Gay and bisexual men (GBM) are at increased risk of HPV-associated anal cancer. Screening for the precursor high-grade squamous intraepithelial lesions (HSIL) analogous to cervical cytology screening has been proposed but there is currently no agreed algorithm. Anal HSIL is highly prevalent in GBM, however it is clear that most anal HSIL do not progress to cancer. Identification of biomarkers to establish patients at highest risk of cancer are therefore needed. Change in DNA methylation is recognised as an early essential step in carcinogenesis, and is predictive of cervical HSIL and cancer. It is thought that DNA methylation patterns may also predict anal HSIL. Here, DNA methylation was evaluated as a potential tool to identify persistent anal HSIL among GBM.
Samples were obtained as part of the Study of the Prevention of Anal Cancer, a 3-year natural history study of anal HPV and related disease in GBM aged 35 years and older. HSIL was defined as a composite of cytology and histology diagnoses (ie had one or both cytological possible HSIL (pHSIL) and/or histological HSIL). Persistent HSIL was defined as HSIL detected at all of baseline, 6 and 12 months clinic visits. DNA was extracted from anal cytology PreservCyt samples from the baseline clinic visit, and subjected to bisulfite conversion. Methylation-specific quantitative PCR (msqPCR) on promoter regions of CADM1, MAL and miR-124-2 genes were performed, with the positive threshold for each marker set at 1.5%, 0.5% and 0.5% methylation, respectively. Sensitivity and specificity of each marker for detecting prevalent baseline and persistent HSIL were calculated.
Of the 165 participants included, 52 (31.5%) were HIV-positive and 69 (41.8%) had composite HSIL at baseline. In total, 23 (13.9%) had persistent HSIL at 12 months post-baseline. Sensitivity and specificity, positive and negative predictive value (PPV, NPV) of each methylation marker are shown in the table.
| Marker | HSIL diagnosis | % Sensitivity (95% CI) | % Specificity (95% CI) | % PPV (95% CI) | % NPV (95% CI) |
|---|---|---|---|---|---|
| MAL | Baseline | 62 (50-74) | 41 (31-52) | 43 (34-54) | 60 (47-72) |
| MAL | Persistent | 74 (52-90) | 42 (33-51) | 18 (11-28) | 90 (80-96) |
| mi-R-124-2 | Baseline | 52 (40-64) | 41 (31-52) | 39 (29-50) | 54 (42-66) |
| mi-R-124-2 | Persistent | 57 (35-77) | 42 (33-51) | 15 (8-24) | 85 (74-92) |
| CADM1 | Baseline | 36 (25-49) | 73 (63-81) | 49 (35-63) | 61 (51-70) |
| CADM1 | Persistent | 48 (27-69) | 70 (62-78) | 22 (12-36) | 88 (81-94) |
Methylation markers MAL, CADM1 and miR-124-2 in baseline anal swab DNA may have higher sensitivity and similar specificity for the detection of persistent HSIL than for the detection of baseline HSIL, however larger numbers and further studies are needed to evaluate markers for detection of persistent HSIL.