Multiplex methylation marker analysis for ctDNA detection in liquid biopsies from anal cancer patients: an HPV-independent approach

Background Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal cancer linked to high-risk human papillomavirus (HPV) infection in approximately 90% of cases. Current circulating tumor DNA (ctDNA) approaches in ASCC primarily rely on pathogenic HPV-based biomarkers; however, these methods do not detect all HPV strains and are not applicable to HPV-negative tumors. To address this limitation, we developed a ddPCR assay targeting hypermethylated genomic CpG biomarkers, allowing ctDNA detection independent of tumor HPV status. Methods An anal cancer methylation-specific multiplex droplet digital PCR (AnMM-ddPCR) assay was developed to target five previously described CpG biomarkers hypermethylated in ASCC: ASCL1 , LHX8 , WDR17 , ZIC1, and ZNF582, and the ALB reference gene. Patient samples and samples from non-cancer controls were analyzed using the BioRad QX600 ddPCR multiplexing platform. Results CpG-methylated biomarker levels were significantly higher in ASCC tissue compared with normal tissue and whole blood. The AnMM-ddPCR assay successfully detected all five ctDNA markers in plasma from ASCC patients, achieving an AUC of 0.72 (95% CI: 0.55–0.89; P = 0.018) in baseline plasma samples from ASCC patients with T1 or T2 tumors ≤4 cm, versus 0.90 (95% CI: 0.76–1.00; P = 0.0005) in plasma from patients with T2 tumors >4 cm or T3 tumors. At a specificity of 91.30%, sensitivity increased with stage from 52.94% (95% CI: 30.96–73.83) to 88.89% (95% CI: 56.50–99.43). Conclusion The AnMM-ddPCR assay enables HPV-independent ctDNA detection in plasma samples from anal cancer patients and supports its evaluation in future liquid biopsy applications.