A pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations in hepatocellular carcinoma

Abstract

Cellular components of solid tumors including DNA are released into the bloodstream, but data on circulating-free DNA (cfDNA) in hepatocellular carcinoma (HCC) are still scarce. This study aimed at analyzing mutations in cfDNA and their correlation with tissue mutations in patients with HCC. We included 8 HCC patients treated with surgical resection for whom we collected paired tissue and plasma/serum samples. We analyzed 45 specimens, including multiregional tumor tissue sampling (n = 24), peripheral blood mononuclear cells (PMBC, n = 8), plasma (n = 8) and serum (n = 5). Ultra-deep sequencing (5500× coverage) of all exons was performed in a targeted panel of 58 genes, including frequent HCC driver genes and druggable mutations. Mutations detected in plasma included known HCC oncogenes and tumor suppressors (e.g., TERT promoter, TP53, and NTRK3) as well as a candidate druggable mutation (JAK1). This approach increased the detection rates previously reported for mutations in plasma of HCC patients. A thorough characterization of cis mutations found in plasma confirmed their tumoral origin, which provides definitive evidence of the release of HCC-derived DNA fragments into the bloodstream. This study demonstrates that ultra-deep sequencing of cfDNA is feasible and can confidently detect somatic mutations found in tissue; these data reinforce the role of plasma DNA as a promising minimally invasive tool to interrogate HCC genetics.

Conflict of interest statement

Conflicts of interests: The authors declare no potential conflicts of interest.

Figures

Figure 1

a) Distribution of DNA length fragments in plasma and serum samples. b) Differential coverage for next-generation sequencing in tissue and plasma. Sequencing depth was significant higher in plasma to identify low-frequency mutations.

Figure 2

a) Heat-map of detected mutations with their concordance in tissue and plasma. Each column represents one tumor region and each row is a mutated gene found in tissue. b) Performance of ultra-deep sequencing to detect plasma mutations, on a mutation-basis and on a patient-basis. c) Impact of tumor size, AFP levels and variant allele fraction of the mutation in tissue in detection rate of cfDNA.

Figure 3

TP53 mutations in patient #5: Left panels illustrate read alignments using Integrative Genomics Viewer. Right panels show the percentage of alternative reads found in each compartment [i.e., tissue (top), PBMC (middle), plasma (bottom)]. Two adjacent mutations of TP53, located 3 base pairs apart, with a variant allele fraction of 80% were detected in tissue. The corresponding alternative alleles of these 2 mutations were not detected in PBMC, thus excluding germline events. Ultra-deep sequencing of plasma DNA detected the same 2 mutations in approximately 4% of circulating DNA fragments. Mutations in plasma are found in the exact same reads, identifying their shared origin from their tissue counterparts.

Figure 4

Mutation assay with ddPCR. Displayed are 1D amplitudes for mutant probe (upper lane) and wild-type probe (lower lane) for each mutation assay. Panels from left to right: Plasma and corresponding HCC tissue of same patient, blank (negative control), wild-type DNA (negative control). Blue: single positive droplets for mutant, green: single positive droplets for wild-type, grey: double negative droplets. Detected positive droplets for mutant alleles in plasma are circled in red.