The detection of cervical intraepithelial neoplasia (CIN) is of increasing importance to cervical cancer screening, and has been fundamental to the overall decrease in the rate of cervical cancer incidence in the United States and around the world. The most common method for investigating CIN levels is immunohistochemistry (IHC). As has been shown in the literature, p16 and Ki-67 have been the leading biomarkers for high-risk HPV infections, which can lead to cancers of the cervix. Infections of HPV genotypes 16, 18, 31, 33, and 51 have been shown to be the leading causes of cervical cancer, and it has further been shown that there is a significant difference in diagnostic result when scoring between a CIN1 sample and a known negative. In this article, we will show how histologists and pathologists can help detect and identify CIN with these two critical biomarkers.
In a clinical histology lab, tissue blocks are received from biopsy specimens, mainly as part of the accessioning and grossing process. Briefly, the patient is entered into the clinical system and is assigned a unique case number. The tissue received is fixed in formalin and set in paraffin; the paraffin blocks are then sectioned into slides. The slides can then be subjected to an immunohistochemical stain.
In IHC, the tissue slide is incubated with a series of antibodies, with the ultimate goal of selectively staining a specific antigen that is expressed by the patient’s cells. The principle of the test involves using antibodies specific to the antigen of interest, followed by an antibody conjugated to an enzyme (for example, horseradish peroxidase, HRP) that can react with a substrate to create an insoluble colored product for visualization of the biomarker. Immunohistochemistry is widely used in the clinical lab because the test is done directly on a biopsy of the tissue in question, can be completed in under three hours, and is relatively inexpensive, and because a qualified pathologist can interpret the results as a diagnostic and/or prognostic result for the patient.
Since 1993, when p16 was first identified as a cyclin-dependent kinase inhibitor, this tumor suppressor protein has gained near-universal recognition among pathologists and oncologists alike. p16 was first implicated in human cancer cell lines through genomic analysis; researchers observed that p16 was frequently mutated, which suggested an important role in cancer development. Liggett and Sidrasky1 showed that in tumors, p16 can be inactivated by homozygous mutation, methylation of the promoter, or a point mutation. Furthermore, it has been suggested that the deletion of p16 is an early event in the progression of cancer, because the deletion of one copy of the gene is found in pre-cancerous cells. However, the expression of p16 is increased under abnormal situations, yet this increase doesn’t result in the suppression of cancer because p16 protein is inactive. Interestingly, under pre-cancerous conditions (CIN1-CIN3), p16 is upregulated and used as a biomarker for the detection of cervical cancer.
While p16 can suppress tumor progression, tumor growth and proliferation are dependent on another key transcription factor, Ki-67. During cellular interphase, Ki-67 is exclusively found in the nucleus, which gives the advantage of making it easy to “spot” in an IHC stain. Ki-67 is present during active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells. During tumorigenesis and cell growth, Ki67 is unregulated, therefore mediating the uncontrolled cell growth. These facts make Ki-67 an antigen of choice when evaluating the growth fraction of a patient’s tissue section. The approximate fraction of Ki-67 positive cells has been shown to correlate with the clinical diagnostic result. A higher percentage of proliferating cells indicates a fast-growing tumor, while a low number of Ki-67 positive cells usually indicates a pre-cancerous lesion or an early-stage cancer.2
In a routine clinical lab, p16 and Ki-67 IHC tests are often run together, side-by-side, to allow pathologists to have a full understanding of the extent of a patient’s diagnostic and prognostic outcomes. While p16 scoring can commonly be used to identify the grade or progression of the cancerous tissue, Ki-67 is used to measure the rate of cell proliferation. Combining the two offers a superior diagnostic result when compared with p16 alone or Ki-67 alone. This is reflected very well in the literature, as Zhong et al found that p16 and Ki-67 expression significantly increased with disease progression (p16, P < 0.001; Ki-67, P < 0.001).3 Clearly, p16 and Ki-67 are useful in the evaluation of progression of cervical dysplasia. Together, they are a powerful tool for evaluating cancer progression.
References
- Liggett WH Jr, Sidrasky D. Role of the p16 tumor suppressor gene in cancer. J Clin Oncol. 1998;16(3):1197-1206.
- Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182(3):311-322.
- Zhong P, Li J, Gu Y, et al. P16 and Ki-67 expression improves the diagnostic accuracy of cervical lesions but [does] not predict persistent high risk human papillomavirus infection with CIN1. Int J Clin Exp Pathol. 2015; 8(3):2979–2986.
