p16/Ki-67 dual staining as a predictive value for cervical cancer compared to other conventional triage tools: a descriptive literature review

Cervical cancer (CC) poses a significant global health concern, ranking as the fourth most frequently diagnosed cancer and the fourth leading cause of death among women worldwide. Ecuador bears a substantial burden of CC, with a considerable number of new cases and deaths reported annually. The primary cause of CC is the human papillomavirus (HPV), a sexually transmitted virus that is usually eliminated by cell immunity. However, around 5% of infections persist and can lead to invasive cancer. This literature review assessed the predictive value of p16 and Ki-67 dual staining (DS) as a standalone method or combined with conventional triage methods to improve CC screening programs. A total of 42 relevant articles were analyzed, evaluating the performance of DS in predicting cervical intraepithelial neoplasia (CIN) of varying severities. DS exhibited a median sensitivity and specificity of 87.7% and 76.7% for detecting CIN2+ and 89.7% and 79.6% for CIN3+. When combined with liquid-based (LB) cytology, DS demonstrated superior sensitivity and specificity compared to other screening strategies. This review suggests that p16 and Ki-67 DS alone or in combination with liquid base (LB) could enhance the accuracy and effectiveness of CC screening.

Dual staining; p16; Ki-67; Cytology; Cervical cancer

[1] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2021; 71: 209–249.

[2] World Health Organization. Global strategy to accelerate the elimination of cervical cancer as a public health problem. World Health Organization: Geneva, Switzerland. 2020.

[3] Vega Crespo B, Neira VA, Ortíz Segarra J, Andrade A, Guerra G, Ortiz S, et al. Barriers and facilitators to cervical cancer screening among under-screened women in Cuenca, Ecuador: the perspectives of women and health professionals. BMC Public Health. 2022; 22: 2144.

[4] Ortiz Segarra J, Vega Crespo B, Campoverde Cisneros A, Salazar Torres K, Delgado López D, Ortiz S. Human papillomavirus prevalence and associated factors in indigenous women in Ecuador: a cross-sectional analytical study. Infectious Disease Reports. 2023; 15: 267–278.

[5] Voidăzan ST, Dianzani C, Husariu MA, Geréd B, Turdean SG, Uzun CC, et al. The role of p16/Ki-67 immunostaining, hTERC amplification and fibronectin in predicting cervical cancer progression: a systematic review. Biology. 2022; 11: 956.

[6] Public Health Ministry. National strategy for comprehensive cancer care in Ecuador. 2017. Available at: https://aplicaciones.msp.gob.ec/salud/archivosdigitales/documentosDirecciones/dnn/archivos/ac_0059_2017.pdf (Accessed: 15 November 2023).

[7] Bakir A, Alacam S, Karabulut N, Beka H, Ozluk Y, Yilmazbayhan D, et al. Evaluation of human papillomavirus genotype distribution in cervical samples. Journal of Cytology. 2021; 38: 44–49.

[8] Luria L; Cardoza-Favarato G. Human papillomavirus. StatPearls Publishing: St. Petersburg. 2023.

[9] Workowski KA, Bachmann LH, Chan PA, Johnston CM, Muzny CA, Park I, et al. Sexually transmitted infections treatment guidelines, 2021. MMWR Recommendations and Reports. 2021; 70: 1–187.

[10] Patel N, Bavikar R, Buch A, Kulkarni M, Dharwadkar A, Viswanathan V. A comparison of conventional pap smear and liquid-based cytology for cervical cancer screening. Gynecology and Minimally Invasive Therapy. 2023; 12: 77–82.

[11] Bebyn MG, Śledzińska P, Wojtysiak J, Jóźwicki W, Mierzwa T, Dziedzic J, et al. HPV RNA and DNA testing in Polish women screened for cervical cancer—a single oncological center study. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2022; 268: 129–134.

[12] Vega Crespo B, Neira VA, Ortíz Segarra J, Rengel RM, López D, Orellana MP, et al. Role of self-sampling for cervical cancer screening: diagnostic test properties of three tests for the diagnosis of HPV in rural communities of Cuenca, Ecuador. International Journal of Environmental Research and Public Health. 2022; 19: 4619.

[13] Costa S, Verberckmoes B, Castle PE, Arbyn M. Offering HPV self-sampling kits: an updated meta-analysis of the effectiveness of strategies to increase participation in cervical cancer screening. British Journal of Cancer. 2023; 128: 805–813.

[14] Rajaram S, Gupta B. Screening for cervical cancer: choices & dilemmas. Indian Journal of Medical Research. 2021; 154: 210–220.

[15] Arbyn M, Simon M, de Sanjosé S, Clarke MA, Poljak M, Rezhake R, et al. Accuracy and effectiveness of HPV mRNA testing in cervical cancer screening: a systematic review and meta-analysis. The Lancet Oncology. 2022; 23: 950–960.

[16] Xhaja A, Ahr A, Zeiser I, Ikenberg H. Two years of cytology and HPV co-testing in Germany: initial experience. Geburtshilfe Und Frauenheilkunde. 2022; 82: 1378–1386.

[17] Dovnik A, Repše Fokter A. The role of p16/Ki67 dual staining in cervical cancer screening. Current Issues in Molecular Biology. 2023; 45: 8476–8491.

[18] Nieh S, Chen S, Chu T, Lai H, Lin Y, Fu E, et al. Is p16INK4a expression more useful than human papillomavirus test to determine the outcome of atypical squamous cells of undetermined significance-categorized Pap smear? A comparative analysis using abnormal cervical smears with follow-up biopsies. Gynecologic Oncology. 2005; 97: 35–40.

[19] Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, et al. Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. International Journal of Cancer. 2001; 92: 276–284.

[20] Trzeszcz M, Mazurec M, Jach R, Mazurec K, Kotkowska-Szeps I, Kania M, et al. p16/Ki67 dual stain triage versus cytology in primary human papillomavirus-based cervical cancer screening with limited genotyping. Journal of Medical Virology. 2023; 95: e29271.

[21] Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan—a web and mobile app for systematic reviews. Systematic Reviews. 2016; 5: 210.

[22] Microsoft Excel. Available at: https://www.microsoft.com/es/microsoft-365/excel?legRedir=true&CorrelationId=a93a8a1d-8610-431d-bc41-eedde48e56ec&rtc=1?legRedir=true&CorrelationId=a93a8a1d-8610-431d-bc41-eedde48e56ec&rtc=1 (Accessed: 14 June 2023).

[23] Posit. 2024. Available at: https://posit.co/ (Accessed: 13 July 2023).

[24] Wickham H. ggplot2: elegant graphics for data analysis. 2nd edn. Springer International Publishing: Switzerland. 2016.

[25] Magri V, Wagenlehner FME, Montanari E, Perletti G. Semen analysis in chronic bacterial prostatitis: diagnostic and therapeutic implications. 2009. Available at: https://www.researchgate.net/figure/Likelihood-ratio-LR-graph-regions-of-comparison-27-The-slopes-tangents-of-the_fig1_24308427 (Accessed: 17 August 2023).

[26] Zhang S, Jia M, Zhao D, Wu Z, Guo Z, Liu Y, et al. Evaluation of p16/Ki-67 dual staining in the detection of cervical precancer and cancer in China. Cancer Epidemiology. 2019; 59: 123–128.

[27] Celewicz A, Celewicz M, Wężowska M, Chudecka-Głaz A, Menkiszak J, Urasińska E. Clinical efficacy of p16/Ki-67 dual-stained cervical cytology in secondary prevention of cervical cancer. Polish Journal of Pathology. 2018; 69: 42–47.

[28] El-Zein M, Gotlieb W, Gilbert L, Hemmings R, Behr MA, Franco EL. Dual staining for p16/Ki‐67 to detect high‐grade cervical lesions: results from the screening triage ascertaining intraepithelial neoplasia by immunostain testing study. International Journal of Cancer. 2021; 148: 492–501.

[29] Ikenberg H, Bergeron C, Schmidt D, Griesser H, Alameda F, Angeloni C, et al. Screening for cervical cancer precursors with p16/Ki-67 dual-stained cytology: results of the PALMS study. JNCI: Journal of the National Cancer Institute. 2013; 105: 1550–1557.

[30] Prigenzi K, Heinke T, Salim R, Focchi G. Dual p16 and Ki-67 expression in liquid-based cervical cytological samples compared to pap cytology findings, biopsies, and HPV testing in cervical cancer screening: a diagnostic accuracy study. Acta Cytologica. 2018; 62: 104–114.

[31] Toliman PJ, Phillips S, de Jong S, O’Neill T, Tan G, Brotherton JML, et al. Evaluation of p16/Ki-67 dual-stain cytology performed on self-collected vaginal and clinician-collected cervical specimens for the detection of cervical pre-cancer. Clinical Microbiology and Infection. 2020; 26: 748–752.

[32] Yu L, Chen W, Lei X, Qin Y, Wu Z, Pan Q, et al. Evaluation of p16/Ki-67 dual staining in detection of cervical precancer and cancers: a multicenter study in China. Oncotarget. 2016; 7: 21181–21189.

[33] Zhang R, Ge X, You K, Guo Y, Guo H, Wang Y, et al. p16/Ki67 dual staining improves the detection specificity of high-grade cervical lesions. Journal of Obstetrics and Gynaecology Research. 2018; 44: 2077–2084.

[34] Amaro-Filho SM, Golub JE, Nuovo GJ, Cunha CB, Levi JE, Villa LL, et al. A comparative analysis of clinical and molecular factors with the stage of cervical cancer in a Brazilian cohort. PLOS ONE. 2013; 8: e57810.

[35] Giorgi Rossi P, Carozzi F, Ronco G, Allia E, Bisanzi S, Gillio-Tos A, et al. p16/ki67 and E6/E7 mRNA accuracy and prognostic value in triaging HPV DNA-positive women. JNCI: Journal of the National Cancer Institute. 2021; 113: 292–300.

[36] Magkana M, Mentzelopoulou P, Magkana E, Pampanos A, Daskalakis G, Domali E, et al. The p16/ki-67 assay is a safe, effective and rapid approach to triage women with mild cervical lesions. PLOS ONE. 2021; 16: e0253045.

[37] Hu Y, Hong Z, Gu L, Xie L, Yang B, Dai H, et al. Evaluation of p16/Ki-67 dual-stained cytology in triaging HPV-positive women during cervical cancer screening. Cancer Epidemiology, Biomarkers & Prevention. 2020; 29: 1246–1252.

[38] Jiang M, Wu Z, Li T, Yu L, Zhang S, Zhang X, et al. Performance of HPV genotyping combined with p16/Ki-67 in detection of cervical precancer and cancer among HPV-positive Chinese women. Cancer Prevention Research. 2020; 13: 163–172.

[39] Luttmer R, Dijkstra MG, Snijders PJF, Berkhof J, van Kemenade FJ, Rozendaal L, et al. p16/Ki-67 dual-stained cytology for detecting cervical (pre)cancer in a HPV-positive gynecologic outpatient population. Modern Pathology. 2016; 29: 870–878.

[40] Wentzensen N, Fetterman B, Castle PE, Schiffman M, Wood SN, Stiemerling E, et al. p16/Ki-67 dual stain cytology for detection of cervical precancer in HPV-positive women. Journal of the National Cancer Institute. 2015; 107: djv257.

[41] Ovestad IT, Dalen I, Hansen E, Loge JLD, Dybdahl BM, Dirdal MB, et al. Clinical value of fully automated p16/Ki‐67 dual staining in the triage of HPV-positive women in the Norwegian cervical cancer screening program. Cancer Cytopathology. 2017; 125: 283–291.

[42] Gustinucci D, Giorgi Rossi P, Cesarini E, Broccolini M, Bulletti S, Carlani A, et al. Use of cytology, E6/E7 mRNA, and p16INK4a-Ki-67 to define the management of human papillomavirus (HPV)-positive women in cervical cancer screening. American Journal of Clinical Pathology. 2016; 145: 35–45.

[43] Ebisch RM, van der Horst J, Hermsen M, Rijstenberg LL, Vedder JE, Bulten J, et al. Evaluation of p16/Ki-67 dual-stained cytology as triage test for high-risk human papillomavirus-positive women. Modern Pathology. 2017; 30: 1021–1031.

[44] Ziemke P, Marquardt K, Griesser H. Predictive value of the combined p16 and Ki-67 immunocytochemistry in low-grade squamous intraepithelial lesions. Acta Cytologica. 2014; 58: 489–494.

[45] Pirtea L, Secosan C, Margan M, Moleriu L, Balint O, Grigoras D, et al. p16/Ki-67 dual staining has a better accuracy than human papillomavirus (HPV) testing in women with abnormal cytology under 30 years old. Bosnian Journal of Basic Medical Sciences. 2019; 19: 336–341.

[46] Bergeron C, Ikenberg H, Sideri M, Denton K, Bogers J, Schmidt D, et al. Prospective evaluation of p16/Ki-67 dual‐stained cytology for managing women with abnormal Papanicolaou cytology: PALMS study results. Cancer Cytopathology. 2015; 123: 373–381.

[47] Wright TC Jr, Stoler MH, Ranger-Moore J, Fang Q, Volkir P, Safaeian M, et al. Clinical validation of p16/Ki-67 dual-stained cytology triage of HPV-positive women: results from the IMPACT trial. International Journal of Cancer. 2022; 150: 461–471.

[48] White C, Bakhiet S, Bates M, Keegan H, Pilkington L, Ruttle C, et al. Triage of LSIL/ASC-us with p16/Ki-67 dual staining and human papillomavirus testing: a 2-year prospective study. Cytopathology. 2016; 27: 269–276.

[49] Uijterwaal MH, Polman NJ, Witte BI, van Kemenade FJ, Rijkaart D, Berkhof J, et al. Triaging HPV-positive women with normal cytology by p16/Ki-67 dual-stained cytology testing: baseline and longitudinal data. International Journal of Cancer. 2015; 136: 2361–2368.

[50] Qian Q, Zhang X, Ding B, Jiang S, Li Z, Ren M, et al. Performance of p16/Ki67 dual staining in triaging Hr-HPV-positive population during cervical Cancer screening in the younger women. Clinica Chimica Acta. 2018; 483: 281–285.

[51] Stoler MH, Baker E, Boyle S, Aslam S, Ridder R, Huh WK, et al. Approaches to triage optimization in HPV primary screening: Extended genotyping and p16/Ki-67 dual‐stained cytology—retrospective insights from ATHENA. International Journal of Cancer. 2020; 146: 2599–2607.

[52] Koo Y, Hahn H, Lee I, Lim K, Lee K, Kim H, et al. Dual immunostaining of cervical cytology specimens with atypical squamous cells for p16/Ki-67 does not exclude the existence of a high-grade squamous intraepithelial lesion. Virchows Archiv. 2013; 463: 689–696.

[53] Solares C, Velasco J, Álvarez-Ruiz E, González-Fernández L, Encinas AI, Astudillo A, et al. Expression of p16/Ki-67 in ASC-US/LSIL or normal cytology with presence of oncogenic HPV DNA. Anticancer Research. 2015; 35: 6291–6295.

[54] Trutnovsky G, Kolovetsiou-Kreiner V, Reich O. p16/Ki-67 dual-stained cytology testing may predict postpartum outcome in patients with abnormal papanicolaou cytology during pregnancy. Acta Cytologica. 2014; 58: 293–296.

[55] Ordi J, Sagasta A, Munmany M, Rodríguez‐Carunchio L, Torné A, del Pino M. Usefulness of p16/Ki67 immunostaining in the triage of women referred to colposcopy. Cancer Cytopathology. 2014; 122: 227–235.

[56] Fujii T, Saito M, Hasegawa T, Iwata T, Kuramoto H, Kubushiro K, et al. Performance of p16INK4a/Ki-67 immunocytochemistry for identifying CIN2+ in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion specimens: a Japanese gynecologic oncology group study. International Journal of Clinical Oncology. 2015; 20: 134–142.

[57] Killeen JL, Dye T, Grace C, Hiraoka M. Improved abnormal pap smear triage using cervical cancer biomarkers. Journal of Lower Genital Tract Disease. 2014; 18: 1–7.

[58] 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 Archiv. 2018; 473: 599–606.

[59] Lorenzi NPC, Termini L, Ferreira-Filho ES, Nunes RAL, Silva GAF, Lepique AP, et al. A positive HPV test with positive p16/Ki‐67 double staining in self‐sampled vaginal material is an accurate tool to detect women at risk for cervical cancer. Cancer Cytopathology. 2022; 130: 41–54.

[60] Clarke MA, Cheung LC, Castle PE, Schiffman M, Tokugawa D, Poitras N, et al. Five-year risk of cervical precancer following p16/Ki-67 dual-stain triage of HPV-positive women. JAMA Oncology. 2019; 5: 181–186.

[61] Mandal R, Ghosh I, Banerjee D, Mittal S, Muwonge R, Roy C, et al. Correlation between p16/Ki-67 expression and the grade of cervical intraepithelial neoplasias. International Journal of Gynecological Pathology. 2020; 39: 384–390.

[62] Miyamoto S, Hasegawa J, Morioka M, Hirota Y, Kushima M, Sekizawa A. The association between p16 and Ki-67 immunohistostaining and the progression of cervical intraepithelial neoplasia grade 2. International Journal of Gynecology & Obstetrics. 2016; 134: 45–48.

[63] Zhong P, Li J, Gu Y, Liu Y, Wang A, Sun Y, et al. p16 and Ki-67 expression improves the diagnostic accuracy of cervical lesions but not predict persistent high risk human papillomavirus infection with CIN1. International Journal of Clinical and Experimental Pathology. 2015; 8: 2979–2986.

[64] Yu L, Guo H, Lei X, Qin Y, Wu Z, Kang L, et al. p16/Ki-67 co-expression associates high risk human papillomavirus persistence and cervical histopathology: a 3-year cohort study in China. Oncotarget. 2016; 7: 64810–64819.

[65] Calil LN, Edelweiss MIA, Meurer L, Igansi CN, Bozzetti MC. P16INK4a and Ki67 expression in normal, dysplastic and neoplastic uterine cervical epithelium and human papillomavirus (HPV) infection. Pathology—Research and Practice. 2014; 210: 482–487.

[66] Shi Q, Xu L, Yang R, Meng Y, Qiu L. Ki-67 and p16 proteins in cervical cancer and precancerous lesions of young women and the diagnostic value for cervical cancer and precancerous lesions. Oncology Letters. 2019; 18: 1351–1355.

[67] Rodríguez-Trujillo A, Martí C, Angeles MA, Sierra A, Esteve R, Saco A, et al. Value of HPV 16/18 genotyping and p16/Ki-67 dual staining to predict progression to HSIL/CIN2+ in negative cytologies from a colposcopy referral population. American Journal of Clinical Pathology. 2018; 150: 432–440.

[68] Huo X, Sun H, Cao D, Yang J, Peng P, Kong L, et al. Evaluation of cervical high-grade squamous intraepithelial lesions-correlated markers as triage strategy for colposcopy after co-testing. OncoTargets and Therapy. 2021; 14: 2075–2084.

[69] Bowden SJ, Ellis LB, Kalliala I, Paraskevaidi M, Tighe J, Kechagias KS, et al. Protocol for a systematic review and meta-analysis of the diagnostic test accuracy of host and HPV DNA methylation in cervical cancer screening and management. BMJ Open. 2023; 13: e071534.

[70] Nayar R, Wilbur DC. The Bethesda System for reporting cervical cytology: a historical perspective. Acta Cytologica. 2017; 61: 359–372.

[71] Peeters E, Wentzensen N, Bergeron C, Arbyn M. Meta-analysis of the accuracy of p16 or p16/Ki‐67 immunocytochemistry versus HPV testing for the detection of CIN2+/CIN3+ in triage of women with minor abnormal cytology. Cancer Cytopathology. 2019; 127: 169–180.

[72] Qayoom S, Bharti A, Jaiswal R, Agarwal P, Singh R, Agarwal S, et al. Can dual staining with p16 and Ki67 be biomarkers of epithelial dysplasia in oral lesions? Journal of Cancer Research and Therapeutics. 2022; 18: 1003–1008.

[73] Hosseini MS, Talayeh M, Afshar Moghaddam N, Arab M, Farzaneh F, Ashrafganjoei T. Comparison of Ki67 index and p16 expression in different grades of cervical squamous intraepithelial lesions. Caspian Journal of Internal Medicine. 2023; 14: 69–75.

[74] Prevodnik VK, Marinsek ZP, Zalar J, Rozina H, Kotnik N, Jerman T, et al. Evaluation of the training program for p16/Ki-67 dual immunocytochemical staining interpretation for laboratory staff without experience in cervical cytology and immunocytochemistry. Radiology and Oncology. 2020; 54: 201–208.

[75] Wentzensen N, Fetterman B, Tokugawa D, Schiffman M, Castle PE, Wood SN, et al. Interobserver reproducibility and accuracy of p16/Ki-67 dual-stain cytology in cervical cancer screening. Cancer Cytopathology. 2014; 122: 914–920.

[76] Øvestad IT, Dalen I, Andersland MS, Vintermyr OK, Moltu P, Berland JM, et al. Triaging HPV-positive cervical samples with p16 and Ki-67 dual stained cytology within an organized screening program—a prospective observational study from western Norway. International Journal of Molecular Sciences. 2023; 24: 7158.

[77] Pimple SA, Mishra GA, Deodhar KK. Evidence based appropriate triage strategies for implementing high risk HPV as primary technology in cervical cancer screening. Minerva Ginecologica. 2020; 72: 96–105.

[78] Tantitamit T, Khemapech N, Havanond P, Termrungruanglert W. Cost-effectiveness of primary HPV screening strategies and triage with cytology or dual stain for cervical cancer. Cancer Control. 2020; 27: 1073274820922540.

[79] Li T, Chen S, Li X, Wu Z, Zhao Y, Cui J, et al. The features of high-risk human papillomavirus infection in different female genital sites and impacts on HPV-based cervical cancer screening. Journal of Virology. 2023; 20: 116.

[80] Kundrod KA, Jeronimo J, Vetter B, Maza M, Murenzi G, Phoolcharoen N, et al. Toward 70% cervical cancer screening coverage: technical challenges and opportunities to increase access to human papillomavirus (HPV) testing. PLOS Glob Public Health. 2023; 3: e0001982.

[81] Yu L, Chen X, Liu X, Fei L, Ma H, Tian T, et al. Significance of triple detection of p16/ki-67 dual-staining, liquid-based cytology and HR HPV testing in screening of cervical cancer: a retrospective study. Frontiers in Oncology. 2022; 12: 915418.

[82] Sengupta M, Das D, Basu K, Tirkey M, Datta C, Chatterjee U. Role of p16/Ki-67 dual immunostaining in detection of cervical cancer precursors. Journal of Cytology. 2018; 35: 153–158.

[83] Han C, Huang W, Ye M, Zou R, Lan J, Chen J, et al. HPV prevalence and genotype distribution in 2,306 patients with cervical squamous cell carcinoma in central and eastern China. Frontiers in Public Health. 2023; 11: 1225652.

[84] Egawa N. Papillomaviruses and cancer: commonalities and differences in HPV carcinogenesis at different sites of the body. International Journal of Clinical Oncology. 2023; 28: 956–964.