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


  • Additional European CINtec Cytology Study group participants: Donatella Caraceni, MD, Ospedale civile, Lanciano, Italy; Bruno Ghiringhello, MD, Ospedale S. Anna, Torino, Italy; Thomas Keller, PhD, Acomed Statistik, Leipzig, Germany; Frieder Kommoss, MD, Institute of Pathology, Mannheim, Germany; Giovanni Negri, MD; Zentralkrankenhaus, Bolzano, Italy; Guiseppe Pizzicannella, MD, Ospedale civile, Lanciano, Italy; Sigrid Regauer, MD, Landeskrankenhaus, Graz, Austria; Heinz Jürgen Roth, Labor Limbach, Heidelberg, Germany; Sophia Taylor, MD, Viollier, Geneva, Switzerland; Alfonso Visonà, MD, Azienda ULSS 4, Thiene, Italy; Jonathan Weintraub, MD, Viollier, Geneva, Switzerland.

  • The excellent support of A. Duwe, S. Niess, S. Rehm, and A. Schrödel (Heidelberg) is acknowledged. We are grateful to S. Sauerbrey (Gehrden/Hannover) for performing the cytotechnologist reading of the dual-stain cytology slides.

  • §

    See editorial on pages 145–7, this issue.



The objective of this study was to analyze the diagnostic performance of a newly established immunocytochemical dual-stain protocol, which simultaneously detects p16INK4a and Ki-67 expression in cervical cytology samples, for identifying high-grade cervical intraepithelial neoplasia (CIN2+) in women with Papanicolaou (Pap) cytology results categorized as atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (LSIL).


Residual liquid-based cytology material from 776 retrospectively collected ASCUS/LSIL cases that were available from a recent study evaluating p16 cytology and HPV testing were subjected to p16/Ki-67 dual staining. The presence of 1 or more double-immunoreactive cell(s) was regarded as a positive test outcome, irrespective of morphology. Test results were correlated to histology follow-up.


Sensitivity of p16/Ki-67 dual-stain cytology for biopsy-confirmed CIN2+ was 92.2% (ASCUS) and 94.2% (LSIL), while specificity rates were 80.6% (ASCUS) and 68.0% (LSIL), respectively. Similar sensitivity/specificity profiles were found for both age groups of women aged <30 years versus women aged ≥30 years. Dual-stain cytology showed comparable sensitivity, but significantly higher specificity, when compared with human papillomavirus (HPV) testing.


The results of this study show that p16/Ki-67 dual-stain cytology provided a high sensitivity for the detection of underlying CIN2+ in women with ASCUS or LSIL Pap cytology results, comparable to the rates previously reported for HPV testing and p16 single-stain cytology. However, the specificity of this morphology-independent interpretation of p16/Ki-67 dual-stain cytology testing was further improved compared with the earlier p16 single-stain cytology approach, which required morphology interpretation, and it is significantly higher when compared with HPV testing. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.