# Multistep ctDNA Monitoring of Minimal Residual Disease in Colorectal Cancer Liver Metastases: From Tissue NGS to Highly Sensitive Digital PCR Platforms

## TL;DR

This study shows that digital PCR can detect tiny amounts of cancer DNA in blood, helping predict cancer recurrence in colorectal cancer patients after surgery.

## Contribution

A personalized multistep ctDNA monitoring approach using digital PCR for MRD detection in CRC liver metastases is introduced.

## Key findings

- Digital PCR platforms showed 100% qualitative concordance in detecting ctDNA, outperforming traditional methods.
- Ultra-low Variant Allele Frequencies were detected, identifying high molecular burdens correlating with rapid disease progression.
- MRD-negative status was found in 70% of patients, indicating potential for recurrence-free survival prediction.

## Abstract

Background/Objectives: Colorectal cancer (CRC) liver metastases present a significant clinical challenge due to high recurrence risks post-resection. Traditional diagnostics often fail to detect early-stage minimal residual disease (MRD). This preliminary pilot study evaluated ctDNA dynamics in 10 patients with liver metastases using a personalized multistep approach. Methods: Following primary tumor Next-Generation Sequencing (NGS) to identify somatic mutations in KRAS, NRAS, TP53, RET, APC, and WRN, custom TaqMan assays were designed for longitudinal plasma analysis. Four methodologies were compared: HRM-PCR, PNA-enhanced qPCR, and two digital platforms (dPCR and ddPCR). Results: While HRM-PCR sensitivity was limited in plasma, digital platforms demonstrated 100% qualitative concordance. MRD-negative status (VAF 0.00%) was identified in 70% of cases (P01, P03, P06, P07, P08, P09, P10), while detectable ctDNA in patients P02, P04, and P05 strongly correlated with aggressive progression. Digital PCR enabled the ultra-low detection of Variant Allele Frequencies (VAFs), identifying high molecular burdens (e.g., P05, VAF 49%) correlating with rapid decline, and capturing early molecular residue in P04 (VAF 0.62%). Conclusions: Our preliminary findings confirm that personalized longitudinal VAF tracking via digital PCR provides superior prognostic value, serving as a robust tool for recurrence monitoring in personalized CRC therapy.