mRNA biomarker detection in liquid-based cytology: a new approach in the prevention of cervical cancer

Abstract

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 ¼ 28), or harboring low-grade SIL (n ¼ 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. Modern Pathology (2015) 28, 312-320; doi:10.1038/modpathol.2014.106; published online 5 September 2014Screening 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...

Reader Reaction: A Note on the Evaluation of Group Testing Algorithms in the Presence of Misclassification

“…The largest efficiency gains occur when specificity is low (), sensitivity is high (), and prevalence is approximately . For example, messenger RNA has a high sensitivity and low specificity for diagnosing early stage cervical cancer (Del Pino et al, ).…”

mRNA Detection in Anal Cytology: A Feasible Approach for Anal Cancer Screening in Men Who Have Sex with Men Living With HIV

“…For this validation study we considered the following three diagnostic categories as concordant results: (a) negative (control group): normal anal cytology result and negative HPV test; (b) LSIL: anal cytology result of LSIL and biopsy confirming LSIL/AIN1; and (c) HSIL/AIN: anal cytology result of HSIL and biopsy confirming HSIL/AIN2-3 [12,19]. The following were considered as exclusion criteria: (1) previous history or histological diagnosis of anal cancer in the simultaneous biopsy; (2) previous treatment for HSIL/AIN2-3 performed within the previous 3 years; and (3) previous HPV vaccination.…”

“…The detection of host-cell mRNA biomarkers involved in DNA replication and cell cycle progression, such as TOP2A, and other HPV-induced cell molecules involved in cell cycle control, such as MKi67 or p16 (CDKN2A), has proved to be feasible in cervical samples preserved in liquid-based cytology media [12]. Moreover, previous studies have shown that these biomarkers are potentially useful in the detection of intraepithelial lesions of the uterine cervix.…”

“…Moreover, previous studies have shown that these biomarkers are potentially useful in the detection of intraepithelial lesions of the uterine cervix. Preliminary results suggest that this strategy might be useful for the detection of cervical HSIL/CIN2-3 [12]. However, there are no data on the use of mRNA-based techniques to detect these host-cell biomarkers in anal liquid-based cytology samples.…”

Rejoinder to “Reader Reaction: A Note on the Evaluation of Group Testing Algorithms in the Presence of Misclassification”

“…The authors do cite a potential application involving screening for human papillomavirus (HPV) where is low. However, the study described in del Pino et al () does not use group testing, and we are unaware of any assay that tests for HPV in pools.…”