RASSF1A Methylation and Survival Outcome in Retroperitoneal Leiomyosarcoma

Introduction The utilisation of liquid biopsy in the detection of methylation status in the cell-free DNA is a new approach in the field of soft tissue sarcoma. We performed a proof-of-concept experiment utilising the cell-free DNA (cfDNA) of retroperitoneal leiomyosarcoma and aimed to investigate the correlation between methylatedRASSF1A in leiomyosarcoma with tumour burden and overall survival.

Material and method Plasma-derived DNA was extracted and eluted in 50 mL of nuclease-free water (NFW). Buffy coat’s and tissue sample’s DNA were also extracted using the DNeasy® Blood and Tissue Kit. DNA was eluted in 100 mL of Buffer AE. Every sample was subjected to two ddPCR reactions – one with the MSRE and the other without the MSRE. Droplets were generated using the QX200™Droplet Generator (Bio-Rad). Incubation and thermal cycling were performed using the C1000 Touch Thermal Cycler (Bio-Rad), with the following program. Following PCR, droplets were read and quantified using the QX200Droplet reader and QuantaSoft™ Software. Methylation ratio {[RASSF1A (Methylated)]: [RASSF1A(Un-methylated)]} was calculated and correlation withtumour burden and survival analysis was made.

Result and discussion A total of twenty-four patients (8 males and 16 females)were included in the study, with a mean age of 55.33years. Cell-free DNA (cfDNA) was successfully detected in all 24 samples. Six plasma-derived DNA samples from patients with LMS, confirmed by immunohistochemistry as positive for either desmin, smooth muscle actin, and/or heavy caldesmon were selected, and these include four samples taken pre-operatively and two sets of paired samples (including post-operative samples) were analysed. Methylated RASSF1A was detected across all samples. We did not observe a consistent reduction in percentage of DNA methylation in the post-operative samples. The correlations between methylation ratio and tumour burden (cm³) (p = 0.246) as well as disease-free survival (days) (p = 0.108) were positive but not statistically significant.

Conclusion DNA methylation can be detected using liquid biopsy and ddPCR approach. We optimised the DNA methylation protocol in detecting methylated RASSF1A.A larger sample is required to improve the sensitivity and specificity of the assay.