(P 104) CIRQUESARC: A NATIONAL PROSPECTIVE OBSERVATIONAL STUDY OF CIRCULATING TUMOR DNA AND QUALITY OF LIFE IN PATIENTS WITH SARCOMA – PRELIMINARY RESULTS FROM THE SURGICAL COHORT

Objective: A comprehensive analysis of circulating tumor DNA (ctDNA), and its role to detect early disease relapse and monitor treatment effect correlated with quality of life, are yet to performed in patients with sarcoma. The CIRQUESARC study will investigate the level of ctDNA in patients with sarcoma. Study 1 will include patients with metastatic disease receiving doxorubicin-based chemotherapy, and study 2 will include patients with localized/oligometastatic disease undergoing surgery. We hereby present the preliminary results of study 2.

Methods: We will include 40 patients with either localized or oligometastatic soft tissue sarcoma, osteosarcoma or Ewings sarcoma at the Department of Oncology, Herlev University Hospital or Aarhus University Hospital; or the Department of Orthopedics, Aarhus University Hospital or Rigshospitalet.
Four STRECK tubes (9 ml) will be collected for ctDNA detection before neoadjuvant treatment, before and after surgery, before adjuvant treatment and during a two-year follow-up period or until disease relapse. A quality-of-life questionnaire (Toronto Extremity Salvage Score) will be filled out during specific timepoints. A fresh tumor tissue sample (2 x 2 mm) will be collected during surgery and assessed by next-generation-sequencing (NGS) with the TruSight Oncology 500 High-Throughput (TSO500 HT) Illumina assay and Illuminas total RNA sequencing. The TSO500 HT assay is covering 523 genes for single nucleotide variants, insertions/deletions, copy number variations, and 55 genes for fusions. Plasma ctDNA will be detected by the QX200 digital droplet polymerase chain reaction (ddPCR) system from Bio-Rad. The patient-specific primers used for ddPCR will be selected based on the NGS results on tumor tissue.

Results: To date, 25 patients have been included (15 female; mean age of 55 years) with 11 different histological subtypes: Ewings sarcoma (5); undifferentiated pleomorphic sarcoma (3); osteosarcoma (3); angiosarcoma (3); myofibroblastic sarcoma/atypical osteosarcoma (2); dedifferentiated liposarcoma (2); myxoid liposarcoma (2); leiomyosarcoma (2); synovial sarcoma (1); pleomorphic liposarcoma (1); and myxofibrosarcoma (1).
Fresh tumor tissue was collected during surgery in 16 patients. In 18 patients fresh or formalin-fixed paraffin-embedded tumor tissue was assessed using the TSO500 HT assay and total RNA sequencing. In seven patients the following two fusions and five mutations were detected: YWHAE-CRK (undifferentiated pleomorphic sarcoma); SS18-SSX1 (synovial sarcoma); TP53/RB1 (angiosarcoma); TP53 (myofibroblastic/atypical osteosarcoma); TP53/ATRX (myxofibrosarcoma); TP53/ATRX (pleomorphic liposarcoma); and TP53/PIK3C2G (undifferentiated pleomorphic sarcoma). In four patients no fusions or mutations were detected and in seven patients the results are still pending. Mean tumor mutational burden was 2.95 mut/Mb ranging from 0.8 - 9.5 mut/Mb.
In a 36-year-old male with angiosarcoma of the spleen a TP53 c.358A>G p.K120E mutation was detected in tumor tissue. The patient received two cycles of neoadjuvant doxorubicin and ifosfamide, radical surgery and one cycle of adjuvant doxorubicin and ifosfamide. Four months after surgery disease relapse in the lungs was confirmed. The TP53 c.358A>G p.K120E mutation was detected in plasma at baseline, before and after surgery and at disease relapse. See figure 1-4.
In four other patients, a TP53 mutation was also detected in tumor tissue. Furthermore, patient-specific TP53 ctDNA was detected in plasma at both baseline and longitudinal follow-up.

Conclusion: Mutations and fusions may be detected in patients with sarcoma using the TSO500 HT assay and total RNA sequencing. Furthermore, it is feasible to detect patient-specific TP53 ctDNA with ddPCR, and the plasma levels detected corresponded to the clinical picture observed in each patient.