P16 immunohistochemistry (IHC) is a diagnostic method that identifies specific cellular changes in tissue samples, primarily to assess the risk of conditions linked to human papillomavirus (HPV) infection. By detecting the p16 protein, it serves as a marker in diagnosing and managing various diseases.
What is p16 and Immunohistochemistry?
The p16 protein (p16INK4a) is a tumor suppressor that regulates the cell cycle by inhibiting cyclin-dependent kinases (CDK) 4 and 6. These kinases are involved in cell progression from the G1 to the S phase. Normally, p16 prevents uncontrolled cell division by pausing the cell cycle.
When high-risk human papillomavirus (HPV) infects cells, its E7 oncoprotein inactivates the retinoblastoma (pRb) protein, which interacts with p16. This inactivation disrupts normal cell cycle control, leading to p16 overexpression. Therefore, abundant p16 protein serves as a marker for high-risk HPV-driven cellular abnormalities.
Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to detect specific proteins within tissue sections. It relies on antibodies binding specifically to their target protein (antigen) within the tissue. Once the antibody-antigen complex forms, it is visualized using a color-producing reaction.
Visualization involves tagging the antibody with an enzyme (e.g., horseradish peroxidase) that reacts with a chromogen to produce a colored product visible under a microscope. Pathologists use this technique to identify the presence and location of the p16 protein in cells from a tissue sample. The combination of p16 and IHC detects p16 overexpression, indicating potential HPV-associated changes.
When is p16 IHC Used?
P16 IHC is frequently used in cervical cancer screening and diagnosis as a biomarker for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. It is used with Pap tests and HPV testing to clarify abnormal results. A positive p16 immunostain supports a diagnosis of high-grade squamous intraepithelial lesion (HSIL), particularly when hematoxylin and eosin (H&E) slide morphology is consistent with CIN 2 or CIN 3.
The American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines recommend p16 IHC for specific indications, especially with H&E morphology, to aid differential diagnosis. P16/Ki-67 dual-stain biomarkers help determine if an HPV infection is progressing towards cervical precancer or cancer. This dual-stain test is used for triage of HPV-positive results, especially when initial screening has limited genotyping.
P16 IHC aids in diagnosing head and neck cancers, particularly human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). These HPV-related OPSCCs often show strong p16 expression and have distinct clinical characteristics. The College of American Pathologists (CAP) guidelines recommend p16 IHC as a surrogate marker for high-risk HPV in newly diagnosed OPSCC, with a cutoff of at least 70% nuclear and cytoplasmic expression.
Beyond cervical and oropharyngeal cancers, p16 IHC has other, less common applications. It diagnoses anal cancer and other HPV-related conditions. P16 expression can also be observed in high-grade serous carcinoma of tubo-ovarian origin through non-HPV related mechanisms, requiring contextual interpretation.
Understanding Your p16 IHC Results
A positive p16 IHC result typically shows strong and diffuse staining within the nucleus and cytoplasm of cells, often indicating high-risk HPV-associated high-grade dysplasia or carcinoma. In cervical lesions, this “block-positive” pattern, where nearly all epithelial cells show strong, continuous positivity, supports a precancer diagnosis. For oropharyngeal squamous cell carcinoma (OPSCC), p16 positivity (≥70% of tumor cells with strong nuclear and cytoplasmic staining) is associated with HPV-driven carcinogenesis.
Conversely, a negative p16 IHC result or one showing only focal or patchy staining suggests the absence of high-risk HPV-driven high-grade disease. Patchy p16 staining in normal cervical squamous epithelium or immature squamous metaplasia is a non-specific finding. In cases of low-grade squamous intraepithelial lesion (LSIL), inconsistent or patchy p16 positivity supports an interpretation of low-grade disease or a non-HPV-associated pathology.
Occasionally, p16 IHC results can be ambiguous, meaning they do not fully meet “block-positive” criteria. These ambiguous results may require correlation with other tests or clinical findings for diagnosis. P16 IHC is an ancillary test; its results must be interpreted by a qualified pathologist who considers staining intensity, extent, continuity, location of p16 expression, and other clinical and pathological findings for a comprehensive diagnosis.
How the p16 IHC Test is Performed
The p16 IHC test begins with tissue sample collection, typically via biopsy or surgery. The tissue is then fixed (usually in 10% neutral buffered formalin) to preserve cellular structure. After fixation, it’s embedded in paraffin wax to create a solid block.
Thin sections (3-5 micrometers thick) are cut from the paraffin block and mounted on glass slides. Slides are deparaffinized to remove wax and rehydrate tissue. An antigen retrieval step (often using heat) exposes the p16 protein for antibody binding.
A specific p16 antibody is applied to the tissue sections to bind to p16 protein. After washing, a secondary antibody, labeled with an enzyme, is applied to detect the primary antibody. Finally, a chromogenic substrate is added, which reacts with the enzyme to produce a visible color, indicating p16 protein presence and location. A pathologist then examines the stained tissue under a microscope to interpret the results.