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Keywords:

  • cervical intra-epithelial neoplasia;
  • screening;
  • cytology;
  • HPV DNA testing;
  • randomized controlled trial

Abstract

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

Since infection with oncogenic human papillomavirus (HPV) has been considered a necessary cause of cervical cancer, tests for oncogenic HPV types have been proposed as adjuncts or replacements to Pap cytology. We designed the Canadian Cervical Cancer Screening Trial (CCCaST) to compare the relative efficacy of HPV DNA testing and Pap cytology in primary screening for cervical cancer and its high-grade precursors. CCCaST randomized women aged 30–69 years in Montreal (Quebec) and in St. John's (Newfoundland) to 1 of 2 screening groups: focus on Pap (conventional) or focus on HPV testing (Hybrid Capture 2). Women in both arms received both tests, but were randomized as to their order, the first test being the index test. Women with an abnormal Pap test or a positive HPV test underwent colposcopy and biopsy, as did a random sample of women with a negative index test. CCCaST enrolled 9,667 women between October 2002 and October 2004. At enrolment, 2.8% had an abnormal Pap test, 6.1% had a positive HPV test and 1.1% were abnormal in both tests. ASC-US was the most frequent cytological abnormality, representing 64% of abnormal Pap results. The frequency of abnormal Pap and HPV results decreased with increasing age and the proportion of HPV-positive results increased with the severity of Pap abnormality. Efficacy analysis will determine if the extra referrals with HPV DNA testing will translate into a relevant increase in high-grade cervical cancer precursor detection. Because of its design, CCCaST will provide sound evidence for formulating cervical cancer screening strategies. © 2006 Wiley-Liss, Inc.

Universal deployment of organized or opportunistic screening with Pap cytology in high and middle-income countries has been the primary reason for the substantial reductions in cervical cancer morbidity and mortality during the last 50 years. However, these reductions have slowed down since the mid 1980s, and there is an indication that this strategy has reached a point of diminishing return.1 While the Pap test is the best screening tool ever introduced for any malignancy, it has well-recognized limitations, especially its limited sensitivity, estimated at only 51%.2

Since infection with oncogenic human papillomaviruses (HPV) has been identified as the underlying cause of cervical cancer,3, 4, 5 there has been substantial interest to apply this knowledge to improve cervical cancer prevention efforts. Primary prevention by HPV vaccines has shown promising results in proof-of-principle efficacy studies.6, 7 In secondary prevention, HPV DNA testing has been found to be a cost-effective triage method for equivocal cytology.8 On the other hand, the exact role HPV DNA testing could play in screening women in the general population (i.e., primary screening) remains controversial. The studies that have examined the performance of HPV DNA testing have found it to be more sensitive than Pap cytology in identifying high-grade precancerous lesions (cervical intraepithelial neoplasia grade 2 [CIN2] and grade 3 [CIN3]) summarized in Ref.9. However, HPV DNA testing is consistently less specific than Pap cytology, which implies that its use as a sole screening test without any changes to screening interval would increase the number of referrals, possible adverse events from unnecessary diagnostic procedures and costs to the health care system.

The Canadian Cervical Cancer Screening Trial (CCCaST) is a randomized controlled trial (RCT) that was designed to examine the performance of HPV DNA testing relative to Pap cytology in primary cervical cancer screening. Its primary objective is to compare the ability of HPV DNA testing and Pap cytology to identify cervical cancers and their high-grade precancerous lesions among women aged 30–69 years who present for routine cervical cancer screening.

Methods

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

Design

The CCCaST study is an RCT of a screening intervention. Figure 1 gives a schematic outline of the trial, which includes both a cross-sectional and a longitudinal component. Participants were randomized 1:1 to 1 of 2 arms: the Pap screening arm or the HPV screening arm. To compare the efficacy of HPV DNA testing to that of Pap testing, the simplest design would have been to randomize women to 1 of 2 screening strategies: Pap or HPV DNA testing. However, at the onset of CCCaST, there was insufficient evidence regarding the efficacy of HPV DNA testing as a stand-alone screening test to withhold Pap cytology from women in the trial. For this reason, we included both tests in each arm, but randomized the order in which the test samples would be collected. In the Pap screening arm, women received a Pap test (the index test) followed by an HPV DNA test (the secondary test). In the HPV screening arm, women received an HPV DNA (the index test) followed by a Pap test (the secondary test). This design will give us, at the analysis stage, the possibility to assess the performance of the 2 tests as if they had been done alone, while giving all women in the trial access to the established standard in cervical cancer screening: the Pap test. It will also enable us to investigate the performance of the 2 tests when used in combination and evaluate any biasing effects due to the test sampling order.

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Figure 1. Outline of study design (see text for abbreviations).

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Women with an abnormal Pap test or a positive HPV test at enrolment underwent a colposcopic examination. If cancer, CIN2 or CIN3 was found they were managed as per standard practice, and followed outside of the trial for convenience to the patient. If CIN1 or no lesion was found, a colposcopic examination was repeated after 6 months. A random sample of women testing negative with the index test also underwent colposcopy, to allow for correction of verification bias. Women with normal test results who were not selected for colposcopy were invited for a second round of screening 12–18 months after their initial visit, in which the initial screening arm allocation is maintained. Women who have had colposcopic examinations and were not found to have CIN2, CIN3 or cancer were also recalled for a second round of screening, 12–18 months after their last colposcopy.

Participants

The study population was comprised of women aged 30–69 years, from Montreal and surrounding municipalities (province of Quebec) and St. John's (province of Newfoundland), enrolled through 30 selected medical practices. The study was limited to women older than 30, since in sexually active women younger than 30 years, transient HPV infections are common, which gives HPV DNA testing an unacceptably low specificity in identifying cervical cancer precursors. The upper age limit (69 years) was set in accordance with current Canadian guidelines.10

Cytology laboratories in the study regions provided us with a list of physicians requesting Pap tests. This allowed us to identify and invite physicians from medical practices that were active in cervical cancer screening. This list included university-affiliated physicians and those in private practice, from family medicine and gynecology practices of different sizes that focus on primary care. This diverse base for recruitment ensured that the screening test efficacy measured in the study corresponded to a broad cross-section of providers.

Women aged 30–69 years consulting in the target medical practices were given an information brochure and a self-administered enrolment card to determine eligibility. Excluded from the study were those (i) attending a colposcopy clinic for evaluation, treatment or follow-up of a cervical lesion, (ii) without a cervix, (iii) who were pregnant, (iv) with a previous history of invasive cervical cancer and (v) who were unable to provide informed consent. To maintain our focus on women undergoing routine screening (and not on women consulting for symptoms or a previous Pap abnormality who would receive a Pap test as part of a diagnostic work-up), we also excluded women who had received a Pap test within 12 months. Physicians and assistants were trained to clarify the study procedures and obtain written informed consent from interested eligible women.

Participants completed a self-administered questionnaire that elicited data on baseline demographics, risk factors for HPV infection and cervical cancer and information that will enable us to conduct a subsequent in-depth cost analysis.

CCCaST was approved by the ethical review boards of each participating hospital and clinic, and of McGill and Memorial Universities.

Randomization

We used a computer-generated block randomization algorithm with block sizes that varied randomly. Randomization was stratified by practice 1:1 to 1 of the 2 screening arms. Randomization was carried out at the study coordination centre, and opaque and sealed envelopes were left in each practice. After an eligible woman had agreed to participate, physicians opened consecutive envelopes so as to determine arm allocation. The following study characteristics ensured a bias-free comparison: (i) all women received both screening tests, (ii) follow-up was the same in the 2 arms (iii) physicians did not favor any one particular order of sampling.

An additional clinic-stratified, randomly selected 10% of women in St. John's and 20% of women in Montreal who had a negative result in the index test in each arm were invited to attend colposcopy. Our target was to have colposcopic evaluation of 10% of the study sample that tested negative on the index tests to obtain adequate precision in correcting for verification bias. We expected up to 30–50% of women to refuse colposcopy despite counseling both at enrolment and when contacted for colposcopy. For this reason, 20% of index test negative women were invited for colposcopy in Montreal, but limited colposcopy resources precluded the same approach for St. John's. An important but tenable assumption for the correction approach to be valid is that refusal to undergo colposcopy is not linked to screening group allocation or risk of outcome. However, this assumption will be verified by comparing baseline demographic and risk factors between women who accepted and those who refused random colposcopy.

Blinding

Patients were blinded to arm allocation. Cytotechnologists and cytopathologists evaluating the Pap smears were not aware of inclusion of women into our study to ensure that the study samples would be treated no differently than other routine tests. They had no access to HPV results and HPV DNA testing sites were unaware of other results. Colposcopists and pathologists evaluating the biopsy specimens were blinded to initial screening test results.

Study interventions

Pap tests

The CCCaST study was designed to provide an evaluation of screening tests in a community setting. For this reason, Pap test collection and preparation were not submitted to a specific protocol. Conventional Pap tests were used in both study sites and results were reported according to locally prevailing nomenclature and forms. At the onset of the study in October 2002, most cytology laboratories were using the Bethesda 2001 terminology, with only a few Montreal laboratories still using the Bethesda 1991 nomenclature. All such tests were reclassified according to Bethesda 2001 terminology.11

HPV tests

The Hybrid Capture 2 test (HC-2) (Digene, Gaithersburg, MD) was selected as the HPV DNA testing format for our study. Specimens were collected with the Digene cervical sampler kit. The HC-2 assays were performed using probe B, a pool of full length HPV RNA probes against types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, according to the manufacturer's recommendations in 2 Digene certified clinical laboratories (Dr. Ratnam's in St. John's and Dr. Coutlée's in Montreal). Specimens were considered positive for HPV DNA if the ratio of Relative Light Units (RLUs) of the specimen to the mean RLUs of triplicates of a positive control was equal to or greater than 1. This is equivalent to 1 pg HPV DNA per ml or 5,000 copies of HPV genome per test.

Outcome definition and assessment

Histological CIN2, CIN3 and cervical cancer are the primary outcomes for our study. Diagnosis was obtained through colposcopically guided biopsies. The following groups of women were referred for a colposcopic evaluation: those with ASC-US, ASC-H, AGC, LSIL, HSIL, AIS10 or cancer on the Pap test; those with a positive HPV test as described earlier; those having been randomly selected for colposcopy despite the fact that both screening test results were negative. Colposcopies took place at pre-selected centers, following a standardized protocol to reduce verification bias. The protocol included (i) exo-cervical biopsies of all abnormal-appearing cervical regions, (ii) at least 1 exocervical biopsy of normal appearing cervices (to reduce the risk that CIN giving an impression of metaplasia would not be biopsied and missed) and (iii) ECC. The integration of all colposcopy results (impression, cytology, ecto and endocervical histology) was done at the study-coordinating center and diagnostic excisional procedures were carried out in cases of significant discrepancies. Follow-up colposcopy after 6 months, adhering to the same protocol, reduced further the possibility of missing lesions.

Pathologists at each hospital where the colposcopy was done made the histological diagnosis for all biopsy specimens following standard histological criteria.

Statistical methods

We set the total sample size to 12,000 women so as to have an 80% power to detect an absolute difference of 20% between the sensitivity of Pap and of HPV DNA testing at an alpha level of 0.05, using a 2-sided hypothesis and assuming lesion prevalence as 1.5% and up to 20% losses to follow-up.

Possible differences in categorical data between study arms and centers were investigated using Fisher's and Chi square test, and differences in the medians of continuous data with the Kruskal–Wallis test. Retesting of the 2 samples of 80 specimens by each laboratory generated 160 pairs of RLU results. Linear regression was used to assess the correlation between results (and departure from equivalency of results) on the basis of log-transformed data. The Kappa statistic was used to ascertain the agreement between the 2 laboratories using RLUs recoded as negative, low positive (1 < RLU < 10) and high positive (RLU ≥ 10). All tests were 2-sided. SAS software version 8.0 was used for all analyses.

Planned analysis of efficacy

We will compare the screening indices (sensitivity, specificity and positive and negative predictive values) of the 2 screening tests, Pap and HPV. The calculation of the 4 indices will be conducted independently for each study arm, respecting original arm allocation (“intention to treat”), taking only into account results of the index test and ignoring results of the secondary test. We will construct 2 × 2 tables compiling the joint results of testing procedures and disease ascertainment in each arm, allowing us to compute the 4 indices with their respective asymptotic 95% confidence intervals based on the binomial distribution. Missing index screening test results will be excluded from the specific 2 × 2 table pertaining to that analysis; women with abnormal screening test result(s) and without verification of true disease status (losses to follow-up and refusals), will be assumed to have the same probability of disease as women who had the same initial screening test results and underwent colposcopic examination. Sensitivity analysis will further explore the robustness of those 2 assumptions. By doing so, each of the 4 cell frequencies in the 2 × 2 table will be corrected. This technique was described in detail in our previous study.12 In the primary analysis, only the cross-sectional data from the initial diagnostic evaluation will be used. Subsequent analyses will take into account the longitudinal, cumulative assessment of disease.

CCCaST will provide a wealth of data to answer ancillary research questions regarding use of Pap and HPV DNA testing either as stand-alone tests or in combination. For example, the fact that participants had both screening tests will enable us to investigate if the order of the test has an effect on its performance. If we conclude that there is no impact of sampling order, we will collapse the 2 arms of the study and repeat the calculations as if the 2 tests were the primary focus of the analysis. This final analysis simulates the conditions of all split-sample studies conducted so far. A planned ROC curve analysis will shed light into optimal test combinations and most effective HPV test signal thresholds. Finally, exploratory analyses will focus on the impact of age, smoking and hormonal status on the performance of the 2 tests. Although power will be limited in such analyses they may generate hypotheses for future research.

Results

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

Between October 1st, 2002 and October 31st, 2004, 14,482 women were assessed for eligibility in the CCCaST trial (Fig. 2). A total of 4,815 were ineligible or refused, leaving 9,667 women (4,204 in Montreal and 5,463 in St. John's) who were randomized to 1 of the 2 trial arms and are the focus of the present report. Four thousand eight hundred and twenty three women were randomized to the Pap arm and 4,844 women to HPV arm. As shown in Figure 2, over 98% of the participants received the allocated intervention as intended (99.1% in the Pap arm, 97.5% in the HPV arm). In the Pap arm, 375 women needed colposcopy because of an abnormal screening test compared to 384 in the HPV arm. In the Pap and HPV arms, 674 and 634 women randomly selected from among those with negative screen results were invited to attend a colposcopic examination.

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Figure 2. Diagram for study progress. Status of colposcopy follow-up as of February 20th, 2005.

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Table I summarizes the distribution for selected characteristics of the study sample by arm and center. As expected, there were comparable distributions across study arms, and the only noteworthy differences were between study centers. The most striking differences were with respect to ethnicity; these differences reflect the concentration of French Canadians in Quebec and the tendency for immigrants to settle in large cities like Montreal. Participants tended to be somewhat younger in St. John's than in Montreal (medians 42 vs. 44; p < 0.0001). Marital status differed between centers with more single women in Montreal than in St. John's (17% vs. 11%; p < 0.0001). Smoking was more common in Montreal than in St. John's (23% vs. 17%; p < 0.001). Contraceptive choices differed between centers, with more participants in St. John's reporting no contraceptive use, and more women in Montreal reporting use of barrier methods. Over 90% of participants reported having had a Pap test in the past. Overall, 26% of participants reported having been told that their Pap test was abnormal at least once in the past. A longer questionnaire with items on reproductive health and sexual practices was administered only to Montreal participants. Of the latter, the median age at first intercourse was 18 and over 80% had been pregnant at least once. 29% were menopausal and of those, approximately half were taking some form of substitutive hormonal therapy.

Table I. Distribution of Selected Characteristics of Participants Enrolled into the CCCaST Study by Arm and Centre
CharacteristicCategoriesMontrealSt. John's
  Pap arm (N = 2,096)HPV arm (N = 2,108)Pap arm (N = 2,727)HPV arm (N = 2,736)
  • Unless stated otherwise, numbers in parentheses represent percentages in all tables.

  • 1

    Percentages may not add up to 100% because of rounding.

  • 2

    Except for the age variable, there are some missing data, which were simply omitted in this table.

  • 3

    Proportion of different contraceptive categories based on: in Montreal, total number of women reporting not being menopaused (n = 2633); in St. Jonh's, total number of women younger than 51 (median age at menopause for Canadian women) (n = 4382).

  • 4

    Tubal ligation and vasectomy.

  • 5

    Oral and parenteral.

  • 6

    Intra-uterine device.

  • 7

    Male and female condom, diaphragm, cervical cap, contraceptive sponge.

  • 8

    Fertility awareness and coitus interruptus.

  • 9

    Inter-Quartile range.

  • 10

    Not available, a shorter questionnaire was administered in St. John's.

Age (y)130–39699 (33.4)712 (33.8)1,151 (42.2)1,144 (41.8)
 40–49733 (35.0)734 (34.8)977 (35.8)955 (34.9)
 50–59496 (23.7)491 (23.3)468 (17.2)524 (19.2)
 60–69168 (8.0)171 (8.1)131 (4.8)113 (4.1)
Ethnicity2French Canadian1,730 (84.6)1,750 (84.7)17 (0.6)16 (0.6)
 English Canadian52 (2.5)47 (2.3)2,655 (97.8)2,262 (98.2)
 Other264 (12.9)270 (13.1)43 (1.6)34 (1.3)
SchoolingElementary school229 (11.1)203 (9.8)281 (10.3)281 (10.3)
 High school463 (22.4)478 (23.0)604 (22.2)631 (23.1)
 College566 (27.4)551 (26.5)826 (30.4)823 (30.2)
 University807 (39.1)846 (40.7)1,007 (37.0)989 (36.3)
Marital StatusSingle370 (17.9)366 (17.6)205 (7.6)250 (9.2)
 Married/in union1,334 (64.6)1,373 (66.0)2,174 (80.2)2,205 (81.1)
 Separated/widowed362 (17.5)342 (16.4)331 (12.2)264 (9.7)
Contraception3Sterilization4321 (24.7)332 (25.5)572 (26.1)581 (26.9)
 Hormonal5298 (22.9)254 (19.5)466 (21.3)451 (20.9)
 IUD668 (5.3)82 (6.3)31 (1.4)33 (1.5)
 Barrier7247 (19.0)239 (18.4)208 (9.5)210 (9.7)
 Natural811 (0.8)9 (0.7)6 (0.3)8 (0.4)
 None355 (27.3)384 (29.5)908 (41.4)879 (40.7)
Age at first intercourseMedian (IQR9)18 (16–20)18 (17–20)NA10NA
 Never had intercourse9 (0.004)7 (0.003)NANA
Lifetime no. ofMedian (IQR)5 (2–10)4 (2–10)NANA
sexual partnersNever had intercourse9 (0.004)7 (0.003)NANA
Number of pregnancies0378 (18.6)393 (19.2)NANA
 1–21,002 (49.3)1,013 (49.4)NANA
 3–4538 (26.5)511 (24.9)NANA
 >4114 (5.6)134 (6.5)NANA
MenopausedYes588 (28.6)594 (29.0)NANA
 No1,316 (64.3)1,317 (64.2)NANA
 Don't know142 (6.9)139 (6.8)NANA
Menopaused, taking HRTYes271 (47.5)298 (52.2)NANA
 No300 (52.5)273 (47.8)NANA
Current smokersYes487 (23.6)466 (22.4)470 (17.3)442 (16.2)
 No1,574 (76.4)1,612 (77.6)2,251 (82.7)2,282 (83.8)
Had Pap smearYes1,919 (93.7)1,927 (93.4)2,715 (99.8)2,720 (99.9)
 No92 (4.5)102 (4.9)4 (0.2)3 (0.1)
 Don't know38 (1.8)34 (1.6)1 (0.04)0 (0.0)
Self-report of ever havingYes447 (23.8)426 (22.5)762 (29.2)806 (30.9)
abnormal PapNo1,389 (73.8)1,401 (74.0)1,723 (65.9)1,663 (63.8)
 Don't know46 (2.4)67 (3.5)128 (4.9)138 (5.3)

Table II summarizes screening test results. There was a similar distribution between study arms, but results differed between centers. Overall, 2.8% of participants had an abnormal Pap test result (ASC-US or worse). This proportion was 3.9% in Montreal but only 2.0% in St. John's (p < 0.001). In both centers, ASC-US Pap results represented 64% of abnormal cytology results (66.2% in Montreal vs. 60.7% in St. John's; p = 0.67). The proportion of Pap tests deemed unsatisfactory to yield a cytological interpretation was low in both centers (1.4% overall; 1.2% in Montreal and 1.5% in St. John's). A higher number of participants had an abnormal HPV test result compared to abnormal Pap tests, 6.1% vs. 2.8% overall (p < 0.001). A higher proportion of Montreal women had positive HPV tests compared to participants from St. John's (7.7% vs. 4.9%; p < 0.001). Over 97% of women with negative results on HPV DNA testing had RLU readings below 0.75.

Table II. Distribution of Screening Test Results in the CCCaST Study by Arm and Centre
GroupPap arm N = 4,823HPV arm N = 4,844Montreal N = 4,194St. John's N = 5,463OverallN = 9,667
Test or Test formatResultN (%)N (%)N (%)N (%)N (%)
  • 1

    ≥ASC/AGC.

  • 2

    Squamous carcinoma.

  • 3

    RLU ≥ 1.0.

Pap test (dichotomous)Negative4,605 (95.5)4,640 (95.8)3,978 (94.6)5,267 (96.4)9,245 (95.6)
 Positive1145 (3.0)128 (2.6)166 (3.9)107 (2.0)273 (2.8)
 Unsatisfactory66 (1.4)69 (1.4)51 (1.2)84 (1.5)135 (1.4)
 Not taken7 (0.1)7 (0.1)9 (0.2)5 (0.1)14 (0.1)
Pap test (detail of categories)Negative4,605 (95.5)4,640 (95.8)3,978 (94.6)5,267 (96.4)9,245 (95.6)
 ASC-US90 (1.9)85 (1.8)110 (2.6)65 (1.2)175 (1.8)
 ASC-H5 (0.1)4 (0.1)3 (0.1)6 (0.1)9 (0.1)
 AGC24 (0.5)20 (0.4)27 (0.6)17 (0.3)44 (0.4)
 LSIL17 (0.4)13 (0.3)18 (0.4)12 (0.2)30 (0.3)
 HSIL8 (0.2)6 (0.1)7 (0.2)7 (0.1)14 (0.1)
 Sq Ca21 (0.02)01 (0.02)01 (0.01)
HPV test (dichotomous)Negative4,517 (93.7)4,522 (93.4)3,844 (91.4)5,195 (95.1)9,039 (93.5)
 Positive3283 (5.9)308 (6.4)325 (7.7)266 (4.9)591 (6.1)
 Not taken23 (0.5)14 (0.3)35 (0.8)2 (0.0)37 (0.4)
HPV test (RLU results)<0.754,423 (91.7)4,408 (91.0)3,714 (88.3)5,117 (93.7)8,831 (91.4)
 0.75–0.9994 (1.9)114 (2.4)130 (3.1)78 (1.4)208 (2.2)
 1.00–1.9954 (1.1)66 (1.4)65 (1.5)55 (1.0)120 (1.2)
 2.00–3.9941 (0.9)43 (0.9)42 (1.0)42 (0.8)84 (0.9)
 4.00–9.9937 (0.8)37 (0.8)44 (1.0)30 (0.5)74 (0.8)
 10–39.9954 (1.1)62 (1.3)63 (1.5)53 (1.0)116 (1.2)
 ≥4097 (2.0)100 (2.1)111 (2.6)86 (1.6)197 (2.0)
Pap and HPV(dichotomous)Both neg.4,379 (90.8)4,389 (90.6)3,721 (88.5)5,047 (92.4)8,768 (90.7)
 Only HPV pos.226 (4.7)251 (5.2)257 (6.1)220 (4.0)477 (4.9)
 Only Pap pos.92 (1.9)76 (1.6)101 (2.4)67 (1.2)168 (1.7)
 Both pos.53 (1.1)52 (1.1)65 (1.5)40 (0.7)105 (1.1)

Table III explores the relationship between test results and age. The St. John's population was younger (77.4% below 50 years of age compared to 68.6% in Montreal). It is interesting to note that the higher proportion of abnormal test results (both Pap and HPV) in Montreal held true for each age group. In both centers, the proportion of positive Pap tests decreased with age (from 4.3% to 2.9% in Montreal, and from 2.2% to 0.8% in St. John's). However, HPV positivity decreased dramatically with age, in both centers (from 12.7% to 3.8% in Montreal, and from 7.0% to 1.2% in St. John's). The difference in overall HPV prevalence between centers (7.9% vs. 5.1%) occurred not because of a difference in age structure, but despite this difference. In fact, when a combined population structure is used for the purposes of direct age standardization, the adjusted HPV positivity rates are 8.2% and 4.7%, for Montreal and St. John's, respectively.

Table III. Proportion of Positive Screening Tests by Age Group and Center
Age group (y)MontrealSt. John's
n% Pap positive% HPV positiven% Pap positive% HPV positive
30–391,4114.312.72,2952.27.0
40–491,4674.55.91,9322.34.0
50–599872.94.89921.02.5
60–693392.93.82440.81.2

That the differences in HPV positivity are real and not the artefact of a detection bias related to testing laboratory is further illustrated in Figure 3, which shows a scatter plot with results from the retesting of 80 specimens from each center by the laboratory serving the other center, for a total of 160 pairs of results. There was a strong correlation between test results in 1 lab and retest by the other, with a near equivalency in RLU readings, as shown by the best fitting regression equation (Predicted Log St. John's RLU = −0.0542 + 1.0403 × observed Log Montreal RLU, R2 = 0.9671). There was a high agreement between the 2 laboratories (κ = 0.878) when results were treated in 3 categories (negative, 1–10, >10) based on recoding the RLUs.

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Figure 3. Correlation between original and retest HPV results (RLU readings) for 2 samples of 80 specimens, 1 from each study center. Samples included ∼40 positive and 40 negative results randomly chosen from each centre. Retesting of specimens from 1 center was performed by the laboratory from the other center. The thick line represents the regression model that best fits the data points. The thin line shows the expected equivalency between the 2 sets of results.

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Finally, we focused on the association between cytology results and HPV positivity (Table IV). Only 6.5% of women with normal Pap tests in Montreal and 4.2% in St. John's tested positive for HPV DNA. HPV positivity increased dramatically in women with abnormal cytology results: 75% of women with LSIL cytological diagnoses had positive high-risk HPV tests, while over 90% of women with HSIL cytological diagnoses tested positive for high-risk HPV DNA. The equivocal nature of ASC and AGC categories is reflected in important variations in HPV positivity between categories and centers.

Table IV. HPV Positivity by Cytological Result and by Center1
CentreCytology resultsStudy groupsTotal by centre
Focus on PapFocus on HPV
n% HPV positiven% HPV positiven% HPV positive
  • 1

    This table includes the data from the 9,617 participants with both Pap and HPV results available.

MontrealNegative1,9586.21,9876.83,9456.5
ASC-US6227.44738.310932.8
ASC-H2100.010.0366.7
AGC1225.01118.22321.7
ASC and AGC333.31100.0450.0
LSIL977.8966.71872.2
HSIL3100.04100.07100.0
Squamous carcinoma1100.0001100.0
Unsatisfactory205.0316.4515.9
St. John'sNegative2,6254.02,6404.44,2654.2
ASC-US2817.93735.16527.7
ASC-H333.3366.7650.0
AGC922.2812.51717.6
LSIL875.04100.01283.3
HSIL5100.0250.0785.7
Unsatisfactory466.5387.9847.1

Discussion

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

The CCCaST trial was designed to provide an unbiased comparison of Pap to HPV efficacy in the identification of high-grade cervical cancer precursors. This trial has several attractive design features that underscore its importance in furthering the cause of evidence-based decisions in the assessment of emerging technologies in cervical cancer screening. Firstly, it targets a North American population of average risk women, who have had previous access to cervical cancer screening. Genetic characteristics, social and cultural markers have been shown to influence the natural history of HPV infection and cervical intra-epithelial lesions reviewed in Refs.13, 14, 15. It is thus important to assess the performance of HPV DNA testing as a candidate-screening tool in a variety of settings; the differences between the 2 populations covered in this trial will provide insights with respect to the generalizability of the efficacy findings. Secondly, in our study, both Pap and HPV tests were collected and processed in the usual community setting where screening takes place. This ensures that our results truly represent what can be expected of these tests in everyday practice, making it easier to formulate public health recommendations. This is different from trials in communities where Pap testing is not usually available or which, by design, have tests collected and sent elsewhere for expert reading. The evaluation of Pap testing in such circumstances is closer to the ideal rather than to the usual performance. Thirdly, the design minimized bias due to disease verification. The randomization process and similarities in screening procedures ensure symmetry in disease detection across arms. Colposcopists and pathologists were blinded to initial test results. A standard colposcopy protocol ensured that all participants undergoing colposcopy received the same level of clinical scrutiny. The randomization scheme achieved a balanced distribution of key patient characteristics between the 2 study arms, which will provide for an unbiased and credible assessment of the differences in efficacy of the 2 screening tests. Finally, cervical pathology practice standards in Canada are comparable to those of many European countries, which underscores the generalizability of the study.

We recruited participants in 2 provinces and found that these 2 groups differed in regard to certain risk factors known to be associated with HPV acquisition and persistence (age, contraception method, marital status and tobacco use). Future studies will complement the CCCaST to explore in-depth the importance of such differences on screening test performance and on the natural history of HPV and cervical lesions.

As was described in previous studies comparing HPV to Pap testing, we found that abnormal HPV test results are more frequent than abnormal Pap test results.16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 We also found that this holds true for all age categories, and both study centers. Some will argue that if HPV DNA testing is to become a standard primary screening tool for cervical cancer precursors, the fact that more women are referred to colposcopy will have a significant impact on health care resource utilization. However, it is only through this targeted increase in diagnostic procedures that we may identify and treat more high-grade intra-epithelial lesions, hoping ultimately to reduce the incidence of cervical cancer. The economic analysis attached to CCCaST will help evaluate the costs associated with this increase in resource utilization. The longitudinal component of CCCaST with extended follow-up will provide information on risks associated with lengthening the testing interval to reduce the costs of screening associated with HPV DNA testing.

Our experience with CCCaST confirms that improving cervical cancer screening is a concern not only for researchers, but also for clinicians and Canadian women. Indeed, their enthusiastic interest has enabled us to recruit close to 10,000 women in just over 2 years, despite the delays imposed by ethical approval at various levels, academic and clinical, and the lack of a financial incentive to recruit subjects. The lack of a formal organized screening program was no deterrent to successful accrual as we found that working with community medical practices provided an efficient strategy to make the study available to women attending opportunistic screening. Less than 10% of the women approached declined to participate.

Adherence to the study protocol has been excellent and physicians have had no difficulty incorporating HPV DNA testing in their everyday practice. It is not surprising, however, that more protocol violations occurred in the HPV arm, and that the majority of those violations were errors in the order of collection of the 2 screening tests. Collecting a Pap test is one of the most common activities for Canadian general practitioners and gynecologists, which explains why the Pap specimen sometimes ended up being collected first as a reflexive act, even in the HPV arm. However, such mistakes were infrequent enough that they do not affect study validity. Moreover, the fact that we collected information on the adherence to study protocol will enable us to quantify the impact of such mishaps through sensitivity analysis.

Recruitment in CCCaST was terminated in February 2005, after 10,171 women had been enrolled, and after an interim analysis had shown that we had reached adequate power to test our main hypothesis. Colposcopy results for women recruited toward the end of the enrolment period are being compiled and the efficacy analysis will soon follow. The second phase of our study dealing with the 12–18 month follow-up of the cohort is already underway: women recruited in the early phases of CCCaST have already returned for their follow-up screening visits. We expect that this trial will provide valuable information to assist evidence-based practice guidelines for cervical cancer screening in Canada and internationally.

Acknowledgements

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

CCCaST is funded by Canadian Institutes of Health Research (CIHR) grant MCT-54063 to E.L.F as Principal Investigator. M.H.M is supported through a fellowship from the National Cancer Institute of Canada; E.L.F is recipient of a Distinguished Scientist award from the CIHR; F.C. is recipient of a National Scholar award from the Fonds de la Recherche en Santé du Québec.

References

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