94P - A one-tube multiplex methylation-specific droplet digital PCR assay for identification of ctDNA biomarkers in anal squamous cell carcinoma

Background

Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal cancer associated with high-risk human papillomavirus (HPV) infection. ASCC is one of the fastest accelerating causes of cancer and mortality all over the Western world. Studies indicate that alterations in DNA methylation is associated with anal carcinogenesis. Liquid biopsy assays for detecting cancer-specific alterations in circulating tumor DNA (ctDNA) is a promising diagnostic approach due to its non-invasive nature. This study aims to develop and validate a novel multiplex assay to detect ASCC methylation-specific ctDNA biomarkers with high sensitivity and specificity.

Methods

A multiplex droplet digital PCR (ddPCR) assay was designed to target five previously described CpG biomarkers hypermethylated in ASCC: ASCL1, LHX8, WDR17, ZIC1 and ZNF582 and a reference gene. Specificity was assessed in anal normal and cancer tissue samples. The limit of blank was determined in plasma samples from control persons. Sensitivity was tested in plasma from 26 anonymized patients diagnosed with ASCC. 17 patients had T1 or T2 tumors ≤ 4 cm, and 9 patients had T2 tumors > 4 cm or T3 tumors. Quantification was performed on the six-color multiplexing QX600 ddPCR system from BioRad.

Results

The multiplex assay detected increased levels of all targeted hypermethylated biomarkers in anal cancer tissues compared to normal tissue. Furthermore, it detected methylated ctDNA biomarkers in plasma from patients with ASCC, achieving detection rates of 76% (13/17) in patients with T1 or T2 tumors ≤ 4 cm, and 89% (8/9) in patients with T2 tumors > 4 cm or T3 tumors.

Conclusions

The results confirm that our multiplex ddPCR assay detects ctDNA biomarkers in plasma from ASCC patients with high sensitivity and specificity. The assay demonstrates potential as a rapid, cost-effective, and high throughput tool for monitoring ASCC progression over time. However, further studies are required to explore the full potential of the assay for detecting recurrence or early cancer events in patients with ASCC.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

K.G. Spindler: Other, Lecture fee: Daiichi Sankyo Northern Europe GmbH, BMS Norway; Other, Fee for teaching: Incyte Biosciences Denmark ApS. All other authors have declared no conflicts of interest.