Abstract 267: microRNA expression analysis in hrHPV positive FFPE tissues for the detection of cervical disease
Authors: Gonzalez Martha I. , Garai Jone , Zabaleta Jovanny , Sanchez Gloria I. 
Published: September 2020

Abstract
Cervical cancer ranks first in cancer mortality among women of low-middle income countries where 80% of the 570,000 cases and 311,000 worldwide deaths estimated for 2018 occurred. Persistent infections with high-risk HPV (hrHPV) genotypes can lead to cervical high-grade lesions (Cervical Intraepithelial Neoplasia grade 2 or more severe disease, CIN-II+), that if left untreated progress to cancer. hrHPV test has high sensitivity but because many women infected with hrHPV genotypes will clear the infection spontaneously, it has low specificity to detect CIN-II+. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and show differential profiles in cervical intraepithelial lesions. Our aim was to identify miRNAs differentially expressed in CIN-II+ and to evaluate their potential use as biomarkers to distinguish low from high-grade lesions in hrHPV positive women. Methods We compared the miRNAs expression pattern between Formalin-Fixed Paraffin-Embedded (FFPE) tissues from hrHPV positive women with low-grade lesions (n=10) and women with high-grade lesions (n=10) of Medellin, Colombia, using the QIAseq miRNA Library Kit (Qiagen) and sequencing reagents and protocols from Illumina. The miRNAs with low coefficient of variation and high fold change were further validated by RT-PCR (miR-133a-3p, 143-3p, 143-5p, 29a-3p and 30b-5p) using miRCURY LNA miRNA PCR kit (Qiagen) and SNORD44 as housekeeping gene. Receiver Operating Characteristic (ROC) analyses with 95% confidence interval of the area under the curve were used to evaluate the diagnostic accuracy of the 5 miRNA and their combinations and the maxim Youden index to determine the optimal specificity and sensitivity. Results The differential miRNAs expression analysis identified 25 miRNAs overexpressed in high- versus low-grade lesions. The relative expression by RT-PCR of 5 of those miRNAs was higher in high-grade when compared to low-grade, and the levels of miR-143-3p, 143-5p and 30b-5p increased with disease progression (from healthy, CIN-I, CIN-II, CIN-III+). ROC analyses showed that miR-143-5p and the combination with miR-133a-3p exhibits the highest AUC 79% (95% CI: 65-93). However, the highest sensitivity is given by miR-29a-3p, 87.50% (95% CI: 67.64 - 97.34), and the highest specificity is given by miR-30b-5p, 95.45% (95% CI: 77.16-99-88), with cut-offs of &gt;1,157 and &gt;3,345 respectively. In conclusion, we identified 25 candidate miRNAs biomarkers and validated 5 of them with good diagnostic performance to distinguish high-grade from low-grade cervical lesions. Further validation on a larger cohort of samples is needed to confirm the potential role of these microRNAs to triage HPV positive women.
Citation Format: Martha I. Gonzalez, Jone Garai, Jovanny Zabaleta, Gloria I. Sanchez. microRNA expression analysis in hrHPV positive FFPE tissues for the detection of cervical disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 267.