High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants

Abstract

Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding the various biologic compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white-blood cell (WBC) DNA covering a large genomic region (508 genes, 2Mb, >60,000X raw-depth) in a prospective study of 124 metastatic cancer patients, with contemporaneous matched tumor tissue biopsies, and 47 non-cancer controls. The assay displayed a high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in cancer patients) had features consistent with clonal hematopoiesis (CH). This cfDNA sequencing approach revealed that CH constitutes a pervasive biological phenomenon emphasizing the importance of matched cfDNA-WBC sequencing for accurate variant interpretation.

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

“…During the process of aging different mutations accumulate in hematopoietic stem cells. This phenomenon occurs frequently in the elderly and its prevalence has been estimated at 31% [39]. The mutations resulting from clonal hematopoiesis are often detected during cfDNA sequencing analysis, since the majority of cfDNA is derived from leukocytes.…”

“…The mutations resulting from clonal hematopoiesis are often detected during cfDNA sequencing analysis, since the majority of cfDNA is derived from leukocytes. Recently, it has been demonstrated that 53.2% of all mutations detected by cfDNA sequencing analysis result from clonal hematopoiesis, indicating the need for collection and sequencing of leukocytes as a reference [39].…”

Enhanced specificity of high sensitivity somatic variant profiling in cell-free DNA via paired normal sequencing: design, validation, and clinical experience of the MSK-ACCESS liquid biopsy assay

“…Several recent studies have suggested that CH mutations present a challenge for proper filtering in highly sensitive NGS-based liquid biopsy assays [26][27][28] . We observed that the use of patient-matched normal WBC DNA in MSK-ACCESS eliminated 7,760 (77%) of variant calls below 10% VAF (Figure 4A-IV).…”

The potential of combined mutation sequencing of plasma circulating cell‐free DNA and matched white blood cells for treatment response prediction

“…In summary, the recent achievements in high-sensitive sequencing methodologies of pretreatment plasma ccfDNA have proven to become an useful tool to detect and map tumor-derived mutations and offer opportunities as those reported by Kruger and colleagues, to investigate the clinical value for the prediction of therapy response and clinical outcome. However, these same high-sensitive sequencing methodologies now also visualize that most variants detected in ccfDNA of cancer patients represent especially CHIP and that CHIP is more prevalent than was previously anticipated (Chen et al, 2019;Razavi et al, 2019). In particular, this high prevalence of CHIP emphasizes the importance of parallel high-sensitive sequencing of DNA derived from WBCs of the same patient for appropriate variant interpretation.…”