Overexpression of p16^INK4A in liquid‐based specimens (SurePath™) as marker of cervical dysplasia and neoplasia

Abstract

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.

Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16 expression and human papillomavirus testing

“…Several studies using p16 have corroborated the above results and confirmed that nearly all high-grade CIN lesions and invasive carcinomas immunohistochemically express very high levels of p16, whereas normal and hyperplastic cervical epithelia usually do not express p16. 21,30,31 Moreover, these findings also have been confirmed in cytologic specimens using liquid-based Pap smears. However, to our knowledge, there are no systematic reports published to date examining immunohistochemical p16 expression and HPV status in both cytology and tissue samples obtained simultaneously from the same patient.…”

“…Previous studies have revealed that interobserver reproducibility of cytologic and histologic interpretations is lower in low-grade cervical lesions than in high-grade lesions. 21,22 Recently, a study on interobserver reproducibility in the histologic diagnosis of CIN reported that significant discrepancies occur in the diagnostic interpretation of low-grade lesions. 23 Therefore, both cytologic and histologic diagnoses are limited with regard to an exact diagnosis in low-grade CIN lesions, and a new biomarker for dysplastic or abnormal cervical cells is required to aid in the exact diagnosis and to evaluate a patient's prognosis.…”

“…31 In LSIL cases, the frequency of p16 expression was found to be relatively low in the current study compared with previously published data. 21,29 This may be attributed most likely to the different primary antibody used for the study.…”

Immunoreactivity of p16 in anal cytology specimens

“…Also, most prior studies in cervical cytology specimens employed antibodies from other manufacturers. 34,35,37,40 To our knowledge, the use of Hybrid Capture 2 (Digene, Gaithersburg, MD) HPV testing is not as well established in anal specimens as in cervical specimens in our institution. Therefore, we were unable to evaluate the HPV status in our patients and assess the correlation between p16 overexpression.…”

Immunochemical analysis of HPV L1 capsid protein and p16 protein in liquid-based cytology samples from uterine cervical lesions

“…11,12 Recently, a study on interobserver reproducibility for histological diagnosis of cervical intraepithelial neoplasia revealed that overexpression of p16, a tumor suppressor protein, was frequently detected in precursors and in carcinoma. 13 Therefore, p16 is a new biomarker for dysplastic or carcinoma cells of the cervix and useful for making an exact diagnosis and evaluating the patient prognosis.…”