Detection of Circulating Tumour DNA(ctDNA) with ddPCR and NGS in Liposarcoma

Introduction Cell-free DNA (cfDNA) is naturally released during cell death processes like apoptosis or necrosis. In the tumour context, it is known as circulating tumour DNA (ctDNA).We harness the power of ddPCR to determine the copy number variation (CNV) of mouse double minute 2(MDM2) and cyclin-dependent kinase 4 (CDK4) gene amplifications, which is one of the molecular characteristics of both well-differentiated liposarcomas(WDLPS) and dedifferentiated liposarcomas (DDLPS).

Material and method Demographic data for all included patients were included in a prospective cross-sectional analysis. A total of 32 adult patients with primary and recurrent retroperitoneal sarcomas were assessed. Blood samples were collected prospectively during treatment. cfDNA was isolated from plasma and quantified. All patients who had tumour in situ during blood sampling and were histologically positive for MDM2 amplification via immunohistochemistry (IHC) were selected for inclusion in this study. A total of 15 plasma-derived DNA samples (Eleven non-paired samples plus four paired samples – pre- and post-surgery) were included between June 2023 - June 2024. ctDNA detection and copy number determination were performed using Bio‐Rad QX200 Droplet Digital PCR (ddPCR) (Bio‐Rad Laboratories) and Oxford NanoporeTechnologies (ONT).

Result and discussion MDM2 was detectable in both tissue tumour samples and plasma-derived samples (ctDNA). The average concentration was 2.5 ng/µl and 5 ng/µl, respectively. Aduplex MDM2:RPP30 ddPCR assay revealed one sample had an amplified copy number of 18.9, which was confirmed by ONT. This positive sample occurred in apatient who had unresectable disease, therefore, open and close laparotomy (stage AJCC IIIB). The remainder negative copy number samples were staged AJCC IB. For the longitudinal paired samples, the was no difference in CNV, however the median follow-up was only 40-month. On the other hand, a repeated experiment with duplex CDK4:RPP30 assay yields a lower amplified copy number of 9.87 for the same sample (E01), but overall consistent findings compared to MDM2 assay.

Conclusion Analysis of ctDNA is a novel approach for cancer screening, diagnosis, monitoring treatment response, and identifying mutations. Our analysis shows that ctDNA detection is possible in patients with both retroperitoneal WDLPS and DDLPS using liquid biopsy ddPCR and NGS approaches, however this appears to be restricted to patients with locally advanced disease. A larger sample size to improve sensitivity in ctDNA MDM2 and CDK4detection is required for clinical utility.