Whole exome sequencing of cell-free DNA – A systematic review and Bayesian individual patient data meta-analysis

Abstract

A B S T R A C TMolecular profiling of tumor derived cell free DNA (cfDNA) is gaining ground as a prognostic and predictive biomarker. However to what extent cfDNA reflects the full metastatic landscape as currently determined by tumor tissue analysis remains controversial. Though technically challenging, whole exome sequencing (WES) of cfDNA enables thorough evaluation of somatic alterations. Here, we review the feasibility of WES of cfDNA and determine the sensitivity of WES-detected single nucleotide variants (SNVs) in cfDNA on individual patient data level using paired tumor tissue as reference ( × 100%shared SNVs All tissue SNVs ). The pooled sensitivity was 50% (95% credible interval (CI): 29-72%). The tissue mutant allele frequency (MAF) of variants exclusively identified in tissue was significantly lower (12.5%, range: 0.5-18%) than the tissue MAF of variants identified in both tissue and cfDNA (23.9%, range: 17-38%), p = 0.004. The overall agreement ( × 100%)shared SNVs All SNVs between SNVs in cfDNA and tumor tissue was 31% (95% CI: 15-49%). The number of detected SNVs was positively correlated with circulating tumor DNA (ctDNA) fraction (p = 0.016). A sub analysis of samples with ctDNA fractions ≥ 25% improved the sensitivity to 69% (95% CI: 46-89%) and agreement to 46% (95% CI: 36-59%), suggesting that WES is mainly feasible for patients with high ctDNA fractions. Pre-and post-analytical procedures were highly variable between studies rendering comparisons problematic. In conclusion, various aspects of WES of cfDNA are largely in its investigative phase, standardization of methodologies is highly needed to bring this promising technique to its clinical potential.

Next Generation Sequencing-Based Profiling of Cell Free DNA in Patients with Advanced Non-Small Cell Lung Cancer: Advantages and Pitfalls

“…Several researchers are trying to apply these techniques to cfDNA analysis, but the high costs, the long turnaround times, and the requirement of qualified bioinformatics experts for the analysis of the large quantity of data generated from WGS and WES, limit their large-scale use (Table 2). For these reasons, WGS and WES are being explored in the field of advanced research and they are not yet applicable in the clinical setting and on large cohorts of patients for clinical research purposes [30][31][32][33]. On the other hand, targeted sequencing of defined regions of the genome is able to identify somatic genetic alterations with a high level of sensitivity and a low number of false negatives.…”

Utility of Circulating Tumor DNA for Detection and Monitoring of Endometrial Cancer Recurrence and Progression

“…As with CTCs, numerous methods are available for total cfDNA extraction from both serum and plasma, with studies illustrating that several pre-analytical factors affect yield and downstream analysis post-venepuncture, including the blood tube used, number of centrifugation steps, centrifugal speed, cfDNA extraction method, and ctDNA detection methods [19][20][21]. Methods employed for detection of ctDNA depend upon the question being asked, with whole genome/exome next generation sequencing (NGS) being performed to obtain a global view of genomic changes associated with disease progression [22,23] (however, this is hampered by low sensitivity), targeted sequencing to obtain higher sensitivity (1 ctDNA molecule in 1000 cfDNA molecules) in a smaller number of genes [6,15,17,24,25], or digital droplet PCR (ddPCR) (usually analysis of one or two mutations to a sensitivity of one in 100,000) to detect early stage disease [14,26]. To date, only a handful of ctDNA tests have been approved by the U.S Food and Drug Administration (FDA) for use in metastatic and locally advanced NSCLC, and HR+, HER2-negative, advanced breast cancer in progression on or after endocrine therapy.…”

Approaching Cancer Evolution from Different Angles

“…This reveals the importance of rapid, inexpensive and frequent assays to monitor cancers during treatment. I'm hopeful that, in the near future, cell free DNA in the blood ( Bos et al., 2020 ; Cervena et al., 2019 ; Jamal-Hanjani et al., 2016 ; Stastny et al., 2020 ) might be used to monitor how the clones are evolving in our patients. Cancers evolve in response to our interventions.…”