Published: 2021-06-28

Efficiency of p16 and Ki-67 immunostaining for detecting premalignant cervical lesions in high risk population

Bugra Sahin, Dagistan Tolga Arioz, Mine Kanat-Pektas, Cigdem Ozdemir, Gizem Cura


Background: This study aimed to evaluate the efficiency of immunostaining with p16 and Ki-67 in cervical cytology specimens for the detection of cervical intraepithelial neoplasia (CIN) in a high risk population.

Methods: This was a prospective review of 287 women who underwent pap smear, human papilloma virus (HPV) testing and colposcopy examination, respectively. There were cervical smear abnormalities in 108 women (37.6%) and 141 patients (49.1%) tested positive for HPV. Cervical biopsy revealed normal cervix in 28 patients (9.75%), cervicitis in 48 patients (16.72%), CIN1 in 178 patients (62.02%), CIN2 in 26 patients (9.05%) and CIN3 in 7 patients (2.43%).

Results: Positive staining for p16 had a sensitivity of 78.2% and a specificity of 97.4% while positive staining for Ki67 had a sensitivity of 80.6% and a specificity of 57.9% for distinguishing CIN lesions in cervical cytology specimens (p=0.001 for both). Concurrent positive staining for p16 and Ki67 in cervical cytology specimens had a sensitivity of 80.6% and a specificity of 97.4% for CIN lesions (p=0.001). Positive staining for p16 had a sensitivity of 94% and a specificity of 90.6% whereas positive staining for Ki67 had a sensitivity of 97% and a specificity of 33% for differentiating CIN lesions in colposcopic biopsy specimens (p=0.001 for both). Concurrent positive staining for p16 and Ki67 in colposcopic biopsy specimens had a sensitivity of 91% and a specificity of 94% for CIN lesions (p=0.001).

Conclusions: p16/Ki-67 immunostaining applied on cervical cytology specimens can screen CIN lesions with high sensitivity and specificity in a low risk population.


Cervical intraepithelial neoplasia, Colposcopy, Human papilloma virus, Immunohistochemistry, Pap smear

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Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.

Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet. 2007;370(9590):890-907.

Stiasny A, Kuhn C, Mayr D, Alexiou C, Janko C, Wiest I, et al. Immunohistochemical evaluation of E6/E7 HPV oncoproteins staining in cervical cancer. Anticancer Res. 2016;36(6):3195-8.

Hu D, Zhou J, Wang F, Shi H, Li Y, Li B. HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo. Arch Gynecol Obstet. 2015;292(6):1345-54.

Curry SJ, Krist AH, Owens DK, Barry MJ, Caughey AB, Davidson KW, et al. Screening for cervical cancer: US preventive services task force recommendation statement. JAMA. 2018;320(7):674-86.

Saslow D, Solomon D, Lawson HW, Killackey M, Kulasingam SL, Cain J, et al. American cancer society, American society for colposcopy and cervical pathology, and American society for clinical pathology screening guidelines for the prevention and early detection of cervical cancer. Am J Clin Pathol. 2012;137(4):516-42.

Wang W, Zhang XH, Li M, Hao CH, Zhao ZM, Liang HP. Association between viral loads of different oncogenic human papillomavirus types and the degree of cervical lesions in the progression of cervical Cancer. Clin Chim Acta. 2018;483:249-55.

Lambert AF, Anshau F, Schmitt. P16 expression in cervical premalignant and malignant lesions. Exp Mol Pathol. 2006;80(2):192-6.

Mills AM, Dirks DC, Poulter MD, Mills SE, Stoler MH. HR-HPV E6/E7 mRNA in situ hybridization: validation against PCR, DNA in situ hybridization, and p16 immunohistochemistry in 102 samples of cervical, vulvar, anal, and head and neck neoplasia. Am J Surg Pathol. 2017;41(5):607-15.

Brown CA, Bogers J, Sahebali S, Depuydt CE, Prins F, Malinowski DP. Role of protein biomarkers in the detection of high-grade disease in cervical cancer screening programs. J Oncol. 2012;2012:289315.

Iaconis L, Hyjek E, Ellenson LH, Pirog EC. p16 and Ki-67 immunostaining in atypical immature squamous metaplasia of the uterine cervix: correlation with human papillomavirus detection. Arch Pathol Lab Med. 2007;131(9):1343-9.

Nam EJ, Kim JW, Hong JW, Jang HS, Lee SY, Jang SY, et al. Expression of the p16 and Ki-67 in relation to the grade of cervical intraepithelial neoplasia and high-risk human papillomavirus infection. J Gynecol Oncol. 2008;19(3):162-8.

Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002;287(16):2114-9.

Wentzensen N, Schwartz L, Zuna RE, Smith K, Mathews C, Gold MA, et al. Performance of p16/Ki-67 immunostaining to detect cervical cancer precursors in a colposcopy referral population. Clin Cancer Res. 2012;18(15):4154-62.

Areán-Cuns C, Mercado-Gutiérrez M, Paniello-Alastruey I, Mallor-Giménez F, Córdoba-Iturriagagoitia A, Lozano-Escario M, et al. Dual staining for p16/Ki67 is a more specific test than cytology for triage of HPV-positive women. Virchows Arch. 2018;473(5):599-606.

Schiffman M, Wentzensen N. From human papillomavirus to cervical cancer. Obstet Gynecol. 2010;116(1):177-85.

Koeneman MM, Kruitwagen RF, Nijman HW, Slangen BF, Gorp TV, Kruse AJ. Natural history of high-grade cervical intraepithelial neoplasia: a review of prognostic biomarkers. Expert Rev Mol Diagn. 2015;15(4):527-46.

Schmidt D, Bergeron C, Denton KJ, Ridder R, European CINtec Cytology Study Group. p16/ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results from the European equivocal or mildly abnormal Papanicolaou cytology study. Cancer Cytopathol. 2011;119(3):158-66.

Waldstrom M, Christensen RK, Ornskov D. Evaluation of p16(INK4a)/Ki-67 dual stain in comparison with an mRNA human papillomavirus test on liquid-based cytology samples with low-grade squamous intraepithelial lesion. Cancer Cytopathol. 2013;121(3):136-45.

Loghavi S, Walts AE, Bose S. CINtec(R) plus dual immunostain: a triage tool for cervical pap smears with atypical squamous cells of undetermined significance and low grade squamous intraepithelial lesion. Diagn Cytopathol. 2013;41(7):582-7.

Petry KU, Schmidt D, Scherbring S, Luyten A, Reinecke-Lüthge A, Bergeron C, et al. Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki67 dual-stained cytology. Gynecol Oncol. 2011;121(3):505-9.

Sahasrabuddhe VV, Luhn P, Wentzensen N. Human papillomavirus and cervical cancer: biomarkers for improved prevention efforts. Future Microbiol. 2011;6(9):1083-98.

Ordi J, Sagasta A, Munmany M, Rodriguez-Carunchio L, Torné A, Pino MD. Usefulness of p16/Ki67 immunostaining in the triage of women referred to colposcopy. Cancer Cytopathol. 2014;122(3):227-35.

Wang SS, Trunk M, Schiffman M, Herrero R, Sherman ME, Burk RD, et al. Validation of p16INK4a as a marker of oncogenic human papilloma virus infection in cervical biopsies from a population-based cohort in Costa Rica. Cancer Epidemiol Biomarkers Prev. 2004;13(8):1355-60.

Xing Y, Wang C, Wu J. Expression of geminin, p16, and Ki67 in cervical intraepithelial neoplasm and normal tissues. Medicine (Baltimore). 2017;96(26):7302.

Kanthiya K, Khunnarong J, Tangjitgamol S, Puripat N, Tanvanich S. Expression of the p16 and Ki67 in cervical squamous intraepithelial lesions and cancer. Asian Pac J Cancer Prev. 2016;17(7):3201-6.

Aslani FS, Safaei A, Pourjabali M, Momtahan M. Evaluation of Ki67, p16 and CK17 markers in differentiating cervical intraepithelial neoplasia and benign lesions. Iran J Med Sci. 2013;38(1):15-21.

Conesa-Zamora P, Doménech-Peris A, Orantes-Casado FJ, Ortiz-Reina S, Sahuquillo-Frías L, Acosta-Ortega J, et al. Effect of human papillomavirus on cell cycle-related proteins p16, Ki-67, Cyclin D1, p53, and ProEx C in precursor lesions of cervical carcinoma: a tissue microarray study. Am J Clin Pathol. 2009;132(3):378-90.

Cavalcante DM, Linhares LM, Pompeu ML, Giraldo PC, Eleuterio J. The utility of p 16INK4a and Ki-67 to identify hig-grade squamous intraepithelial lesion in adolescents and young women. Indian J Med Microbiol. 2012;55(3):339-42.

Jackson JA, Kapur U, Ersahin C. Utility of p16, Ki67, and HPV test in diagnosis of cervical intraepithelial neoplasia and atrophy in women older than 50 years with 3- to 7-year follow-up. Int J Surg Pathol. 2012;20(2):146-53.

Zhu Y, Ren C, Yang L, Zhang X, Liu L, Wang Z. Performance of p16/Ki67 immunostaining, HPV E6/E7 mRNA testing, and HPV DNA assay to detect high-grade cervical dysplasia in women with ASCUS. BMC Cancer. 2019;19(1):271.