Whole genome deep sequencing analysis of cell-free DNA in samples with low tumour content

Abstract

Background 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). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA. Methods We 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. Results The 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. Conclusions Deep 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.

Cell-free DNA fragmentomics and second malignant neoplasm risk in patients with PTEN hamartoma tumor syndrome

“…provide evidence that ctDNA is enriched within the dinucleosome fraction in early- and late-stage tumors, respectively. 27 , 28 Ganesamoorthy et al. investigated plasma samples with low tumor content and did not detect enrichment in cfDNA fragments <150 bp, but reported significant enrichment of tumor-derived fragments within the dinucleosome fraction.…”

Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer

“…Previous studies revealed higher fragmentation of tumor-derived cfDNA fragments and their enrichment with somatic mutations, particularly in colorectal cancer patients (Diehl et al, 2005;Mouliere et al, 2011;2013), while at the same time some evidence conversely support the presence of longer molecules in tumor cfDNA fraction (Umetani et al, 2006;Ganesamoorthy et al, 2022). The complexity of cfDNA size profile seems to be the result of a balance between several biological processes including apoptosis, necrosis, senescence and active release that may be altered in pathology (Rostami et al, 2020;Ungerer et al, 2021;Ungerer et al, 2022).…”

The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients

“…Interestingly, 90% of somatic cfDNA alterations were not detected in matched tumor tissues and were due to two background mutational signatures. Intriguingly, cfDNA fragments ranging from 300 bp to 350 bp in size (e.g., di-nucleosomal plasma DNA) had a much higher proportion (30%) of mutations in common with the tumor [ 43 ]. The findings of this study show the complexity of ctDNA analysis using WGS.…”