NEW YORK – HKG Epitherapeutics has developed an epigenetic biomarker-based diagnostic to screen patients for cervical cancer and potentially augment traditional Pap testing and human papillomavirus (HPV) assays.
The Hong Kong-based firm hopes its test, called epiCervix, can more reliably detect cervical cancer and premalignant cervical cells than HPV testing alone using the same samples collected during a routine Pap test. The test uses next-generation sequencing to screen samples for epigenetic changes in four genes associated with cancer.
HPV infection can sometimes progress into cancer, but the limitation of HPV testing is that it often cannot differentiate between temporary HPV infections, which usually clear on their own, and HPV infections that may progress into cancer. HPV screenings are PCR-based tests that can detect the presence of several high-risk HPV infections, such as HPV16 and HPV18, in cervical cell samples. If these tests come back positive, gynecologists can be faced with a dilemma about whether to treat patients right away or to wait and see if the HPV infection clears on its own.
The traditional Pap test for cervical cancer relies on a pathologist's analysis of the same cervical cell samples, but this test can also miss cancer cells that exist but are not present in the Pap test samples.
"The question in the field is how do we increase the [test] sensitivity so that the women who are tested get a meaningful result," HKG Epitherapeutics CEO Moshe Szyf said. "We believe that combining the traditional pathology or cytology analysis with HPV testing and the DNA methylation test can increase our ability to detect cancer."
This month, HKG and collaborators at McGill University published a validation study of the epiCervix test in the International Journal of Cancer. The researchers examined whether the test could accurately identify cervical cancer in patients regardless of HPV status by measuring DNA methylation markers in four genes: CA10, DPP10, FMN2, and HAS1.
HKG and McGill researchers identified these biomarkers and trained the test in a previous study, called the MARKER study, which pulled samples from a study called CASSIS (Cervical And Self-Sample In Screening). Szyf and three other McGill researchers, one of whom is also on HKG's staff, hold the patent related to the discovery of these methylation biomarkers.
The latest study validated the test's ability to screen for the biomarkers and determine whether a sample contained cervical cancer cells, precancerous cells, or no cancer. The validation cohort included more than 300 cervical colposcopy samples randomly selected from the Biomarkers of Cervical Cancer Risk (BCCR) study, a case-control study carried out at McGill University-affiliated hospitals in Montreal.
The validation cohort included normal samples, cervical cancer samples, and samples from patients with low- and high-grade cervical intraepithelial neoplasia (CIN), a premalignant stage of cervical cancer associated with HPV infection. Cervical cancer was histologically confirmed by hospital pathologists. The normal cervical samples were collected from women who underwent their annual cytology with no previous cervical abnormalities.
The epiCervix test uses Illumina's MiSeq next-generation sequencing bisulfite mapping platform to profile DNA methylation at a single DNA molecule resolution.
In the validation cohort, epiCervix had 67.7 percent sensitivity to detect cervical cancer at 100 percent specificity. At 95 percent specificity, the test had 84.3 percent sensitivity to detect cervical cancer. Detection of high-grade CIN at 100 percent specificity was 8.2 percent for grade 2 CIN and 28.3 percent for grade 3 CIN. The sensitivity for grade 2 and grade 3 CIN also increased as specificity was lowered. At 95 percent specificity, 56.6 percent of grade 3 CIN samples and 39.3 percent of grade 2 CIN samples were detected.
The researchers also explored epiCervix's ability to identify both cancers and premalignant CIN cells compared to HPV testing and Pap test analysis. To compare, the BCCR samples used in the validation study also included data on HPV positivity for these patients. The overall HPV positivity rate for the validation cohort was 68.6 percent, inclusive of both low- and high-risk HPV strains.
Szyf said his team developed the test to screen for genes that had "absolute" differences in methylation between cancerous cells and noncancerous cells to provide a "black and white signal."
"We wanted to examine whether the methylation biomarkers could detect the premalignant stages in cancers that were not detected by regular pathology," Szyf said. "Remarkably, we could see cancer not just in cancer samples, but we could see cancer DNA even in the premalignant stages in some of the women."
The study does have some limitations, however, according to Jason Ross, a senior researcher scientist focused on epigenomics, bioinformatics, and statistical genetics at the Australian science agency Commonwealth Scientific and Industrial Research Organization (CSIRO), who was not involved with the research.
Ross noted that the validation results were encouraging, but a prospective study focused on a specific indication, such as general population screening or cancer detection after abnormal cytology result, is needed to determine how the test would perform in clinical practice. He also sees value in a longitudinal study exploring whether the high methylation score across CIN is "correlated with a higher chance of progression to carcinoma."
Nevertheless, the research "is further evidence of the power of DNA methylation assays for inexpensive population screening," Ross said. "Epigenetic changes happen early in neoplasia and we can see from the data that a panel of only four genes captures a large fraction of the tumors. NGS-based somatic mutation panels are ideal for providing information on clinical actions given a diagnosis, but epigenetic tests are well suited for primary screening and recurrence testing."
Ross also suggested that this epigenetic test could be "complimentary" to HPV screening. "Since they are both molecular tests, it would also be non-challenging from a technical point of view to use the same biospecimen and screen for both at once," he said.
In fact, that's how HKG is expecting the test to be used. Szyf said ideally the same sample could be partially used for the Pap test, HPV screening, and epiCervix. "We believe that the combination of these three tests will provide much higher sensitivity, and all the clinic needs is what they already have," Szyf added.
There are several other epigenetic cervical cancer detection tests, but not all are available in the US. Qiagen's test, called QiaSure, also measures DNA methylation markers to differentiate patients' risk of developing cervical cancer after a positive test for high-risk HPV but is only available in Europe. German diagnostics firm Oncgnostics GmbH also has a methylation test, called GynTect, for cervical cancer screening that is also available in Europe.
Ross noted that these other methylation-based tests examine different biomarkers to identify cancer. Qiagen's test detects hypermethylation of FAM19A4 and mir124-2 in either clinician-collected or self-collected samples, and Oncgnostics' test gauges six DNA methylation markers, including ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Both tests are also intended to be used as a follow-up after a positive HPV test or abnormal cytology findings, he added.
"Considering these are established tests, the HKG Epitherapeutics team needs to carefully consider the indication and jurisdictions for their forthcoming prospective study," he said.
Szyf said that HKG does hope to conduct a prospective study of epiCervix in a large population in the future, but, for now, the firm is focusing its effort on building its testing platform.
Other firms, such as Roche and Becton Dickinson, have refined their tests for HPV infection to better stratify cancer risk for patients who are positive for HPV. Roche's FDA-approved CINtec PLUS Cytology test screens HPV-positive samples for p16 and Ki-67 biomarkers that are associated with transforming HPV infections. Becton Dickinson's Onclarity HPV assay, also approved by the FDA, is able to identify 14 high-risk HPV genotypes to better determine which patients need intervention for HPV infection.
HKG began offering the epiCervix test in the US in December as a laboratory-developed test (LDT) through its CLIA-registered and CAP-accredited laboratory. Clinicians can send a sample collected during a Pap test to HKG's Hong Kong lab to be screened for the DNA methylation markers. Turnaround time is about two weeks, and the test costs between $150 and $300.
Going forward, the firm aims to build out more CAP- and CLIA-certified labs in other countries that can process the epiCervix test, Szyf said, along with its suite of other cancer early detection tests in development.
HKG has three other epigenetic tests in development: a liver cancer test, a breast cancer test, and a blood-based pan-cancer detection test. Szyf said the company recently published results of a validation study conducted in Bangladesh for its liver cancer test and that more research of the breast and pan-cancer assay is expected this year. The firm also plans to follow the LDT approach for these tests.