Diagnostic Performance of Host and Viral DNA Methylation Analysis in the Identification of Anal Intraepithelial Neoplasia and Cancer: Systematic Review and Meta-Analysis

Abstract

Introduction: DNA methylation-based biomarkers have been investigated as useful tools in the carcinogenesis process, including the triage of HPV-associated cancers. In this context, we conducted a systematic review and meta-analysis focused on evaluating the changes in the level of DNA methylation in cases of pre-cancerous (i.e., anal intraepithelial neoplasia, AIN-1, -2., -3) and cancerous (i.e., squamous cell carcinoma, SCC) anal lesions.

Methods: A research in the PubMed, Scopus, and Web of Science databases was carried out, following the PRISMA 2020 protocol, using the following keywords: "anal cancer", "anal intraepithelial neoplasia", "methylation", and "epigenetic". All observational studies that reported the level of DNA methylation by grade of anal lesions and for different target genes were included. The QUADAS-2 tool was used to assess the studies' quality, whereas pooled prevalence, sensitivity, specificity, and diagnostic odds ratio (DOR) were employed to verify the accuracy of the test in the detection of high-grade lesions.

Results: Eight studies met the inclusion criteria, involving a total of 1555 clinical samples. The prevalence of methylation-positive samples by histological grading was 27%, 45%, 54%, and 98% for AIN1, AIN2, AIN3, and SCC, respectively. Similar results were observed for the DOR, with higher ORs in more severe lesions. The pooled AUC (95%CI) for the diagnosis of ≥AIN2 was 0.68 (0.63-0.73).

Conclusions: The present review and meta-analysis support the introduction of DNA methylation-based biomarkers in the triage of subjects with low-grade anal lesions and in the monitoring of treatment outcomes. Standardized protocols and a prospective study design are needed to implement methylation tests in clinical practice.

Keywords: DNA methylation test; HPV; anal cancer; biomarkers; epigenetic.

Figure 1

PRISMA flowchart of the search strategy results.

Figure 2

Forest plot of DNA methylation positivity in AIN1 and LSIL clinical specimens. The size of the gray squares is proportional to the weight that each study contributes to the meta-analysis. The red diamond represents the overall estimated pooled prevalence. The vertical dark line indicates the threshold for statistical significance (when the 95%CI includes the vertical line of “no effect”, the result is not statistically significant) [13,14,15,16,17,18,19,20].

Figure 3

Forest plot of DNA methylation positivity in AIN2 and HSIL clinical specimens. The size of the gray squares is proportional to the weight that each study contributes to the meta-analysis. The red diamond represents the overall estimated pooled prevalence. The vertical dark line indicates the threshold for statistical significance (when the 95%CI includes the vertical line of “no effect”, the result is not statistically significant) [13,14,15,16,17,18,19,20].

Figure 4

Forest plot of DNA methylation positivity in AIN3 clinical specimens. The size of the gray squares is proportional to the weight that each study contributes to the meta-analysis. The red diamond represents the overall estimated pooled prevalence. The vertical dark line indicates the threshold for statistical significance (when the 95%CI includes the vertical line of “no effect”, the result is not statistically significant) [13,14,17,18,19,20].

Figure 5

Forest plot of DNA methylation positivity in SCC clinical specimens. The size of the gray squares is proportional to the weight that each study contributes to the meta-analysis. The red diamond represents the overall estimated pooled prevalence. The vertical dark line indicates the threshold for statistical significance (when the 95%CI includes the vertical line of “no effect”, the result is not statistically significant) [13,17,18,19,20].

Figure 6

Forest plot of the pooled sensitivity of the methylation test in the detection of ≥2 AIN lesions among the included studies. The size of the blue dots is proportional to the weight that each study contributes to the meta-analysis. The blue diamond represents the overall estimated pooled sensitivity. The vertical red lines indicate the 95%CI for the pooled sensitivity [13,14,15,16,17,18,19,20].

Figure 7

Forest plot of the pooled specificity of the methylation test in the detection of ≥2 AIN lesions among the included studies. The size of the blue dots is proportional to the weight that each study contributes to the meta-analysis. The blue diamond represents the overall estimated pooled specificity. The vertical red lines indicate the 95%CI for the pooled specificity [13,14,15,16,17,18,19,20].