TOP2A/MCM2, p16INK4a, and cyclin E1 expression in liquid-based cytology: a biomarkers panel for progression risk of cervical premalignant lesions

Abstract

Background To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. Methods We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. Results We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). Conclusions We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.

Expression analysis of p16 and TOP2A protein biomarkers in cervical cancer lesions and their correlation with clinico-histopathological characteristics in a referral hospital, Tanzania

“…TOP2A overexpression was observed in 60.2% of squamous cell carcinoma and 83.3% of adenocarcinoma. Our findings agree with those reported by Del Moral-Hernández et al [ 24 ], which showed that increased expression of TOP2A/MCM2 biomarkers to approximately 3-times increased progression risk of HSIL to cervical cancer lesions. Similarly, TOP2A expression levels discriminated dysplastic and non-dysplastic FFPE cervical tissues with improved detection of CIN2+ [ 15 , 36 ].…”

“…In a multivariate logistic regression model, the age-adjusted OR for predicting cervical cancer lesions were independently significant for p16/TOP2A biomarkers in archived FFPE cervical tissues [p16: OR = 1.142 (95% CI: 1.059–1.232, p <0.001) and TOP2A: OR = 1.046 (95% CI: 1.008–1.085, p = 0.015)]. These findings corroborate with previously reported studies that used cervical scraps/ FFPE tissues, which showed that progression of LSIL/CIN-1 to cervical cancer lesions was conferred by p16, TOP2A, p16 and TOP2A [ProEx C] [ 24 , 38 , 39 ]. However, our results herein disagree with those reported by Peres et al [ 22 ], that high TOP2A expression levels were observed in cervical smear samples than in cervical biopsies.…”

“…Similarly, TOP2A expression levels discriminated dysplastic and non-dysplastic FFPE cervical tissues with improved detection of CIN2+ [ 15 , 36 ]. In addition, a 40% difference in the expression of p16 (high) versus TOP2A (high) biomarkers (100% versus 60%) observed in the squamous cell carcinoma may imply that the p16 biomarker is more specific for squamous cell carcinoma than the TOP2A biomarker [ 22 , 24 , 31 , 37 ]. Furthermore, immunoscoring of the cut-off threshold for positive cells for both p16 and TOP2A biomarkers might vary between pathologists resulting in significant differences.…”

“…During the pathogenesis of cervical cancer, the expression of E7 viral oncoprotein inactivates retinoblastoma proteins (pRb), which consequently increases the E2F activation factors in the cell, which leads to increased p16 in the S-phase of the cell cycle. Likewise, the TOP2A gene encodes for DNA topoisomerase, a nucleic enzyme responsible for unwinding supercoiled DNA strands during its replication in the S-phase of the cell cycle [ 22 , 24 ]. Expression levels of p16 and TOP2A biomarkers have been reported for early diagnosis of cervical cancer in high-resource countries, [ 14 , 15 , 22 , 25 ] which necessitates the need for their feasibility study in low-resource settings.…”

“…These cellular biomarkers (p16 and TOP2A) are emerging as novel biomarkers for early diagnosis and prognosis of cervical cancer [ 16 , 20 , 26 ]. However, very limited studies have been conducted to investigate their application as early diagnostic markers in improving the accuracy of clinico-histopathological diagnosis of cervical lesions in resources limited settings with a high burden of cervical cancer [ 4 , 9 , 10 , 23 , 24 ]. This may contribute to insufficient data being reported on the prevalence of cervical cancer in Tanzania.…”