HPV and Cervical Cancer

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Human papillomaviruses (HPVs) are small, nonenveloped, double-stranded circular DNA viruses. They cannot be cultured by conventional microbiological methods, and are classified based on similarities in nucleic acid sequences.

HPV Epidemiology

Infections with HPV can lead to benign and malignant tumors of the skin and mucous membranes; these viruses are responsible for at least 5% of the yearly human cancer burden. They are not only responsible for almost all cervical cancers, but are also associated with other anogenital and oropharyngeal cancers. Of the approximately 40 mucosal HPVs commonly transmitted through sexual contact, there are about 15 high-risk genotypes detected in anogenital cancer biopsy specimens.¹ Of these, HPV-16 and -18 are associated with about 70% of cervical cancers and 50% of vaginal, vulvar, and penile cancers. HPV-16 is also associated with about 85% of anal cancers.²

Most genital HPV infections in women produce transient squamous cell abnormalities of the cervix that completely resolve, and the probability of any one HPV infection progressing to cervical cancer is quite small. In addition, cofactors such as tobacco use, multiparity, and immunosuppression are thought to play a role in malignant transformation.³ Nevertheless, cervical cancer represents the second most common cancer in women, with about 530,000 new cases and 275,000 deaths worldwide reported in 2008.⁴ A large majority of the deaths due to cervical cancer occur in developing countries or resource-poor communities in wealthy countries.

Vaccines for HPV

In 2006, two vaccines, a bivalent and a quadravalent, were first approved for use in the United States and several other countries. Both target the high-risk HPV-16 and -18, and the quadrivalent also targets HPV-6 and -11, which cause anogenital warts and respiratory papillomatosis. A nonavalent vaccine targeting the HPVs that cause about 90% of cervical cancers worldwide is in clinical trials.⁵ All of these vaccines will require additional follow-up to resolve questions regarding effects on cross-protection and long-term prevention of cancer.⁶

The first countries to initiate vaccine coverage included the United States, Canada, the United Kingdom, Australia, and Mexico (a regional pilot program).⁷ As of September 2012, HPV immunization was being implemented in 77 countries either through national public-sector programs or pilot programs.⁷ The recent “Report to the Nation on the Status of Cancer” reported that in the United States in 2010, complete HPV vaccine coverage (three doses) in girls was 32%, and was significantly lower in the uninsured and in some southern states. Coverage in the U.S. was much lower than that for the other countries that had introduced the vaccine, probably due to a variety of reasons, ranging from issues with provider reimbursement to cultural concerns.⁸

Prevention of Cervical Cancer

Since the broad implementation of vaccine coverage and assessment of the effect of HPV vaccination on the incidence of cervical cancer will take years, if not decades, public health prevention of cervical cancer still includes secondary prevention through cervical cancer screening in addition to primary prevention through HPV vaccination.^7,9 The U.S. Preventive Services Task Force 2012 screening recommendations include screening women aged 21 to 65 every three years using the Pap smear (cytology), or screening women aged 30 to 65 every five years using both the Pap smear and an HPV DNA test. HPV DNA testing is not recommended for women in their twenties because infections are very common in this age group and most resolve spontaneously.

Conventional screening for cervical cancer consists of the Pap smear and colposcopy/biopsy, which is followed by treatment if needed. In some countries, repeated screening using this approach has achieved up to a 70% reduction in cervical cancer incidence and mortality.⁶ However, in countries with limited resources, this approach has not been sustainable. HPV DNA testing, which has been consistently much more sensitive than Pap smears, can be used in regions that have a developed laboratory infrastructure. Because HPV infection is a broad-based infection of basal epithelial cells in the cervix, it is easier to obtain an adequate sample for HPV DNA testing than an adequate sample for PAP smear. Indeed, a self-sampled specimen has provided sensitivity comparable to professional sampling.⁶ Another option for low-income countries is visual inspection of the cervix after it has been stained with acetic acid or Lugol’s iodine. If a suspicious lesion is found, cryotherapy can be immediately performed; therefore there is no need for repeated follow-up, which is especially important in areas where women may have to travel long distances for medical care.

HPV Tests

Techniques for detection of HPV DNA include signal amplification and target amplification. Although HPV DNA testing is very sensitive, it is clinically not very specific, that is, it cannot differentiate transient from persistent infections leading to cancer. Alternative diagnostic approaches, including new cytologic/histologic biomarkers for transforming HPV infections, methylation of host and viral genes, and detection of overexpressing HPV E6 and E7 proteins, are being evaluated.¹⁰

Signal Amplification

• Qiagen (Germantown, MD) offers the FDA-approved digene HC2 High-Risk HPV DNA Test, an in vitro nucleic acid hybridization assay employing signal amplification and chemiluminescence for the qualitative detection of 13 HPV genotypes (16/18/31/33/35/39/45/51/52/56/58/59/68) having confirmed association with cervical cancer development. It is approved for use in the United States for reflex testing of cytology showing atypical squamous cells of undetermined significance (ASC-US), and also in conjunction with the Pap smear for the routine testing of women aged 30 years or older. The HC2 assays utilize cocktails of specific RNA probes that are directed toward target HPV DNA sequences. An antibody to DNA–RNA hybrids is used for both capture and detection, which uses labeled antibody for signal amplification in a microplate chemiluminescence detection system. The digene HC2 High-Risk HPV DNA Test has been approved for use with the digene HC2 DNA Collection Device, digene HC2 Sample Conversion Kit, digene Cervical Sampler, digene Specimen Transport Medium, and PreservCyt^® Solution (Hologic, Inc., Sunnyvale, CA), depending on country regulations. Automated options for the test include the Rapid Capture System (for high-volume sample throughput testing); guidelines vary among countries. According to the manufacturer, the HC2 High-Risk HPV DNA Test provides clinically relevant sensitivity for high-risk HPV-induced illness, at around 5000 copies of HPV DNA, as opposed to PCR, with an analytical sensitivity of <10 copies of HPV DNA. The test accommodates 88 cervical samples.

Target Amplification

• Abbott Laboratories (Abbott Park, IL) offers the CE-marked RealTime High Risk (HR) HPV Assay, a qualitative in vitro polymerase chain reaction (PCR) assay that utilizes homogeneous target amplification and detection technology for the detection of high-risk HPV DNA in cervical cells collected in liquid cytology media. The Abbott RealTime HR HPV Assay is intended to detect 14 high-risk HPV genotypes (16/18/31/33/35/39/45/51/52/56/58/59/66/68), and to partially genotype HPV-16 and -18 from the other 12 high-risk genotypes. That is, this assay simultaneously detects 14 high-risk genotypes and identifies HPV genotypes 16 and 18, thereby consolidating screening and genotyping. It can detect single and mixed infections, and accommodates up to 96 tests per run. It can be performed in low-, medium-, and high-throughput laboratories using, respectively, manual sample preparation, the m24sp extraction instrument, or the m2000sp for sample preparation plus the m2000rt for real-time amplification and detection.
• Hologic Gen-Probe (San Diego, CA) offers the Aptima^® HPV Assay for the qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk type HPVs (16/18/31/33/35/39/45/51/52/56/58/59/66/68) in cervical specimens. The assay is approved for use in the United States for reflex testing of ASC-US cytology results, and also in conjunction with the Pap smear for the routine testing of women aged 30 years or older. The assay is used with the TIGRIS DTS System, an automated nucleic acid testing system. The cervical specimen is transferred to a tube containing specimen transport media that lyses cells to release the mRNA, which is then protected from degradation during storage. The assay consists of three main steps, which take place in a single tube: capture of target mRNA, transcription-mediated amplification, and detection of the amplified products using a hybridization protection assay. The kit contains 250 tests. Positive results can be further tested by the APTIMA HPV 16 18/45 Genotype Assay.

Testing in Remote Areas and Alternative Approaches

• Qiagen’s recently CE-marked careHPV™ test was specifically designed for use in remote areas, and can be performed in the absence of running water and electricity. The system consists of the assay, a small portable instrument, and collection devices. It has color-coded, easy-to-understand menus, contains reagents, and tolerates temperature variations. Cervical cell specimens can be obtained by a health-care worker or by the patient herself. The test requires minimal training, and easy-to-interpret results are available within 2.5 hours. The assay detects DNA from 14 high-risk HPVs.
• Arbor Vita (Fremont, CA) offers the recently CE-marked OncoE6™ test, which is the first approved and validated alternative approach to direct testing. It directly detects the HPV viral protein E6, which is associated with cellular transformation and is not found in normal tissue. The test was designed as a near point-of-care test for use in physicians’ offices and clinical laboratories to detect HPV-16 and -18. It does not require complex equipment or refrigeration, can be performed with minimal training, and provides results in about 2.5 hours. The test is a lateral-flow sandwich assay, employing monoclonal antibodies for both capture and detection, and requires a minimum of 1000 cells. A study in China of 7500 women aged 25–64 years compared the performances of the OncoE6, careHPV, and the High-Risk HPV DNA tests on cervical specimens and found that the OncoE6 test had 99% specificity and a positive predictive value (PPV) of >50%, which were much higher values than those of the other tests (5% PPV).¹¹

Among the approximately 11,000 new cases of cervical cancer that occurred annually in the United States from 2004 to 2008, about 3.2% of them were in young women between the ages of 20 to 29 years.⁹ Because the current HPV tests are not very specific and would lead to unnecessary procedures and psychological stress in this frequently infected group, this young population cohort may benefit from the OncoE6 test. The results of the study conducted in China indicate that if the OncoE6 test were to be used for primary screening, only 1.7% of women in the general population would need to be referred for colposcopic evaluation and further diagnosis if needed. Although the highest sensitivity was obtained by Qiagen’s digene HC2 High-Risk HPV DNA and careHPV tests (98%), their specificity was lower, and use of those tests would lead to the additional follow-up of approximately 14% of women in the general population.¹⁰

Conclusion

The worldwide prevention of cervical cancer includes ongoing implementation of HPV vaccination and the continued development of sensitive and specific screening tests that can be used in resource-poor regions and for different age groups.

References

1. Muñoz, N.; Bosch, F.X. et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Eng. J. Med. 2003, 348, 518–27.
2. https://www.labcompare.com/Clinical-Diagnostics/6070-Clinical-Diagnostics-Assays/.
3. http://www.cancerresearchuk.org/cancer-info/cancerstats/causes/infectiousagents/humanpapillomaviruses/human-papillomaviruses.
4. http://globocan.iarc.fr/factsheets/populations/factsheet.asp?uno=900.
5. Van de Velde, N.; Boilly, M.-C. et al. Population-level impact of the bivalent, quadrivalent, and nonavalent human papillomavirus vaccines. A model-based analysis. J. Natl. Cancer Inst.  2012, 104, 1712–23.
6. Bosch, F.X.; Castellsagué, X.; et al. HPV and cervical cancer: screening or vaccination? Br. J. Cancer  2008, 98, 15–21.
7. http://www.cervicalcanceraction.org/pubs/CCA_reportcard_low-res.pdf.
8. Jemal, A.; Simard, E.P. et al. Annual report to the nation on the status of cancer, 1975–2009, featuring the burden and trends in human papillomavirus (HPV)-associated cancers and HPV vaccination coverage levels. JNCI. J. Natl. Cancer. Inst.  2013, 105, 175-201.
9. Wu, X.; Watson, M. et al. Human papillomavirus–associated cancers—United States, 2004–2008. MMWR 2012, 61, 258–61.
10. Cuzick, J.; Bergeron, C. et al. New technologies and procedures for cervical cancer screening. Vaccine 2012, 30S, F107–16.
11. http://www.arborvita.com/index.php?option=com_contact_enhanced&view=contact&id=3&Itemid=115.