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Keywords:

  • Cervical cancer;
  • cervical cytology;
  • follow up;
  • papillomavirus;
  • polymerase chain reaction;
  • screening programme

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective  To determine in a screening population the human papillomavirus (HPV) status in those with cytological abnormalities and to evaluate the presence of high-risk (HR) HPV with a minimum of 5-year follow up.

Design  Retrospective examination of HPV status on prospectively collected and cytologically screened cervical smears.

Setting  Canisius-Wilhelmina Hospital in Nijmegen, the Netherlands.

Population  Three hundred and fifty-seven women aged 30–60 years, from the population screened.

Methods  Three hundred and fifty-seven women with borderline or higher cytological abnormalities were retrospectively examined for HPV with DNA microarray typing. Follow up was through the nationwide Dutch Pathology database (PALGA).

Main outcome measures  For the cytological abnormalities, the CISOE-A classification was used. HPV was scored as negative or positive. In case of positive HPV polymerase chain reaction, the HPV genotype was determined. The occurrence of cervical intraepithelial neoplasia lesions of grade 3 or higher was considered as endpoint for follow up.

Results  The majority of the women with borderline cytology in this study were HPV negative (87%). Among the HPV-positive women in borderline cytology group, 74% had HR-HPV or probable high-risk types. The overall percentage of HR-HPV types increased with progressive cytological abnormalities. The cytological classifications of borderline dyskaryosis and moderate dyskaryosis contain all types of HPVs, e.g. low risk, HR and unknown risk. The samples with severe dyskaryosis or higher contain only HR types. The negative predictive value for HR-HPV typing in the group with borderline cytological abnormalities is more than 99%.

Conclusions  In cervical screening with an interval of 5 years, HPV can be reliably used as triage point in cases of borderline cytological abnormalities.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Screening for preventing cervical cancer is performed in many western countries by cervical cytology, leading to a reduction of the incidence and mortality.1 The fraction of false-positive and false-negative smears is around 10%.2,3 In case of moderate dyskaryosis or higher, participants are referred to the gynaecologist. If no cytological abnormalities are present, the participant will have another cervical examination in the next screening round. The interval varies between 1 and 5 years for different countries.4 In the cytological spectrum, categories of borderline or mild dyskaryosis are present, comparable to atypical squamous cells of undetermined significance (ASCUS) or atypical glandular cells of undetermined significance (differences in terminology between countries are depicted in Table 1). The group of participants with borderline cytology is much larger than the group with mild dyskaryosis or higher. If the fraction of borderline cases can be reduced by adding another test, this may be beneficial for women and the healthcare system.

Table 1.  The distribution of HPV-PCR-positive (subdivided in single and multiple infections) and HPV-PCR-negative samples over the cytological classifications of the USA38, Europe and the Netherlands39
USA, Bethesda 2001EuropeEurope, Netherlands; CISOE-AHPV positiveHPV negativeTotal
SingleMultipleTotal (%)Total (%)
  1. Twenty-seven percent of all HPV-positive samples contained a multiple infection. CISOE-A is the standard cytology classification system (Dutch, KOPAC-B).

ASCUS/atypical glandular cells of undetermined significance + low-grade squamous intraepithelial lesionsBorderlinePap 225934 (12.5)237 (87.5)271
Mild dyskaryosisPap 3a111516 (44.4)20 (55.6)36
HSILModerate dyskaryosisPap 3a27512 (80.0)3 (20.0)15
Severe dyskaryosisPap 3b12416 (88.9)2 (11.1)18
In situ carcinomaPap 4909 (90.0)1 (10.0)10
Total 642387 (24.6)263 (75.1)350

Human papillomavirus (HPV) infection precedes the occurrence of cytological abnormalities.7 HPV analysis alone for cervical cancer screening or in combination with cytology has been advocated before.8 In the USA, the ASCUS-LSIL study showed evidence for the use of HPV as triage.9,10

In 2003, the various types of HPV have been redefined in categories of carcinogenic or high-risk (HR) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82; probable carcinogenic types 26, 53 and 66; and low-risk (LR) types 6, 11, 40, 42, 43, 54, 61, 70, 72, 81 and CP108.11 HR types are associated with high-grade squamous intraepithelial lesions (HSIL) with high potential for the development of cervical carcinoma. Large prospective studies on the implementation of HR-HPV testing are currently underway.12

The aim of this study was to determine in a screening population the baseline HPV status in those with cytological abnormalities and to evaluate the presence of histological severe dyskaryosis, carcinoma in situ or squamous cell carcinoma (cervical intraepithelial neoplasia [CIN] lesion of grade 3 or more) with 5-year follow up. To this end, we retrospectively examined the cases of cytological abnormalities prospectively collected during 1997–99. For baseline HPV typing, we used a rapid DNA microarray method.13 The negative predictive value for HR-HPV in the group with borderline dyskaryosis is more than 99%.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sample collection

In the Netherlands, women aged 30–60 years are enrolled in the screening programme with a 5-year interval. During 1997–99, 9085 women were examined with the ThinPrep Pap Test method (Cytyc, Boxborough, MA, USA) for population screening of cervical cytology in the Canisius-Wilhelmina Hospital. Three hundred and fifty-seven women (4%) were shown to have Papanicolaou classification class 2, i.e. borderline cytologic abnormalities or higher. The relationship between cytological classifications of the USA, Europe and the Netherlands is shown in Table 1. The cellular residues (in PreservCyt; Cytyc) of the samples were prospectively collected and stored at room temperature in the original collection vials. Baseline HPV detection was retrospectively performed on these samples. The follow up was according to the Dutch screening programme. Thus, women with borderline and mild dyskaryosis (BMD) abnormalities had in the subsequent smear either normal cytology or were referred to the gynaecologist with subsequent histological examination. Follow up was through the nationwide Dutch Pathology database (PALGA). If histology was available, this was used (n= 77), else the highest cytological abnormality was used as outcome. The second smear may have been classified to a lesser category than that of the original smear, e.g. no cytological abnormalities. This study has been approved by the local medical ethical committee.

DNA extraction

PreservCyt content of the vials is ±20 ml before addition of the cervical sample. For the prospectively collected samples, molecular HPV analysis was performed retrospectively.

From all vials, the amount of residual fluid was estimated. In vials without visible fluid, 3 ml of fresh PreservCyt medium was added and mixed vigorously. From each vial, 1.5 ml of the suspension was used for DNA extraction with the QIAamp DNA Blood Mini Kit (QIAGEN, Venlo, the Netherlands), according to the protocol of the supplier. DNA was eluted in 100 μl volumes, and 5 μl of it was used for spectrophotometric measurement of absorption at 260 nm to calculate the DNA concentration of samples.

Beta globin polymerase chain reaction

To check if the DNA was of sufficient quality in the cases where the HPV polymerase chain reaction (PCR) was negative, human β-globin PCR was performed with quantitative PCR on a LightCycler (Roche Diagnostics, Almere, The Netherlands) according to the method described.14

HPV amplification

From human genomic DNA extracted from the ThinPrep residue, 100 ng was used in a HPV-E1-specific PCR test. A set of five pairs of PCR primers was used to include HPV types of HR, LR and undetermined risk for cervical cancer.13 Each PCR reaction mix contained 100 ng of template DNA, 1 unit of HotGoldStar DNA polymerase (Eurogentec, Seraing, Belgium), 2 mM MgCl2, 15 mM Tris–HCl pH 8.0/50 mM potassium chloride/0.01% (v/v) Tween 20. The PCR conditions for amplification of the five clusters are as follows: 10 minutes, 95°C; 30 seconds, 95°C/30 seconds, 52°C/1 minute, 72°C for 40 cycles; and 10 minutes, 72°C. Amplification products were analysed by agarose gel electrophoresis (2%).

Preparation of microarrays, Digoxigenin labelling of the HPV sequences, microarray hybridisation, immunodetection and visualisation were as described.13,15 The array contains biotin-labelled HPV probes spotted on streptavidin-coated slides (Dot Diagnostics, Beuningen, the Netherlands) for HR types (16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 68, 73 and 82), probable high-risk (P-HR) types (26, 53 and 66), LR types (6, 11, 40, 42, 44, 54, 61, 70 and 72) and unknown risk (UR) types (2, 3, 7, 10, 13, 28, 32, 34, 54, 55, 57, 67, 69 and 85). Samples tested positive in HPV PCR but negative on microarray were genotyped by DNA sequence analysis (ABI; Perkin Elmer, Groningen, the Netherlands).

Statistical analysis

The occurrence of CIN3 lesions or higher was considered as endpoint for follow up.12 Assessment of the reliability of the test was carried out by calculation of the sensitivity, specificity and negative predictive value in Microsoft Office Excel 2003.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In total, 9085 women were examined with liquid-based cytology for population screening in the Canisius-Wilhelmina hospital during 1997–99, and 357 of them (3.9%) showed borderline or higher cytological abnormalities. The vials of 52 clinical samples contained <1 ml residue, of which three samples were negative in β-globin PCR and HPV PCR. These three samples were omitted from further analysis. All other HPV-negative samples in this study were β-globin positive. After storage for 4 or more years, 354 samples were analysed for HPV. Four samples were omitted because the participants appeared twice in the study; in such cases, the first sample was used. In 4 out of 13 women, CIN3 was detected based on biopsy specimen, performed 3, 6, 11 and 16 years before baseline analysis of the current study, respectively.

HPV analysis

The analytical sensitivity of the microarray test for the detection of HPV 16 is between 167–333 copies per PCR reaction of the HPV 16 genome (data not shown). In 86 (24%) samples analysed, one or more of the primer combinations resulted in amplification of HPV sequences as detected by agarose gel electrophoresis analysis and genotyped by HPV DNA microarray. With the microarray analyses, multiple HPV types are simultaneously detected, categorised as HR, P-HR, LR or UR.

The distribution of HPV-positive cases for the different cytological categories increased with severity of the abnormalities: 13% in borderline, 44% in mild, 80% in moderate, 89% in severe dyskaryosis and 90% in carcinoma in situ (Table 1). The majority of women with borderline cytology in this study were HPV negative (87%). The overall percentage of HR-HPV types increased with progressive cytological abnormalities, in contrast to LR-HPV and UR-HPV. Among the HPV-positive women in cytological borderline group, 74% had HR-HPV or P-HR types (data not shown). The cytological classifications of mild–moderate dyskaryosis contained all types of HPVs, e.g. LR, HR and UR. In carcinoma in situ HPV-positive samples, only HR-HPV types were detected. HPV screening of the BMD group (n= 307) resulted in 269 cases (88%) of LR-HPV or HPV-negative individuals, which was 3% of the total screened population. The number of mixed HPV infections in this study was 23 (6.6% of total) (Table 1). The distribution of multiple HPV infections, divided according to their associated risk of squamous cell cervical cancer, is as follows: 8 (35%) HR/HR; 2 (9%) HR/P-HR; 7 (30%) HR/LR and 6 (25%) HR/UR. All multiple infection combinations detected in this study contained a HR-HPV type. The distribution of HPV types is shown in Figure 1.

image

Figure 1. Distribution of HPV types. Number of samples infected with individual HPV types as determined by HPV PCR and HPV microarray. Each type includes single and multiple infections. HPV types are grouped in UR, LR, P-HR and HR for cervical cancer.

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Follow up

Follow up was obtained in 332 out of 350 cases (94.9%) by repeated cytological examination at the next screening interval term of 5 years and/or earlier in time examination due to the indication(s) resulting from baseline cytological screening examination. The minimum follow-up period was 5 years (range 5.0–7.9 years; median 6.5). The mean age of participants with follow up is 42.3 years. All participants older than 55 years attended follow-up screening, i.e. the last screening round for this age cohort. CIN3 and squamous cell carcinomas were detected during follow up in 32 and 3 women, respectively. All three women with squamous cell carcinoma had HR type HPV 16, and the cytology showed severe dyskaryosis (n= 2) or carcinoma in situ (n= 1). The four women with CIN3 biopsy diagnosis before baseline were cytological or histological negative at this screening interval.

HPV status

The relation between HPV status and follow up is shown in Table 2. In 263 women (75%), no HPV was detected. In 29 out of 70 women with HR-HPV, follow up revealed severe dyskaryosis or higher. In 6 out of 264 women without HR-HPV, follow up revealed CIN3: these women had atypical primary smears (four with BMD, one with moderate dyskaryosis and one with in situ carcinoma). It is noteworthy that the nine women who had <CIN3 in follow-up histology and ‘positive cytology’ had Pap 3a2 (n= 6) and Pap 4 (n= 3). The sensitivity was 82.9% and specificity 86.9%. The negative predictive value for the whole group is 97.7%. The total group also contains women who should have been directly referred to the gynaecologist. In the borderline cytology group with follow up (n= 254), 24 were HR-HPV positive, and in two of them, the follow up was positive. Of the 230 borderline cytological cases without HR-HPV, the follow up was positive in one case. The negative predictive value for HR-HPV in this group is 99.6%. Note that in 18 borderline cases, no further follow-up examination was performed.

Table 2.  The outcome of cytological and HPV analyses in relation to follow up for all samples (n= 350)
Follow up≥CIN3< CIN3No FUTotal
CytologyCytologyCytology
PositiveNegativePositiveNegativeNegative
  1. ≥CIN3, histological severe dysplasia, carcinoma in situ or squamous cell carcinoma; No FU, no follow up available; cytology positive, cytological classification ≥ moderate dyskaryosis (HSIL); cytology negative, borderline or mild dyskaryosis.

HPV
No HPV21424115263
LR alone10111
UR33 6
P-HR2 2
HR alone232916252
HR + LR17 8
HR + UR312 6
HR + P-HR11 2
Total2871528218350

Interestingly, most of the women with severe dyskaryosis or higher were shown to have HR-HPV or HSIL at baseline. CIN3 diagnosis was made in 32 of the 35 women within 18 months after baseline screening. The remaining three women turned up in subsequent years. The total number of women with multiple HPV types was 23, where the presence of HR-HPV was the determining factor: none of the women with multiple HPV types, who were lacking HR-HPV, showed CIN3 during follow up. This demonstrates that within 5-year follow up, only the presence of HR-HPV, irrespective of the presence of additional HPV types, is discriminating in the group with BMD.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The aim of this study was to determine HPV status at baseline when screening women with cytological abnormalities and the additional value of HPV testing after 5 years of follow up. The results clearly show that the absence of HR-HPV is discriminating, even in case of multiple HPV infections, for the chance of development of CIN3 or squamous cell carcinoma. The negative predictive value in the group with borderline cytological abnormalities (Pap 2) is more than 99%. This implies that in this large subgroup, the combination with HR-HPV is likely to be an efficient triage point. Up to a 5-year follow-up interval will be adequate instead of a 6-month recall. Extrapolation from this study implies that only 1.6% instead of 4% from the screened population would require short-term action at 6 months, and the other 2.4% could safely wait further examination until the next screening interval of 5 years. Alternatively, in case of HR-HPV, direct referral may be an option. Note that this triage point holds only for ‘atypical’ or borderline cytological abnormalities and not for more severe abnormalities (HSIL).

In a meta-analysis, HPV DNA testing appeared more sensitive and equally specific in comparison to follow-up cytology for triage of ASCUS and for predicting recurrence of CIN.16 The negative predictive value of >99% for developing high-grade cervical lesions after combined cytological and HPV testing has been reported in several studies.17–23 This outcome has also been used to suggest an increase of the screening interval to a period more than 1 year.17,24 Using liquid-based cytology analysis, HPV may be determined on the same sample as that for the smear, and thus, a second sampling procedure can be avoided (also called reflex HPV). The liquid-based procedure is cost-effective in this respect.25 Prolonging the screening interval may reduce at least a large part of the increased costs of HPV analysis.17,26

The use of the negative predictive value of HPV for conservative management is also advocated in the follow up of women treated for CIN3 of the cervix.27,28

ThinPrep (liquid based) cytology samples that have been stored for 3–5 years were used for this retrospective study. There is a manufacturer-recommended testing life for ThinPrep samples of a few weeks for regular HPV testing in the clinical setting. An internal control (β-globin DNA) was included with each negative sample to diagnose technical problems with the samples. Only 3 out of the 357 cases were inadequate and omitted from further analysis. Other studies have described good stability of HPV DNA in liquid-based cytology samples for prolonged storage at ambient conditions.29

In technical terms, the HPV microarray approach was initially performed for HR-HPV in three PCR tubes. After a recent modification, the HR-HPV types are amplified in a single-tube multiplex PCR, validated on the same cases as in the previous study.13 After re-examination, HR-HPV was shown in DNA of all cases with ≥CIN3, rendering the negative predictive value for borderline cytology to 100% and for BMD to 99.2%.

The ratio of borderline versus mild cytological abnormalities in our study is 8:1, while this is 5:1 in more recent studies. This difference is explained by the fact that in the Netherlands, initially a high fraction of women were diagnosed with borderline abnormalities. The adjustment to more stringent cytological criteria led to a nationwide reduction from more than 8% before 1997 to around 3–4% after 1999. This also explains the relatively low prevalence rate of HPV in women with borderline cytology in our study (16%) and in other studies (12%).30 The prevalence is higher in more recent studies, 72.7%8 and 34.6%.12

In the literature, many different HPV tests have been used. When comparing different tests, small fractions of discrepancies usually occur.31 The question of which HPV test is the best may arise. A more appropriate approach, in our viewpoint, is to wonder what requirements have to be met with regards to the analytical sensitivity of the HPV test in relation to the pathophysiology of the disease process. Women with persistent HR-HPV infection, even without initial cytological abnormalities, have a high propensity for the development of CIN3 or invasive carcinoma.32 In addition, the risk of progression to cervical carcinoma (in situ) is associated with an increased viral load, i.e. high viral DNA copy number.33 The highest risk association is for HPV type 16.34 The issue of analytical and clinical sensitivities has been reviewed by Snijders et al.35 PCR-based methods usually require a lower number of viral DNA copies, i.e. higher analytical sensitivity, to obtain a positive test result than that required for non-PCR-based HPV detection methods. The presence of the same HR-HPV type on repeated testing is an indicator for the development of cervical HSIL.36 In cohort studies, a higher analytical sensitivity will lead towards an increased fraction of HPV-positive cases as well as an increase of the negative predictive value (up to the maximum of 100%). In the setting of cervical screening, the required analytical sensitivity to a HPV test is dependent on the following factors: viral copy number, progression time of viral incubation to development of carcinoma and screening interval time (Figure 2). In other words, women prone to develop high-grade intraepithelial neoplasia with recent infection of HR-HPV will initially have a low copy number (below the detection limit of the technique) and insufficient time to develop borderline or higher cytological abnormalities. These women will be HPV negative on the first screening test but would turn out positive on the next screening round before an invasive squamous cell carcinoma has emerged. Thus, in comparing test X with a lower analytical specificity than test Y, the HPV test with a higher detection limit may be preferred because of gain in clinical specificity without reduction of clinical sensitivity. Moreover, this test X is likely to have a more optimal cost-effectiveness ratio due to less referral. The analytical sensitivity of the microarray HPV test is sufficient for cervical screening with a 5-year interval, taking into account the negative predictive value of >99% in combination with borderline cytology. By using borderline cytology without HR-HPV as the triage point, the reduction in the fraction of referrals (back to the population-based screening) may differ depending on the analytical sensitivity of the test: for the current study, this is around 85%, while for others, this number may be 55%.19

image

Figure 2. Model assumption where HPV infection has occurred before baseline HPV test (time = 0) and progression time for development of CIN3/carcinoma is assumed to be constant (asterisk-line). The required analytical sensitivety of a HPV test performed is related to the screening interval. In a program me with a 5-year interval, the analytical sensitivity should be lower (arrow A) than that with a 1-year interval (arrow B).

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In case of multiple HPV types, it was typed as HR in the general analysis if at least one of the types was a HR type. The spectrum of genotypes of the current study is not at variance with previous reports,37–39 but a few genotypes reported here as ‘unknown risk’ (HPV 32, 44, 67, 74 and 85) have not been reported before. Occasionally, a small fraction of the HPV-positive cases are reported ‘untypable’.37 Interestingly, the multiple infections were found in the categories of borderline to moderate cytological abnormalities. In contrast, HPV-positive cases with high-grade lesions were positive for a single HR-HPV type only.

In conclusion, in cervical screening with a screening interval of 5 years, the lack of HR-HPV in case of borderline cytology is a reliable triage point.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The critical reading of the manuscript by A. Uyterlinde is greatly appreciated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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