Detection of Circulating Tumor DNA in Patients With Leiomyosarcoma With Progressive Disease

Abstract

Purpose: Leiomyosarcoma (LMS) is a soft tissue sarcoma characterized by multiple copy number alterations (CNAs) and without common recurrent single nucleotide variants. We evaluated the feasibility of detecting circulating tumor DNA (ctDNA) with next-generation sequencing in a cohort of patients with LMS whose tumor burden ranged from no evidence of disease to metastatic progressive disease.

Patients and methods: Cell-free DNA in plasma samples and paired genomic DNA from resected tumors were evaluated from patients with LMS by ultra-low passage whole genome sequencing (ULP-WGS). Sequencing reads were aligned to the human genome and CNAs identified in cell-free DNA and tumor DNA by ichorCNA software to determine the presence of ctDNA. Clinical data were reviewed to assess disease burden and clinicopathologic features.

Results: We identified LMS ctDNA in eleven of sixteen patients (69%) with disease progression and total tumor burden over 5 cm. Sixteen patients with stable disease or low disease burden at the time of blood draw were found to have no detectable ctDNA. Higher ctDNA fraction of total cell-free DNA was associated with increasing tumor size and disease progression. Conserved CNAs were found between primary tumors and ctDNA in each case, and recurrent CNAs were found across LMS samples. ctDNA levels declined following resection of progressive disease in one case and became detectable upon disease relapse in another individual patient.

Conclusion: These results suggest that ctDNA, assayed by a widely available sequencing approach, may be useful as a biomarker for a subset of uterine and extrauterine LMS. Higher levels of ctDNA correlate with tumor size and disease progression. Liquid biopsies may assist in guiding treatment decisions, monitoring response to systemic therapy, surveying for disease recurrence and differentiating benign and malignant smooth muscle tumors.

Keywords: Leiomyosarcoma; circulating tumor DNA; copy number alteration; liquid biopsy.

Fig 1.

Identification of circulating tumor DNA (ctDNA) in plasma from patients with leiomyosarcoma (LMS). (A) Plot of percent ctDNA in active disease (n = 16) and indolent disease (n = 16) LMS groups. The active disease group consists of patients with tumors > 5 cm in size and with progressive disease at the time of blood draw as indicated by computed tomography (CT) scan or clinical report. The indolent disease group consists of patients with tumors < 5 cm in size and/or no evidence of disease progression by CT scan. Groups were compared using Mann-Whitney test. (*) P < .001. (B) Scatterplot of log₂ tumor fraction and tumor burden. Labels indicate the active disease subgroup (red) with tumor size > 5 cm and progressive disease (n = 16), or indolent disease subgroups (gray) with tumor size > 5 cm and stable disease (n = 2, diamond), tumor size < 5 cm and progressive disease (n = 4; triangle), and tumor size < 5 cm and stable disease (n = 10; circle). Tumor burden was determined by adding the diameters of the tumor lesions reported on CT scan at the time of ctDNA assessment. Pearson correlation coefficient between tumor fraction and tumor burden is shown for all patients with measurable disease. (C) Scatterplot of log₂ total extracted cell-free DNA from all plasma samples (n = 32) and tumor burden. Pearson correlation coefficient across all samples is shown.

Fig 2.

Concordant copy number alterations in leiomyosarcoma tumors and circulating tumor DNA. (A-E) Copy number plots generated from ultra-low passage whole-genome sequencing of five representative tumor surgical (left panels) and plasma (right panels) sample pairs. The x-axis indicates chromosome and y-axis copy number (log₂ ratio). Tumor fraction is indicated for each plot.

Fig 3.

Recurrent copy number alterations in leiomyosarcoma (LMS). (A-D) GISTIC2.0 analysis identifying recurrent focal amplified (A and B) and deleted (C and D) regions in LMS tumors (n = 37; left panels) and cell-free DNA samples with detectable circulating tumor DNA (n = 11; right panels). LMS-associated genes and/or putative oncogenes are labeled in panel A, whereas putative tumor suppressor genes are labeled in panel C. The x-axis indicates chromosome and y-axis false discovery rate (FDR). The green line indicates an FDR of 0.25.

Fig 4.

Leiomyosarcoma circulating tumor DNA (ctDNA) correlates with changes in disease burden. (A and B) Change in tumor fraction in the preoperative and postoperative setting, with images indicating tumor burden at each time point. (C and D) Change in tumor fraction after unifocal recurrence of disease, with images indicating tumor burden at each time point. The interval between surgical date and cell-free DNA collection is indicated.