Cervical cytology screening has reduced cervical cancer morbidity and mortality but shows important shortcomings in terms of sensitivity and specificity. Infection with distinct types of human papillomavirus (HPV) is the primary etiologic factor in cervical carcinogenesis. This causal relationship has been exploited for the development of molecular technologies for viral detection to overcome limitations linked to cytologic cervical screening. HPV testing has been suggested for primary screening, triage of equivocal Pap smears or low-grade lesions and follow-up after treatment for cervical intraepithelial neoplasia. Determination of HPV genotype, viral load, integration status and RNA expression could further improve the effectiveness of HPV-based screening and triage strategies. The prospect of prophylactic HPV vaccination stresses the importance of modification of the current cytology-based screening approach. (Cancer Epidemiol Biomarkers Prev 2008;17(4):810–7)

Cervical cancer is the second most common malignancy and cause of cancer-related death in women worldwide (1, 2). Invasive cervical carcinoma is preceded by precursor lesions, which are characterized by disturbances of cellular maturation, stratification, and nuclear atypia (3, 4) and can be classified histologically as cervical intraepithelial neoplasia (CIN) or cytologically as squamous intraepithelial lesions according to the Bethesda terminology (5, 6). Because of this well-defined premalignant phase, cervical cancer is particularly amenable to screening (6). The classic screening method is based on the cytologic evaluation of Papanicolaou (Pap)–stained cervical smears, in which cervical cells are scraped from the transformation zone and transferred to a glass slide (7, 8). During the last 50 years, screening programs based on Papcytology have undoubtedly reduced cervical cancer morbidity and mortality (4, 6, 9-11). In spite of its success, the Paptest is a subjective method with a limited sensitivity of 50% and high susceptibility to intraindividual and interindividual variability (6, 11, 12). Introduction of liquid-based cytology (LBC) has contributed to mitigating the problem of efficiency in processing samples, but the diagnostic validity in terms of sensitivity and specificity still shows important shortcomings (11, 13-15). Despite the substantial resources spent in cytologic screening and follow-up, cervical cancer still is the 10th most common cause of cancer death in European women (16). Because cervical cancer is the only cancer that is almost completely preventable through regular screening and thus early treatment, improvement and expansion of existing screening strategies and technologies constitutes a main target of the European Council Recommendation on Cancer Screening (17).

Over the last decade, astonishing progress has been made in understanding the pathogenesis of cervical cancer. An overwhelming body of evidence shows that infection with distinct types of the human papillomavirus (HPV) is the primary risk factor for the development of cervical cancer and its precursor lesions (2, 17-19). HPVs are small circular double-stranded DNA viruses that belong to the Papovaviridae family. The HPV genome is ∼8,000 bp in length and encodes eight open reading frames, which are transcribed as polycistronic mRNAs (20). The gene products can be divided into “early” (E) and “late” (L) proteins, depending on the time of expression during the viral life cycle (Fig. 1; ref. 21). The critical molecules in viral replication are E6 and E7, which functionally inactivate the products of two important tumor suppressor genes, p53 and pRb, respectively. Both oncoproteins induce proliferation, immortalization, and malignant transformation of the infected cells (21, 22).

Figure 1.

Schematic representation of the HPV genome, highlighting the regions important in PCR-based HPV analysis. L1 region, consensus PCRs, such as SPF, GP5+/6+, and MY09/11, target different conserved regions of the HPV L1 gene, amplifying several genital HPV types in one reaction. Amplicons can be analyzed to discriminate between HPV types by hybridization with type-specific probes or DNA sequencing. E6/E7 region, type-specific PCR assays target specific sequences of viral early genes, usually E6 and E7, and exclusively amplify a single HPV genotype. Application of HPV type–specific PCRs allows immediate discrimination between different HPV types. Quantitative PCR allows simultaneous assessment of HPV presence, genotype, and viral load. E1/E2 region, quantification of the E2 gene allows determination of the HPV integration status.

Figure 1.

Schematic representation of the HPV genome, highlighting the regions important in PCR-based HPV analysis. L1 region, consensus PCRs, such as SPF, GP5+/6+, and MY09/11, target different conserved regions of the HPV L1 gene, amplifying several genital HPV types in one reaction. Amplicons can be analyzed to discriminate between HPV types by hybridization with type-specific probes or DNA sequencing. E6/E7 region, type-specific PCR assays target specific sequences of viral early genes, usually E6 and E7, and exclusively amplify a single HPV genotype. Application of HPV type–specific PCRs allows immediate discrimination between different HPV types. Quantitative PCR allows simultaneous assessment of HPV presence, genotype, and viral load. E1/E2 region, quantification of the E2 gene allows determination of the HPV integration status.

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Of the >100 different HPV types, 40 are known to infect the genital tract (2). These mucosal types are classified as “low-risk” and “high-risk” based on their prevalence in cervical cancer and its precursors. Low-risk HPV types, such as 6 and 11, induce benign lesions with minimum risk of progression to malignancy. By contrast, high-risk HPVs (HR-HPV) have higher oncogenic potential (21). The tremendous importance of HPV in cervical carcinogenesis has opened up possibilities for cancer prevention.

The establishment of HPV as central and necessary cause of cervical cancer was exploited for the development of molecular technologies for viral detection (19, 23) to overcome limitations linked to cytologic cervical screening. HPV DNA testing identifies women at risk for developing cervical neoplasia without the inherent subjectivity of cervical cytologic assessment (11, 24).

Therefore, HPV testing has been suggested for primary screening (16, 25, 26), triage of equivocal Pap smears or low-grade lesions (25, 27, 28), and follow-up after treatment for CIN (25, 29-31).

Primary Screening

Recent independent studies indicate that HPV testing, as a primary screening method, has a higher sensitivity (25-35% higher) and a higher negative predictive value for the detection of preinvasive disease than cytology (11, 32-34). Therefore, in the United States, it was recently concluded to add HPV testing to cytology screening after the age of 30 at an interval of 3 years if both tests are negative (11, 25).

An important drawback of HPV screening, compared with cytologic screening, is its lower specificity (5-10% lower) and low positive predictive value for high-grade CIN due to the high prevalence of transient infections (15, 25). HPV acquisition peaks near the late teens or early 20s and these HPV infections and associated mild lesions almost always clear spontaneously, rendering HPV screening at young age less efficient. Therefore, age plays a tremendous role in the determination of the target population (25, 35).

To date, the applicability of HPV as sole primary screening modality has only been evaluated in cross-sectional comparisons or epidemiologic studies. Large trials comparing this approach to cytology alone are needed to assess the effect of primary HPV screening on cancer incidence and mortality (35). In Europe, several randomized controlled trials are being conducted to establish the performance of HPV testing as a primary cervical cancer screening test. The main postulated outcome of these trials is a reduction in the cumulative incidence of high-grade CIN 3 to 5 years after screening among baseline HPV-negative compared with baseline cytology-negative women. Until the results are published in 2008, the Pap smear continues to be the standard screen test in the European Union (11, 16, 25). The approach of using HPV as the sole primary screening modality has several advantages: HPV assays provide an automated, objective, and highly sensitive test; the need for cytology would be reduced, improving its quality; unnecessary triage of equivocal and low-grade lesions would be avoided; and the screening interval could be safely prolonged, improving cost-efficiency and convenience of screening (35).

Triage of Equivocal Pap smears

Low-grade squamous intraepithelial lesion (LSIL) and atypical squamous cells of undetermined significance (ASCUS) represent the largest fraction of abnormalities in cervical cancer screening and comprise the most histologically confirmed high-grade abnormalities. HPV testing was proposed to achieve cost-effective management of these diagnostic categories (36, 37). The ASCUS-LSIL Triage Study has investigated in a prospective, randomized fashion the optimal management of LSIL and ASCUS by three strategies: immediate colposcopy, HPV triage, and repeated cytology. HPV triage seemed to be at least as sensitive as immediate colposcopy in the detection of high-grade CIN, whereas the number of women referred for colposcopy was halved (38). Therefore, HPV triage emerges as the best strategy for management of women with ASCUS. As other studies strengthened the effectiveness of this approach (28), the use of HPV detection in triage of ASCUS has been introduced into many international guidelines.

On the other hand, because cytologic interpretation of LSIL is fairly reproducible and the majority of LSIL cases (>80%) are HPV positive, the use of HPV testing for the management of LSIL is not cost-effective, as the majority of women would still be referred for colposcopy (27). As yet, it is still unclear whether determination of HPV genotype or viral load would be useful in triage of LSIL.

Follow-up after Treatment

Women treated for CIN should be followed up regularly to monitor their outcome. HPV testing was suggested to predict residual or recurrent CIN in women treated for high-grade cervical lesions. Current data show that HPV testing indicates residual disease more quickly, with higher sensitivity and similar specificity than follow-up cytology or histological assessment of section margins. A negative HPV test allows shortening of the posttreatment surveillance period, but more long-term data are necessary to present detailed evidence-based follow-up algorithms (29-31).

HPV Detection

The two methodologies most widely used for HPV DNA detection are PCR and the Hybrid Capture II system (HC2, Digene Corp.). HC2 is a nucleic acid hybridization assay for the qualitative detection of DNA of 13 carcinogenic (probe A) and 5 benign HPV types (probe B) in cervical specimens. It is the only HPV test which has been Food and Drug Administration (FDA)–approved for ASCUS triage and cervical cancer screening in combination with cytology after the age of 30 and is used in clinical settings worldwide as a robust and reproducible screening assay (14, 39). However, this test shows a number of disadvantages, such as the inability to identify specific HPV genotypes and the risk of cross-hybridization of additional HPV types with the probe mix (40, 41). Moreover, its applicability in routine screening is hampered by cost implications, clinical effectiveness of repeated testing in follow-up, and its lower sensitivity compared with PCR (12, 23).

There are two relevant approaches for detection of HPV DNA by PCR: consensus PCR and type-specific PCR. The most widely used PCR assays use consensus primers, such as GP5+/6+, MY09/11, and SPF, which target a highly conserved region of the HPV L1 gene, amplifying numerous genital HPV types in one reaction (Fig. 1; refs. 42, 43). The GP5+/6+ PCR set consists of two primers that detect a broad range of HPVs at lowered annealing temperature (23). MY09/11 PCR, on the other hand, is synthesized with several degenerate nucleotides in each primer, generating a mixture of 25 primers that are capable of amplifying a wide spectrum of HPV types (44, 45). Although the MY09/11 and GP5+/6+ systems yield a nearly identical prevalence of HPV in a set of clinical samples, the MY09/11 primers detect twice as many multiple infections (46).

PGMY09/11 primers were designed to improve MY09/11 sensitivity across the type spectrum with increased detection of multiple infections and improved reproducibility and specificity (42, 47). Surprisingly, one report shows similar analytic sensitivities for MY09/11 and PGMY09/11, with better detection of several important HPV types, including HPV16, by MY09/11. As such, caution should be exerted and reproducibility should be monitored when comparing performances of different primer systems (48).

SPF primers are technically analogue to PGMY09/11, as they also include inosine, which matches with any nucleotide and allows PCR at optimum annealing temperature, leading to higher HPV detection rates than those of MY09/11 (49, 50).

Because type-specific PCRs often target specific sequences of viral early genes and exclusively amplify a single HPV genotype, multiple PCRs must be done to detect the presence of HPV DNA in one sample (Fig. 1; ref. 23). Recently, a comparison between MY09/11 consensus PCR and type-specific PCRs showed that consensus PCR frequently missed clinically important HPV infections. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration, or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. When type-specific PCRs are combined with fluorescent probes for real-time detection, multiplexing of several primers allows high-throughput, type-specific HPV detection with excellent cost-effectiveness and turnaround times (51).

A commercial PCR-based assay for HPV detection in cervical scrape specimens is the Roche Amplicor HPV test, which involves a pool of primers amplifying the same 13 HR-HPV types as included in HC2 assay (52, 53).

HPV Typing

HPV types differ in their ability to induce cervical carcinogenesis. HPV16 and HPV18 are the most prevalent types in invasive cervical cancer (54). Therefore, HPV genotyping approaches could be more appropriate for the identification of individuals at risk of disease than a presence/absence test (55).

After consensus PCR amplification, HPV types can be discriminated by reverse hybridization with type-specific probes, but also DNA sequencing can be done (Fig. 1). However, the latter technique is inappropriate for genotyping of multiple infections, undermining its clinical applicability (56). The most frequently used reverse hybridization technology is the line probe or line blot assay, comprising multiple probes immobilized as parallel lines on a membrane strip. The commercial Roche Linear Array HPV genotyping test, which was developed based on the PGMY09/11 PCR in combination with a line blot assay, has become a convenient tool in epidemiologic studies for the detection and typing of HPV DNA (57).

SPF PCR served to develop the commercial INNO-LiPA HPV assay, which is capable of genotyping 25 different HPV types simultaneously and has proved to be sensitive, specific, simple, and rapid in the assessment of HPV (52, 58).

Although these assays are capable of typing a relatively large spectrum of HPV genotypes, they cannot be automated or deployed in a high-throughput platform. Therefore, improved genotyping methods have recently been developed, such as the Luminex xMAP system in which microspheres coated with HPV type-specific probes provide a rapid and cost-effective method to simultaneously detect 26 different HPV genotypes (59).

Also the application of HPV type-specific PCRs allows immediate discrimination between different HPV types in a high-throughput clinical setting (23, 43).

HPV Viral Load

The amount of HR-HPV DNA in a cervical sample, i.e., the viral load, has been suggested as a variable to distinguish HPV infections of clinical relevance. A high HPV load could be considered as a type-dependent risk marker for high-grade cervical lesions or carcinoma (60-63). However, the initial optimism regarding its clinical value was tempered by inconsistencies in the association between viral load and duration of infection, HPV clearance, and subsequent risk of acquisition or progression of disease (2).

The amount of HPV DNA can be determined by quantitative real-time PCR, and a multiplex format allows simultaneous assessment of HPV presence, genotype, and viral load (Fig. 1; refs. 51, 64, 65). Most studies using quantitative HPV PCR methods show a substantial overlap of viral load values among women with and without high-grade CIN, especially in the range of high viral loads. This approach precludes cutoff values for high-grade CIN on the basis of high viral loads, as such limiting the clinical applicability of viral load analysis (35). A recent publication concludes that high viral load is associated with prevalent cervical cancer precursors for most HR-HPV genotypes, but only HPV16 load predicts the development of incident disease (66).

HPV Integration

In cervical cells, HPV can occur in episomal form, integrated form, or both. Viral integration in the human genome often happens in the viral E1 or E2 region and can result in the loss of negative feedback control of oncogene expression by the regulatory E2 protein. Moreover, HPV integration increases the stability of integrant-derived E6 and E7 transcripts (67).

The physical state of the virus can be determined by the failure to amplify full-length E2 using PCR, but also by using more comprehensive Southern blot hybridization. Real-time PCR assays, which simultaneously measure E2 and E6 copy numbers, have recently been developed to determine the integration status (Fig. 1). However, unavoidable technical limitations related to the abundance of episomal forms, low viral load, or the length of the E2 amplicon should be considered when interpreting integration studies (2, 68). For now, it remains largely unclear whether the measurement of HPV integration status could be a useful biomarker for progressive disease. Several studies suggest that identification of integrated viral forms could support HPV-based screening and triage strategies (2, 69-75).

HPV RNA Detection

In cervical carcinogenesis, expression of viral oncogenes is a prerequisite for progression toward malignancy and maintenance of the cancerous phenotype, with E6 and E7 as the main arbitrators (21, 22). Therefore, detection of RNA transcripts of genes involved in oncogenesis enables differentiation between asymptomatic HPV infections and infections associated with high-risk lesions and cervical carcinoma and could therefore be considered as a better risk factor than mere DNA detection (12, 35, 55, 76). RNA as a potential target for routine clinical diagnostics may improve sensitivity, reproducibility, and specificity compared with DNA. As fixation of cells can interfere with RNA quality, it would be favorable that fixatives used for routine collection and processing of cervical specimens maintain RNA integrity (77, 78).

Currently, one RNA-based HPV assay is commercially available, the PreTect HPV Proofer (Norchip AS; ref. 79). This assay incorporates nucleic acid sequence-based amplification (NASBA) of E6/E7 mRNA transcripts before type-specific detection for HPV16, HPV18, HPV31, HPV33, and HPV45 (ref. 23; Fig. 2). It has been suggested for triaging HPV DNA-positive women and women with equivocal cytology (73, 80, 81). Compared with HPV DNA detection, the presence of E6/E7 mRNA transcripts was less sensitive, but more specific for the detection of disease and follow-up (55). Application of NASBA technology is not restricted to the use of the PreTect HPV proofer kit. In-house primers and molecular beacons can be developed to detect HPV types of interest. As NASBA is a sensitive and fast technique with commercially available basic reagents, it is well suitable for application in a routine screening setting. In principle, RNA detection can be applied as a primary screening test, but this has never been evaluated. Sensitivity is limited when only five HR-HPV types are detected. A broad spectrum mRNA test (15 types), Aptima (GenProbe), is currently under development (35). Preliminary evaluation of the prototype assay showed that HR-HPV E6/E7 mRNA detection, compared with HR-HPV DNA detection, improved the association of positive test results with cervical precancer and cancer by reducing the number of tests positive in women without precancer without reducing clinical sensitivity for cervical precancer and cancer (82).

Figure 2.

NASBA amplification reaction. NASBA is a sensitive transcription-based amplification method that specifically targets RNA and has been applied for the detection of viral genomes, viroids, rRNAs, and mRNAs. The technology relies on three enzymes: avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase, which uses the T7 promotor at the 5′ end of the forward primer. In NASBA, only nucleic acids which are single-stranded in the primer binding regions function as a template. Because the reaction is isothermal (41°C), specific amplification of single-stranded RNA in the presence of double-stranded DNA is possible, as long as DNA denaturation is prevented in the sample preparation procedure. This makes the method useful for specific mRNA detection in a background of genomic DNA, even for intronless genes. The resulting single-stranded RNA amplicons can be easily detected by hybridization with sequence-specific probes, such as molecular beacons.

Figure 2.

NASBA amplification reaction. NASBA is a sensitive transcription-based amplification method that specifically targets RNA and has been applied for the detection of viral genomes, viroids, rRNAs, and mRNAs. The technology relies on three enzymes: avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase, which uses the T7 promotor at the 5′ end of the forward primer. In NASBA, only nucleic acids which are single-stranded in the primer binding regions function as a template. Because the reaction is isothermal (41°C), specific amplification of single-stranded RNA in the presence of double-stranded DNA is possible, as long as DNA denaturation is prevented in the sample preparation procedure. This makes the method useful for specific mRNA detection in a background of genomic DNA, even for intronless genes. The resulting single-stranded RNA amplicons can be easily detected by hybridization with sequence-specific probes, such as molecular beacons.

Close modal

High RNA quality is required for the application of real-time reverse transcriptase PCR to evaluate E6/E7 mRNA expression levels (83). The biological importance of the extent of oncogene expression in cervical carcinogenesis suggests that quantification of E6 and E7 transcription may be useful as a prognostic tool to identify women at increased risk of developing cervical cancer.

HPV infection and its consequences are associated with changes in expression levels and/or function of host genes (6). Improved understanding of the molecular pathways of cervical carcinogenesis led to the discovery of clinically useful biomarkers. This translational research approach identified markers that reflect deregulation of the cell cycle in cervical neoplasia, such as p16, Ki-67, MCM proteins, and cyclin E (6, 84, 85). However, many of these biomarkers are only indicative of the presence of aberrant S-phase induction and lack specificity for cervical malignancy (6). Alternative candidate markers can emerge from an approach focused on proteomic analysis of cervical cancer samples. Proteomics is widely accepted as a powerful tool in the development of molecular diagnosis and the identification of disease biomarkers in the postgenomic era (86-89). Proteomic profiling of altered proteins may provide new insights into cervical carcinogenesis and yield new biomarkers, serving as molecular signposts to detect early cancer and people at risk for developing disease (90).

The strong relationship between HPV and cervical cancer has also opened up the possibility of primary prevention by the development of prophylactic vaccines against HR-HPV infections. HPV vaccines are based on virus-like particles (VLP) assembled from recombinant HPV capsid proteins L1 and L2 (91). To date, two effective prophylactic VLP L1 vaccines, which are capable of inducing virion neutralizing antibodies, have been developed (37, 92-98). Cervarix is a bivalent HPV16/18 vaccine developed by GlaxoSmithKline (GlaxoSmithKline Biologicals), and the FDA–approved vaccine Gardasil is a quadrivalent HPV16/18/6/11 vaccine developed by Merck (Merck and Co. Inc.). These four HPV types cause the vast majority of anogenital disease (99, 100). HPV16 and HPV18 account for 62% to 77% of all cervical cancers, depending on the geographic region (54). HPV6 and HPV11 infections are responsible for over 90% of the low-risk HPV-associated disease (100). Ongoing clinical trials are currently investigating the long-term efficacy of both bivalent and quadrivalent vaccines (92).

The establishment of vaccination programs and reasonable levels of coverage do not imply that cervical screening programs can be discontinued (15, 92, 101). One reason is that the primary target population consists of 9-year-old to 13-year-old females and the “catch-up” vaccination of older women will likely be achieved at much lower coverage rates. Moreover, vaccination will not protect against all oncogenic HPV types, although some cross-protection against other HR-HPV types will be achieved by vaccinating against HPV16 and HPV18. Inevitably, screening and prevention strategies should be adapted to one another. Cervical cytology screening programs will require modification to attain cost-effective cervical cancer control and surveillance of vaccinated populations. They could assume a new role in monitoring the long-term effectiveness of vaccination and the changes in the natural history of HPV malignancy (11, 15, 102).

Cervical cancer shows the greatest burden in developing countries, which suffer with considerable barriers to set up cytology-based screening programs (35, 103). After all, high-quality cytology requires trained personnel and specialized equipment, and Pap-based algorithms entail multiple visits. Although HPV vaccines may eventually provide the best possible solution for prevention of cervical cancer in these countries, innovative HPV-based approaches might enable the establishment of a feasible and effective screening policy. However, HPV testing mostly requires high-technology laboratory-based molecular analyses, involving high costs. Currently, several initiatives aim at the development of rapid, simple, accurate and affordable HPV tests. One test, based on Digene's HC2, allows testing for oncogenic HPVs in 96 samples in <2 h. Another assay by Arbor Vita Corporation targets HR-HPV E6 and yields results in 20 min. Because these tests offer rapid results, women could ultimately be screened and treated during the same visit. Because the need for a good sample is less imperative for HPV testing than for cytology, the possibility of self-sampling has been explored. Optimistic results concerning the diagnostic accuracy of self-collected vaginal specimens and cost effectiveness indicate that this strategy might be valuable for the improvement of population coverage (35).

The organization of an effective public health prevention program is complex. The scientific knowledge about cervical cancer and HPV, which has accumulated over the last decades, has opened the possibility to improve existing prevention strategies and screening practices. HPV testing could reduce incidence and mortality from cervical cancer. Sensitive molecular testing techniques can bypass the limitations of cervical cytology screening and can offer women greater protection against cervical cancer at lower cost (11). Ongoing randomized control trials of primary HPV screening will yield the degree of evidence necessary for public health policymakers to make informed decisions about the future of cervical cancer screening programs (15). Especially in an era of HPV vaccination, it makes public health sense to develop a screening system based on HPV monitoring. One of the most neglected aspects of the potential effect of prophylactic HPV vaccines is the evaluation of existing screening practices to permit synergy between primary and secondary prevention efforts (104). Therefore, the biggest issue that should be debated upon the screening community is not whether to incorporate HPV testing into screening programs, but how to incorporate it (11, 14).

In our opinion, HPV detection, with a higher sensitivity and negative predictive value for the detection of preinvasive disease than cytology, is without a doubt the preferred primary test in a routine screening setting. The more specific test, cytologic reflex testing, should be done to triage HPV-positive samples and guide the clinical response. Both tests can be done on the same LBC sample. In the early phase of adopting HPV primary screening, both HPV detection and cytology could be done simultaneously, mainly to increase the confidence of cytopathologists/cytotechnologists in HPV screening. Clinicians should properly educate their patients about the effect of their HPV positivity, an aspect which is overlooked when commercial interests of vaccine-producing companies seem to surpass the importance of patients' tranquillity of mind.

Beyond the appropriate introduction of HPV testing, an improvement of population health can be expected from an increased coverage of the target population and quality control of the different steps in the screening process. In Belgium, the 3-year Pap screening coverage in women 25 to 64 years old currently amounts to only 59%, whereas many of the women are overscreened (105). These data underline the importance of implementing cancer screening programs with a call/recall system, appropriate quality control at all levels, with effective diagnostic, treatment, and after-care service following evidence-based guidelines.

Grant support: Fund for Scientific Research Flanders (FWO-Vlaanderen G.0205.04), Belgian Cancer Foundation (Belgische Stichting tegen Kanker), and Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: G.A.V. Boulet and C.A.J. Horvath contributed equally to this work.

1
Parkin DM, Bray F. Chapter 2: the burden of HPV-related cancers.
Vaccine
2006
;
24
:
S11
–25.
2
Woodman CB, Collins SI, Young LS. The natural history of cervical HPV infection: unresolved issues.
Nat Rev Cancer
2007
;
7
:
11
–22.
3
Cannistra SA, Niloff JM. Cancer of the uterine cervix.
N Eng J Med
1996
;
334
:
1030
–8.
4
Stanley MA. Prognostic factors and new therapeutic approaches to cervical cancer.
Virus Res
2002
;
89
:
241
–8.
5
Smith JFH. Bethesda 2001.
Cytopathology
2002
;
13
:
4
–10.
6
Baldwin P, Laskey R, Coleman N. Translational approaches to improving cervical screening.
Nat Rev Cancer
2003
;
3
:
217
–26.
7
Koss LG. The Papanicolaou test for cervical cancer detection.
JAMA
1989
;
261
:
737
–43.
8
Bekkers RLM, Massuger LFAG, Bulten J, et al. Epidemiological and clinical aspects of human papillomavirus detection in the prevention of cervical cancer.
Rev Med Virol
2004
;
14
:
95
–105.
9
Michalas SP. The Pap test: George N. Papanicolaou (1883–1962) A screening test for the prevention of cancer of uterine cervix.
Eur J Obstet Gynecol Reprod Biol
2000
;
90
:
135
–8.
10
Kitchener HC, Castle PE, Cox TJ. Chapter 7: achievements and limitations of cervical cytology screening.
Vaccine
2006
;
24S3
:
S3/63
–S3/70.
11
Wright TC. Cervical cancer screening in the 21st century: is it time to retire the PAP smear?
Clin Obst Gynecol
2007
;
50
:
313
–23.
12
Lie AK, Risberg B, Borge B, et al. DNA- versus RNA-based methods for human papillomavirus detection in cervical neoplasia.
Gynecol Oncol
2005
;
97
:
908
–15.
13
Arbyn M, Herbert A, Schenck P, et al. European guidelines for quality assurance in cervical cancer screening: recommendations for collecting samples for conventional and liquid-based cytology.
Cytopathology
2007
;
18
:
133
–9.
14
IARC Working Group, on the Evaluation of Cancer Prevention Strategies. Cervix Cancer Screening, volume 10. Lyon: IARC Press; 2005.
15
Franco EL, Ferenczy A. Cervical cancer screening following the implementation of prophylactic human papillomavirus vaccination.
Future Oncol
2007
;
3
:
319
–27.
16
Davies P, Arbyn M, Dillner J, Kitchener HC, et al. A report on the current status of European research on the use of human papillomavirus testing for primary cervical cancer screening.
Int J Cancer
2005
;
118
:
794
–6.
17
Commission of the European Communities. Proposal for a council recommendation on cancer screening. 2003/0093 (CNS). Brussels, 5 May, 2003.
18
Walboomers JMM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.
J Pathol
1999
;
189
:
12
–9.
19
Bosch FX, Lorincz A, Muñoz N, et al. The causal relation between human papillomavirus and cervical cancer.
J Clin Pathol
2002
;
55
:
244
–65.
20
Hebner CM, Laimins LA. Human papillomavirus: basic mechanisms of pathogenesis and oncogenicity.
Rev Med Virol
2006
;
16
:
83
–97.
21
Fehrmann F, Laimins LA. Human papillomaviruses targeting differentiating epithelial cells for malignant transformation.
Oncogene
2003
;
22
:
5201
–7.
22
Boulet G, Horvath C, Vanden Broeck D, et al. Human papillomavirus: E6 and E7 oncogenes.
Int J Biochem Cell Biol
2007
;
39
:
2006
–11.
23
Molijn A, Kleter B, Quint W, et al. Molecular diagnosis of human papillomavirus (HPV) infections.
J Clin Virol
2005
;
32S
:
S43
–51.
24
Villa L, Denny L. Methods for detection of HPV infection and its clinical utility.
Int J Gynecol Obstet
2006
;
94
:
S71
–80.
25
Arbyn M, Sasieni P, Meijer CJLM, et al. Chapter 9: clinical applications of HPV testing: a summary of meta-analyses.
Vaccine
2006
;
24S3
:
S3/78
–S3/89.
26
Koliopoulos G, Arbyn M, Martin-Hirsch P, et al. Diagnostic accuracy of human papillomavirus testing in primary cervical screening: a systematic review and meta-analysis of non-randomized studies.
Gynecol Oncol
2007
;
104
:
232
–6.
27
ASCUS-LSIL Triage Study Group. Results of a randomized trial on the management of cytology interpretations of atypical squamous lesions, follow-up of women treated for high-grade CIN.
Am J Obstet Gynecol
2003
;
188
:
1383
–92.
28
Arbyn M, Buntinx F, Van Ranst M, et al. Virologic versus cytologic triage of women with equivocal Pap smears: a meta-analysis of the accuracy to detect high-grade intraepithelial neoplasia.
J Natl Cancer Inst
2004
;
96
:
280
–93.
29
Paraskevaidis E, Arbyn M, Sotiriadis A, et al. The role of HPV DNA testing in the follow-up period after treatment for CIN: a systematic review of the literature.
Cancer Treat Rev
2004
;
30
:
205
–11.
30
Zielinski GD, Bais AG, Helmerhorst TJ, et al. HPV testing and monitoring of women after treatment of CIN3: review of the literature and meta-analysis.
Obstet Gynecol Surv
2004
;
59
:
543
–53.
31
Arbyn M, Paraskevaidis E, Martin-Hirsch P, et al. Clinical utility of HPV-DNA detection: triage of minor cervical lesions, follow-up of women treated for high-grade CIN: an update of pooled evidence.
Gynecol Oncol
2005
;
99
:
7
–11.
32
Cuzick J, Beverley E, Ho L, et al. HPV testing in primary screening of older women.
Br J Cancer
1999
;
81
:
554
–8.
33
Schiffman M, Herrero R, Hildesheim A, et al. HPV DNA testing in cervical cancer screening: results from women in a high-risk province of Costa Rica.
JAMA
2000
;
283
:
87
–93.
34
Clavel C, Masure M, Bory JP, et al. Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women.
Br J Cancer
2001
;
84
:
1616
–23.
35
Cuzick J, Mayrand MH, Ronco G, et al. Chapter 10: new dimensions in cervical cancer screening.
Vaccine
2006
;
24S3
:
S3/90
–S3/97.
36
Manos M, Kinney WK, Hurley LB, et al. Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results.
JAMA
1999
;
281
:
1605
–10.
37
Wheeler CM. Advances in primary and secondary interventions for cervical cancer: human papillomavirus prophylactic vaccines and testing.
Nat Clin Pract Oncol
2007
;
4
:
224
–35.
38
ALTS 2000 & Anonymous. Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial.
J Natl Cancer Inst
2000
;
92
:
397
–402.
39
Stoler MH, Castle PE, Solomon D, et al. American Society for Colposcopy and Cervical Pathology: the expanded use of HPV testing in gynaecologic practice per ASCCP-guided management requires the use of well-validated assays.
Am J Clin Pathol
2007
;
127
:
335
–7.
40
Peyton CL, Schiffman M, Lörincz AT, et al. Comparison of PCR- and hybrid capture-based human papillomavirus detection systems using multiple cervical specimen collection strategies.
J Clin Microbiol
1998
;
36
:
3248
–54.
41
Vernon SD, Unger ER, Williams D. Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture.
J Clin Microbiol
2000
;
38
:
651
–5.
42
Coutlee F, Gravitt P, Kornegay J, et al. Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples.
J Clin Microbiol
2002
;
40
:
902
–7.
43
Clifford G, Franceschi S, Diaz M, et al. Chapter 3: HPV-type distribution in women with and without cervical neoplastic diseases.
Vaccine
2006
;
23S3
:
S3/26
–S3/24.
44
Manos MM, Ting MY, Wright DK, et al. The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses.
Cancer Cells
1989
;
7
:
209
–14.
45
Hildesheim A, Schiffman MH, Gravitt PE, et al. Persistence of type-specific human papillomavirus infection among cytologically normal women.
J Infect Dis
1994
;
169
:
235
–40.
46
Qu W, Jiang G, Cruz Y, et al. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems.
J Clin Microbiol
1997
;
35
:
1304
–10.
47
Gravitt PE, Peyton CL, Alessi TQ, et al. Improved amplification of genital human papillomaviruses.
J Clin Microbiol
2000
;
38
:
357
–61.
48
Castle PE, Schiffman M, Gravitt PE, et al. Comparisons of HPV DNA detection by MY09/11 PCR methods.
J Med Virol
2002
;
68
:
417
–23.
49
Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.
Am J Pathol
1998
;
153
:
1731
–9.
50
Perrons C, Kleter B, Jelley R, et al. Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11 primers in cervical cells taken form women attending a colposcopy clinic.
J Med Virol
2002
;
67
:
246
–52.
51
Depuydt CE, Boulet GAV, Horvath CAJ, et al. Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.
JCMM
2007
;
11
:
881
–91.
52
Van Ham MA, Bakker JM, Harbers GK, et al. Comparison of two commercial assays for detection of human papillomavirus (HPV) in cervical scrape specimens: validation of the Roche AMPLICOR HPV test as a means to screen for HPV genotypes associated with a higher risk of cervical disorders.
J Clin Microbiol
2005
;
43
:
2662
–7.
53
Monsonego J, Bohbot JM, Pollini G, et al. Performance of the Roche AMPLICOR human papillomavirus (HPV) test in prediction of cervical intraepithelial neoplasia (CIN) in women with abnormal PAP smear.
Gynecol Oncol
2005
;
99
:
160
–8.
54
Clifford GM, Smith JS, Aguado T, et al. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis.
Br J Cancer
2003
;
89
:
101
–5.
55
Cuschieri KS, Whitley MJ, Cubie HA. Human papillomavirus type specific DNA and RNA persistence-implications for cervical disease progression and monitoring.
J Med Virol
2004
;
73
:
65
–70.
56
Giuliani L, Coletti A, Syrjänen K, Favelli C, Ciotti M. Comparison of DNA sequencing and Roche Linear array in human papillomavirus (HPV) genotyping.
Anticancer Res
2006
;
26
:
3939
–41.
57
Coutlée F, Rouleau D, Petignat P, et al. Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the linear array HPV genotyping test.
J Clin Microbiol
2006
;
44
:
1998
–2006.
58
Kleter B, Van Doorn L, Schrauwen L, et al. Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.
J Clin Microbiol
1999
;
37
:
2508
–17.
59
Jiang H, Zhu H, Zhou L, Chen F, Chen Z. Genotyping of human papillomavirus in cervical lesions by L1 consensus PCR and the Luminex xMAP system.
J Med Microbiol
2006
;
55
:
715
–20.
60
Ho GY, Bierman R, Beardsley L, et al. Natural history of cervicovaginal papillomavirus infection in young women.
N Engl J Med
1998
;
338
:
423
–8.
61
Josefsson AM, Magnusson PK, Ylitalo N, et al. Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested case-control study.
Lancet
2000
;
355
:
2189
–93.
62
Dalstein V, Riethmuller D, Pretet JL, et al. Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal French cohort study.
Int J Cancer
2003
;
106
:
396
–403.
63
Moberg M, Gustavsson I, Wilander E, et al. High viral loads of human papillomavirus predict risk of invasive cervical carcinoma.
Br J Cancer
2005
;
92
:
891
–4.
64
Moberg M, Gustavsson I, Gyllensten U. Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer.
J Clin Microbiol
2003
;
41
:
3221
–8.
65
Depuydt CE, Benoy IH, Bailleul EJ, et al. Improved endocervical sampling and HPV viral load detection by Cervex-Brush Combi.
Cytopathology
2006
;
17
:
1
–8.
66
Gravitt P, Kovacic M, Herrero R, et al. High load for most high risk human papillomavirus genotypes is associated with prevalent cervical cancer precursors but only HPV16 load predicts the development of incident disease.
Int J Cancer
2007
;
121
:
2787
–93.
67
Jeon S, Lambert PF. Integration of human papillomavirus type 16 DNA into human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis.
Proc Natl Acad Sci
1995
;
92
:
1654
–8.
68
Arias-Pulido H, Peyton C, Joste N, Vargas H, Wheeler C. Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer.
J Clin Microbiol
2006
;
44
:
1755
–62.
69
Tonon SA, Picconi MA, Bos PD, et al. Physical status of the E2 human papillomavirus 16 viral gene in cervical preneoplastic and neoplastic lesions.
J Clin Virol
2001
;
21
:
129
–34.
70
Badaracco G, Venuti A, Sedati, et al. HPV16 and HPV18 in genital tumors: significantly different levels of viral integration and correlation to tumor invasiveness.
J Med Virol
2002
;
67
:
574
–82.
71
Alazawi W, Pett M, Strauss S, et al. Genomic imbalances in 70 snap-frozen cervical squamous intraepithelial lesions: associations with lesion grade, state of the HPV16 E2 gene and clinical outcome.
Br J Cancer
2004
;
91
:
2063
–70.
72
Hudelist G, Manavi M, Pischinger KI, et al. Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade.
Gynecol Oncol
2004
;
92
:
873
–80.
73
Andersson S, Safari H, Mints M, et al. Type distribution, viral load and integration status of high-risk human papillomaviruses in pre-stages of cervical cancer (CIN).
Br J Cancer
2005
;
92
:
2195
–200.
74
Cheung JL, Lo KW, Cheung TH, et al. Viral load, E2 gene disruption status, and lineage of human papillomavirus type 16 infection in cervical neoplasia.
J Infect Dis
2006
;
194
:
1706
–12.
75
Guo M, Sneige N, Silva EG, et al. Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.
Mod Pathol
2007
;
20
:
256
–66.
76
Molden T, Kraus I, Karlsen F, et al. Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
367
–72.
77
Powell N, Smith K, Fiander A. Recovery of human papillomavirus nucleic acids from liquid-based cytology media.
J Virol Methods
2006
;
137
:
58
–62.
78
Horvath CA, Boulet G, Sahebali S, et al. Effects of fixation on RNA integrity in a liquid based cervical cytology setting.
J Clin Pathol
2008
;
61
:
132
–7.
79
Molden T, Kraus I, Skomedal H, et al. PreTect HPV-proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses.
J Virol Methods
2007
;
142
:
204
–12.
80
Molden T, Kraus I, Karlsen F, et al. Human papillomavirus E6/E7 mRNA expression in women younger than 30 years of age.
Gynecol Oncol
2006
;
100
:
95
–100.
81
Rosini S, Zappacosta R, Di Bonaventura G, et al. Management and triage of women with human papillomavirus infection in follow-up for low-grade cervical disease: association of HPV-DNA and RNA-based methods.
Int J Immunopathol Pharmacol
2007
;
20
:
341
–7.
82
Castle P, Dockter J, Giachetti C, et al. A cross-sectional study of a prototype carcinogenic human papillomavirus E6/E7 messenger RNA assay for the detection of cervical precancer and cancer.
Clin Cancer Res
2007
;
13
:
2599
–605.
83
Wang-Johanning F, Lu DW, Wang Y, et al. Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou test using real-time polymerase chain reaction analysis.
Cancer
2002
;
94
:
2199
–210.
84
Sahebali S, Depuydt CE, Segers K, et al. Ki-67 immunocytochemistry in liquid-based cervical cytology: useful as an adjunctive tool?
J Clin Pathol
2003
;
56
:
681
–6.
85
Sahebali S, Depuydt CE, Segers K, et al. P16INK4A as an adjunct marker in liquid-based cervical cytology.
Int J Cancer
2004
;
108
:
871
–6.
86
Kolch W, Mischak H, Pitt AR. The molecular make-up of a tumour: proteomics in cancer research.
Clin Sci
2005
;
108
:
369
–83.
87
Kuramitsu Y, Nakamura K. Proteomic analysis of cancer tissues: shedding light on carcinogenesis and possible biomarkers.
Proteomics
2006
;
6
:
5650
–61.
88
Brusic V, Marina O, Wu CJ, et al. Proteome informatics for cancer research: from molecules to clinic.
Proteomics
2007
;
7
:
976
–91.
89
Cho WCS. Contribution of oncoproteins to cancer biomarker discovery.
Mol Cancer
2007
;
6
:
1
–13.
90
Yim EK, Park JS. Role of proteomics in translational research in cervical cancer.
Exp Rev Proteomics
2006
;
3
:
21
–36.
91
zur Hausen H. Perspectives of contemporary papillomavirus research.
Vaccine
2006
;
24S3
:
S3/iii
–S3/iv.
92
Chan JK, Berek JS. Impact of the human papillomavirus vaccine on cervical cancer.
J Clin Oncol
2007
;
25
:
2975
–82.
93
Koutsky LA, Ault KA, Wheeler CM, et al. A controlled trial of a human papillomavirus type 16 vaccine.
N Engl J Med
2002
;
347
:
1645
–51.
94
Harper DM, Franco EL, Wheeler C, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomized control trial.
Lancet
2004
;
364
:
1757
–65.
95
Villa LL, Costa RL, Petta CA, et al. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16 and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial.
Lancet Oncol
2005
;
6
:
271
–8.
96
Harper DM, Franco EL, Wheeler CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomized control trial.
Lancet
2006
;
367
:
1247
–55.
97
Mao C, Koutsky LA, Ault KA, et al. Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized control trial.
Obstet Gynecol
2006
;
107
:
18
–27.
98
Koutsky LA, Harper DM. Chapter 13: current findings from prophylactic HPV vaccine trials.
Vaccine
2006
;
24S3
:
S3/114
–S3/121.
99
Muñoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer.
N Engl J Med
2003
;
348
:
518
–27.
100
Bryan JT. Developing an HPV vaccine to prevent cervical cancer and genital warts.
Vaccine
2007
;
25
:
3001
–6.
101
Dillner J, Arbyn M, Dillner L. Translational mini-review series on vaccines: monitoring of human papillomavirus vaccination.
Clin Exp Immunol
2007
;
148
:
199
–207.
102
Adams M, Jasani B, Fiander A. Human papilloma virus (HPV) prophylactic vaccination: challenges for public health and implications for screening.
Vaccine
2007
;
25
:
3007
–13.
103
Denny L, Michael Q, Sankaranarayanan R. Chapter 8: screening for cervical cancer in developing countries. Vaccine 2006;2453:S3/71-S3/77.
104
Franco EL, Cuzick J, Hildesheim A, et al. Chapter 20: issues in planning cervical cancer screening in the era of HPV vaccination.
Vaccine
2006
;
24S3
:
S3/171
–S3/177.
105
Arbyn M, Van Oyen H. Analysis of individual health insurance data pertaining to Pap smears, colposcopies, biopsies and surgery on the uterine cervix (Belgium, 1996–2000). Brussels: Scientific Institute of Public Health; 2004.