Detection of Cervical Lesions and Cancer in Air-Dried Cytologic Smears by Combined Analysis of mRNA and miRNA Expression Levels.

Cervical cancer screening is based on cytologic analysis and high-risk human papillomavirus (HR-HPV) testing, each having their drawbacks. Implementation of new biomarker-based methods may improve screening accuracy. Here, the levels of 25 microRNAs (miRNAs, miRs) and 12 mRNAs involved in cervical carcinogenesis in 327 air-dried Papanicolaou-stained cervical smears from patients with cervical precancerous lesions, cancer, or without the disease were estimated by real-time PCR. Using logistic regression analysis, small-scale miRNA-based, mRNA-based, and combined molecular classifiers were built based on paired ratios of miRNA or mRNA concentrations; their ability to detect high-grade cervical lesions and cancer was then compared. Combined mRNA-miRNA classifiers manifested a better combination of sensitivity and specificity than miRNA- and mRNA-based classifiers. The best classifier, combining miR-375, miR-20, miR-96, CDKN2A, TSP4, and ECM1, predicted high-grade lesions with diagnostic sensitivity of 89.0%, specificity of 84.2%, and a receiver-operating characteristic area under the curve of 0.913. Additionally, in a subsample of the same specimens, the levels of MIR124-2 and MAL promoter methylation, HR-HPV genotypes, and viral loads were analyzed. The relative high-grade lesion risk estimated by the classifier correlated with the frequency of MAL and MIR124-2 methylation but not with the HR-HPV genotype or viral load. The results support the feasibility of cellular biomarker-based methods for cervical screening and patient management.

Detection of high-grade neoplasia in air-dried cervical PAP smears by a microRNA-based classifier.

Recent studies have shown that changes in the expression levels of certain microRNAs correlate with the degree of severity of cervical lesions. The aim of the present study was to develop a microRNA-based classifier for the detection of high-grade cervical intraepithelial neoplasia (CIN ≥2) in cytological samples from patients with different high-risk human papillomavirus (HR-HPV) viral loads. For this purpose, raw RT-qPCR data for 25 candidate microRNAs, U6 snRNA and human DNA in air-dried PAP smears from 174 women with different cervical cytological diagnoses, 144 of which were HR-HPV-positive [40 negative for intraepithelial lesion or malignancy (NILM), 34 low-grade squamous intraepithelial lesions (L-SIL), 57 high-grade squamous intraepithelial lesions (H-SIL), 43 invasive cancers], were statistically processed. The expression level changes of various individual microRNAs were found to be significantly correlated with the cytological diagnosis but the statistical significance of this correlation was critically dependent on the normalization strategy. We developed a linear classifier based on the paired ratios of 8 microRNA concentrations and cellular DNA content. The classifier determines the dimensionless coefficient (DF value), which increases with the severity of cervical lesion. The high- and low-grade CINs were better distinguished by the microRNA classifier than by the measurement of individual microRNA levels with the use of traditional normalization methods. The diagnostic sensitivity of detecting high-grade lesions (CIN ≥2) with the developed microRNA classifier was 83.4%, diagnostic specificity 81.2%, ROC AUC=0.913. The analysis can be performed with the same nucleic acid preparation as used for HPV testing. No statistically significant correlation of the DF value and HR-HPV DNA load was found. The DF value and the HR HPV presence and viral DNA load may be regarded as independent criteria that can complement each other in molecular screening for high-grade cervical intraepithelial neoplasia. Although it has several limitations, the present study showed that the small-scale analysis of microRNA signatures performed by simple PCR-based methods may be useful for improving the diagnostic/prognostic value of cervical screening.

Divergent non-LTR retrotransposon lineages from the genomes of scorpions (Arachnida: Scorpiones).

We screened across the taxonomic diversity of order Scorpiones (22 species belonging to 21 genera and 10 families) for the presence of seven different clades of non-LTR retrotransposons in their genomes using PCR with newly designed clade-specific consensus-degenerate hybrid oligonucleotide primers. Scorpion genomes were found to contain four known non-LTR retrotransposon clades: R1, I, Jockey, and CR1. In total, 35 fragments of reverse transcriptase genes of new elements from 22 scorpion species were obtained and analyzed for three clades, Jockey, I, and CR1. Phylogenies of different clades of elements were built using amino acid sequences inferred from 33 non-LTR retrotransposon clones. Distinct evolutionary lineages, with several major groups of the non-LTR retroelements were identified, showing significant variation. Four lineages were revealed in Jockey clade. The phylogeny of I clade showed strong support for the monophyletic origin of such group of elements in scorpions. Three separate lineages can be distinguished in the phylogenetic tree of CR1 clade. The large fraction of the isolated elements appeared to be defective.