p16INK4a (p16) is a cyclin-dependent kinase inhibitor which has been demonstrated in published literature to show marked over-expression in cancerous and precancerous cervical tissue. mtm has developed its patent protected CINtec® products based on the p16 biomarker.

Cervical cancer is known to be caused by persistent infections of high-risk Human Papillomavirus (HR-HPV) types.However, the vast majority of HPV infections are transient and typically clear within 6-12 months. Some HR-HPV infections persist and can mediate oncogenic transformation which can lead to the development of high-grade cervical dysplasia (CIN 2/3), adenocarcinoma in situ (AIS), and invasive cervical carcinomas.

The over-expression of p16 is linked to the oncogenic transformation caused by persistent HR-HPV infection. While testing for HR-HPV cannot differentiate transient infections, which make up a majority of cases, from transforming infections, which occur less frequently, the over-expression of p16 is correlated with the start of the oncogenic transformation process which is necessary for the development of cervical cancer and its precursor lesions.

Scientific background to cellular proliferation

Cell replication is controlled through a complex mechanism involving many regulatory pathways. One of these is the retinoblastoma protein (pRB) pathway which controls cell cycle progression and cellular proliferation. Under normal conditions, the bound complex of transcription factor E2F and pRB is a critical control mechanism that prevents cells from continuously replicating and proliferating (Fig 1A). p16 normally serves as a negative regulator of cell cycle progression that blocks proliferation by promoting the interaction between E2F and pRb (Fig 1B).

A


B

Fig 1 Panel A: Normal regulation of cell cycle arrest. pRB binds to the transcription factor E2F. This pRB-E2F protein complex blocks the transcription of the genes that promote cell cycle progression and proliferation, as well as the gene that encodes for p16, a negative regulator of cell cycle progression. Panel B: Normal regulation of cell cycle progression. The release of E2F from pRB results in cell cycle progression, mitotic replication, and activation of the p16 gene, enabling p16 protein expression. p16 protein in turn facilitates the re-binding of pRB to E2F, leading to cell cycle arrest. This feedback control mechanism is key to maintaining the balance between cell cycle progression/proliferation and cell cycle arrest.

The role of p16 in oncogenic transformation

Some HR-HPV infections persist and produce levels of viral E7 oncoprotein that can mediate oncogenic transformation through the disruption of the cell cycle regulatory mechanism (Fig 2).

A

B

Fig 2. Panel A: HR-HPV E7 mediates oncogenic transformation. Panel B: Fully transformed cells are characterized by unregulated cell cycle progression, disrupted maturation, and the ability to invade underlying cervical stroma.


This transformation can lead to the development of high-grade cervical dysplasia (CIN 2/3), adenocarcinoma in situ (AIS), and invasive cervical carcinomas. The over-expression of p16 is correlated with oncogenic transformation (Fig 3).



Fig 3. In transforming cells, HPV viral oncoprotein E7 impairs the function of pRB, disrupting its ability to bind to transcription factor E2F. This leads to deregulated cell proliferation, genetic instability and p16 protein over-expression which is detectable by immunohistochemistry.

p16 over-expression is independent of HR-HPV type and patient age

The over-expression of p16 is directly correlated with oncogenic transformation regardless of which individual high-risk HPV type has infected the cervix. While there are several types of HR-HPVs, in each case the effect of their E7 oncoprotein is the same. E7 blocks pRB leading to the over-expression of p16.

Testing for HPV cannot differentiate transient infections, which make up a majority of cases, from transforming infections, which occur less frequently. Therefore, testing for HPV is of little use in pinpointing clinically significant disease especially in younger women where the prevalence of HPV infection can be as high as 30%.

p16 over-expression, however, results from E7-mediated inactivation of the pRB-E2F control mechanism, which is a central hallmark of the oncogenic process. Thus, in contrast to HPV DNA positivity, the over-expression of p16 is correlated directly with oncogenic transformation of the cervical mucosa.

CINtec p16 Kits

In order to selectively detect p16 in cervical tissue, mtm's kits make use of the clinically validated proprietary E6H4™ antibody clone, which is highly selective and sensitive to the presence of p16. Detection with E6H4™, as compared to other p16 antibody clones, does not show the cross reactivity with Trichomonas (a protozoal infection of the vagina) which gives rise to a high rate of false positives. The GMP manufactured kit components in combination with the optimized antibody ensures the reproducible quality and results in assessing a wide variety of biological specimens.