RASSF1A Methylation and Survival Outcome in Retroperitoneal Leiomyosarcoma

Background: Liquid biopsy for detecting DNA methylation in cell-free DNA (cfDNA) is an emerging approach in soft tissue sarcoma. We conducted a proof-of-concept study using cfDNA from patients with retroperitoneal leiomyosarcoma (LMS) to assess the correlation between RASSF1A methylation, tumour burden, and overall survival.

Materials and Methods: Plasma-derived cfDNA was extracted and eluted in 50 μL of nuclease-free water. DNA from buffy coat and tumour tissue was extracted using the DNeasy® Blood and Tissue Kit and eluted in 100 μL of Buffer AE. Each sample underwent two droplet digital PCR (ddPCR) reactions—one with and one without methylation-sensitive restriction enzyme (MSRE). Droplets were generated using the QX200™ Droplet Generator, and thermal cycling was performed using the C1000 Touch Thermal Cycler. Droplet reading and quantification were conducted with the QX200 Droplet Reader and QuantaSoft™ Software (v1.7.4). The methylation ratio was calculated as RASSF1A (methylated) to RASSF1A (unmethylated), and correlated with tumour burden and survival.

Results: Twenty-four patients (8 males, 16 females; mean age 55.3 years) were enrolled. cfDNA was detected in all samples. Six LMS plasma-derived samples (four pre-operative, two paired pre-/post-operative) were analysed. Methylated RASSF1A was detectable in all cases. No consistent reduction in methylation was observed post-operatively. Correlations between methylation ratio and tumour burden (p = 0.246) and disease-free survival (p = 0.108) were positive but not statistically significant. Sequencing data revealed a distinctive methylation signature in locus RASSF1A.

Conclusions: Liquid biopsy and ddPCR can detect RASSF1A methylation, and its profile appears to be a promising LMS biomarker.