By Samantha Black, Ph.D.

A new technique called HPV RNA-Seq can provide a second-line test in HPV-positive patients to reduce unnecessary colposcopies and even be used as a two-in-one test combining HPV typing with triage capabilities. Researchers from Institut Pasteur and the Pathogen Discovery Laboratory in Paris, France published their findings in The Journal of Molecular Diagnostics on August 12, 2019.

Human papillomavirus (HPV) infections are associated with the development of cervical carcinoma. HPVs are the etiological agents responsible for >99% of all cervical cancers. HPVs are small, nonenveloped DNA viruses commonly transmitted through sexual contact, which infect basal cells and replicate in the nucleus of squamous epithelial cells. HPVs include >200 genotypes characterized by their oncogenic potential.

Cervical cancer screening allows detection and treatment of precancerous lesions before the development of cervical cancer. Current cervical cancer screening techniques is based on different algorithms, some allowing detection of HPV and others identifying abnormal cells. mainly based on the Papanicolaou cytology test (Pap test) and do not include molecular virology tests.

Molecular tests typically have a low positive predictive value (PPV). Because they rely on the detection of HPV genome (DNA) and do not address the patterns of viral expression (RNA), they are weak predictors of the evolution from low-grade squamous intraepithelial lesion (LSIL) to high-grade squamous intraepithelial lesion (HSIL). HPV RNA tests, specifically the detection of E6 and E7 mRNAs have been proposed as better molecular markers of cancer development, but E6 and E7 are also expressed during HPV transient infection. It is difficult to define a threshold of expression associated with the persistence and evolution to high-grade lesions and cancer.

Therefore, analysis of cervical cells with the Pap test, even when combined with molecular detection of high-risk HPV, is not very effective. Researchers have developed a novel molecular diagnostic test that not only can detect HPV infection, but also have the ability to accurately predict precancerous stages to offer a better and cost-saving medical benefit.

"We have developed HPV RNA-Seq, a novel in vitro molecular diagnostics procedure for the detection of high-risk HPV infection and the identification of patients with high-grade squamous intraepithelial lesions (HSIL), which is a precancerous stage of the cervix. HPV RNA-Seq is a unique test that combines the advantages of molecular assays (HPV typing) and cervical cytology (cell phenotyping)," explained lead investigator Marc Eloit, DVM, PhD, of the Pathogen Discovery Laboratory, Biology of Infection Unit, Institut Pasteur, Paris, and the National Veterinary School of Alfort, Paris-Est University, Maisons-Alfort, France.

The system was developed on multiplexed amplification (reverse transcription PCR), targeting the virus splice junctions, coupled with next-generation sequencing (NGS) analysis, which allows describing fine equilibrium among transcript species of 13 high-risk HPVs plus 3 putative high-risk HPVs, in a single reaction. HPV RNA-Seq was used to analyze samples from 55 patients, 28 with LSIL and 27 patients with precancerous HSIL.

HPV RNA-Seq was evaluated in comparison to the HPV DNA PapilloCheck kit. It was able to detect and determine the type of HPV infection among a panel of 16 high-risk HPVs with results comparable to a widely used and officially approved HPV DNA molecular diagnostic kit. In fact, HPV RNA-Seq detected two more HPV-positive patients than the DNA test and identified more patients with multiple HPV infections. HPV RNA-Seq had higher sensitivity (97.3 % and negative predictive value (NPV), likelihood of not having HPV (93.8 %) compared to DNA-based tests. "Effective cervical cancer screening requires high sensitivity and NPV for high-risk HPV infection, since women with a negative HPV test are usually tested again only after several years," noted Prof. Eloit.

The researchers suggest a second application of HPV RNA-Seq, as a triage test. It would be used as a logistic regression model for the prediction of high-grade cytology was built on the basis of a combination of the number of reads captured at specific HPV RNA spliced junctions, using the grade found by cytology as a reference. They also found the PPV (likelihood of having HPV infection) of HPV RNA-Seq vs. histology was always greater than the PPV of cytology vs. histology. Histology is considered the gold standard for cervical cancer diagnosis but is often invasive and requires more time for results. Therefore, HPV RNA-Seq shows potential as a medical triage test.

HPV RNA-Seq makes it possible to take a snapshot of the early versus late populations of HPV transcripts and to define a model based on a combination of reads that reflects the biology of the virus, which can then be correlated with the evolution of cervical lesions. Moreover, it has recently been shown that only one third of women recommended for colposcopy after primary HPV testing (DNA) and cytology actually had HSIL. By increasing the PPV in detecting HSIL, HPV RNA-Seq could significantly increase the medical benefit/cost ratio of colposcopies and eliminate the need for unnecessary colposcopies.



Do you have a unique prospective on your research related to molecular diagnostics and human diesase. The Science Advisory Board wants to highlight your research! Contact the editor today to learn more.


---

Join The Science Advisory Board today!




Copyright © 2019 scienceboard.net


Register below for our weekly Letter from the Editor to receive the latest Science news and insights.
Email
Conferences
American Chemical Society Meeting & Expo
August 25-29
San Diego, California United States
2019 Peptide Drug Design & Delivery Summit
August 27-29
Philadelphia, Pennsylvania United States
AOAC Annual Meeting
September 8-11
Denver, Colorado United States
Connect
Science Advisory Board on LinkedIn
Science Advisory Board on Facebook
Science Advisory Board on Twitter