This story has been updated to include comments from Roche Diagnostics.
NEW YORK – A new study in the Journal of Molecular Diagnostics has demonstrated an HPV assay from startup Atila Biosystems performs comparably to standard-of-care testing from Roche Diagnostics. The low-cost, high-throughput, multiplex isothermal system was also shown to perform well using formalin-fixed, paraffin-embedded cervical and orophyrengeal samples without any nucleic acid extraction.
Although Atila Biosystems AmpFire system is being used in research settings by a number of prominent groups, as previously reported, a lack of published data has made it somewhat challenging for most labs to assess its effectiveness. As such, the JMD study is the first technical paper describing the system and its performance, and should open the door for more uptake, according to Youxiang Wang, Atila's founder and CEO.
For example, while labs in eight countries in Africa are using the AmpFire system for HPV screening, having switched from Qiagen's CareHPV, Wang said, other locations have been waiting for a peer-reviewed publication.
AmpFire HPV uses a proprietary multiplex isothermal nucleic acid amplification chemistry and real-time fluorescence to detect 15 high-risk HPV genotypes in a single-tube reaction. The firm's AmpFire instrument can run 94 samples at a time. In a recent report from a site in Africa using the system, the instrument cost $16,000 with a cost per test of less than $20.
In the JMD study, the test was evaluated by a team of researchers affiliated with the US Centers for Disease Control and Prevention, Memorial Sloan Kettering Cancer Center, Weill Medical College of Cornell University, Hackensack University Medical School, and universities in Spain, Brazil, Canada, and China.
According to the study, the limits of detection for the assay, as determined using plasmids cloned with HPV genotype-specific sequences, were two copies per reaction for HPV16, HPV18, and some of the high risk HPV genotypes, and 20 copies per reaction for the remaining HR HPV genotypes.
The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens.
The international team also evaluated the Atila AmpFire HPV test on 214 FFPE samples, comparing it to gold-standard testing, the Roche Cobas HPV and LinearArray tests.
The AmpFire test had a 100 percent positive agreement for HPV16 and HPV18, and 95 percent for non-16/18 HR-HPV, as well as a 100 percent negative agreements for HPV16, HPV18, and non-16/18 HR-HPV.
Importantly, 53 of the FFPE clinical samples were biopsies of the oropharynx. In the US, 70 percent of oropharyngeal cancers are HPV-related, and fresh and preserved biopsy samples are frequently tested for HPV.
Currently, the HPV tests that have been approved by the United States Food and Drug Administration are intended for use in cervical cancer testing and screening.
Although the College of American Pathologists and the American Society of Clinical Oncology guided in 2018 that HPV tumor status should be determined for newly diagnosed oropharyngeal squamous cell carcinomas, it does not explicitly guide the use of PCR-based testing. There are no HPV diagnostic tests with regulatory approval for oropharyngeal cancer.
Specifically, as noted in a 2018 review on HPV testing in the context of orophyrengeal cancer, there are a number of different commercially available HPV assays that use different types of detection, including in situ hybridization and immunohistochemistry.
In terms of molecular testing, commercial tests include the Onclarity HPV assay from BD Diagnostics Systems, the Cervista HPV High Risk, HPV 16/18 Test, and Aptima HPV assays from Hologic, and the Abbott RealTime High Risk HPV assay. There is also the INNO‐LiPA HPV Genotyping from Fujirebio, and the Digene Hybrid Capture 2 (HC2) High‐Risk HPV DNA Test and the Digene HPV Genotyping PS Test from Qiagen.
Roche markets two HPV tests — the Cobas HPV Test for detection of high risk HPV genotypes, and the LinearArray test, which performs comprehensive genotyping and is most frequently used for epidemiological research, as previously reported.
However, it is notable that the molecular tests that are cleared in the US are indicated for cervical testing.
"Because of its inhibition tolerance, the [Atila] assay will be able to be used for other specimen types such as FFPE," Tang said in an email. Furthermore, the test's "simplicity and rapidity will allow it to be used in the field as a real-time point-of-care test."
Tang performed the Atila evaluation while at MSKCC, but became Cepheid's chief medical officer for China two months ago. Although there are no explicit plans to meld the two systems, Tang commented that "the combination of [Atila's] isothermal multiplex amplification with Cepheid's integrated cartridge would be a powerful tool."
Atila's Wang also noted that sample prep of PCR-based detection using FFPE samples takes a lot of expertise, time, and reagents, and can also lead to sample loss.
"With ours, you don't do DNA extraction — you directly incubate the FFPE sample, and in two and a half hours you have a result," Wang said.
Wang said the publication pump has been primed, and soon there will be more studies of the AmpFire coming out. He suggested there are as many as six studies of the system, with manuscripts already submitted, that should be published this year.
Atila is based in Mountain View, California, but Wang has numerous global connections, including in China.
Wang said that he has been contacted by an expert who had evaluated the AmpFire HPV and noninvasive prenatal tests previously and is now working at China's Centers for Disease Control and Prevention. The expert, whom Wang declined to name, has asked Atila to develop a test for novel coronavirus 2019-nCoV. Currently used methods require RNA extraction, so theoretically Atila's extraction-free methods could offer some time saving.
"We will provide a simplified isothermal assay, without extraction, hopefully with results within hours," Wang said. A highly-sensitive and specific test would be ideal in order to detect infection early, even before symptoms appear. Atila began work on a 2019-nCoV assay earlier this week, he said.