Rate of cervical cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ in primary HPV DNA screening with cytology triage: randomised study within organised screening programme

Recruitment

The study was based on follow-up during 2003-7 in women randomised (1:1) to primary HPV DNA screening with cytology triage or conventional cytological screening within the population based screening programme for cervical cancer in southern Finland between 1 January 2003 and 31 December 2005. We used data from eight municipalities and two screening laboratories. In total 58 282 women were invited to participate in the screening programme (fig 1⇓). The screening invitations at the recruitment phase included the five yearly invitations to the screening programme. Information on study design, screening methods used, and cross sectional screening findings have been previously published.11 12 13 14

Fig 1 Enrolment, random allocation, completeness of follow-up, and analysis

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Randomisation and exclusions

Of the 58 282 women, 29 144 (50%) were individually randomised to screening with the HPV DNA test and 29 138 to conventional cytological screening as the control arm. Randomisation was done two to 12 months before invitation. After randomisation, 206 women were excluded, 107 from the HPV arm and 99 from the conventional arm (fig 1).⇑ We included data from 58 076 women in the analysis.

Conventional cytology screening protocol

The national screening programme for cervical cancer in Finland was started in the early 1960s, and women aged 30-60 are invited by letter to attend screening every five years. Trained nurses or midwives usually take screening samples in primary healthcare facilities in the municipality. In the trial, cytological sampling was the same in both arms: a VCE smear (vaginal, cervical and endocervical samples) was taken with a cytobrush from the junction and endocervix, and with two spatulas from the ectocervix and vaginal fornices, and spread on the same objective glass. The fixative was 95% ethylalcohol and Papanicolaou staining was used. A cytotechnician reviewed all the screening smears and referred any abnormal findings to a cytopathologist. The cytopathologist also re-evaluated 10-20% of the smears with normal results. The cytopathologist was responsible for the final diagnosis and recommendations.

In conventional practice, a referral for colposcopy and biopsy is based on a cytology result equal to Papanicolaou class III-V, roughly corresponding to low grade squamous epithelial lesions or a worse (LSIL+) in the Bethesda 2001 reporting system. A borderline cytology (Papanicolaou class II, corresponding to atypical squamous cells of undetermined significance (ASC-US) or reactive cytological abnormality, or both, in the Bethesda 2001) usually leads to a recommendation for intensified screening, meaning a repeated screening invitation after 12-24 months. Women with negative results on colposcopy and histology are also recommended for intensified screening. If women have borderline results on cytology two or three times during intensified screening, they are referred for colposcopy.15 The threshold for treatment in the current programme is a CIN I lesion. In Finland, CIN I lesions in women aged 30 or more are treated, but women aged under 30 tend to be followed up without treatment.

HPV screening protocol

In the HPV screening protocol women underwent an HPV DNA test instead of conventional cytology as the primary screening test. After the conventional cytological smear was prepared (see above), the rest of the cellular material from the endocervical sample was used for an HPV test.11 12 13 14 The transport medium containing the screening sample was processed with the supplies and reagents of the hybrid capture II assay (Qiagen Finland, Helsinki). Results of the detection assay were expressed as a ratio of relative light units (RLU ratio) with 1.00 (equivalent to HPV DNA concentration of 1 pg/ml) as the cut off for a positive result. The test cut off and internal quality assurance procedures were according to the manufacturer’s instructions.

In women with a negative HPV result, we did not evaluate cytology and the next invitation for screening was scheduled after five years as in the cytology based programme. If the HPV result was positive, cytology was evaluated with a triage method to decide about the management. The cytotechnician and thereafter the cytopathologist carried out the cytology triage as in the conventional arm. The technician analysing the smear and the physicians at the clinical phase were aware of the result of HPV DNA test. The treatment threshold was the same as for conventional screening.

In the HPV screening protocol, any decision for immediate referral was based on a clearly positive cytological finding. In cases of borderline cytology, intensified screening was recommended. Intensified screening was also recommended if the HPV result was positive but the result of cytological triage was negative. During the intensified screening in the HPV arm, women were referred for colposcopy after repeated borderline findings at cytological triage or after three consecutive positive HPV test results even if cytology was normal.

A small group of women, about 8.5%, in the HPV arm underwent a conventional cervical smear test because an HPV test was not taken, mainly for technical reasons (the proper brush or tube was missing or the sample was not taken because of error). Few women refused the HPV test. The HPV test was not available to women in the conventional screening arm, even though in a few cases (<0.1% of visits) it was carried out in error.

Follow-up data and linkage

The screening database was linked with the population register and cancer register to obtain information on vital status, emigration, cervical cancer, cervical intraepithelial neoplasia grade III (CIN III), and adenocarcinoma in situ. All the outcomes in our study were based on data from the cancer registry. Within the studied municipalities, all screening invitations and visits were recorded. Many women were also screened outside the screening programme. Each year about the same number of cervical smears are taken opportunistically as in the organised programme, but these data are not registered.15 16

The follow-up started at the time of the first valid invitation of the current randomised study period (2003-5) and closed at emigration, death, diagnosis of CIN III/adenocarcinoma in situ, or cervical cancer or at 31 December 2007, whichever was earlier. The screening data did not include the exact date of invitation, so we used the month and year of the screening visit to approximate invitation time among attendees. Among non-attendees we estimated the month of invitation as the median month of visits in their municipality during the invitational year.

Sample size

The a priori planned outcome included the incidence of invasive cervical cancer and CIN III/adenocarcinoma in situ at 5, 10, and 15 years through record linkage between screening and cancer registers. We needed about a million woman years at risk for an 80% power to detect a 50% decrease in the subsequent risk of cervical cancer.12 Considering data on CIN III+ the statistical power to detect a 1.5-fold difference among all invitees was about 70% (relative rate 1.5, α 0.05, one sided) and for detecting a twofold difference among those with positive results was 95% (2.0, α 0.05, one sided) in current phase of follow-up.

Definitions of summary screening findings

We used the following terms: invitee—a woman who got an invitation (a personal letter) to attend screening; attendee—a woman who took part in screening (that is, a cervical sample was taken); and non-attendee—a woman who was invited for screening but did not attend.

In attendees, the main groups consisted of women with positive or negative results on primary screening tests. An RLU ratio <1.00 on an HPV test and Papanicolaou class I on a cervical smear test were classed as negative results. An RLU ratio ≥1.00 on an HPV test or an abnormal result of the cervical smear was classed as a positive result. Women with a positive result were further grouped into those with a positive screening episode and those who were recommended for intensified screening.

Women with a positive screening episode were referred for colposcopy and thereafter any histologically confirmed cervical intraepithelial neoplasia or cervical cancer was detected at the index screening round. Women were recommended for intensified screening if the results of colposcopy or histology were negative (that is, no cervical intraepithelial neoplasia diagnosed at the index episode among recommended women) or if the result of the cervical smear was class II (in conventional screening) or the cytology triage result was normal or gave a borderline result (in HPV screening).

Statistical methods

We compared patterns of CIN III and respective sensitivities between arms with Poisson regression; the numerator was the number of detected lesions of interest, and the denominator was the woman years at risk in all women who were invited or attended. We calculated relative risk estimates (relative rate) for the HPV screening arm using the conventional screening arm as the reference. The 95% confidence intervals for the relative risk estimates and potential effect modification with age were assessed with likelihood ratio statistics. The analyses were based on the intention to screen principle throughout, both among invitees as well as in more detailed subgroups of attendees.

The invitees were further grouped into attendees and non-attendees and the attendees into those with positive and negative test results during the first or “index” screening round (see the definitions above). Women remained in the same category throughout the current follow-up period.