mRNA Biomarker Detection in Liquid-based Cytology: A New Approach in the Prevention of Cervical Cancer

Marta del Pino; Cecilia Svanholm-Barrie; Aureli Torné; Lorena Marimon; Jina Gaber; Amaia Sagasta; David H Persing; Jaume Ordi


Mod Pathol. 2015;28(2):312-320. 

In This Article

Abstract and Introduction


Several high-risk human papillomavirus (HPV)-induced cell biomarkers have been proposed as possible candidates to identify patients harboring high-grade squamous intraepithelial lesions (HSILs) of the uterine cervix. We aimed to determine the feasibility of the detection of the mRNA of six biomarkers in cervical smear specimens obtained by liquid-based cytology and to evaluate whether this approach might be useful in the identification of patients with HSIL. One-hundred and twenty three women referred to colposcopy in the Hospital Clinic of Barcelona were included in the study. After a thorough study, including Pap test, high-risk HPV testing (Hybrid Capture 2 test), and colposcopy with directed biopsy and/or endocervical curettage, 48 patients were diagnosed with HSIL, whereas 75 were classified as negative (n=28), or harboring low-grade SIL (n=47). CDKN2A/p16, BIRC5, MMP9, TOP2A, MCM5, and MKI67 mRNA expression was analyzed by reverse transcription quantitative polymerase chain reaction in liquid-based cytology after the Pap test and Hybrid Capture 2 performance. The tissue expression of these biomarkers was analyzed by immunohistochemistry in the biopsy material. One-hundred and thirteen out of 123 (92%) liquid-based cytology yielded adequate material for mRNA analysis. TOP2A was the most sensitive (97%) biomarker for the detection of HSIL and CDKN2A/p16 the most specific (78%). The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 96% (95% confidence interval (CI): 88–99) and a specificity of 71% (95% CI: 55–82). In the immunohistochemistry analysis, all biomarkers showed a high sensitivity but low specificity for HSIL, except CDKN2A/p16 which had a sensitivity of 100% and a specificity of 63%. The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 100% (95% CI: 91–100) and a specificity of 43% (95% CI: 32–55). The detection of mRNA of cell biomarkers in liquid-based cytology material is feasible. The combination TOP2A and CDKN2A/p16 has a good balance between sensitivity and specificity for the detection of women with HSIL.


Screening programs based on cervical cytology (Pap test) have led to a decrease in the incidence and the mortality by cervical cancer. However, the efficacy of cytological screening is hampered by the suboptimal sensitivity and interobserver variability of the conventional Pap test.[1,2] Although the introduction of liquid-based cytology has not led to improvements in terms of relative sensitivity for the detection of cervical cancer precursors,[3] this technique has enabled the use of adjunctive tests that may help to reduce the rate of false-negative cytological results and add objectivity to the classical morphological evaluation.

High-risk human papillomaviruses (HPV) are the causative agents of cervical cancer, and high-risk HPV testing has shown to improve the sensitivity of the Pap test to over 95%.[4,5] However, high-risk HPV tests have a lower specificity than the Pap test, and positive results are frequently observed in women without cervical lesions or who show mild abnormalities, such as atypical squamous cells of unknown significance (ASC-US) or low-grade squamous intraepithelial lesions (LSILs). In most women with these low-risk lesions, both high-risk HPV infection and the epithelial abnormalities are cleared within a few months or years (transient infections). Therefore, there is a need to explore new more specific techniques that may help to identify the subset of women harboring high-grade squamous intraepithelial lesions (HSIL),[6] the true precursor of CC, from the broad group of women testing positive for a high-risk HPV.

In recent years, several high-risk HPV-induced cellular molecules involved in replication, transcription, DNA repair, apoptosis, proliferation, and invasion and metastasis have been analyzed as possible biomarkers to detect women at risk of cervical cancer development. The list of candidates who have shown potential utility in cervical cancer screening includes p16 INK4a (CDKN2A), survivin (BIRC5), metalloproteinase 9 (MMP9), topoisomerase 2 alpha (TOP2A), minichromosome maintenance 5 (MCM5), or MKi67 proteins (MKI67).[7–9] Most studies focused on these biomarkers have used immunohistochemistry to detect protein expression. However, several shortcomings of immunohistochemistry, in particular the interobserver variation inherent to all morphological techniques and the difficulty to obtain reproducible quantification, hamper proper evaluation of the clinical usefulness of these biomarkers. mRNA detection has the advantage of being less subjective than morphologic assessment. However, very little data are available on the use of mRNA-based techniques to detect these cell biomarkers in liquid-based cytology cervical samples, and their possible value in cervical cancer prevention has not been proven.

In the present study we aimed to determine the feasibility of the detection of the mRNA of six biomarkers in liquid-based cytology preserved samples and to evaluate whether this approach might be useful in the identification of patients with HSIL.