Utility of p16^sup Ink4a^ in Discriminating Between Cervical Intraepithelial Neoplasia 1 and Nonneoplastic Equivocal Lesions of the Cervix, The

Archives of Pathology & Laboratory Medicine,  May 2008  by Redman, Rachel,  Rufforny, Irina,  Liu, Chen,  Wilkinson, Edward J,  Massoll, Nicole A

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Context.-The protein p16^sup ink4a^ is overexpressed in cervical lesions associated with high-risk human papillomavirus (HPV) subtypes 16 and 18, but not in low-risk HPV subtypes 6 and 11 or non-HPV-associated cervical lesions.

Objective.-To determine whether p16^sup ink4a^ expression in equivocal cervical lesions helps distinguish atypical non- HPV changes from HPV-related changes.

Design.-One hundred ninety-one cervical lesions, including 81 cervical intraepithelial neoplasia 1, 52 squamous metaplasia, 33 cellular features suggestive of HPV-related change, 9 reserve cell hyperplasia, 4 microglandular hyperplasia, and 12 inflammatory cervicitis, were randomly selected from archival cervical biopsy specimens. All 191 samples were studied with p16^sup ink4a^ (JC8 monoclonal antibody). Reactivity for p16^sup ink4a^ was scored on a 3-tier system as follows: negative, 0% to 5% cells reactive; focal/scattered positive, greater than 5% and less than or equal to 80% cells reactive; diffuse positive, greater than 80% cells reactive. Reactivity was based on normal/reactive cervical specimens where anti-p16 antibody was negative/weakly expressed in non-cervical epithelial cells. Cervical intraepithelial neoplasia 1 lesions not reactive for p16^sup ink4a^ were investigated for the presence of high-risk HPV by real-time polymerase chain reaction.

Results.-No p16^sup ink4a^ reactivity was detected in the cervical lesions associated with atypical non-HPV change. Eleven of the cervical intraepithelial neoplasia 1 lesions showed focal/scattered reactivity expression for p16^sup ink4a^, and 19 of the CIN 1 lesions had diffuse reactivity. Fifty of 51 of the CIN 1 lesions negative for p16^sup ink4a^ were real-time polymerase chain reaction negative for the presence of high-risk HPV; 1 was real-time polymerase chain reaction positive for high-risk HPV.

Conclusions.-The data support the routine use of p16^sup ink4a^ immunohistochemical evaluation of cervical biopsy specimens for better discrimination of non-HPV-associated lesions from HPV-related lesions.

(Arch Pathol Lab Med. 2008;132:795-799)

Current literature suggests that the protein p16^sup Ink4a^ may be used as a biomarker for the identification of cervical intraepithelial neoplasia (CIN) as well as a test to decrease interobserver variability in making the diagnosis of CIN lesions.1-9 p16^sup Ink4a^ is a cell-cycle regulatory protein that negatively influences cell proliferation through a reciprocal relationship with another tumor suppressor protein, retinoblastoma gene product (pRb).1,5 The action of pRb is to inhibit cells from entering the S phase of the cell cycle and proliferating.5 It is modified via phosphorylation by cyclin D1 complexed with other cyclin-dependent kinases (cdk 4 through 6).1-3,5 In the presence of the human papillomavirus (HPV) oncoprotein E7, pRb is inactivated.5 This inactivation of pRb allows for the transcription of genes required for DNA replication and the inappropriate shifting of the cell cycle past the G1/S restriction point into the S phase.2,3,5 As pRb is functionally inactivated by HPV oncoprotein E7, there is a reciprocal overexpression of p16^sup Ink4a^.1-3,5

Marked overexpression of the p16^sup Ink4a^ protein can be demonstrated immunohistochemically using monoclonal antibodies. Studies of cervical carcinomas and preneoplastic lesions of the cervix resulting from high-risk human papillomavirus (HR-HPV) subtypes, specifically subtypes 16 and 18, have demonstrated a strong and diffuse nuclear and cytoplasmic immunoreactivity of p16^sup Ink4a^ in these lesions.2,3,5,8-10 Subtypes 16 and 18 are representative of HR-HPV and are the most clinically important HPV subtypes because infection by these subtypes is associated with an increased risk of cervical carcinoma.2,4 Cervical lesions associated with low-risk HPV subtypes, specifically HPV 6 and 11, express focal and weak p16^sup Ink4a^ immunohistochemical reactivity and are not associated with an increased risk of cervical carcinoma.2,8,9

The utilization of p16^sup Ink4a^ immunohistochemistry in the assessment of cervical intraepithelial lesions may be applicable in reducing interobserver variability in the diagnosis of CIN. Studies on interobserver variability in the histological grading and diagnosis of CIN lesions have demonstrated an overall poor interobserver agreement.11-14 Although experienced pathologists did well with invasive lesions and moderately well with high-grade CIN lesions (CIN 2/3), significant disagreement occurred in differentiating CIN 1 lesions from reactive squamous proliferations. 11,15,16

It is recognized that certain metaplastic and reactive lesions unrelated to HPV or neoplasia can mimic CIN 1 lesions.7 Some of the histologic findings that may resemble CIN 1 include squamous metaplasia, reserve cell hyperplasia, microglandular hyperplasia, and inflammatory conditions such as cervicitis.17

In order to avoid overinterpretation of CIN 1 in nonneoplastic cervical biopsy specimens, strict criteria for the diagnosis of CIN 1 lesions on cervical biopsy and cone specimens have been proposed.17 In addition to the presence of koilocytes, which has been correlated with the presence of fully assembled intranuclear HPV particles,18 other criteria, including the maintenance of an intact basement membrane with a loss of the usual maturation of the squamous epithelial cells at the surface, a 3-fold increase in nuclear size, conspicuous nuclear hyperchromasia with irregular contour of nuclear membranes, and multinucleation of cells in some cases, have been suggested.15 Despite these suggested criteria, however, discrimination of CIN 1 lesions from nonneoplastic equivocal cervical lesions is still not optimal.15,16,18