Abstract
This study aimed to investigate DNA methylation in anal pap specimens in male
who have sex with men ( MSM) populations who are at risk for anal cancer
development due to persistent infection with high-risk type human papillomavirus
(HPV) infection, especially in people with human immunodeficiency virus (HIV). In this
study, 180 male volunteers were recruited into the study, which included 152 MSM
and a control group of 28 men who have sex with women (MSW). Demographic and
behavioral data were collected by Computer Assisted Self-interview. HIV testing, anal
cytology and anal pap DNA extraction for HPV testing and genotyping were conducted.
In addition, DNA methylation studies were performed using Methylation-sensitive high
resolution melting assay (MS HRM) developed in this study that were validated with
Whole genome bisulfite sequencing (WGBS).
Result: Anal cytology examination in the MSM group showed a significant
proportion of abnormalities than the control group (51/152 (33.55%) vs 3/27 (11.11%),
p < 0.05 Fisher's exact test). The prevalence of HPV infection in the MSM population
was 90. 13% (137/152), significantly higher than that of the MSW population with
21.43% (6/28) HPV infection (p < 0.001, Fisher's exact test). Also, significantly higher
HIV prevalence was found among MSM population than that in in MSW ( 70/ 152
(46.05%) vs 1/28 (3.57%), p < 0.001, Fisher's exact test). DNA methylation assessment
using MSHRM developed in this study was consistent with the standard method using
WGBS (R2 = 0. 9460 P < 0. 0001 Pearson correlation) and visualization of the BlandAltman plot revealed an agreement of DNA methylation between the two methods.
The acceptant reproducibility of MS HRM DNA methylation was shown by the
coefficient of variations, 9.77% and 8.58%, as assessed by the Intra- and inter-assay
measurement, respectively. ANOVA analysis and multiple comparison with Holm-Sidak
revealed the significantly high level of CBLN4 DNA methylation especially among the
group infected with HPV and HIV , i.e . ASCUS+ HPV+HIV+ (adjusted P < 0.005), NILM
HPV+HIV+ (adjusted P < 0.05) compared to NILM HPV-HIV-. In addition, ROC curve
analysis in comparison to NILM HPV+HIV-, the ASCUS+ HPV+HIV+ group had AUC 0.9548
(P < 0.0001 ) and the NILM HPV+HIV+ group had AUC 0.7764 P (< 0.0001 ). After
following the population in the at-risk group (ASCUS+ HPV+) regardless of HIV status
for at least 1 year, the heat map analysis of CBLN4 DNA methylation at baseline,
revealed that those who tested negative histology for cancer had relatively low levels
of CBLN4 DNA methylation, while those with relatively high levels of CBLN4 DNA
methylation, especially those who were also infected with HIV, there is a chance of
abnormal biopsy results such as LSIL or HSIL.
In conclusion, this study confirms that the MSM population is a group at risk for anal
cancer with the high prevalence of HPV and HIV infection, as well as the detection of
abnormal anal pap that were common in the MSM group compared to the MSW group.
DNA methylation analysis by MS HRM in the CBLN4 target gene is interesting as it
indicates people infected with HPV and HIV will be at risk of anal cancer regardless of
anal cytology testing which may be useful as an adjunct screening test for anal cancer
or cancers associated with HPV infection. The analyses of DNA methylation by MS HRM
in RASSF1A, SFRP2, CDKN2A target genes remain underway to examine the correlation
with risk factor variables such as HPV infection, HIV status and anal cytology/histology
clinical factors.
