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Transcriptome-Based HPV Assay Shows Correlation With High-Grade Cytology

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NEW YORK – Colposcopies and cervical biopsies can be intimidating, as well as painful and costly, but are often the best way for detecting cervical cancer or precancerous cervical lesions because less invasive diagnostic methods aren't as accurate or predictive. 

But researchers in France have developed a sequencing-based test that detects transcription levels of certain genes in high-risk human papillomavirus subtypes, and preliminary evaluations suggest it might someday serve as a surrogate for pathology and reduce unnecessary testing.

The new assay, called HPV RNA-Seq, was described earlier this month in the Journal of Molecular Diagnostics. It detects high-risk HPV subtypes but also provides additional molecular markers of precancerous cells, specifically using the number of sequencing reads at specific early and late HPV transcripts as a biomarker for high-grade pathologies.

According to Marc Eliot, an author of the study and a researcher at the Institute Pasteur, "HPV RNA-Seq is the first test that combines the sensitivity of HPV genotyping assays … with a good positive predictive value for the detection of precancerous lesions, comparable to cytology.

Eloit and his colleagues in the Pathogen Discovery Laboratory, Biology of Infection Unit, Bioinformatics and Biostatistics Hub, and the French Human Papillomavirus Reference Laboratory at Institut Pasteur — as well as colleagues at other hospitals in France who participated in the research — believe tests like the one they've developed may someday obviate the need for cytological analysis.

The HPV RNA-Seq test consists of multiplexed RT-PCR coupled with next-generation amplicon sequencing that can be performed on Thermo Fisher Scientific's Ion Torrent platforms or Illumina's systems. It uses a custom AmpliSeq panel designed for both the Ion Torrent PGM and Ion Proton instruments. In total, 750 target sequences are included in the panel, amplified with a pool of 525 unique primers, according to the JMD study.

"The originality lies in the fact that the whole HPV transcriptome is amplified and sequenced, as opposed to current molecular tests in which only one or two genes or transcripts are detected," Eloit explained in an email.

This approach made it possible "to take a snapshot of the population of viral mRNA" and to define a model based on a combination of reads that reflected the biology of the virus, which was then correlated to the evolution of cervical lesions, he said.

There are 16 high-risk strains of HPV that are thought to cause more than 99 percent of all cervical cancers. However, simply detecting a high-risk strain does not mean someone is on the path to developing cancer. Infection is quite common among young women while cervical cancer is rare at younger ages, and most infections can be cleared within a year by a women's own immune system.

While molecular HPV tests based on the detection of viral DNA or RNA are very sensitive for viral detection, they have low positive predictive value for the identification of cancer or precancerous lesions, Eloit said. Because of the low PPV, complementary diagnostic methods, such as cytology using a Pap test, colposcopic visual examination of the cervix, or histology from biopsy samples, have to be used, although these ultimately "may prove unnecessary and invasive" in many cases, he said.

A proof-of-concept evaluation of 55 patient samples showed that the PPV of HPV RNA-Seq for the identification of precancerous lesions was approximately 82 percent. In comparison, the PPV of other HPV DNA or RNA tests has "never exceeded 50 percent in similar populations of women referred for colposcopy," Eloit noted.

Furthermore, the HPV RNA-Seq test detects and types HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82, and the precancerous markers are assessed for all HPV types detected, meaning that in the case of a patient infected with multiple HPV strains it is possible to characterize the contribution of each HPV genotype individually to the lesion, Eloit added.

The researchers compared the HPV RNA-Seq test to pathology and to a molecular comparator test, the PapilloCheck test from Greiner Bio-One, a CE-marked kit for the qualitative detection and genotyping of 18 high-risk and six low-risk types of HPV via detection and identification of a fragment of the viral E1 gene using a DNA microarray, Eloit said. The researchers chose this as a comparator because it is routinely used by the French Human Papillomavirus Reference Laboratory.

Other sequencing-based HPV typing tests have been described — the BGI HPV sequencing test and the TypeSeq assay developed by the National Cancer Institute, for example — but these typically assess HPV DNA, and Eloit pointed out that while DNA tests are very sensitive for viral detection, they have low positive predictive value for the detection of cancers or precancerous lesions.

In the US, cervical cancer screening guidelines currently recommend testing women aged 30 to 65 every three years with cervical cytology alone, every five years with high-risk HPV testing alone, or every five years with HPV testing in combination with cytology, also known as cotesting.

In 2014, a high-risk HPV test from Roche was the first to be cleared by the US Food and Drug Administration for primary cervical cancer screening, and HPV tests from Hologic, Becton Dickinson, Qiagen are also frequently used, although they differ in certain respects. The test from Hologic detects messenger RNA, which one study has suggested has advantages in terms of yielding fewer false-positive results.  

But Eloit emphasized that while molecular tests for viral DNA are very sensitive to detect virus, "the presence of the virus per se is not indicative of the risk of developing precancerous lesions," in part because "persistent viral infection, for months or even years," is the main risk factor for developing cervical lesions that can lead to cancer.

Molecular tests that detect viral RNA, such as the Hologic HPV test in which E6/E7 mRNA are targeted, have been proposed as better molecular markers of cancer development because of the oncogenic properties of HPV E6 and E7 genes, Eloit said.

However, E6 and E7 genes are also expressed during transient HPV infection, he said, "so it remains difficult to define a threshold of expression associated with the persistence and evolution to high-grade lesions and cancer."

On the other hand, HPV infection during the development of cervical cancer is associated with a shift from a productive, acute infection, toward a nonproductive, persistent, and transforming infection that is characterized by a high level of E6 and E7 mRNA and low expression of E2 and late genes such as L1, he said. Therefore, the HPV RNA-Seq method seeks to look at the HPV transcriptomic balance as a whole, rather than focusing only on E6/E7 mRNA.

The L1 gene is also the most pertinent gene for HPV typing, but it can drop out upon integration into the human genome in persistent infection and precancerous lesions. For this reason, several HPV DNA tests target the L1 gene of high-risk HPVs using consensus PCR primers, Eloit said. HPV RNA-Seq detects the HPV transcriptome, so in the event of HPV integration and L1 dropout, other HPV transcripts such as E6, E7, E2 or E4 will still be detected. The logistic regression model that the researchers developed was trained by including such examples, Eloit explained

"With the help of a logistic regression model, we were able to define a molecular signature which better reflects the biology of the virus and thus predicts cervical lesions," he said.

The researchers now plan to license the technology or enter into an industrial partnership, but note that to ultimately be commercialized the test will require further clinical validation in larger cohorts.

Eloit said that the HPV RNA-Seq might someday be useful as a cervical cancer screening test — as a second-line test to reduce unnecessary colposcopies in patients who have gotten positive DNA-based HPV typing results, and as a "two-for-one test" combining HPV typing with triage capabilities.

Specifically, once the performance and medical benefit have been evaluated in large cohorts, a broad-range HPV transcriptome assay could eventually replace first-line cytology and DNA-based molecular tests, he said, providing both HPV genotyping and a molecular marker of precancerous lesions in a single procedure.