Iben Lyskjær1, Neesha Kara2, Solange De Noon3, Christopher Davies3, Ana Maia Rocha3, Anna-Christina Strobl4, Inga Usher5, Craig Gerrand6, Sandra J Strauss7, Daniel Schrimpf8, Andreas von Deimling8, Stephan Beck2, Adrienne M Flanagan9. 1. Research Department of Pathology, University College London, London, UK; Medical Genomics Research Group, University College London, London, UK. 2. Medical Genomics Research Group, University College London, London, UK. 3. Research Department of Pathology, University College London, London, UK; Department of Histopathology, Royal National Orthopaedic Hospital, Stanmore, HA7 4LP, UK. 4. Department of Histopathology, Royal National Orthopaedic Hospital, Stanmore, HA7 4LP, UK. 5. Research Department of Pathology, University College London, London, UK. 6. Bone Tumour Unit, Royal National Orthopaedic Hospital, Stanmore, HA7 4LP, UK. 7. Department of Oncology, University College London, London, UK. 8. Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, and CCU Neuropathology, German Cancer Institute, Heidelberg, Germany. 9. Research Department of Pathology, University College London, London, UK; Department of Histopathology, Royal National Orthopaedic Hospital, Stanmore, HA7 4LP, UK. Electronic address: a.flanagan@ucl.ac.uk.
Abstract
AIM: Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Circulating free (cfDNA) and circulating tumour DNA (ctDNA) are promising biomarkers for disease surveillance and prognostication in several cancer types; however, few such studies are reported for OS. The purpose of this study was to discover and validate methylation-based biomarkers to detect plasma ctDNA in patients with OS and explore their utility as prognostic markers. METHODS: Candidate CpG markers were selected through analysis of methylation array data for OS, non-OS tumours and germline samples. Candidates were validated in two independent OS datasets (n = 162, n = 107) and the four top-performing markers were selected. Methylation-specific digital droplet PCR (ddPCR) assays were designed and experimentally validated in OS tumour samples (n = 20) and control plasma samples. Finally, ddPCR assays were applied to pre-operative plasma and where available post-operative plasma from 72 patients with OS, and findings correlated with outcome. RESULTS: Custom ddPCR assays detected ctDNA in 69% and 40% of pre-operative plasma samples (n = 72), based on thresholds of one or two positive markers respectively. ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients, which in four cases were associated with or preceded disease relapse. Both pre-operative cfDNA levels and ctDNA detection independently correlated with overall survival (p = 0.0015 and p = 0.0096, respectively). CONCLUSION: Our findings illustrate the potential of mutation-independent methylation-based ctDNA assays for OS. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers in the management of OS. Crown
AIM: Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Circulating free (cfDNA) and circulating tumour DNA (ctDNA) are promising biomarkers for disease surveillance and prognostication in several cancer types; however, few such studies are reported for OS. The purpose of this study was to discover and validate methylation-based biomarkers to detect plasma ctDNA in patients with OS and explore their utility as prognostic markers. METHODS: Candidate CpG markers were selected through analysis of methylation array data for OS, non-OS tumours and germline samples. Candidates were validated in two independent OS datasets (n = 162, n = 107) and the four top-performing markers were selected. Methylation-specific digital droplet PCR (ddPCR) assays were designed and experimentally validated in OS tumour samples (n = 20) and control plasma samples. Finally, ddPCR assays were applied to pre-operative plasma and where available post-operative plasma from 72 patients with OS, and findings correlated with outcome. RESULTS: Custom ddPCR assays detected ctDNA in 69% and 40% of pre-operative plasma samples (n = 72), based on thresholds of one or two positive markers respectively. ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients, which in four cases were associated with or preceded disease relapse. Both pre-operative cfDNA levels and ctDNA detection independently correlated with overall survival (p = 0.0015 and p = 0.0096, respectively). CONCLUSION: Our findings illustrate the potential of mutation-independent methylation-based ctDNA assays for OS. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers in the management of OS. Crown