Pathology & Oncology Research

, Volume 25, Issue 2, pp 477–486 | Cite as

Dual-Stained Cervical Cytology and Histology with Claudin-1 and Ki67

  • Tímea Szekerczés
  • Ádám Galamb
  • Adrienn Kocsis
  • Márta Benczik
  • Tibor Takács
  • Attila Martonos
  • Balázs Járay
  • András Kiss
  • Csaba Jeney
  • Miklós Nyíri
  • Zsuzsa SchaffEmail author
  • Gábor Sobel
Original Article


Several biomarkers are in use to improve the sensitivity and specificity of cervical cancer screening. Previously, increased expression of tight junction protein claudin-1 (CLDN1) was detected in premalignant and malignant cervical lesions and applied for cytology screening. To improve the specificity, a double immunoreaction with CLDN1/Ki67 was developed in the recent study. Parallel p16/Ki67 (CINtec® PLUS) and CLDN1/Ki67 dual-stained cytology and histology were performed and compared. p16/Ki67 immunoreaction showed positivity in 317 out of 1596 smears with negativity in 1072 and unacceptable reactions in 207 samples. CLDN1/Ki67 dual staining was positive in 200 of 1358 samples, negative in 962, whereas 196 smears could not be evaluated due to technical reasons. Considering the high-grade squamous intraepithelial lesion cytology as gold standard, sensitivity of CLDN1/Ki67 reaction was 76%, specificity was 85.67%, while for p16/Ki67 sensitivity was 74% and specificity was 81.38%. Comparison of CLDN1/Ki67 and p16/Ki67 dual stainings showed the results of the two tests not to be significantly different. Analysing histological slides from 63 cases, the results of the two tests agreed perfectly. As conclusion the sensitivity and specificity proved to be similar using p16/Ki67 and CLDN1/Ki67 double immunoreactions both on LBC samples and on histological slides.


Cervical cancer Claudin-1/Ki67 immunochemistry p16/Ki67 reaction Claudins 


Author Contributions

TSz performed and evaluated the immunreactions and statistics, ÁG and AM collected the clinical samples and data, BJ and AKi evaluated the cytology, ZsS and GS designed and coordinated the study and wrote the manuscript. AKo and MB are clinical experts of the TRACE clinical study (Cellcall). TT is the inventor of the HPV detection technology applied in the CONFIDENCE HPV™ (NEUMANN Diagnostics) testing. CsJ was former head of the TRACE clinical study (Cellcall). ZsS accepts full responsibility for the work. All authors have read and approved the final manuscript.


This work was supported by grant #NKFP_07_2-SPE-SAFE, Jedlik Ányos 2. subprogram KMR_12–1–2012-0032 by the Hungarian National Research and Development Fund and grant #OTKA PD105019 by the Hungarian National Research Foundation.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest with respect to the research, authorship, and/or publication of this article.

AKo is an employee of Cellcall and NEUMANN. MB receives consultancy fees from Cellcall and NEUMANN, she is an employee of SYNLAB GenoID Laboratory, which participated in the TRACE clinical study (all related expenses were charged to Cellcall and NEUMANN). TT is a part time employee of Cellcall, he receives consultancy fees from NEUMANN, he is minority owner of NEUMANN, the inventor of the HPV detection technology described in the manuscript covering which Cellcall submitted a patent application (license now owned by NEUMANN Diagnostics Ltd.); he is a part time employee of SYNLAB GenoID Laboratory. CsJ was a former employee of Cellcall. MNy is an employee of Cellcall and NEUMANN.


  1. 1.
    Hillemans P, Soergel P, Hertel H, Jentschke M (2016) Epidemiology and early detection of cervical cancer. Oncol Res Treat 39:1–6CrossRefGoogle Scholar
  2. 2.
    Sawaya GF, Smith-McCune K (2016) Cervical cancer screening. Obstet Gynecol 127:459–467CrossRefGoogle Scholar
  3. 3.
    Lees BF, Erickson BK, Huh WK (2016) Cervical cancer screening: evidence behind the guidelines. Am J Obstet Gynecol 214:438–443CrossRefGoogle Scholar
  4. 4.
    Ocque R, Austin RM (2016) Follow-up of women with negative pap test results and abnormal clinical signs or symptoms. Am J Clin Pathol 145:560–567CrossRefGoogle Scholar
  5. 5.
    Ikenberg H, Bergeron C, Schmidt D, Griesser H, Alameda F, Angeloni C, Bogers J, Dachez R, Denton K, Hariri J, Keller T, von Knebel Doeberitz M, Neumann HH, Puig-Tintore LM, Sideri M, Rehm S, Ridder R, PALMS Study Group (2013) Screening for cervical cancer precursors with p16/Ki-67 dual-stained cytology: results of the PALMS study. J Natl Cancer Inst 105:1550–1557CrossRefGoogle Scholar
  6. 6.
    Wentzensen N, Fetterman B, Castle PE, Schiffman M, Wood SN, Stiemerling E, Tokugawa D, Bodelon C, Roitras N, Lorey T, Kinney W (2015) p16/Ki-67 dual stain cytology for detection of cervical precancer in HPV-positive women. J Natl Cancer Inst 107:1–8CrossRefGoogle Scholar
  7. 7.
    Cuzick J, Clavel C, Petry KU, Meijer CJ, Hoyer H, Ratnam S, Szarewski A, Birembaut P, Kulasingam S, Sasieni P, Iftner T (2006) Overview of the European and north American studies on HPV testing in primary cervical cancer screening. Int J Cancer 119:1095–1101CrossRefGoogle Scholar
  8. 8.
    Plummer M, Schiffman M, Castle PE, Maucort-Boulch D, Wheeler CM, ALTS Group (2007) A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion. J Infect Dis 195:1582–1589CrossRefGoogle Scholar
  9. 9.
    Benczik M, Galamb Á, Koiss R, Kovács A, Járay B, Székely T, Szekerczés T, Schaff Z, Sobel G, Jeney C (2016) Claudin-1 as a biomarker of cervical cytology and histology. Pathol Oncol Res 22:179–188CrossRefGoogle Scholar
  10. 10.
    Bergeron C, Ikenberg H, Sideri M, Denton K, Bogers J, Schmidt D, Alameda F, Keller T, Rehm S, Ridder R, PALMS Study Group (2015) Prospective evaluation of p16/Ki-67 dual-stained cytology for managing women with abnormal Papanicolaou cytology: PALMS study results. Cancer Cytopathol 123:373–381CrossRefGoogle Scholar
  11. 11.
    Kocsis A, Takács T, Jeney C, Schaff Z, Koiss R, Járay B, Sobel G, Pap K, Székely I, Ferenci T, Lai HC, Nyíri M, Benczik M (2017) Performance of a new HPV and biomarker assay in management of hrHPV positive women: Subanalysis of the ongoing multicenter TRACE Clinical Trial (n>6,000) to evaluate POU4F3 as a potential biomarker of cervical precancer and cancer. Int J Cancer 140:1119–1133CrossRefGoogle Scholar
  12. 12.
    Sobel G, Páska C, Szabó I, Kiss A, Kádár A, Schaff Z (2005) Increased expression of claudins in cervical squamous intraepithelial neoplasia and invasive carcinoma. Hum Pathol 36:162–169CrossRefGoogle Scholar
  13. 13.
    Szabo I, Kiss A, Schaff Z, Sobel G (2009) Claudins as diagnostic and prognostic markers in gynecological cancer. Histol Histopathol 24:1607–1615Google Scholar
  14. 14.
    Lee JW, Lee SJ, Seo J, Song SY, Ahn G, Park CS, Lee JH, Kim BG, Bae DS (2005) Increased expressions of claudin-1 and claudin-7 during the progression of cervical neoplasia. Gynecol Oncol 97:53–59CrossRefGoogle Scholar
  15. 15.
    Lal-Nag M, Morin PJ (2009) The claudins. Genome Biol 10:235CrossRefGoogle Scholar
  16. 16.
    Nayar R, Wilbur DC (2015) The pap test and Bethesda 2014. Cancer Cytopathol 123:271–281CrossRefGoogle Scholar
  17. 17.
    Solomon D (2015) Foreword. In: Nayar F, Wilbur DC (ed) The Bethesda system for reporting cervical cytology. Definitions, criteria, and explanatory notes, 3rd ed. Springer International Publishing Switzerland, Cham, pp v-viiGoogle Scholar
  18. 18.
    Wentzensen N, von Knebel Doeberitz M (2007) Biomarkers in cervical cancer screening. Dis Markers 23:315–330CrossRefGoogle Scholar
  19. 19.
    Lee S, Rose MS, Sahasrabuddhe VV, Zhao R, Duggan MA (2017) Tissue-based immunohistochemical biomarker accuracy in the diagnosis of malignant glandular lesions of the uterine cervix: a systematic review of the literature and meta-analysis. Int J Gynecol Pathol 36:310–322CrossRefGoogle Scholar
  20. 20.
    Polman NJ, Uijterwaal MH, Witte BI, Berkhof J, van Kemenade FJ, Spruijt JW, van Baal WM, Graziosi PG, van Dijken DK, Verheijen RH, Helmerhorst TJ, Steenbergen RD, Heideman DA, Ridder R, Snijders PJ, Meijer CJ (2017) Good performance of p16/ki-67 dual-stained cytology for surveillance of women treated for high-grade CIN. Int J Cancer 140:423–430CrossRefGoogle Scholar
  21. 21.
    Yu LL, Guo HQ, Lei XQ, Qin Y, Wu ZN, Kang LN, Zhang X, Qiao YL, Chen W (2016) p16/Ki-67 co-expression associates high risk human papillomavirus persistence and cervical histopathology: a 3-year cohort study in China. Oncotarget 7:64810–64819Google Scholar
  22. 22.
    Possati-Resende JC, Fregnani JHTG, Kerr LM, Mauad EC, Longatto-Filho A, Scapulatempo-Neto C (2015) The accuracy of p16/Ki67 and HPV test in the detection of CIN2/3 in women diagnosed with ASC-US or LSIL. PLoS One 10:e0134445CrossRefGoogle Scholar
  23. 23.
    Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–1550CrossRefGoogle Scholar
  24. 24.
    Morin PJ (2005) Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res 65:9603–9606CrossRefGoogle Scholar
  25. 25.
    Soini Y (2005) Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology 46:551–560CrossRefGoogle Scholar
  26. 26.
    Cunniffe C, Brankin B, Lambkin H, Ryan F (2014) The role of claudin-1 and claudin-7 in cervical tumorigenesis. Anticancer Res 34:2851–2857Google Scholar
  27. 27.
    Akimoto T, Takasawa A, Murata M, Kojima Y, Takasawa K, Nojima M, Aoyama T, Hiratsuka Y, Ono Y, Tanaka S, Osanai M, Hasegawa T, Saito T, Sawada N (2016) Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, −4, −7, occludin and JAM-A, in human cervical adenocarcinoma. Histol Histopathol 31:921–931Google Scholar
  28. 28.
    Győrffy H, Holczbauer Á, Nagy P, Szabó Z, Kupcsulik P, Páska C, Papp J, Schaff Z, Kiss A (2005) Claudin expression in Barrett’s esophagus and adenocarcinoma. Virchows Arch 447:961–968CrossRefGoogle Scholar
  29. 29.
    Tőkés AM, Kulka J, Paku S, Szik Á, Páska C, Novák PK, Szilák L, Kiss A, Bögi K, Schaff Z (2005) Claudin-1, −3 and −4 proteins and mRNA expression in benign and malignant breast lesions: a research study. Breast Cancer Res 7:R296–R305CrossRefGoogle Scholar
  30. 30.
    Lódi C, Szabó E, Holczbauer Á, Batmunkh E, Szíjártó A, Kupcsulik P, Kovalszky I, Paku S, Illyés G, Kiss A, Schaff Z (2006) Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas. Mod Pathol 19:460–469CrossRefGoogle Scholar
  31. 31.
    Szász AM, Nyírády P, Majoros A, Szendrői A, Szűcs M, Székely E, Tőkés AM, Romics I, Kulka J (2010) Beta-catenin expression and claudin expression pattern as prognostic factors of prostatic cancer progression. BJU Int 105:716–722CrossRefGoogle Scholar
  32. 32.
    English DP, Santin AD (2013) Claudins overexpression in ovarian cancer: potential targets for Clostridium Perfringens enterotoxin (CPE) based diagnosis and therapy. Int J Mol Sci 14:10412–10437CrossRefGoogle Scholar
  33. 33.
    Vázquez-Ortíz G, Ciudad CJ, Pina P, Vazquez K, Hidalgo A, Latorre B, Garcia JA, Salamanca F, Peralta-Rodriguez R, Rangel A, Salcedo M (2005) Gene identification by cDNA arrays in HPV-positive cervical cancer. Arch Med Res 36:448–458CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2018

Authors and Affiliations

  • Tímea Szekerczés
    • 1
  • Ádám Galamb
    • 2
  • Adrienn Kocsis
    • 3
    • 4
  • Márta Benczik
    • 3
    • 4
    • 5
  • Tibor Takács
    • 3
    • 4
    • 5
  • Attila Martonos
    • 2
  • Balázs Járay
    • 1
  • András Kiss
    • 1
  • Csaba Jeney
    • 3
    • 6
  • Miklós Nyíri
    • 3
    • 4
  • Zsuzsa Schaff
    • 1
    Email author
  • Gábor Sobel
    • 2
  1. 1.2nd Department of PathologySemmelweis UniversityBudapestHungary
  2. 2.2nd Department of Obstetrics and GynecologySemmelweis UniversityBudapestHungary
  3. 3.Cellcall Ltd.BudapestHungary
  4. 4.NEUMANN Diagnostics Ltd.BudapestHungary
  5. 5.SYNLAB Hungary Ltd., GenoID Molecular Diagnostic LaboratoryBudapestHungary
  6. 6.Department of Medical MicrobiologySemmelweis UniversityBudapestHungary

Personalised recommendations