Objective: This study evaluated the usefulness of p16INK4a/Ki-67 as a new biomarker in the diagnosis of human papillomavirus (HPV)-related cervical lesions. Study Design: From 69 women with previous positive cytology, clinician-collected (CC) samples were obtained using a Cervex-Brush®. One month later, self-collected (SC) material was acquired using a Rovers® Viba-Brush. Liquid-based cytology specimens were prepared from both samples, and then the grades of squamous intraepithelial lesions (SIL) were determined; following immunostaining with CINtec® PLUS, HPV status was analyzed using a linear array. Results: The mean double-positive cell scores (SCORE) in the CC samples were 3.2 in samples negative for intraepithelial lesions or malignancy, 1.3 in atypical squamous cells of undetermined significance, 87.1 in low-grade SIL, and 367 in high-grade SIL. According to HPV risk type, the mean SCORE was 16 in samples negative for HPV, 8.4 in low-risk HPV, 143 in high-risk HPV other than type 16, and 420 in HPV16 – with a statistical significance between high-risk HPV and type 16 (p < 0.05). The SCORE of the SC group was lower than that of the CC group because of the limited number of cells collected. Conclusions: The SCORE showed a significant correlation with HPV16 compared with lesser-degree lesions. CINtec PLUS is useful as a diagnostic marker of progression of lesions and a surrogate marker of an elevated risk of high-grade lesions with HPV16.

Koutsky LA: Epidemiology of genital human papillomavirus infection. Am J Med 1997;102:3–8.
Association of Reproductive Health Professionals: Health and Sexuality. 2005, p 10. www.arhp.org.
National Institutes of Health. NIH consensus Statement: Cervical Cancer. 1996, vol. 14. http://consensus.nih.gov/1996/1996CervicalCancer102PDF.pdf.
Mathew A, George PS: Trends in incidence and mortality rates of squamous cell carcinoma and adenocarcinoma of cervix-worldwide. Asian Pac J Cancer Prev 2009;10:645–650.
Vizcaino AP, Moreno V, Bosch FX,Muñoz N, Barros-Dios XM, Borras J, Parkin DM: International trends in incidence of cervical cancer. II. Squamous-cell carcinoma. Int J Cancer 2000;86:429–435.
Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJF, Vaccarella S, Anh PTH, Ferreccio C, Hieu NT, Matos E, Molano M, Rajkumar R, Ronco G, de Sajose S, Shin HR, Sukvirach S, Thomas JO, Tunsakul S, Meijer CJLM, Franceschi S, The IARC HPV Prevalence Surveys Study Group: Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366:991–998.
Dutra I, Santos MR, Soares M, Couto AR, Bruges-Armas M, Trixeira F, Monjardino L, Hodgson S, Bruges-Armas J: Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island. Infect Agent Cancer 2008;3:1–5.
Clifford GM, Smith JS, Plummer M, Muñoz N, Franceschi S: Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63–73.
Jacob MV, Walboomers JM, Snijders PJF, Voorhorst FJ, Verheijen RHM, Fransen-Daalmeijer N, Meijer CJML: Distribution of 37 mucosotropic HPV types in women with cytologically normal cervical smears: the age-related patterns for high-risk and low-risk types. Int J Cancer 2000;87:221–227.
Inoue M, Sakaguchi J, Sasagawa T, Tango M: The evaluation of human papillomavirus DNA testing in primary screening for cervical lesions in a large Japanese population. Int J Gynecol Cancer 2006;16:1007–1013.
Sasagawa T, Dong Y, Saijoh K, Satake S, Tateno M, Inoue M: Human papillomavirus infection and risk determinants for squamous intraepithelial lesion and cervical cancer in Japan. Jpn J Cancer Res 1997;88:376–384.
Cuzick J, Sasieni P, Davies P, Adams J, Normand C, Frater A, van Ballegooijen M, van den Akker E: A systematic review of the role of human papillomavirus testing within a cervical screening programme. Health Technol Assess 1999;3:1–196.
Schiffman M, Herrero R, Hidesheim A, Sherman ME, Bratti M, Wacholder S, Alfaro M, Hutchinson M, Morales J, Greenberg MD, Lorincz AT: HPV DNA testing in cervical cancer screening: results from women in a high-risk province of Costa Rica. JAMA 2000;283:87–93.
Herrero R, Castle PE, Schiffman M, Bratti MC, Hildesheim A, Moraled J, Alfaro M, Sherman ME, Wacholer S, Chen S, Rodriguez AC, Burk RD: Epidemiologic profile of type-specific human papillomavirus infection and cervical neoplasia in Guanacaste, Costa Rica. J Infect Dis 2005;191:1796–1807.
de Villers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Classification of papillomaviruses. Virology 2004;324:518–527.
Coutlée F, Rouleau D, Petignat P, Georges G, Kornegay JR, Schlag P, Boyle P, Hankins C, Vezina S, Cote P, Macleod J, Voyer H, Forest P, Walmsley S, The Canadian Women’s HIV Study Group, Franco E: Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the linear array HPV genotyping test. J Clin Microbiol 2006;44:1998–2006.
Dunn ST, Allen RA, Wang S, Walker J, Schiffman M: DNA extraction: an understudied and important aspect of HPV genotyping using PCR-based methods. J Virol Methods 2007;143:45–54.
Steinau M, SwanDC, Unger ER: Type-specific reproducibility of the Roche linear array HPV genotyping test. J Clin Virol 2008;42:412–414.
Woo YL, Damay I, Stanley M, Crawford R, Sterling J: The use of HPV linear array assay for multiple HPV typing on archival frozen tissue and DNA specimens. J Virol Methods 2007;142:226–230.
Serrano M, Hannon GJ, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704–707.
Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, Dallenbach-Hellweg G, Schmidt D, von Knebel M: Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer 2001;92:276–284.
Sano T, Oyama T, Kashiwabara K, Fukuda T, Nakajima T: Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions. Am J Pathol 1998;153:1741–1748.
Nakao Y, Yang X, Yokoyama M, Ferenczy A, Tang SC, Pater MM, Pater A: Introduction of p16 during immortalization by HPV 16 and 18 and not during malignant transformation. Br J Cancer 1997;75:1410–1416.
zur Hauzen H: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2:342–350.
Meyer JL, Hanlon DW, Rasmussen OF, Bisgaard K: Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay. Caner Cytopathol 2007;111:83–92.
Bibbo M, Klump WJ, DeCocco J, Kovatich AJ: Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens. Acta Cytologica 2002;46:25–29.
Saqi A Pasha TL, McGrath CM, Yu GH, Zhang P, Gupta P: Overexpression of p16INK4a in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia. Diagn Cytopathol 2002;27:365–370.
Yoshida T, Sano T, Kanuma T, Owada N, Fukuda T, Nakajima T: Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16INK4a expression and human papillomavirus testing. Cancer Cytopathol 2004;25:100–108.
Sahebali S, Depuydt CE, Segers K, Moeneclaey LM, Vereecken AJ, van Marck E, Bogers JJ: P16INK4a as an adjunct marker in liquid-based cervical cytology. Int J Cancer 2004;108:871–876.
Keating JT, Cviko A, Riethdorf S, Riethdorf L, Quade BJ, Sun D, Duensing S, Sheets EE, Munger K, Crum CP: Ki-67, cycline E, and p16INK4A are complementary surrogate biomarkers for human papillomavirus-related cervical neoplasia. Am J Surg Pathol 2001;25:884–891.
Bulten J, van der Laak JA, Gemmink JH, Pahlplatz MMM, de Wilde PC, Hanselaar AGJM: MIB, a promising marker for the classification of cervical intraepithelial neoplasia. J Pathol 1996;178:268–273.
Zeng Z, Del PG, Cohen JM, Mittal K: MIB-1 expression in cervical Papanicolaou tests correlates with dysplasia in subsequent cervical biopsies. Appl Immunohistochem Mol Morphol 2001;10:15–19.
Dunton CJ, Van Hoeven KH, Kavatich AJ, Oliver RE, Scacheri RQ, Cater JR, Carlson Jr JA: Ki-67 antigen staining as an adjunct to identifying cervical intraepithelial neoplasia. Gynecol Oncol 1997;64:451–455.
Sahebali S, Depuydt CE, Segers K, Moeneclaey LM, Vereecken AJ, van Marck E, Bogers JJ: Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool? J Clin Pathol 2003;56:681–686.
Wright TC, Schiffman M: Adding a test for human papillomavirus DNA to cervical-cancer screening. N Eng J Med 2003;348:489–490.
Kruse AJ, Skaland I, Janssen EA, Buhr-Widhagen S, Kios J, Arends MJ, Baak JP: Quantitative molecular parameters to identify low-risk and high-risk early lesions: role of marker proliferative activity and differentiation and Rb availability. Int J Gynecol Pathol 2004;23:100–109.
Maeda MY, Simoes M, Wakamatsu A, Longatto Filho AL, Oyafuso M, de Mello ES, Otta MM, Alves VA: Relevance of the rates of PCNA, Ki-67 and p53 expression according to the epithelial compartment in cervical lesions. Pathologica 2001;93:189–195.
dePutte GV, Kristensen GB, Lie K, Baekelandt M, Holm R: Cyclins proliferation markers in early squamous cervical carcinoma. Gynecol Oncol 2004;92:40–46.
McCluggage WG, Maxwell P, McBride HA, Hamilton PW, Bharucha H: Monoclonal antibodies Ki-67 and MIB-1 in the distinction of tuboendometrial metaplasia from endocervical adenocarcinoma and adenocarcinoma in situ in formalin-fixed material. Int J Gynecol Pathol 1995;14:209–214.
Al-Saleh W, Devenne P, Greimers R, Fridman V, Doven J, Boniver J: Assessment of Ki-67 antigen immunostaining in squamous intraepithelial lesions of the uterine cervix: correlation with the histologic grade and human papillomavirus type. Am J Clin Pathol 1995;104:154–160.
Pirog EC, Baergen RN, Soslow RA, Tam D, DeMattia AE, Chen YT, Isacson C: Diagnostic accuracy of cervical low-grade squamous intraepithelial lesions is improved with MIB-1 immunostaining. Am J Surg Pathol 2002;26:70–75.
Geng L, Connolly DC, Issacson C, Ronnett BM, Cho KR: Atypical immature metaplasia (AIM) of the cervix: is it related to high-grade squamous intraepithelial lesion (HSIL)? Hum Pathol 1999;30:345–351.
Pirog EC, Chen YT, Isacson C: MIB-1 immunostaining is beneficial adjunct test for the accurate diagnosis of vulvar condyloma acuminatum. Am J Surg Pathol 2000;24:1393–1399.
Ansari-Lari MA, Staebler A, Zaino RJ, Shah KV, Ronnett BM: Distinction of endocervical and endometrial adenocarcinomas: immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection. Am J Surg Pathol 2004;28:160–167.
Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA: P16INK4a expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol 2003;16:665–673.
Filho AL, Utagawa ML, Ghirata NK, Pereira SM, Namiyama GM, Kanamura CT, Santos G da C, de Oliveria MA, Wakamatsu A, Nonogaki S, Roteli-Martins C, di Loreto C, de Castro Ferraz M da GM, Sakamoto Maeda MY, Alves VAF, Syrjanen K: Immunocytochemical expression of p16 INK4a and Ki-67 in cytologically negative and equivocal Pap smears positive for oncogenic human papillomavirus. Int J Gynecol Pathol 2005;24:118–124.
Pirog EC, Quint KD, Yantiss RK: P16INK4a/CDKN2A and Ki-67 enhance the detection of anal intraepithelial neoplasia and condyloma and correlate with human papillomavirus detection by polymerase chain reaction. Am J Surg Pathol 2010;34:1449–1455.
Akpolat I, Smith DA, Ramzy I, Cgirala M, Mody DR: The utility of p16INK4A and Ki-67 staining on cell blocks prepared from residual thin-layer cervicovaginal material. Cancer 2004;102:142–149.
Schimidt D, Bergeron C, Denton KJ, Ridder R, for the European CINtec Cytology Study Group: p16/Ki67 dual-stain cytology in the triage of ASCUS and LSIL papnicolaou cytology. Cancer Cytopathol 2011;119:158–166.
Atkins K: p16/Ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology. Cancer Cytopathol 2011;119:145–147.
Petry KU, Schimidt D, Scherbring S, Luyten A, Reinecke-Luthge A, Bergeron C, Kommoss F, Loning T, Ordi J, Regauer S, Ridder R: Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67 dual stained cytology. Gynecol Oncol 2011;121:505–509.
Yoshida T, Sano T, Takada N, Kanuma T, Inoue H, Itoh T, Yazaki C, Obara M, Fukuda T: Comparison of self-collected and clinician-collected materials for cervical cytology and human papillomavirus genotyping: analysis by linear array assay. Acta Cytol 2011;55:106–112.
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