Human papillomavirus and cervical cancer : detection of potential markers of disease progression using liquid-based cytology

Abstract

This protect aims to evaluate alternative strategies to screen for cervical cancer including liquid-based cytology (LBC) with supplementary reflex testing for human papillomavirus (HPV), p16INK4a, HPV-L1 capsid protein, and miR-205 for use as possible diagnostic and prognostic markers in women with abnormal findings detected through the organized cervical cancer screening program.

This split-sample study enrolled 137 women with atypical Pap smear findings to compare the effectiveness of conventional cytology (CC) with LBC cytology for cervical cancer screening. Sensitivity to detect cervical intraepithelial neoplasia (CIN 2+) was 47% for CC compared with 66% for LBC. These results from these two sampling techniques agreed with histological findings in 37% and 53% of cases, respectively, a statistically significant difference. The significant advantage of improved sensitivity combined with the ability to carry out reflex testing for diagnostic and prognostic markers such as HPV DNA or p16 INK4a without repeated sampling favors LBC for a possible future role as a screening technique.

In cases of low-grade cytological abnormalities combining LBC with HPV triage (LBC+HPV triage) may improve detection of CIN compared with CC. Our study found that CC and LBC+HPV triage produced similar detection rates of CIN2+. When comparing CIN detection rates the adjusted OR for CIN2+ was 0.87 (95% CI: 0.60- 1.26) and for CIN3+ 1.00 (95% CI: 0.64-1.58). Consequently, the use of LBC+HPV triage offered no advantage over conventional cytology regarding sensitivity and specificity or positive and negative predictive value for detection of histologically confirmed CIN2+. Nevertheless, LBC+HPV triage may potentially reduce unnecessary medical procedures and testing among HR-HPV-negative women with findings of abnormal cytology.

We analyzed 118 patient samples of dysplastic cells using immunocytochemical staining to assess the expression of p16INK4a. Expression levels of p16INK4a, correlated with CIN grade, showing stronger reactivity in higher grade lesions. We found a stronger correlation between severity of cytological abnormality and p16INK4a, immunoreactivity when the diagnosis was simultaneously confirmed by routine cytology (p<0.001, χ2 exact test for trend). This staining technique may be able to serve as a complementary prognostic marker when used as a reflex test to identify women at risk of cervical cancer.

We also used immunocytochemistry to detect L1 capsid protein in HR-HPV-positive women who displayed minor cytological abnormalities. Progression to CIN2+ occurred in 2 of 13 L1-positive women (15.0%) infected with HPV16, compared with 4 L1- positive women infected with other HR-HPV types. Among all L1-positive women with CIN2+, 35.7% were infected with both HR and low-risk (LR) HPV types, 25.0% with HR-HPV types only and 13.3% with HPV16. Loss of previously positive L1 expression may serve as a prognostic marker for preinvasive cervical lesions.

We carried out reverse transcription quantitative PCR (RT-qPCR) on our LBC samples to assess miR-205 expression and correlate these results with histology. Regardless of whether findings were based on histology or cytology, our results showed no significant changes in miR-205 expression relating to different stages of disease progression. It may be that the increased expression of miR-205 is not stage-specific, but could represent a continuous process throughout disease progression, although we cannot conclude this with certainty.Cervical cancer could become one of our most preventable cancers by effectively combining vaccination programs against HR-HPV with improved detection of precursors by integrating molecular markers into screening programs.