Next generation sequencing-based analysis of mitochondrial DNA characteristics in plasma extracellular vesicles of patients with hepatocellular carcinoma

Emerging evidence has revealed that mitochondrial DNA (mtDNA) is encapsulated in plasma extracellular vesicles (EVs). However, the characteristics of mtDNA in EVs from patients with cancer remain largely unexplored, which greatly limits its clinical application. Whole genome and capture-based sequencing found that EV mtDNA covered the whole mitochondrial genome. The medium fragment size in EV mtDNA was significantly larger compared with that in cell-free mtDNA [cfmtDNA; 159 vs. 109 base pairs (bp); P<0.001]. EV DNA appeared to have a higher mtDNA copy number compared with cfDNA. Of note, patients with hepatitis had >300-bp fragments in EV mtDNA compared with patients with hepatocellular carcinoma (HCC) and healthy controls. EV mtDNA fragments >300 bp in length exhibited a significantly higher proportion of EV mtDNA fragment ends than those that were ≤300 bp in length in patients with hepatitis. The EV mtDNA copy number in patients with HCC and hepatitis were significantly lower compared with those in healthy controls. Furthermore, inconsistencies in the mtDNA heteroplasmic variant were observed among HCC tissues, plasma and EVs. In conclusion, EV mtDNA exhibited different characteristics among patients with HCC, hepatitis and healthy controls, indicating the potential value of EV mtDNA as a diagnostic biomarker that complements cfmtDNA.