Role of host cell DNA Methylation Analysis in predicting non-Regression or regression of high-grade anal Intraepithelial NEoplasia in HIV+ men (MARINE) trial

Rationale: Human papillomavirus (HPV)-induced anal cancer precursors, high-grade anal
intraepithelial neoplasia (HGAIN), are known to have a spontaneous regression rate of 28%.
Current histopathological assessment is unable to distinguish between HGAIN likely to
regress and HGAIN likely to persist or progress to cancer. To prevent anal cancer, currently
all HGAIN is treated by electrocautery, which leads to substantial overtreatment.
Objective: In this study we will clinically validate if previously identified promising host cell
DNA methylation markers and other biomarkers can predict (non-)regression of HGAIN, thus
determining the need of immediate treatment versus active surveillance, and the safety of
withholding treatment. This could prevent overtreatment and the associated anal and
psycho-sexual morbidity, thus improving anal cancer screening efficacy and quality of life of
HIV+ MSM.
Study design: A multicentre active monitoring cohort study in Amsterdam, the Netherlands,
in which HIV+ MSM with HGAIN will not be treated during a 24-months follow-up.
Study population: HIV+ MSM (n=200) diagnosed with HGAIN (<50% of anal circumference)
will be recruited.
Intervention: Participants will be monitored by six-monthly high-resolution anoscopy (HRA)
with biopsies and anal swabs for cytology. Baseline tissue samples will be tested with host
cell DNA methylation markers (ASCL1, ZNF582) and other biomarkers: HPV genotyping,
HPV-E4, p16INK4A, Ki-67, and immunological markers.
Main study parameters/endpoints: The primary study endpoint is regression or nonregression
of each individual HGAIN lesion at the end of the study, based on the
histopathological outcome. Regression and non-regression are defined as respectively
≤LGAIN and ≥HGAIN in the exit biopsy at 24 months, taken within the same octant or shifted
up to one octant on either side of the baseline lesion.
Main secondary endpoints are: clinical outcome, reflected as the number of octants affected
by each individual HGAIN lesion, overall disease: the histological and clinical outcomes of all
HGAIN lesion combined; and the health-related quality of life (HRQoL) of the study
population compared to a control group of 50 HIV+MSM receiving HGAIN treatment.
We will calculate regression rates in lesions with low versus high methylation levels at
baseline.
Nature and extent of the burden and risks associated with participation, benefit and
group relatedness: Participating patients will be withheld potential treatment for anal cancer
prevention. Currently, treatment of HGAIN for anal cancer prevention is not yet evidencebased.
Efficacy of treatment is suboptimal and patients experience considerable side effects.
We therefore already offer patients the possibility to choose between active monitoring at a
similar interval or treatment.
The associated risk of this study is acceptable because: (1) the study follow-up will be two
years, and the risk of progression to cancer will be particularly low in that period, (2) patients
at very high risk for cancer (i.e. patients with more than 50% of anal canal affected, patients
with clinical suspicion for cancer and with abnormalities on digital ano-rectal examination
(DARE) plus MRI) will not be included and (3) participants will be closely monitored during
the trial and excluded for treatment when clinical suspicion for cancer arises.

The hypothesis is that methylation marker panel, potentially combined with additional biomarkers, can predict (non-)regression of HGAIN, thus determining the need of immediate treatment versus active surveillance, and the safety of withholding treatment. This could prevent overtreatment and the associated anal and psycho-sexual morbidity, and improve anal cancer screening efficacy and quality of life of HIV+ MSM.

The primary outcome will be measured at the 24-month follow-up visit and compared to baseline findings using the histological outcome of the biopsies taken during HRA, as described above. The secondary outcomes will be measured at the 6-, 12-, 18-, and 24-month follow-up visits and compared to baseline findings using the histological outcome of the biopsies taken during HRA and the clinical outcome observed during HRA, defined as a change in size measured by the number of octants of the anal surface affected. The HRQoL will be measured at baseline, 6- and 12-month follow-up using the ANCHOR Health-Related Symptom Index (A-HRSI), which is a validated assessment form specifically designed for our target population.

At baseline socio-demographic, medical, AIN, HIV, and sexual history will be recorded in an electronic Case Report Form (eCRF). At baseline and at six-monthly (±2 weeks) visits, during a total follow-up of 24-months, participants will undergo an anal swab, digital anal-rectal examination (DARE) and HRA (including photo documentation) after at least 1-2 minutes application of acetic acid (5% solution), followed by repeated application during the exam, and biopsies of all suspected lesions after staining with Lugol’s iodine when indicated by experienced HRA providers adhering to the International Anal Neoplasia Society Guidelines. Lesion characteristics, localisation and size will be recorded.

Anal swab specimens will be collected using a wetted nylon-flocked swab, that will be slowly retracted after insertion while rotating and applying firm lateral pressure, and transferred and stored in ThinPrep PreservCyt Solution (Hologic, Marlborough, Massachusetts, USA). Anal cytology will be performed as a quality control measure: in case of an indication for a high-grade lesion on cytology while no high-grade lesions were found at HRA, the HRA will be repeated to make sure no lesions were missed. Residual anal swab specimens will be stored at -20°C.

At the last follow-up visit, an exit biopsy is taken from every lesion, or at random if there is no visible lesion at the location of the previous lesion. After the 24-month follow-up visit patients will return to standard care.

For safety and per standard of care, clinical suspicion of cancer and/or palpable abnormalities found using DARE will be followed up with MRI to rule out progression to cancer. In case of invasion seen at MRI, or in a biopsy obtained during a follow-up HRA, the participant will be excluded from the study, for treatment of anal cancer (cancer will then be diagnosed at a very early stage) according to local guidelines.