Using All Our Genomes: Blood-based Liquid Biopsies for the Early Detection of Cancer

Abstract

The pursuit of highly sensitive and specific cancer diagnostics based on cell-free (cf) nucleic acids isolated from minimally invasive liquid biopsies has been an area of intense research and commercial effort for at least two decades. Most of these tests detect cancer-specific mutations or epigenetic modifications on circulating DNA derived from tumor cells (ctDNA). Although recent FDA approvals of both single and multi-analyte liquid biopsy companion diagnostic assays are proof of the tremendous progress made in this domain, using ctDNA for the diagnosis of early-stage (stage I/II) cancers remains challenging due to several factors, such as low mutational allele frequency in circulation, overlapping profiles in genomic alterations among diverse cancers, and clonal hematopoiesis. This review discusses these analytical challenges, interim solutions, and the opportunity to complement ctDNA diagnostics with microbiome-aware analyses that may mitigate several existing ctDNA assay limitations.

Keywords: cancer detection; cancer screening; cell-free DNA; circulating tumor DNA; early detection; microbiome.

Figure 1.. Genomic and metagenomic features for minimally invasive, blood-based detection of cancers.

(Right) Existing commercial liquid biopsy assays focus on single gene or multi-gene analyses of ctDNA mutations present in oncogenes, tumor suppressor genes or validated biomarkers (e.g., EGFR mutations) that aid in drug selection. cfDNA shotgun metagenomic sequencing expands traditional cfDNA analyses to encompass all microbial DNA sequences present in circulation, enabling discovery of cancer-microbiome associations. (Top) Epigenetic analysis of cfDNA has traditionally focused on the methylation state of purified mammalian cfDNA, which serves as a powerful signature of ctDNA tissue of origin. Epigenetic marks on circulating microbial DNA may likewise serve to identify/enrich disease-specific microbial associations.^, Recent work has shown that epigenetic hallmarks of gene expression or suppression on cell-free nucleosomes (e.g., histone acetylation and methylation marks) be used as a surrogate for RNA-based transcriptional analysis. (Left) Single-stranded DNA library preparation methods increase sequencing reads derived from mitochondrial and microbial sources, potentially increasing coverage of cancer-associated mitochondrial DNA (mtDNA) mutations and microbial taxonomies.^, In addition to linear mtDNA, circular mtDNA molecules have been identified in plasma and extrachromosomal circular DNA (eccDNA) have emerged as a fascinating new class of circulating biomarkers.^– (Bottom) cfDNA fragment analysis aimed at elucidating tissue/disease-specific nuclease activity or nucleosome positioning throughout the genome offer new ways of extracting molecular insights that are independent from and complementary to traditional mutation-based analyses.^,,