Harald Zur Hausen was awarded the Nobel Prize for Medicine, in 2008, for the isolation and characterisation (in 1983) of human papillomavirus (HPV) 16 as a causative agent in cervical cancer. The first evaluation of HPV testing in a potential clinical application was published by John Tidy et al, in 1989, on the detection of HPV by polymerase chain reaction (PCR) in normal and dyskaryotic cervical smears from 21 women. They ventured that HPV testing might eventually supplement cytological analysis in cervical screening.
There are approximately 40 different types of HPV that infect the cervix. Some of these viruses have the ability to integrate the HPV genome into the epithelial cell genome and thus become carcinogenic. The International Agency for Research on Cancer (IARC) has classified 12 HPV types as Group 1 carcinogens – HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59. These are known as oncogenic or high-risk (hr) HPV types. HPV 68 is classed by IARC as a Group 2A carcinogen (probably carcinogenic to humans) and there are several HPV types classed by IARC as group 2B (possibly/rarely carcinogenic to humans). HPV 16 is by far the most carcinogenic in terms of numbers of cases of cervical cancers and causes the most cancers linked to HPV in other anogenital epithelia and in the oropharynx.
Screening program and Renewal
HPV testing was first introduced to the Medicare schedule in Australia with the adoption of the 2005 NHMRC guidelines for the management of asymptomatic women with screen-detected abnormalities. It was recommended for the follow up of high-grade squamous intraepithelial lesions (HSIL) at 12- and 24-months post treatment. At that time the method of HPV detection was not prescribed.
In May 2017, Australia will change the National Cervical Screening Program from primary screening by cytology to primary screening with HPV testing. This is despite Australia having an extremely successful screening program based on cytology. The reasons for the change are that, while the program is effective in ensuring low incidence and mortality rates of cervical cancer (among the lowest in the world), it is relatively intensive compared with other countries. There have also been significant advances in our understanding of the development of cervical cancer. It is now known that infection with HPV is a necessary prerequisite for its development. There is new evidence for alternative pathways for cancer screening and prevention. Ian Hammond’s article (see p28) documents the effects of the HPV vaccination on the Australian population, giving further impetus to examining different screening tests, target age range and screening intervals.
Trials on HPV testing
HPV testing has been studied in a number of different contexts:
- information on rates of persistence of HPV infection;
- investigation of regional and international prevalence of HPV types;
- identification of HPV type distribution in cancers;
- triage of women with equivocal or low-grade cytology;
- follow up of women after treatment for cervical intraepithelial neoplasia; and
- primary screening for cervical cancer precursors, either alone or in combination with cytology.
As a result of points one to three, we know the following
- The majority of HPV infections are transient, most clearing within a few years. However, some persist and it is these persistent infections that may lead to development of precancerous changes.
The prevalence of different HPV types is different in different regions of the world, but worldwide HPV 16 and 18 are the most common high-risk types and approximately 70 per cent of cervical cancers are caused by one of these two genotypes.
HPV as primary screening
Trials examining HPV testing as a primary screening tool have included studies in high-resource settings, such as Australia, and also a study in India, comparing a single HPV test with a single cytology test in a previously never-screened population in a low-resource setting. The latter showed that a single HPV test could significantly reduce the incidence and mortality of cervical cancer compared with the single cytology test. The trials in high resource settings have shown that at five years the incidence of HSIL is lower after a negative HPV test than a negative Pap smear. Also, negative predictive value of a negative HPV test lasts twice as long as a negative Pap smear; thus allowing for longer screening intervals.
In late 2012, the Netherlands became the first country in the world to announce it would change its screening program from cytology to primary HPV screening. The program is expected to commence in January 2016. In November 2011, the Department of Health and Ageing commenced a review of the Australian program, called the Renewal of the National Cervical Screening Program.
The Medical Services Advisory Committee commissioned a review of the evidence for specified screening pathways and an economic modelling and health outcomes review. After a robust process, they made the following recommendations:
- five-yearly HPV test with partial genotyping;
- reflex liquid based cytology triage;
- age range of screening 25–69 years;
- exit HPV testing age 70–74 years;
- the program remain the same for
- HPV vaccinated and unvaccinated women; and
- self-collection HPV test be available for underscreened and never-screened women.
On 19 September 2014, the Australian Health Ministers’ Advisory Council endorsed the recommendations and a draft interim implementation plan.
Technical aspects of HPV testing
Unlike conventional cytology, HPV testing is not one test, but rather a number of different technologies. Many of the original trials on its validity were done using hybrid capture technology. The hybrid capture technology (HC2, Qiagen) is based on a RNA probe binding to the DNA target to form a RNA:DNA hybrid. Amplification (x3000) and detection of RNA:DNA hybrids by means of multiple labelled antibodies shows the presence of 13 high-risk antibodies (not individually identified).
A refinement of the Hybrid Capture 2 technology makes use of hybridisation followed by signal amplification using invader technology. This third-wave invader HPV test (Cervista, Hologic) allows for partial HPV 16 and 18 genotyping.
The other main method of HPV testing is polymerase chain reaction (PCR), which involves amplification of the target sequences in the L1 (a HPV protein) open reading frame (ORF) of the HPV nucleocapsid. It is flexible, has the greatest sensitivity, can be used for detection, quantitation, sequencing, mutation analysis and there are multiple available formats. As well as in-house laboratory PCRs, there are commercially available PCR technologies, including Abbott real-time HPV test and Roche Cobas 4800. Partial HPV genotyping is available with these tests.
Another means of testing for hrHPV infection is detection of mRNA of E6/E7. This method is used by GenProbe for its APTIMA test, which has shown greater specificity than other HPV tests.
Validated HPV tests
In 2009, Meijer et al published the guidelines for HPV DNA test requirements for primary cervical cancer screening in women aged 30 years and older (this was accepted internationally as the ‘Meijer criteria’ for HPV testing). They require that to be validated the test must demonstrate non-inferiority to the HC2 test in terms of sensitivity and specificity and there should be intra-laboratory reproducibility.
HPV Test for Australian NCSP
Since the strength of the HPV test is its negative predictive value and women will only be tested every five years; it is essential that any HPV test used for screening be a validated test that has demonstrated robust performance in a screening environment. In addition, the program has decided to perform partial genotyping to identify HPV types 16, 18 and, possibly, 45. The reason for this decision is that these types are considerably more oncogenic than the other high-risk HPV types, accounting for more than 70 per cent of all cervical cancers. Thus, the tests accepted as part of the screening program will need to be capable of partial genotyping.
It is known that the majority of infections detected by HPV testing will be transient and will not result in precancerous or cancerous lesions. In the future, in addition to using partial genotyping to improve specificity, biomarkers may be used to differentiate these transient infections from the potentially more serious integrated infections. There have been many markers identified and examined; one such, which has been used in some trials to improve specificity, is p16INK4a. The published literature indicates improved specificity, but at the cost of sensitivity. Other potential biomarkers, such as the loss of L1 capsid expression, have also been explored.
In Australia, we are moving to an exciting situation where we could anticipate the near eradication of cervical cancer. The combination of HPV vaccination as a primary prevention strategy and improved secondary prevention with more sensitive screening tests, longer screening intervals and improved detection rates, augurs well for the future health of Australian women.
J. Thomas Cox. History of the use of HPV testing in cervical screening and in the management of abnormal cervical screening results. J Clinical Virology 2009;45 S1:S3-S12.
Arbyn M et al. Evidence regarding HPV testing in Secondary prevention of cervical cancer. Vaccine 2012; 305:88-F99.
Schiffman M et al. HPV testing in the Prevention of Cervical Cancer. Review JNCI. 2011;103:
Meijer C.J.L.M et al. Guidelines for HPV DNA test requirements for primary cervical cancer screening in women of 30 years and older. Int J Cancer. 2009;124(3):516-520.
Cuzick J et al. Comparing the performance of six HPV tests in a screening population. British Journal of Cancer. 2013;108: 908-913.
Renewal evidence and economic modelling documents including the Executive Summary are available from: www.cervicalscreening.gov.au .