P16INK4a immunohistochemistry improves the reproducibility of the histological diagnosis of cervical intraepithelial neoplasia in cone biopsies

doi:10.1016/j.ygyno.2008.06.032

Gynecologic Oncology

Volume 111, Issue 1, October 2008, Pages 120-124

https://doi.org/10.1016/j.ygyno.2008.06.032 Get rights and content

Abstract

Objective

Cervical cancer is currently the most frequently occurring cancer among women in Mexico. Mexican cervical cancer prevention programs have been unsatisfactory in part because the tests used to diagnose precursor lesions have poor reproducibility. The implementation ofspecific biomarkers may overcome these limitations. Here, we analyzed whether immunohistochemistry for p16^INK4a could improve the reproducibility of histopathological diagnoses of cervical precancerous lesions.

Methods

Serial sections of 78 specimens were stained for H&E and p16^INK4a and independently interpreted by three Mexican pathologists. Specimens were interpreted and categorized in two ways: 1) four diagnostic categories including negative lesions, CIN1, CIN2, and CIN3, or 2) two diagnostic categories; either lesions that do not require therapy (negative, CIN1), or lesions that require therapy (≥ CIN2). The agreement in diagnoses between pairs of observers was evaluated by kappa statistics.

Results

The best concordance in diagnosing was observed with two categories and p16^INK4a staining. Interestingly, the overall diagnostic discordances of higher than one CIN grade were 26.1% for H&E and 9.20% for p16^INK4a (P < 0.001). Using four diagnostic categories, weighted kappa values for each pair of observers were 0.28, 0.15, and 0.36 for H&E and 0.34, 0.35, and 0.60 for p16^INK4a stains. Using two diagnostic categories, kappa values were 0.36, 0.12, and 0.18 for H&E and 0.59, 0.70, and 0.59, p16^INK4a stains.

Conclusion

These data show that p16^INK4a immunohistochemistry substantially improved the reproducibility of interpreting histological slides. This approach may result in more accurate diagnoses and improved clinical management of patients with cervical precancerous lesions in Mexico and elsewhere.

Introduction

In Mexico, cervical cancer is the most frequent cancer among women, with a mortality rate of 8.3 per 100,000 women in 2003 [1].

Early detection and prevention programs for cervical cancer aim to identify asymptomatic pre-neoplastic lesions that require surgical management before progression to invasive cancer. Cervical biopsies are histologically classified as Cervical Intraepithelial Neoplasia grade 1 (CIN1), CIN2 and CIN3 [2]. CIN 1 is usually regarded as benign and may result from an acute Human Papilloma Virus (HPV) infection. CIN2–3 are regarded as precursors of invasive carcinomas and therapy is indicated. Despite clear morphological criteria for histopathological diagnoses of CIN, lesion classification is fraught with rather poor inter- and intra-observer reproducibility [3], [4]. Specific biomarkers represent a potential technical advance that may improve the reproducibility of interpreting staining patterns in histological sections [5].

Neoplastic transformation is triggered by the deregulated expression of two high-risk-HPV encoded oncogenes, E6 and E7, in the basal and parabasal cells of the cervical epithelium. This is accompanied by substantial overexpression of the p16^INK4a gene product [6], [7], [8]. In accordance with these observations, recent studies demonstrated that cervical cancer and most CIN2 and CIN3 expressed high levels of p16 protein (p16^INK4a) that were readily detected by immunohistochemistry [9], [10]. These findings suggested that p16^INK4a immunohistochemistry may improve the reproducibility of histopathological classification of cervical cancer precursor lesions. Several recent studies have addressed this point. A substantial improvement in diagnostic accuracy was reported from studies performed in Western Europe and North America. However, this approach has not been tested in emerging countries that suffer from high cervical cancer incidence due to inappropriate screening technology and programs. In this study, we evaluatedthe inter-observer agreement in diagnoses of lesions stained by either hematoxylin and eosin (H&E) or p16^INK4a immunohistochemistry in 78 cervical cone biopsy samples from colposcopy clinics in Guadalajara, Mexico.

Section snippets

Study design and interpretation of histological slides

Mexican women were recruited from colposcopy clinics at the Mexican Institute for Health Security, located in Guadalajara city, and underwent cervical cone biopsies. The mean age of the patients was 34.5 ± 8.5 years (18–50 years old). Seventy-eight biopsy specimens were fixed in formalin, embedded in paraffin wax, cut into thin sections, and mounted on slides. Consecutive section pairs were stained with H&E and p16^INK4a antibody, as described previously [10].

Three consultant pathologists with

Results

The overall scoring frequency and concordance among pairs of observers were analyzed. The three pathologists interpreted the H&E and p16^INK4a stained sections and classified them into four diagnostic categories: negative, CIN1, CIN2, and CIN3 (Table 1). The primary disagreement among observers was between negative lesions and CIN1. In particular, observer 1 diagnosed a majority of sections negative, independent of the staining method (76.9% of H&E and 76.3% of p16^INK4a). Consequently, observer

Discussion

The clinical management of cervical precancerous lesions is based on the histopathological diagnosis of cervical biopsies. Currently in Mexico there has been little critical evaluation of the standard diagnostic method using H&E staining and even less of diagnostic methods that include additional biomarkers like p16^INK4a. However, some studies have evaluated the agreement in scoring cytological specimens and the results are surprisingly poor [15], [16], [17], [18]. The H&E stain of cervical

Conflict of interest statement

MvKD is a share holder, advisor, and member of the Board of mtm-laboratories Inc. Heidelberg, Germany. This company produces and purchases products related to the context of this article. All other authors have no conflicts of interest to declare.

Acknowledgments

We are very grateful to Ing. Rogelio Troyo Sanroman for help in the statistical analysis. We also thank Gabriela Blancas for technical assistance.

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P16INK4a immunohistochemistry improves the reproducibility of the histological diagnosis of cervical intraepithelial neoplasia in cone biopsies

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Study design and interpretation of histological slides

Results

Discussion

Conflict of interest statement

Acknowledgments

Eur. J. Cancer

Gynecol. Oncol.

Interobserver variation in the reporting of cervical colposcopic biopsy specimens: comparison of grading systems

J. Clin. Pathol.

Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study

JAMA

Clinical applications of immunohistochemistry in gynecological malignancies

Int. J. Gynecol. Cancer

Overexpression of p16 and p14ARF isassociated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia

Pathol. Int.

Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri

Int. J. Cancer

Role of P16(INK4a) expressioninidentifying CIN2 or more severe lesions among HPV-positive patients referred for colposcopy after abnormal cytology

Cancer

p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia

Am. J. Surg. Pathol.

Natural history of cervical intraepithelial neoplasia

Clin. Obstet. Gynecol.

P16^INK4a immunohistochemistry improves the reproducibility of the histological diagnosis of cervical intraepithelial neoplasia in cone biopsies

p16^INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia