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Aims Cell-free DNA (cfDNA) and circulating tumour DNA (ctDNA) are used for prognostication and monitoring in patients with carcinomas, but their utility is unclear in sarcomas. The objectives of this pilot study were to explore the prognostic significance of cfDNA and investigate whether tumour-specific alterations can be detected in the circulation of sarcoma patients. Methods Matched tumour and blood were collected from 64 sarcoma patients (n = 70 samples) prior to resection of the primary tumour (n = 57) or disease recurrence (n = 7). DNA was isolated from plasma, quantified, and analyzed for cfDNA. A subset of cases (n = 6) underwent whole exome sequencing to identify tumour-specific alterations used to detect ctDNA using digital droplet polymerase chain reaction (ddPCR). Results Cell-free was present in 69 of 70 samples above 0.5 ng/ml. Improved disease-free survival was found for patients with lower cfDNA levels (90% vs 48% at one-year for ≤ 6 ng/ml and > 6 ng/ml, respectively; p = 0.005). Digital droplet PCR was performed as a pilot study and mutant alleles were detectable at 0.5% to 2.5% of the wild type genome, and at a level of 0.25 ng tumour DNA. Tumour-specific alterations (ctDNA) were found in five of six cases. Conclusion This work demonstrates the feasibility and potential utility of cfDNA and ctDNA as biomarkers for bone and soft-tissue sarcomas, despite the lack of recurrent genomic alterations. A larger study is required to validate these findings. Cite this article: Bone Joint Res 2021;10(9):602–610.

Objective: Circulating tumour DNA (ctDNA) is an active area of research in many types of cancer due to its value as a non-invasive diagnostic tool. Despite making up a small fraction of total cell-free DNA (cfDNA), detection of ctDNA has been made possible given advances in next generation sequencing and Digital Droplet PCR (ddPCR). CtDNA testing has some key advantages compared to tissue biopsy; ease of repetition provides a means of real-time monitoring of treatment response and signs of recurrence in cancer. This has specific relevance in sarcoma, where early detection of recurrence may provide an opportunity for faster intervention and can potentially lead to better prognostic outcomes. The purpose of this study is to determine if ctDNA is detectable in plasma samples collected from sarcoma patients and to validate our pre-analytical procedures and to identify opportunities to optimize our protocols for future use. Methods: 20 ml of blood samples were collected from 270 soft tissue sarcoma and osteosarcoma patients with or without pre-operative adjuvant treatment and processed to separate the plasma. Cf DNA was isolated from 2ml of plasma and the quantity and quality assessment was performed by qRT-PCR and capillary electrophoresis, respectively. Whole exome sequencing (WES) was performed on 6 matched tumor-blood DNA samples to identify tumour specific single nucleotide alterations. Sequence variants identified from WES analysis were used to design primers and probes to detect the tumor specific alterations in the corresponding ctDNA in those 6 cases using ddPCR. Results: Of the 60 patient samples analysed to date, the majority were positive for cfDNA. Quality assessment of 41cfDNA by capillary electrophoresis showed peaks approximately 170 bp in size, characteristic of cfDNA. WES of 6 patients' matched tumor-blood samples identified many tumor specific variants. Six of those sequence variants on SMAD4, COL19A1, DDX3X, ADGRG4, HECW1 and FOXR2 genes (Table 1) were used to design primers and probes to detect the tumour specific alterations in the corresponding ctDNA using dd PCR. We observed the presence tumour specific mutations in all corresponding ctDNAs tested in this study. Table 1:Identification of tumour specific variants by WESCaseGeneSNVSarcoma SubtypeVariant Allele Frequency1SMAD4T1584CMyxofibrosarcoma86%2COL19A1G1252AUndifferentiated pleomorphic sarcoma50%3DDX3XA1038COsteosarcoma54%4ADGRG4T6361CLiposarcoma65%5HECW1C1874AMyxofibrosarcoma25%6FOXR2C130GMyxofibrosarcoma24% Conclusions: Our ongoing study to evaluate the use of ctDNA as a biomarker in sarcoma monitoring shows promise related to the efficiency of our protocols and assays. Further testing is ongoing to assess the clinical value in sarcoma. Citation Format: Nalan Gokgoz, Paige Darville-O'Quinn, Ainaz Malekoltojari, Patrick Prochazka, Peter C. Ferguson, Jay S. Wunder, Irene L. Andrulis. Investigating circulating tumor DNA as a biomarker of cancer progression and recurrence in sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1984.