Targeted RNA next generation sequencing analysis of cervical smears can predict the presence of hrHPV-induced cervical lesions.

BACKGROUND: Because most cervical cancers are caused by high-risk human papillomaviruses (hrHPVs), cervical cancer prevention programs increasingly employ hrHPV testing as a primary test. The high sensitivity of HPV tests is accompanied by low specificity, resulting in high rates of overdiagnosis and overtreatment. Targeted circular probe-based RNA next generation sequencing (ciRNAseq) allows for the quantitative detection of RNAs of interest with high sequencing depth. Here, we examined the potential of ciRNAseq-testing on cervical scrapes to identify hrHPV-positive women at risk of having or developing high-grade cervical intraepithelial neoplasia (CIN). METHODS: We performed ciRNAseq on 610 cervical scrapes from the Dutch cervical cancer screening program to detect gene expression from 15 hrHPV genotypes and from 429 human genes. Differentially expressed hrHPV- and host genes in scrapes from women with outcome "no CIN" or "CIN2+" were identified and a model was built to distinguish these groups. RESULTS: Apart from increasing percentages of hrHPV oncogene expression from "no CIN" to high-grade cytology/histology, we identified genes involved in cell cycle regulation, tyrosine kinase signaling pathways, immune suppression, and DNA repair being expressed at significantly higher levels in scrapes with high-grade cytology and histology. Machine learning using random forest on all the expression data resulted in a model that detected 'no CIN' versus CIN2+ in an independent data set with sensitivity and specificity of respectively 85 ± 8% and 72 ± 13%. CONCLUSIONS: CiRNAseq on exfoliated cells in cervical scrapes measures hrHPV-(onco)gene expression and host gene expression in one single assay and in the process identifies HPV genotype. By combining these data and applying machine learning protocols, the risk of CIN can be calculated. Because ciRNAseq can be performed in high-throughput, making it cost-effective, it can be a promising screening technology to stratify women at risk of CIN2+. Further increasing specificity by model improvement in larger cohorts is warranted.

Novel high-resolution targeted sequencing of the cervicovaginal microbiome.

BACKGROUND: The cervicovaginal microbiome (CVM) plays a significant role in women's cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. RESULTS: We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes' abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. CONCLUSIONS: CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.

RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues.

Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues (n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.

Mapping actionable pathways and mutations in brain tumours using targeted RNA next generation sequencing.

Many biology-based precision drugs are available that neutralize aberrant molecular pathways in cancer. Molecular heterogeneity and the lack of reliable companion diagnostic biomarkers for many drugs makes targeted treatment of cancer inaccurate for many individuals. Identifying actionable hyperactive biological pathways in individual cancers may improve this situation.To achieve this we applied a novel targeted RNA next generation sequencing (t/RNA-NGS) technique to surgically obtained glioma tissues. The test combines mutation detection with analysis of biological pathway activities that are involved in tumour behavior in many cancer types (e.g. tyrosine kinase signaling, angiogenesis signaling, immune response, metabolism), via quantitative measurement of transcript levels and splice variants of hundreds of genes. We here present proof of concept that the technique, which uses molecular inversion probes, generates a histology-independent molecular diagnosis and identifies classifiers that are strongly associated with conventional histopathology diagnoses and even with patient prognosis. The test not only confirmed known glioma-associated molecular aberrations but also identified aberrant expression levels of actionable genes and mutations that have so far been considered not to be associated with glioma, opening up the possibility of drug repurposing for individual patients. Its cost-effectiveness makes t/RNA-NGS to an attractive instrument to aid oncologists in therapy decision making.