HPV testing and cancer: will E6/E7 change the paradigm?

Virtually all cervical cancers are caused by human papilloma virus (HPV) infections. In 2013, an estimated 12,340 women in the United States are expected to be diagnosed with cervical cancer and 4,030 are expected to die of the disease.^1,2 HPV infections also cause other cancers; the incidence of HPV-associated anal and oropharyngeal cancers has been rising, with new cases of more than 30,000 and 12,000, respectively, in the U.S. each year.³

Approximately 75% of women and 50% of men in the United States will be infected with HPV ⁴ during their lives. Today, more than 42% of women have genital HPV infections, and nearly 7% of adults have oral HPV infections.^5,6 Most HPV infections (~90%) self-resolve within two years.⁷ However, among the many viruses classified as HPV, persistent infections with 14 “high-risk” HPV (hrHPV) types can cause cellular changes that may lead to high-grade cervical disease and cancer.

As our understanding of the natural history of HPV continues to grow, so too does our understanding of the importance of the role of HPV testing in cervical cancer screening and detection. The current challenge is to develop tests that best distinguish self-resolving HPV infections from infections that are progressing to precancer and cancer. Such tests could reduce the number of unnecessary procedures substantially, save millions of women from related fear, anxiety, and pain, and lower healthcare costs.

Current screening methods for cervical cancer

Although cervical cancer was once the leading cause of cancer death for women in the U.S., the number of cases and deaths has declined significantly due to widespread use of screening tests to find and treat precancerous cervical lesions before they turn into cancer.⁸ Today, 50 to 60 million American women are screened annually to find the fewer than 1% who have high-grade cervical disease (known as CIN2 or CIN3) or cancer—the stages at which treatment is useful.⁹

The most common screening test is a liquid-based cytology (LBC), or Papanicolaou (Pap), test, in which cervical cells are collected using a cervical swab and then analyzed under the microscope. The Pap test is subjective, relies upon visual examination and interpretation by the cytopathologist, and has significant inter-laboratory variations. In the U.S., about 8% to 10% of women screened have abnormal Pap test results each year.¹⁰

DNA testing for HPV has become a useful addition for women with abnormal Pap results. HPV DNA tests have excellent sensitivity (around 95%) for CIN2 or higher, but specificity is low (ranging in the 20s and 30s).¹¹ Because persistent HPV infections cause nearly all cervical cancer, a negative HPV DNA test result helps rule out possible cervical cancer development within the next few years. However, since most women have self-resolving HPV infections that will not develop into cancer, a positive HPV test result provides little actionable information. This holds true for newer HPV tests, which offer genotyping for the HPV types (16 and 18) that are responsible for the majority of cervical cancers. HPV DNA testing is currently FDA-approved for screening in conjunction with a Pap test for women age 30 and older, and as a follow-up to mildly abnormal Pap test results for women of all ages. For women under age 30, the HPV DNA test is not recommended for general screening, ¹² as HPV infections among them are common but most will self-resolve. HPV DNA screening of these women would detect many who had only temporary HPV infections, thus possibly leading to unnecessary treatment and morbidity.

In the U.S., the Pap test serves as the primary screening test, and the HPV DNA test is used as a co-test or reflex test to triage Pap-positive patients. Internationally, the trend is toward reversing this sequence. More and more countries, including China, India, Sweden, and the Netherlands, are beginning to establish programs with HPV DNA testing as the primary screen and the Pap test used to triage patients who are HPV-positive.

Regardless of the sequence, current screening methods capture a large population of women who have abnormal Pap and/or HPV-positive results but who do not have clinically significant disease. To further identify women for treatment, physicians typically rely upon repeat testing and/or colposcopy-directed biopsy and histology. Not only is colposcopy-directed biopsy invasive and expensive, but it also requires subjective interpretation and may miss some cancers due to sampling errors or other factors.¹³

Unnecessary repeat testing and procedures burden both patients and the healthcare system. Patients may experience time lost from work, anxiety, and pain related to invasive procedures. One unpublished analysis estimates that more than $1 billion is wasted annually due to inefficiencies in cervical cancer screening.

How HPV vaccination fits in

No vaccine is foolproof, and although the FDA-approved HPV vaccines protect against the major high-risk HPV types (16 and 18), they do not protect against the other hrHPV types that cause approximately 30% of cervical cancers. To be most effective, vaccines should be administered before a person becomes sexually active, yet only about 32% of eligible teenage girls in the U.S. are currently vaccinated.¹⁴ HPV vaccination does not eliminate cancer risk, and leading experts agree that vaccinated women will still need regular cervical cancer screening.¹⁵

Detecting E6 and E7 oncoproteins

The ability to detect E6 and E7 oncoproteins offers new opportunities to improve the efficiency and effectiveness of cervical cancer screening. Data have shown that over-expression of E6 and E7 oncoproteins is a critical and necessary step toward HPV-related disease progression and cancer.¹⁶ Among their oncogenic functions, these oncoproteins inactivate tumor suppressor proteins. This disrupts the normal cell cycle and cell death while promoting cell immortality and proliferation. Continuous over-expression of E6 and E7 proteins causes proliferation of cervical carcinoma cells, and high-grade cervical disease and invasive cervical cancers have demonstrated high levels of E6 and E7.

Recently available commercial tests measure oncogenic activity indirectly by detecting E6E7 mRNA in cervical cells. Compared to HPV DNA tests, the E6E7 mRNA tests have higher specificity for high-grade cervical disease and cancer.¹¹ Although this higher specificity could potentially help reduce colposcopy referrals, widespread adoption of E6E7 mRNA tests will likely be limited by the test’s complexity and need for expensive laboratory instruments. Current E6E7 mRNA tests require 28 probes (14 for each HPV type against E6 and, similarly, 14 against E7) and processing by either polymerase chain reaction (PCR) or a combination of flow cytometry and FISH (fluorescence in situ hybridization).

E6E7 mRNA are precursors to E6E7 oncoproteins, which are functional biological molecules and more directly relevant to disease progression. Over-expression of E6E7 oncoproteins can now be detected with immunoassays due to the recent availability (research use only) of pan anti-E6 and anti-E7 antibodies (each applicable to all 14 high-risk HPV types).¹⁷ Data suggest that simple and easy-to-perform E6E7 immunoassays are now feasible on basic enzyme-linked immunosorbent assay (ELISA) or flow cytometry instruments, which would require minimal training and setup. The assays use only a small amount of cervical cells, and leftover samples from cervical swabs collected for Pap testing are sufficient.¹⁷ Thus, an additional physician visit is not needed.

Looking ahead

The recognition of the importance of HPV testing is growing rapidly, buoyed by increasing global awareness of the link between HPV and cervical cancer. Current screening and diagnostic techniques—using both cytology and HPV testing—have been effective, but too many women still undergo unnecessary testing and procedures. Further, developing countries lack the healthcare infrastructure to support the colposcopies and biopsies that would be needed to triage the large number of women with clinically insignificant HPV infections.

Use of new markers, specifically E6 and E7 oncoproteins, may help to more accurately assess cervical cancer risk in HPV-infected women, and E6E7 immunoassays are expected to become a valuable new addition to the current testing “toolkit.” In the future, E6E7 immunoassay technology may also be applied to anal, oral, and other HPV-related cancers, which lack an effective diagnostic test and affect both women and men.

With the growing focus on efficient and effective healthcare delivery, physicians and laboratorians will face increasing demand for a simple, robust way to differentiate precancerous and cancerous stages from clinically insignificant and self-limited HPV infection. A next-generation HPV test with higher specificity that does not disrupt the clinical regimen or laboratory workflow and is simple and cost-effective to execute will better enable clinicians to assess risk, manage patients, and improve outcomes. Objective, clinically relevant, high-throughput immunoassays may reduce patient anxiety and lost time, unnecessary repeat testing, use of invasive colposcopy/biopsy, unneeded treatments, and related costs. The dual goals of better patient management and major cost savings to the healthcare system are within sight.