Low-pass whole methylome sequencing–based liquid biopsy for metastatic colorectal cancer monitoring in the VALENTINO trial.
Authors: Paolo Manca, Marta Paoli, Adele Busico, Giovanni Randon, Samantha Di Donato, Michele Prisciandaro, Francesca Galardi, Alessandra Raimondi, Agostina Nardone, Margherita Ambrosini, Roberta Fazio, Elisa Sottotetti, Antonia Martinetti, Luca Malorni, Sara Lonardi, Federica Morano, Matteo Benelli, Filippo Pietrantonio
Journal: Journal of Clinical Oncology (2025)

3552 Background: The amount of ctDNA is a proxy for metastatic colorectal cancer (mCRC) disease burden, with potentials for prognostic stratification and treatment monitoring. We investigated two methods based on low-pass whole genome and methylome sequencing (WGS and WMS) for ctDNA detection and quantification in the VALENTINO trial. Methods: All patients from the VALENTINO trial - a phase II trial comparing the addition of 5FU to Panitumamb-based maintenance after first line FOLFOX+Panitumamb induction in RAS wild-type mCRC - were eligible. Baseline (BL) and 8-week (8w) plasma samples were collected for low-pass WGS and WMS analysis. Two methods for ctDNA quantification based on DNA methylation (METER) and copy number alterations (ichorCNA) were assayed. Chi-squared, Wilcoxon and Cox regression tests were used. Performances of WMS and variant allele fraction (VAF) of a 14-gene panel were compared. Results: A BL liquid biopsy was available for 154 patients, with 142 also having an 8w assessment. METER and ichorCNA detected ctDNA in 112 (72.7%) and 94 (59.7%) BL samples, respectively; all discordant cases were METER+ but ichorCNA-. Detection rate increased in the presence of liver metastases (86.0% vs 42.6% for METER, 75.7% vs 23.4% for ichorCNA; both p &lt; 0.001) and decreased with peritoneal metastases (55.6% vs 78.0% for METER, 38.9% vs 66.1% for ichorCNA; p = 0.011, p = 0.006). Tumor fraction (TF) of both BL METER and ichorCNA correlated with the diameter of measurable lesions (both p &lt; 0.001) and CEA (p &lt; 0.001 and p = 0.010). Both PFS and OS were shorter after baseline ctDNA detection with METER (mPFS: 10.6 vs 18.6 months, HR: 1.65, p = 0.010; mOS: 28.7 vs 62.2 months; HR: 2.24, 95%CI: 1.37-3.66; p = 0.001) or ichorCNA (mPFS: 10.6 vs 15.0 months, HR: 1.42, 95%CI: 1.00-2.00, p = 0.047; mOS: 27.8 vs 48.4 months, HR: 1.35, 95%CI: 1.29-2.95; p = 0.002). In the multivariate analysis, METER ctDNA detection was the strongest predictor of both PFS and OS (p = 0.005 and p = 0.001) while ichorCNA ctDNA detection was significantly associated with OS but not with PFS (p = 0.002 and p = 0.093). METER ctDNA TF decreased significantly at 8w in patients with CR, PR, or SD (paired Wilcoxon p = 0.015, p &lt; 0.001, p &lt; 0.001) but not PD (p = 0.560) as the best radiological response. Patients without METER ctDNA clearance at 8w had a higher risks of progression (HR: 2.70, 95%CI: 1.63-4.49; p &lt; 0.001) and death (HR: 3.37, 95%CI: 2.00-5.69; p &lt; 0.001). Among 123 patients with both METER and VAF available, concordance was 78.0% and in 10 and 17 patients, respectively, ctDNA was detected only with METER or only with VAF. The mPFS and mOS of discordant cases were longer than METER+ / VAF+ cases and shorter than METER− / VAF− cases. Conclusions: CtDNA quantification with low-pass WMS by METER retains a prognostic significance, can be used for disease monitoring during treatment and refines ctDNA detection based on a restricted gene panel assay. Clinical trial information: NCT02476045 .