Harnessing the knowledge we have gained on the cell cycle disruption caused by human papillomaviruses (HPV) will likely lead to improved screening modalities for cervical cancer and its precursors. An easily applied biomarker that has high specificity and sensitivity would represent an attractive alternative or complement to cytology and HPV testing. To date, a number of promising markers have been investigated. These include p16INK4A, MIB-1, BD-ProEx C, and L1. Newer possibilities involve a variety of gene products associated with aberrations of chromosome 3q, such as telomerase, p63, and PIK3CA, as well the combination of biomarkers such as p16INK4A and MIB-1 in the same assay. Although none of them has yet been incorporated into screening algorithms or found its way into routine practice, their performance characteristics remain a focus of current investigations. This review summarizes what we know and where we hope to go in translating basic pathobiology into clinical practice.

Keywords:
Papanicolaou, Biomarkers, HPV, Cervical cytology, Human papillomavirus, Immunocytochemistry
1.
Arbyn M, Castellsague X, de Sanjose S, Bruni L, Saraiya M, Bray F, Ferlay J: Worldwide burden of cervical cancer in 2008. Ann Oncol 2011;22:2675-2686.
2.
Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, Schiffman MH, Moreno V, Kurman R, Shah KV: Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87:796–802.
3.
Baldwin P, Laskey R, Coleman N: Translational approaches to improving cervical screening. Nat Rev Cancer 2003;3:217–226.
4.
Samama B, Schaeffer C, Boehm N: p16 expression in relation to human papillomavirus in liquid-based cervical smears. Gynecol Oncol 2008;109:285–290.
5.
Negri G, Moretto G, Menia E, Vittadello F, Kasal A, Mian C, Egarter-Vigl E: Immunocytochemistry of p16INK4a in liquid-based cervicovaginal specimens with modified Papanicolaou counterstaining. J Clin Pathol 2006;59:827–830.
6.
Sung CO, Kim SR, Oh YL, Song SY: The use of p16INK4A immunocytochemistry in ‘Atypical squamous cells which cannot exclude HSIL’ compared with ‘Atypical squamous cells of undetermined significance’ in liquid-based cervical smears. Diagn Cytopathol 2010;38:168–171.
7.
Beccati MD, Buriani C, Pedriali M, Rossi S, Nenci I: Quantitative detection of molecular markers ProEx C (minichromosome maintenance protein 2 and topoisomerase IIa) and MIB-1 in liquid-based cervical squamous cell cytology. Cancer 2008;114:196–203.
8.
Szarewski A, Ambroisine L, Cadman L, Austin J, Ho L, Terry G, Liddle S, Dina R, McCarthy J, Buckley H, Bergeron C, Soutter P, Lyons D, Cuzick J: Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev 2008;17:3033–3042.
9.
Monsonego J, Pollini G, Evrard MJ, Sednaoui P, Monfort L, Quinzat D, Dachez R, Syrjanen K: p16INK4a immunocytochemistry in liquid-based cytology samples in equivocal Pap smears: added value in management of women with equivocal Pap smear. Acta Cytol 2007;51:755–766.
10.
Shidham VB, Mehrotra R, Varsegi G, D’Amore KL, Hunt B, Narayan R: p16 immunocytochemistry on cell blocks as an adjunct to cervical cytology: potential reflex testing on specially prepared cell blocks from residual liquid-based cytology specimens. Cytojournal 2011;8:1.
11.
Norman I, Brismar S, Zhu J, Gaberi V, Hagmar B, Hjerpe A, Andersson S: p16INK4a immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use? Int J Oncol 2007;31:1339–1343.
12.
Nasioutziki M, Daniilidis A, Dinas K, Kyrgiou M, Valasoulis G, Loufopoulos PD, Paraskevaidis E, Loufopoulos A, Karakitsos P: The evaluation of p16INK4a immunoexpression/immunostaining and human papillomavirus DNA test in cervical liquid-based cytological samples. Int J Gynecol Cancer 2011;21:79–85.
13.
Yoshida T, Fukuda T, Sano T, Kanuma T, Owada N, Nakajima T: Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16 expression and human papillomavirus testing: a comparative study using simultaneously sampled histology materials. Cancer 2004;102:100–108.
14.
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.
15.
Bibbo M, DeCecco J, Kovatich AJ: P16INK4A as an adjunct test in liquid-based cytology. Anal Quant Cytol Histol 2003;25:8–11.
16.
Sahebali S, Depuydt CE, Boulet GA, Arbyn M, Moeneclaey LM, Vereecken AJ, Van Marck EA, Bogers JJ: Immunocytochemistry in liquid-based cervical cytology: analysis of clinical use following a cross-sectional study. Int J Cancer 2006;118:1254–1260.
17.
Samarawardana P, Dehn DL, Singh M, Franquemont D, Thompson C, Gaido L, Torkko KC, Homer P, Burke S, Titmus MA, Nayi V, Shroyer KR: p16INK4a is superior to high-risk human papillomavirus testing in cervical cytology for the prediction of underlying high-grade dysplasia. Cancer Cytopathol 2010;118:146–156.
18.
Denton KJ, Bergeron C, Klement P, Trunk MJ, Keller T, Ridder R: The sensitivity and specificity of p16INK4a cytology vs. HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results. Am J Clin Pathol 2010;134:12–21.
19.
Shin EK, Lee SR, Kim MK, Kang EJ, Ju W, Lee SN, Han WS, Kim SC: Immunocytochemical staining of p16ink4a protein as an adjunct test in equivocal liquid-based cytology. Diagn Cytopathol 2008;36:311–316.
20.
Bergeron C, Wentzensen N, Cas F, von Knebel Doeberitz M: The p16INK4a protein: a cytological marker for detecting high grade intraepithelial neoplasia of the uterine cervix (in French). Ann Pathol 2006;26:397–402.
21.
Liu H, Shi J, Wilkerson M, Huang Y, Meschter S, Dupree W, Lin F: Immunohistochemical detection of p16INK4a in liquid-based cytology specimens on cell block sections. Cancer 2007;111:74–82.
22.
Benevolo M, Vocaturo A, Mottolese M, Mariani L, Vocaturo G, Marandino F, Sperduti I, Rollo F, Antoniani B, Donnorso RP: Clinical role of p16INK4a expression in liquid-based cervical cytology: correlation with HPV testing and histologic diagnosis. Am J Clin Pathol 2008;129:606–612.
23.
Tsoumpou I, Arbyn M, Kyrgiou M, Wentzensen N, Koliopoulos G, Martin-Hirsch P, Malamou-Mitsi V, Paraskevaidis E: p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev 2009;35:210–220.
24.
Pinto AP, Crum CP, Hirsch MS: Molecular markers of early cervical neoplasia. Diagn Histopathol (Oxf) 2010;16:445–454.
25.
Wentzensen N, Bergeron C, Cas F, Eschenbach D, Vinokurova S, von Knebel Doeberitz M: Evaluation of a nuclear score for p16INK4a-stained cervical squamous cells in liquid-based cytology samples. Cancer 2005;105:461–467.
26.
Trunk MJ, Dallenbach-Hellweg G, Ridder R, Petry KU, Ikenberg H, Schneider V, von Knebel Doeberitz M: Morphologic characteristics of p16INK4a-positive cells in cervical cytology samples. Acta Cytol 2004;48:771–782.
27.
Mao C, Balasubramanian A, Yu M, Kiviat N, Ridder R, Reichert A, Herkert M, von Knebel Doeberitz M, Koutsky LA: Evaluation of a new p16INK4A ELISA test and a high-risk HPV DNA test for cervical cancer screening: results from proof-of-concept study. Int J Cancer 2007;120:2435–2438.
28.
Balasubramanian A, Hughes J, Mao C, Ridder R, Herkert M, Kiviat NB, Koutsky LA: Evaluation of an ELISA for p16INK4a as a screening test for cervical cancer. Cancer Epidemiol Biomarkers Prev 2009;18:3008–3017.
29.
Boulet GA, Horvath CA, Depuydt CE, Bogers JJ: Biomarkers in cervical screening: quantitative reverse transcriptase PCR analysis of P16INK4a expression. Eur J Cancer Prev 2010;19:35–41.
30.
Yu L, Wang L, Zhong J, Chen S: Diagnostic value of p16INK4A, Ki-67, and human papillomavirus L1 capsid protein immunochemical staining on cell blocks from residual liquid-based gynecologic cytology specimens. Cancer Cytopathol 2010;118:47–55.
31.
Halloush RA, Akpolat I, Jim Zhai Q, Schwartz MR, Mody DR: Comparison of ProEx C with p16INK4a and Ki-67 immunohistochemical staining of cell blocks prepared from residual liquid-based cervicovaginal material: a pilot study. Cancer 2008;114:474–480.
32.
Goto T, Takano M, Sasa H, Tsuda H, Yamauchi K, Kikuchi Y: Clinical significance of immunocytochemistry for PIK3CA as a carcinogenesis-related marker on liquid-based cytology in cervical intraepithelial neoplasia. Oncol Rep 2006;15:387–391.
33.
Longatto Filho A, Utagawa ML, Shirata NK, Pereira SM, Namiyama GM, Kanamura CT, Santos Gda C, de Oliveira MA, Wakamatsu A, Nonogaki S, Roteli-Martins C, di Loreto C, Mattosinho de Castro Ferraz Mda G, Maeda MY, Alves VA, Syrjanen K: Immunocytochemical expression of p16INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus. Int J Gynecol Pathol 2005;24:118–124.
34.
Sahebali S, Depuydt CE, Segers K, 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.
35.
Risse EK, Ouwerkerk-Noordam E, Meijer-Marres EM, Boon ME: Exploiting the residual of cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies. Acta Cytol 2010;54:175–182.
36.
Schmidt D, Bergeron C, Denton KJ, Ridder R: 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;119:158–166.
37.
Kelly D, Kincaid E, Fansler Z, Rosenthal DL, Clark DP: Detection of cervical high-grade squamous intraepithelial lesions from cytologic samples using a novel immunocytochemical assay (ProEx C). Cancer 2006;108:494–500.
38.
Shroyer KR, Homer P, Heinz D, Singh M: Validation of a novel immunocytochemical assay for topoisomerase II-alpha and minichromosome maintenance protein 2 expression in cervical cytology. Cancer 2006;108:324–330.
39.
Depuydt CE, Makar AP, Ruymbeke MJ, Benoy IH, Vereecken AJ, Bogers JJ: BD-ProExC as adjunct molecular marker for improved detection of CIN2+ after HPV primary screening. Cancer Epidemiol Biomarkers Prev 2011;20:628–637.
40.
Tambouret RH, Misdraji J, Wilbur DC: Longitudinal clinical evaluation of a novel antibody cocktail for detection of high-grade squamous intraepithelial lesions on cervical cytology specimens. Arch Pathol Lab Med 2008;132:918–925.
41.
Henderson D, Hall L, Prpic N, Hessling J, Parker M, Sampson S, Simkins S, Brough G, Dixon E, Lenz K, Knapp S, Murphy P, Taylor A, Fischer T, Malinowski DP: The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia. J Immunol Methods 2011;370:1–13.
42.
Hilfrich R, Hariri J: Prognostic relevance of human papillomavirus L1 capsid protein detection within mild and moderate dysplastic lesions of the cervix uteri in combination with p16 biomarker. Anal Quant Cytol Histol 2008;30:78–82.
43.
Xiao W, Bian M, Ma L, Liu J, Chen Y, Yang B, Wu Q: Immunochemical analysis of human papillomavirus L1 capsid protein in liquid-based cytology samples from cervical lesions. Acta Cytol 2010;54:661–667.
44.
Griesser H, Sander H, Hilfrich R, Moser B, Schenck U: Correlation of immunochemical detection of HPV L1 capsid protein in Pap smears with regression of high-risk HPV positive mild/moderate dysplasia. Anal Quant Cytol Histol 2004;26:241–245.
45.
Ungureanu C, Socolov D, Anton G, Mihailovici MS, Teleman S: Immunocytochemical expression of p16INK4a and HPV L1 capsid proteins as predictive markers of the cervical lesions progression risk. Rom J Morphol Embryol 2010;51:497–503.
46.
Sarmadi S, Izadi-Mood N, Pourlashkari M, Yarandi F, Sanii S: HPV L1 capsid protein expression in squamous intraepithelial lesions of cervix uteri and its relevance to disease outcome. Arch Gynecol Obstet 2011, E-pub ahead of print.
47.
Heselmeyer K, Schrock E, du Manoir S, Blegen H, Shah K, Steinbeck R, Auer G, Ried T: Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix. Proc Natl Acad Sci USA 1996;93:479–484.
48.
Redon R, Muller D, Caulee K, Wanherdrick K, Abecassis J, du Manoir S: A simple specific pattern of chromosomal aberrations at early stages of head and neck squamous cell carcinomas: PIK3CA but not p63 gene as a likely target of 3q26-qter gains. Cancer Res 2001;61:4122–4129.
49.
Gollin SM: Chromosomal alterations in squamous cell carcinomas of the head and neck: window to the biology of disease. Head Neck 2001;23:238–253.
50.
Connolly DC, Greenspan DL, Wu R, Ren X, Dunn RL, Shah KV, Jones RW, Bosch FX, Munoz N, Cho KR: Loss of fhit expression in invasive cervical carcinomas and intraepithelial lesions associated with invasive disease. Clin Cancer Res 2000;6:3505–3510.
51.
Kirchhoff M, Rose H, Petersen BL, Maahr J, Gerdes T, Lundsteen C, Bryndorf T, Kryger-Baggesen N, Christensen L, Engelholm SA, Philip J: Comparative genomic hybridization reveals a recurrent pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and in advanced-stage cervical carcinoma. Genes Chromosomes Cancer 1999;24:144–150.
52.
Rao P, Arias-Pulido H, Lu X-Y, Harris C, Vargas H, Zhang F, Narayan G, Schneider A, Terry M, Murty V: Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma. BMC Cancer 2004;4:5.
53.
Kirchhoff M, Rose H, Petersen BL, Maahr J, Gerdes T, Philip J, Lundsteen C: Comparative genomic hybridization reveals non-random chromosomal aberrations in early preinvasive cervical lesions. Cancer Genet Cytogenet 2001;129:47–51.
54.
Umayahara K, Numa F, Suehiro Y, Sakata A, Nawata S, Ogata H, Suminami Y, Sakamoto M, Sasaki K, Kato H: Comparative genomic hybridization detects genetic alterations during early stages of cervical cancer progression. Genes Chromosomes Cancer 2002;33:98–102.
55.
Caraway NP, Khanna A, Dawlett M, Guo M, Guo N, Lin E, Katz RL: Gain of the 3q26 region in cervicovaginal liquid-based Pap preparations is associated with squamous intraepithelial lesions and squamous cell carcinoma. Gynecol Oncol 2008;110:37–42.
56.
Alameda F, Espinet B, Corzo C, Munoz R, Bellosillo B, Lloveras B, Pijuan L, Gimeno J, Salido M, Sole F, Carreras R, Serrano S: 3q26 (hTERC) gain studied by fluorescence in situ hybridization as a persistence-progression indicator in low-grade squamous intraepithelial lesion cases. Hum Pathol 2009;40:1474–1478.
57.
Jalali GR, Herzog TJ, Dziura B, Walat R, Kilpatrick MW: Amplification of the chromosome 3q26 region shows high negative predictive value for nonmalignant transformation of LSIL cytologic finding. Am J Obstet Gynecol 2010;202:581.e1–581.e5.
58.
Cheung AN, Chiu PM, Tsun KL, Khoo US, Leung BS, Ngan HY: Chromosome in situ hybridisation, Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology. J Clin Pathol 2004;57:721–727.
59.
Cheung AN, Tsun KL, Ng KM, Szeto E, Siu MK, Wong ES, Ngan HY: P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology – potential biomarkers for diagnosis and progress prediction of cervical neoplasia. Mod Pathol 2010;23:559–566.
60.
Nieh S, Chen SF, Chu TY, Lai HC, Lin YS, Fu E, Gau CH: 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. Gynecol Oncol 2005;97:35–40.
61.
Meyer JL, Hanlon DW, Andersen BT, Rasmussen OF, Bisgaard K: Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: correlation with biopsy follow-up results. Cancer 2007;111:83–92.
62.
Guo M, Hu L, Baliga M, He Z, Hughson MD: The predictive value of p16INK4a and hybrid capture 2 human papillomavirus testing for high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2004;122:894–901.
63.
Rauber D, Mehlhorn G, Fasching PA, Beckmann MW, Ackermann S: Prognostic significance of the detection of human papilloma virus L1 protein in smears of mild to moderate cervical intraepithelial lesions. Eur J Obstet Gynecol Reprod Biol 2008;140:258–262.
64.
Meijer CJ, Berkhof J, Castle PE, Hesselink AT, Franco EL, Ronco G, Arbyn M, Bosch FX, Cuzick J, Dillner J, Heideman DA, Snijders PJ: Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer 2009;124:516–520.
65.
Petry KU, Schmidt 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.
66.
Reuschenbach M, Clad A, von Knebel Doeberitz C, Wentzensen N, Rahmsdorf J, Schaffrath F, Griesser H, Freudenberg N, von Knebel Doeberitz M: Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results. Gynecol Oncol 2010;119:98–105.
67.
Dunton CJ, van Hoeven KH, Kovatich AJ, Oliver RE, Scacheri RQ, Cater JR, Carlson JA Jr: Ki-67 antigen staining as an adjunct to identifying cervical intraepithelial neoplasia. Gynecol Oncol 1997;64:451–455.
68.
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 2002;10:15–19.
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