particular p16 clone used in one’s laboratory
- p16 expression of weak intensity or limited distribution (<75% of cells) should be staged according to p16-negative guidelines
References
Matt Quick, MD, Univeristy of Arkansas for Medical Sciences, Department of Pathology (personal communication)
Shelton J, Purgina BM, Cipriani NA, Dupont WD, Plummer D, Lewis JS. p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: a comparison of antibody clones using patient outcomes and high-risk human papillomavirus RNA status. Mod Pathol. 2017;30: 1194–1203. doi:10.1038/modpathol.2017.31
Amin, M. B., & Edge, S. B. (2017). AJCC cancer staging manual. Chicago: American Joint Committee on Cancer.
Klaes, et. al. Int. J. Cancer. 2001;92:276-284.