New technologies for cervical cancer screening

Dear Colleagues,

The insight in recent years into the causative link between human papillomavirus (HPV) and cervical cancer has opened the door to a national vaccination programme, which is now underway in the target age groups. The link may also have implications for the way that the NHS cervical screening programme is organised, as this month’s edition of Cancer Services Forum explains.

In his informative article, Jack Cuzick presents evidence for the validity of HPV testing as an adjunct to conventional cervical cytology. Of particular interest to those involved in the commissioning and organisation of cancer services, he proposes an algorithm for cervical screening, in which cytology is reserved for women who are HPV-positive, thereby reducing the demand on cytology services. Professor Cuzick also sheds light on the emerging technology of immunostaining, which may eventually have a role in cervical screening.

Given the practical limitations of cytology—the need for accurate sampling and the subjective nature of reporting—and the large number of women in the at-risk population, these new objective tests would appear to offer an attractive new way of approaching cervical screening.

Ian Watson, National GP Cancer Lead

Key Points

• New methods for cervical screening are expected to improve diagnosis while reducing the demand on cytology and colposcopy services
• A model is proposed in which HPV testing is used as the first screening method, with cytology only for those who are positive for the virus
• Self-sampling is a feasible option with HPV testing, and may help to increase uptake of screening

Introduction

The rate of deaths caused by cervical cancer continues to fall—2.3 per 100,000 in England in 2008, compared with 3.2 per 100,000 in 1999¹—a change largely attributable to the work of the NHS Cervical Screening Programme. At present, screening is dominated by cervical cytology, a process that relies on women accepting regular invitations to undergo smear tests, accurate cell sampling conducted by a trained professional, and the work of skilled cyto-screeners. Women found to have pre-cancerous changes—cervical intraepithelial neoplasia (CIN, Table 1)²—are referred for colposcopy. Of the 134,000 referrals to colposcopy in England in 2008–09,¹ 31% were for CIN grade 1, and 18% for borderline changes.³ New methods for detecting women at risk of cervical cancer are becoming available and, if introduced, may help to reduce the number who need to attend for cervical cytology and colposcopy—with the possibility of financial and organisational benefits for local healthcare services. Some of these methods—and perhaps the most likely to be seen in routine practice in the near future—test for human papillomavirus (HPV), the organism that causes cervical cancer.⁴

This article considers the potential role of HPV testing in cervical screening, and looks ahead at an immunostaining test that may also hold promise in this clinical arena.

*CIN remains only pre-cancerous, even at CIN 3

Table 1: CIN terminology in cervical cancer screening²

The case for HPV testing

Sensitivity and long-term validity

The argument for introducing HPV testing as the primary cervical cancer screening test for women over the age of 30 years is now overwhelming. Numerous cross-sectional studies^5–7 have shown it to be more sensitive than cervical cytology for the detection of CIN 2+ (HPV testing, sensitivity >95%; cytology, sensitivity 19–77%),⁵ albeit at a somewhat lower specificity (Figure 1).⁵ These results have been confirmed in the first rounds of various randomised trials in which women underwent cytology, HPV testing or both, and the findings were compared with those of earlier cross-sectional studies in which all women received both cytology and HPV testing.^6–11 More recently, evidence has emerged that a negative HPV test result is associated with a longer period of low risk than is seen after a negative conventional smear (Figure 2).^12,13

Figure 1: Sensitivity of cytology and HPV testing for CIN 2+. Updated from Cuzicket al (2006)⁵

Figure 2: Kaplan-Meier plots of the cumulative incidence rate of CIN 3+ according to baseline test results in the first 72 months of follow up–from an overview of trials¹²

Practical advantages

Use of HPV testing as the sole primary screening modality has several advantages:

• HPV assay is automated, objective and very sensitive

• Enhances quality control

• Reduces the grounds for medicolegal claims, compared with cytology, which relies on subjective interpretation of cell morphology¹⁴

• Cytology can be reserved for women who are HPV-positive (approximately 6% of the screened population4)

• Facilitates low-volume, high-quality cytology

• Requires the employment of fewer, more focused cyto-screeners than are required for mass cytology-based screening

• Avoids unnecessary colposcopy for women with borderline (atypical cells of undetermined significance) findings or low-grade squamous intraepithelial lesions (LSILs) at cytology but who are HPV-negative

• Reduces demand on colposcopy services

• Avoids anxiety in this patient group

• May enable a longer screening interval,^12,13 leading to:

• Lower overall screening costs

• Less inconvenience for women

HPV testing as the primary screen

A proposed algorithm for cervical screening, based on HPV testing followed by targeted use of cytology, is shown in Figure 3. This model may need modification to satisfy local criteria; in particular, the issues of when to start screening and the appropriate screening interval remain controversial.

However, it illustrates how an initial HPV test can be used to help identify women for whom cytology, and perhaps subsequent colposcopy, is the necessary next step.

Figure 3: Proposed algorithm for cervical cancer screening

Type-specific HPV tests

Approximately 15–18 HPV types are considered to be oncogenic.^15,16 However, HPV-16 and, to a lesser extent, HPV-18 and HPV-45, may confer a greater risk than other types.^16,17 For example, Khan et al have shown a sustained increase in the risk of developing CIN 3 or cervical cancer for up to 10 years after an initial positive HPV-16 result, compared with positivity for other oncogenic types.¹⁷ For this reason, it may be efficient to genotype women who give a positive result when tested with a pooled HPV assay, thus allowing follow up to be tailored to those at greatest risk.

Self-sampling for HPV

The requirements for a good sample are less rigorous for HPV testing than for cytology, and self-sampling may be a feasible option. An overview of various studies has shown that selfsampling produced an overall relative sensitivity of 74% and a specificity of 88%.¹⁸ Although this sensitivity is inferior to that of clinician-taken sampling (81%),¹⁸ self-sampling for HPV compares favourably with cytology, where the sensitivity for detection of CIN 2+ is typically less than 75%.⁵ These results suggest that self-sampling for HPV is a valuable screening method for women who refuse to attend for clinician-based screening, and hence a potential method for improving population coverage.

Several studies are currently evaluating new selfsampling collection devices, which it is hoped will improve performance still further.

mRNA expression of E6 and E7 transcripts

Persistent expression of the viral oncogenes E6 and E7 is a necessary step for HPV-induced carcinogenesis,¹⁹ so detection of E6 and E7 mRNA for high-risk types may be an indicator not only of infection, but also of a further step in the progression towards cancer. Indeed, detection of E6 and E7 transcripts is expected to result in increased specificity for high-grade lesions compared with HPV DNA detection. A high detection rate of E6 and E7 transcripts has been found in cervical cancer tissues,²⁰ and a relationship with histological severity has been observed in cervical biopsies.²¹

Two kits are currently available for conducting mRNA testing. One (Pre-Tect HPV-proofer, Norchip) detects E6 and E7 mRNA from five HPV types (HPV-16, HPV-18, HPV-31, HPV-33 and HPV-45).²² The other (APTIMA, GenProbe) is a broad-spectrum mRNA test for 14 HPV types.²³ These tests were recently compared with a range of DNA tests in 953 women referred for colposcopy because of abnormal smear results.²⁴ The APTIMA test had a very high sensitivity (97.4% for CIN 3+), which was similar to the DNA-based tests, but its specificity (38.8%) was substantially better (e.g. 13.4% better than the Hybrid Capture II HPV DNA test). By contrast, the Norchip test had a lower sensitivity (82.2%), but a substantially better specificity (70.4%).²⁴ It is unclear if the lesions missed by the Norchip test were progressive or not, but its lower sensitivity could be seen to limit its potential use to a second-level test to determine which women with low-grade smears or a positive HPV test need immediate referral. The APTIMA test could, in principle, also be used as a primary screening test, but it has not yet been evaluated in cytologically normal women.

Immunostaining

Expression of the cyclin-dependent kinase inhibitor p16ink4a (hereafter referred to as p16) is negatively controlled by the pRB gene product. p16 is usually expressed at a very low level in normal cells, but is strongly overexpressed in cervical cancer cell lines in which RB has been inactivated by the highrisk HPV E7 oncoprotein.²⁵ p16 overexpression, which can be recognised by immunostaining, may be a good marker not only of HPV infection, but also of activated expression of viral genes and of virus-induced deregulation of the cell cycle.²⁶ Immunostaining for p16 has been found to be associated with intraepithelial or invasive neoplasia in cervical histology specimens.²⁷ p16 staining of liquid-based cytology shows reasonable overall correlation with morphological classification,²⁸ but nondysplastic cells, particularly metaplastic, atrophic and endocervical cells, also display p16 immunoreactivity, therefore reducing specificity.

To improve specificity, the application of a nuclear score to p16-positive cells has been proposed. p16 immunostaining has been suggested as a tool for triaging women with low-grade or borderline cytology. It has been found to have good specificity (65.8%), but its sensitivity (92.7%) is well below that which can be obtained using a DNA-based HPV test, where sensitivity as high as 99.5% has been reported.²⁴ When using HPV testing as the primary screening test, p16 immunostaining may be useful for triaging HPV-positive women to distinguish those who need direct referral to colposcopy from those who can be managed by re-testing in 6–12 months. However, the sensitivity and specificity of p16 testing in cytologic material still needs further evaluation.

Conclusion

Although HPV vaccination is expected to have a significant impact on cervical cancer mortality,¹ this has not been shown in women who are already sexually active,²⁹ and hence will not lessen the need for screening for many decades.

Meanwhile, it will be important to improve screening through more effective testing combined with a high level of population coverage. New testing methods, based on detection of oncogenic HPV types, may not only enhance the sensitivity of screening, but also help to reduce the pressure on diagnostic services though a reduction in unnecessary referral for colposcopy.

References

1. National Health Institute. Cervical screening programme, England, 2008–09. London: NHS, 2009.
2. Cancer Research UK. Cervical cancer screening. Available at: www.cancerhelp.org.uk/type/cervical-cancer/about/cervical-cancerscreening (accessed June 2010).
3. National Health Institute. NHS cervical screening programme. Annual review 2009. London: NHS, 2009.
4. Cuzick J, Szarewski A, Cubie H et al. Management of women who test positive for high-risk types of human papillomavirus: the HART study. Lancet 2003; 362: 1871–1876.
5. Cuzick J, Clavel C, Petry KU et al. Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer 2006; 119: 1095–1101.
6. Ronco G, Segnan N, Giorgi-Rossi P et al. Human papillomavirus testing and liquid-based cytology: results at recruitment from the new technologies for cervical cancer randomized controlled trial. J Natl Cancer Inst 2006; 98: 765–774.
7. Mayrand MH, Duarte-Franco E, Rodrigues I et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 2007; 357: 1579–1588.
8. Ronco G, Giorgi-Rossi P, Carozzi F et al. Results at recruitment from a randomized controlled trial comparing human papillomavirus testing alone with conventional cytology as the primary cervical cancer screening test. J Natl Cancer Inst 2008; 100: 492–501.
9. Kotaniemi-Talonen L, Anttila A, Malila N et al. Screening with a primary human papillomavirus test does not increase detection of cervical cancer and intraepithelial neoplasia 3. Eur J Cancer 2008; 44: 565–571.
10. Bulkmans NW, Berkhof J, Rozendaal L et al. Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial. Lancet 2007; 370: 1764–1772.
11. Kitchener HC, Almonte M, Thomson C et al. HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol 2009; 10: 672–682.
12. Dillner J, Rebolj M, Birembaut P et al. Long term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study. BMJ 2008; 337: a1754.
13. Cuzick J, Szarewski A, Mesher D et al. Long-term follow-up of cervical abnormalities among women screened by HPV testing and cytology—results from the Hammersmith study. Int J Cancer 2008; 122: 2294–2300.
14. Sasieni P, Cuzick J. Could HPV testing become the sole primary cervical screening test? J Med Screen 2002; 9: 49–51.
15. Allan B, Marais DJ, Hoffman M, Shapiro S, Williamson AL. Cervical human papillomavirus (HPV) type infection in South African women: implications for HPV screening and vaccine strategies. J Clin Microbiol 2008; 46: 740–742.
16. Muñoz N, Bosch FX, de Sanjosé S et al for the International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: 518–527.
17. Khan MJ, Castle PE, Lorincz AT et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005; 97: 1072–1079.
18. Ogilvie GS, Patrick DM, Schulzer M et al. Diagnostic accuracy of self collected vaginal specimens for human papillomavirus compared to clinician collected human papillomavirus specimens: a meta-analysis. Sex Transm Infect 2005; 81: 207–212.
19. zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol 1994; 48: 427–447.
20. Nakagawa S, Yoshikawa H, Yasugi T et al. Ubiquitous presence of E6 and E7 transcripts in human papillomavirus-positive cervical carcinomas regardless of its type. J Med Virol 2000; 62: 251–258.
21. Kraus I, Molden T, Erno LE, Skomedal H, Karlsen F, Hagmar B. Human papillomavirus oncogenic expression in the dysplastic portio; an investigation of biopsies from 190 cervical cones. Br J Cancer 2004; 90: 1407–1413.
22. Molden T, Kraus I, Skomedal H, Nordstrøm T, Karlsen F. PreTectTM HPV-Proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses. J Virol Methods 2007; 142: 204–212.
23. Dockter J, Schroder A, Eaton B et al. Analytical characterization of the APTIMA HPV Assay. J Clin Virol 2009; 45 (Suppl 1): S39–S47.
24. Szarewski A, Ambroisine L, Cadman L et al. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev 2008; 17: 3033–3042.
25. Khleif SN, DeGregori J, Yee CL et al. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity. Proc Natl Acad Sci USA 1996; 93: 4350–4354.
26. von Knebel DM. New markers for cervical dysplasia to visualise the genomic chaos created by aberrant oncogenic papillomavirus infections. Eur J Cancer 2002; 38: 2229–2242.
27. Dray M, Russell P, Dalrymple C et al. p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: experience with squamous lesions in 189 consecutive cervical biopsies. Pathology 2005; 37: 112–124.
28. Yoshida T, Fukuda T, Sano T, Kanuma T, Owada N, Nakajima T. Usefulness of liquid-based cytology specimens for the immunocytochemical study of p16 expression and human papillomavirus testing: a comparative study using simultaneously sampled histology materials. Cancer 2004; 102: 100–108.
29. National Health Service. The new HPV vaccine. A Q&A sheet for girls & their parents on the HPV vaccination. London: DH, 2008.