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p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: A systematic review and meta-analysis

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Summary

Background

P16INK4a is a biomarker for transforming HPV infections that could act as an adjunct to current cytological and histological assessment of cervical smears and biopsies, allowing the identification of those women with ambiguous results that require referral to colposcopy and potentially treatment.

Material and methods

We conducted a systematic review of all studies that evaluated the use of p16INK4a in cytological or histological specimens from the uterine cervix. We also estimated the mean proportion of samples that were positive for p16INK4a in cytology and histology, stratified by the grade of the lesion.

Results

Sixty-one studies were included. The proportion of cervical smears overexpressing p16INK4a increased with the severity of cytological abnormality. Among normal smears, only 12% (95% CI: 7–17%) were positive for the biomarker compared to 45% of ASCUS and LSIL (95% CI: 35–54% and 37–57%, respectively) and 89% of HSIL smears (95% CI: 84–95%). Similarly, in histology only 2% of normal biopsies (95% CI: 0.4–30%) and 38% of CIN1 (95% CI: 23–53%) showed diffuse staining for p16INK4a compared to 68% of CIN2 (95% CI: 44–92%) and 82% of CIN3 (95% CI: 72–92%).

Conclusion

Although there is good evidence that p16INK4a immunostaining correlates with the severity of cytological/histological abnormalities, the reproducibility is limited due to insufficiently standardized interpretation of the immunostaining. Therefore, a consensus needs to be reached regarding the evaluation of p16INK4a staining and the biomarker needs to be assessed in various clinical settings addressing specific clinical questions.

Introduction

Since the Papanicolaou (Pap test) cytological screening for cervical precancerous lesions was introduced in the 1940s, there has been a significant reduction in the incidence and mortality from cervical cancer.1 However, the efficacy of the Pap test is hampered by high interobserver variability and high false negative and false positive rates that range between 20–30%2 and 5–70%3, respectively. Technical improvements of the Pap test such as the liquid based cytology (LBC) have not been shown to improve sensitivity or specificity for detection of high-grade cervical intraepithelial neoplasia (CIN) compared to the conventional cytology.4

The introduction of human papillomavirus (HPV) DNA testing in clinical practice raised hopes for further improvements in the efficacy of the primary screening, triage and post-treatment surveillance. Randomized clinical trials published recently have demonstrated that HPV testing can be efficiently integrated into primary screening, either as an adjunct to cytology or as a sole primary test.[5], [6] It has also been shown that HPV DNA testing can be used to triage women with equivocal cytological abnormalities7 and that it has a potential role in identifying women at risk of residual or recurrent disease after treatment for CIN.[8], 9 However, it fails in the triage of low-grade lesions9 and even if implemented as a primary screening test, it would be necessary to have a more disease specific triage marker to identify women that would need to undergo colposcopy. Furthermore, a single HPV DNA test although it could confirm infection by the virus, present in 99% of all cervical cancers10, it does not discriminate between transient and chronic infection. The discrimination between the two types of infection is crucial as it is the persistent infection that predisposes to progression to cervical neoplasia and not the transient one.11

Even the histological assessment of cervical biopsies that is often considered as the “gold standard”, can be significantly hampered by intra- and inter-observer variability.12 Novel markers applied on histological specimens could improve the identification of women with ambiguous results that require treatment.

Research nowadays is focused on the development of objective biomarkers that can distinguish transforming from productive HPV infections and predict disease severity. The cellular tumor suppressor protein p16INK4a (p16) has been identified as a biomarker for transforming HPV infections. Physiologically, p16 blocks the activity of cyclin-dependent kinases CDK4/6. In a transforming HPV infection the viral oncogenes E6 and E7 interfere substantially with apoptosis and cell cycle regulation. Most importantly, E7 disrupts the protein of retinoblastoma (pRb) from its binding to E2F transcription factor and thereby promotes cell cycle progression, a molecular switch that is usually activated by CDK4/6. Affected cells strongly express p16 to counteract the irregular cell cycle activation; however, since E2F is not released through CDK4/6 action, but by E7, p16 expression has no effect on cell cycle activation. Over time, p16 accumulates in the nucleus and cytoplasm of affected cells and can be detected by immunostaining.13

This review represents an attempt to collect, systematically present and analyse the existing evidence on possible clinical applications of p16 in cytological and histological samples from the uterine cervix.

Section snippets

Search strategy

We searched two electronic databases – MEDLINE and EMBASE – targeting reports published between January 1998 and September 2007. The search strategy used terms such as “cancer”, “dysplasia”, “SIL”, “CIN”, “cervix”, “p16” and “cyclin-dependent kinase”. The references of retrieved articles together with the proceedings of relevant conferences were hand-searched in order to identify other potentially eligible studies for inclusion in the analysis missed by the initial search or any unpublished

Results

The electronic search yielded 584 studies that were assessed for inclusion in the review. Of those, 97 were potentially eligible and subsequently scrutinized in full text (Fig. 1).

Discussion

The progress in the understanding of HPV-related cervical carcinogenesis promoted the evaluation of various biomarkers that could potentially improve the current methods of cervical cancer screening. The cyclin-dependent kinase inhibitor protein p16 is one of the most promising and most studied of these biomarkers.

Immunostaining for p16 can be easily applied in both cytology and histology specimens. However, assessment of its clinical applications is seriously hampered by lack of standardized

Conclusion

The very large majority of studies on p16 immunostaining focus on the correlation between the biomarker and the degree of cytological or histological abnormality. Only a few address specific clinical questions such as the role of p16 in primary cervical cancer screening, in the triage of equivocal or low-grade smears versus the HPV-DNA test and its role as a marker of progression risk in low-grade dysplastic lesions of the cervix uteri. Furthermore, the discrepancies in the interpretation of

Conflict of interest statement

None of the authors has any financial or personal relationships with other people or organisations that could inappropriately influence (bias) their work.

Funding sources

Marc Arbyn received funding from the Gynaecological Cancer Cochrane Review Collaboration (Bath, United Kingdom) and the European Commission (Directorate of SANCO, Luxembourg, Grand-Duchy of Luxembourg) through the ECCG (European Cooperation on development and implementation of Cancer screening and prevention Guidelines, IARC, Lyon, France).

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