Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes

Abstract

Background: The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies.

Patients and methods: We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data.

Results: Whole-exome sequencing resulted in 95%-99% of the target regions covered at a mean depth of 133-490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens.

Conclusions: We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

Keywords: exosomes; liquid biopsy; next-generation sequencing; pancreatic cancer.

Figure 1.

LBx01 tumor profiling. (A) Copy number profile comparison between the metastatic lung tissue (top) sampled 15 months before the pleural effusion exoDNA (bottom). The cancer-related genes on the light-red vertical bars have copy number gains and those on the light-blue vertical bars have copy number losses, where the numbered labels represent the estimated copy numbers. The yellow vertical bar annotates putatively actionable copy number variations (CNVs) (e.g. ERBB2). The arrow to the left depicts the progression of cancer-associated CNVs between the 2 time points. These happen to all be amplifications, which were also confirmed to be upregulated in the exoRNA compared with that in the metastatic lung tissue RNA-seq. (B) Mutant KRAS was identified in the mRNA (RNA sequencing) as well as DNA (exome and genome sequencing) of the pleural effusion exosomes. (C) Mutational signature of the plasma exosomes derived from exome sequencing (top) and genome sequencing (middle) compared with the mutational signature of the metastatic lung tissue (bottom). (D) Circos plot illustrating putative gene fusions (blue), lung metastatic copy number profile (inner-most ring), pleural effusion exosomes copy number profile (second inner-most ring) and gene aberrations. Mutations seen in the pleural effusion are black and those seen in both the metastatic lung tissue and pleural effusion are in bolded black.

Figure 2.

LBx02 tumor profiling. (A) Copy number profile of the plasma exoDNA. (B) Mutational signature of the pleural effusion exosomes derived from exome sequencing (top) and genome sequencing (bottom). (C) Circos plot illustrating putative gene fusions (blue), plasma exoDNA copy number profile (inner-most ring) and potential actionable genes (blue, deletions; red, amplifications; black, somatic point mutations).

Figure 3.

LBx03 tumor profiling. (A) Copy number profile of the plasma exoDNA. (B) Mutational signature of the plasma exosomes derived from exome sequencing (top) and genome sequencing (bottom). (C) Circos plot illustrating putative gene fusions (blue), plasma exoDNA copy number profile (inner-most ring) and potential actionable genes.