The role of messenger RNA testing in the screening for cervical cancer

Human papillomavirus (HPV) is a group of more than 150 related viruses, several of which are associated with an increased risk of developing cervical, vaginal, vulvar, and oral cancers. A recent report from the Centers for Disease Control and Prevention (CDC) found that 45 percent of Americans ages 18 to 59 carry some form of HPV.¹ About 20 percent of women in the study had certain HPV strains that carry a higher risk of cancer.

Although the majority of HPV infections spontaneously resolve within two years, individuals with persistent high-risk HPV infection are at high risk of developing pre-malignant squamous intraepithelial lesions (cervical intraepithelial neoplasia, or CIN), cervical adenocarcinoma in-situ, and cervical adenocarcinoma. At least 14 of more than 100 known HPV genotypes can cause cervical cancer and are also associated with other anogenital cancers and cancers of the head and neck. HPV 16 and 18, the two most common high-risk genotypes, cause approximately 70 percent of all cervical cancers.

With such staggering numbers of Americans infected with HPV, and with HPV-related cancers on the rise,² it is becoming increasingly important for clinicians to determine whether their patients are infected, and whether the infection is with one of the transforming, cancer-causing strains.

The importance of screening

With HPV infections on the rise, screening is becoming more important to monitor cervical health. Guidelines are in place to ensure patients are screened appropriately for HPV and cervical cancer. The United States Preventive Services Task Force (USPSTF) recommends screening for cervical cancer in average-risk women age 21 to 65 years with cytology (Pap smear) every three years or, for women age 30 to 65 years who want to lengthen the screening interval, screening with a combination of cytology and HPV testing every five years. HPV screening before the age of 30 is generally not recommended at this time.

There are several types of HPV testing available to clinicians. The two most commonly used are DNA and messenger RNA (mRNA). Unlike HPV DNA testing, which tests for the presence of HPV, mRNA detection allows for the identification of transcriptionally active viruses. Numerous studies have shown that testing for high-risk HPV is more sensitive (high negative predictive value) than cytology for the detection of high-grade squamous intraepithelial lesions (HSIL) (CIN 2 or greater). mRNA testing has been shown to have a higher specificity while maintaining about the same sensitivity to HPV DNA tests by identifying the mRNA transcripts of the HPV E6/E7 oncoproteins from the 14 high-risk HPV genotypes, which mediate the development of cervical cancer. These oncoproteins’ over-expression is associated with a significantly increased risk of CIN and cervical cancer.

Up until now, molecular testing of cytology specimens for HPV has mainly involved the use of DNA-based assays, in particular the HC2 test.³ However, as HPV is highly prevalent and the lifetime risk for HPV is estimated to be up to 75 percent for sexually active people, more specific biological markers for early detection of cervical cancer are needed. Though current high-risk HPV DNA testing methods generally provide very good sensitivity, specificity is limited (low positive predictive value) as they detect infection by the HPV virus in both their transient and persistent (transforming) states. This leads to positive results in patients who harbor non-transforming HPV infections that might lead to unnecessary invasive procedures such as colposcopy and biopsy in some women. In a study of 800 women referred for colposcopy, the sensitivity and specificity of this test were shown to be >92 percent and 99 percent, respectively, for detection of high-risk HPV types and 91 percent and >55 percent, respectively, for the detection of CIN2+.⁴ In another study of 1,373 women undergoing routine screening, the sensitivity and specificity for the detection of CIN2+ were 100 percent and 88.3 percent, respectively.⁵

Why mRNA?

Why is mRNA for HPV E6 and E7 more specific than HPV DNA testing? In order to understand the answer to this question, we need to briefly review the biology of HPV viral infections. Like most viral infections (the common cold or hepatitis B viral infections), the majority of these are self-resolving and eradicated by the immune response. In an immunocompetent host, they are transient. This is the case in more than 90 percent of HPV infections.

A small number of these infections, though, become chronic or persistent. In the case of HPV, this happens less than 10 percent of the time. It is this fraction that develops the ability to escape the immune surveillance mechanism and hijack the replication mechanism of the host cell, establishing itself as a persistent infection. Once this first step is achieved, the other steps of cellular disruption, proliferation, mutations, and eventual transformation into a neoplastic process can occur. In the case of HPV, the oncoproteins responsible for this transformation cascade are the early proteins 6 and 7 (E6 and 7).⁶ Hence, molecules expressed early in this transformation process will have more specificity than just identifying the transient infectious agent. By the same token, molecules identified further downstream (like p16) would be more specific, but would lack the sensitivity to be used as a screening test for premalignant disease, while being extremely useful in the diagnostic biopsy workup of HSIL (CIN2+) cases.⁷

At a time when guidelines are advocating for a better balance between the screening and follow-up in the detection of cervical cancer and its precursors, mRNA testing for E6/E7 provides a high sensitivity, as DNA testing, and better specificity either when used alone or in combination with cytology results to achieve a better balance when evaluating harm versus benefit.

Risks associated with less-specific HPV testing

Despite the proven benefits of mRNA, many clinicians still use DNA testing for several reasons. Some of the reasons are related to the challenges associated with switching lab providers, costs to train staff, etc. With statistics that demonstrate a high prevalence of HPV—most of which will never become cancer—it is imperative that we utilize mRNA testing for HPV, which has demonstrated higher specificity and the ability to identify the transforming E6/E7 viral oncoproteins transcripts. One reason some clinicians may hesitate to switch to mRNA testing is the argument that sending some women for extra treatment is a small price to pay if it means catching more cases of cancer. This argument, however, tilts the balance we must pursue between the physical, emotional, and monetary costs versus additional screening and follow-up procedures.

One risk of follow-up procedures resulting from the screening for cervical cancer is increased fertility risks. While first-line screening provides little to no harm, a tissue biopsy can lead to a weakened cervix. A weak cervix can lead to pregnancy problems, including early labor or miscarriage. Another risk is increased anxiety and fear. Aside from the distress, fear can also preclude women from following up with their OBGYNs, potentially delaying the detection and treatment of a myriad of related or unrelated health conditions.

Finally, there is a clear need to get healthcare costs under control. One way to do this is to implement more effective screening methods, such as mRNA for HPV, which can provide a more specific screening the first time and reduce follow-up procedures. Additionally, of note, the mRNA-based HPV tests receive the same insurance coverage as DNA-based HPV tests, which translates to no additional cost to clinicians and their
patients.

HPV screening is an important component of women’s healthcare. As HPV infection rates rise, it will become even more important to ensure that screening methods can provide the specificity and sensitivity needed to differentiate HPV infections that are cancer -causing from those that aren’t. Reducing HPV-positive results for transient infections and the resultant decrease in follow-up procedures may result in better care and outcomes, and provide patients and their clinicians with peace of mind.

REFERENCES

1. McQuillan G, Kruszon-Moran D, Markowitz LE, Unger ER, Paulose-Ram R. Prevalence of HPV in adults aged 18–69: United States, 2011–2014. Centers for Disease Control and Prevention. NCHS Data Brief No. 280. April 2017. https://www.cdc.gov/nchs/data/databriefs/db280.pdf.
2. Viens LJ, Henley SJ, Watson M, et al. Human papillomavirus–associated cancers—United States, 2008–2012.MMWR. 2016;65(26);661–666. https://www.cdc.gov/mmwr/volumes/65/wr/mm6526a1.htm?s_cid=mm6526a1_w.
3. Centers for Disease Control and Prevention. Epidemiology and prevention of vaccine-preventable diseases. https://www.cdc.gov/vaccines/pubs/pinkbook/hpv.html.
4. Dockter J, Schroder A, Hill C, et al. Clinical performance of the APTIMA HPV assay for the detection of high-risk HPV and high-grade cervical lesions. J Clin Virol. 2009;45(Suppl 1):S55-S61.).
5. Ratnam S, Coutlee F, Fontaine D, et al. Aptima HPV E6/E7 mRNA test is as sensitive as Hybrid Capture 2 assay but more specific at detecting cervical precancer and cancer. J Clin Microbiol. 2011;49(2):557-564.
6. Szarewski A, Mesher D, Cadman L, et al. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the predictors 2 study. J Clin Microbiol. 2012;55(6):1867-1873.
7. Darragh T, Colgan T, Cox JT, et al. The lower anogenital squamous terminology standardization project for HPV-associated lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med. 2012;136(10):1266-1297.

Luis A. Diaz-Rosario, MD, serves as pathologist and Southeast Regional Medical Director for AmeriPath, a business of Quest Diagnostics, which provides a complete menu of cervical cancer testing services, including mRNA E6/E7 testing.