Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study

Abstract

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.

Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

“…After preparation, libraries were quantified and showed a wide range of concentrations between 40.5-1440 pMol (median 450 pMol). As expected, it was observed that library yields increased proportionally with the amount of cfDNA ng employed per reaction (Spearman's correlation coefficient (r s ) = 0.67, p < 0.0001) ( Figure 2), a finding in line with literature [17].…”

“…LoD evaluation through the samples revealed, not surprisingly, that higher quantities of cfDNA input allowed to reach very low levels of LoD (up to 0.02%), as also similarly reported by others [17], with the advantage to preserve specificity of the NGS workflow. It is interesting to note that at least 0.1% LoD was reached in 37/92 cases (40.22%).…”

“…Bearing in mind what is reported by manufacturer to reach the main goal of a 0.1% LoD, we showed a fulfillment of the median value of molecular families (MedMolCov >2500) and the median values of read coverage (MedReadCov >25,000) in more than half of sequenced samples (60.9%). Not surprisingly, a strong correlation between the cfDNA input and MedMolCov was found, since it is expected that the more cfDNA that is introduced for NGS libraries, the higher will be the number of molecular families sequenced, as already reported [17]. By contrast, no significant association was found between DNA quantity (both cfDNA input and cfDNA abundance) and the EGFR mutational status (p > 0.15).…”

“…Nevertheless, the analysis of ctDNA is challenging due to the mainly low representation of mutated tumor-derived DNA molecules in the plasma. Furthermore, although plasma of cancer patients contains more cfDNA compared to healthy individuals [17,22], the proportion of ctDNA originating from the tumor is highly variable, ranging from <0.1% to >50% of total cfDNA [23,24]. Therefore, methods which are able to detect small number of mutated molecules in an abundance of unmutated DNA fragments with high sensitivity and specificity are required.…”

Cell-Free DNA Methylation: The New Frontiers of Pancreatic Cancer Biomarkers’ Discovery

“…In healthy individuals, cfDNA concentration ranges from 0 to 100 ng/mL of blood [33]. In comparison, cancer patients present on average four to 40 times greater levels, with an upper range exceeding 1000 ng/mL [34][35][36][37][38][39]. The higher cellular turnover observed in cancer would explain at least in part the higher plasmatic cfDNA levels observed.…”