Simplified monitoring of post-treatment CIN2/3 women by molecular testing for hrHPV and methylation markers

Despite population based cervical screening still approximately 600 women are
diagnosed with cervical cancer in The Netherlands each year. Another 6000 women
are treated annually for the cervical cancer precursor lesions, named
high-grade Cervical Intraepithelial Neoplasia (CIN2/3). Generally 10-15% of
these women develop residual/recurrent cervical disease after treatment.
According to the Dutch guidelines, women are monitored for residual/recurrent
cervical disease by cervical cytology at 6, 12 and 24 months after treatment.
However, cytology is suboptimal given its low sensitivity and specificity for
residual/recurrent CIN2/3. Furthermore the many follow-up visits result in loss
of adherence of women to the monitoring schedule. Besides, the low positive
predictive value of cytology for post-treatment CIN2/3 leads to unnecessary
diagnostic procedures (repeat smears and colposcopic examinations).
Infection with high-risk human papillomavirus (hrHPV) is necessary for the
development of cervical cancer, and adding testing for high-risk human
papillomavirus (hrHPV) DNA six months after treatment dramatically increased
the sensitivity for post-treatment CIN2/3, while the negative predictive value
of a hrHPV-negative, cytological normal smear was 99%. However, the positive
predictive value of a hrHPV test was still limited, indicating that the
specificity of molecular testing needs further improvement. Methylation
markers, i.e. markers reflecting promoter methylation of host cell genes such
as CADM1 and MAL may enhance the specificity for CIN2/3. We recently found that
silencing of both tumour suppressor genes CADM1 and MAL, primarily resulting
from promoter methylation, is functionally involved in cervical cancer
development. Analysis of cervical biopsies showed significantly more CADM1 and
MAL promoter methylation in >=CIN3 compared with <=CIN1 lesions (p<0.001).
Moreover, CADM1 and MAL promoter methylation was significantly more frequent in
hrHPV-positive scrapings of women who developed >=CIN2 compared to those that
did not and displayed sensitivity for these lesions greater than cytology.
Hence, it can be hypothesized that addition of i.a. CADM1 and MAL promoter
methylation analysis during post-treatment monitoring will markedly increase
the specificity for >=CIN2. Moreover, recent studies have demonstrated that
molecular testing on self-sampled cervical cells offers a reliable alternative
to analysis of conventional cervical scrapings in screening programs.

Our primary objective is to determine whether testing for molecular markers,
i.e. hrHPV, methylationmarkers , i.a. CADM1/MAL and combinations thereof,
yields a higher sensitivity and specificity for the detection of CIN2/3 or
cancer after treatment in comparison with cytology.

The study is designed as a multicenter prospective clinical cohort study.
At treatment a cervical scrape will be taken for cytology and testing of hrHPV
and methylationmarkers , i.a. CADM1/MAL . Six, twelve and twenty-four months
post-treatment cervical cells will be collected by both a self-sampler and by
the gynaecologist and tested for hrHPV and methylation markers. The latter
scrapes will also be analysed by cytology. In case of an abnormal smear (>=BMD)
and/or a hrHPV and methylation marker positive test in the physician obtained
sample, at six months post-treatment, colposcopy will be performed and biopsies
will be taken. At thirteen months, a colposcopy with mandatory biopsy taking
will be performed on test negative women as well. Women with residual/recurrent
>=CIN2/3 disease will be treated. Twenty-four months post-treatment cervical
cells will be collected by both a self-sampler and the cervical smear and
tested for cytology and hrHPV and methylation markers. In case of an abnormal
smear (>=BMD) and/or a hrHPV positive test a colposcopy will be performed. In
case of normal cytology and a hrHPV positive test, cytology and the hrHPV have
to be repeated at 36 months after treatment.In case of a neg smear and a neg
HPV test the women will be sent to population based screening

Risks and burden are linked to protocol procedures, such as cervical sampling
and colposcopy. Although these are routine procedures, carried out by medical
qualified personnel, they may cause side effects or discomfort to the subject.
However, it is expected that these procedures will generally be well
tolerated. The only extra burden involves the self-sampling of
cervical-vaginal cells using a user-friendly self-sampling device.
Self-sampling poses no threats to the physical well-being of a woman.