Abstract
BackgroundCirculating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA.MethodsWe performed WGS analysis of cfDNA from 4 breast cancer patients and 2 patients with benign tumours. We sequenced matched germline DNA for all 6 patients and tumour samples from the breast cancer patients. All samples were sequenced on Illumina HiSeqXTen sequencing platform and achieved approximately 30x, 60x and 100x coverage on germline, tumour and plasma DNA samples, respectively.ResultsThe mutational burden of the plasma samples (1.44 somatic mutations/Mb of genome) was higher than the matched tumour samples. However, 90% of high confidence somatic cfDNA variants were not detected in matched tumour samples and were found to comprise two background plasma mutational signatures. In contrast, cfDNA from the di-nucleosome fraction (300 bp–350 bp) had much higher proportion (30%) of variants shared with tumour. Despite high coverage sequencing we were unable to detect sCNAs in plasma samples.ConclusionsDeep sequencing analysis of plasma samples revealed higher fraction of unique somatic mutations in plasma samples, which were not detected in matched tumour samples. Sequencing of di-nucleosome bound cfDNA fragments may increase recovery of tumour mutations from plasma.
Highlights
Circulating cell-free DNA in the plasma of cancer patients contains cell-free tumour DNA derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer
Cell-free DNA is an emerging non-invasive biomarker for diagnosis and prognosis of various acute and chronic disorders. cell-free DNA (cfDNA) has been detected in many body fluids, including plasma, serum, urine and cerebrospinal fluid [1]. cfDNA is predominantly of hematopoietic origin [2], recent studies have showed release of cfDNA from other organs and tissues into the extracellular compartments [3,4,5]
Tumour-specific alterations such as somatic copy number aberrations and single nucleotide variants (SNVs) have been detected in the plasma of cancer patients [8]. cell-free tumour DNA (ctDNA) has been detected in both early and late stage tumours [9] and the utility of ctDNA as a biomarker has been assessed for various cancer types with promising results [10]
Summary
Circulating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). These approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA. CfDNA with several tissues and organs in the body makes it an attractive non-invasive biomarker for various diseases including cancer. As ctDNA is derived from various tumour clones and sites, it provides a comprehensive representation of the tumour heterogeneity in the patient [5] These features make them an ideal biomarker for cancer diagnosis and monitoring
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