4.7 Article

NGS-based accurate and efficient detection of circulating cell-free mitochondrial DNA in cancer patients

期刊

MOLECULAR THERAPY-NUCLEIC ACIDS
卷 23, 期 -, 页码 657-666

出版社

CELL PRESS
DOI: 10.1016/j.omtn.2020.12.017

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资金

  1. National Natural Science Foundation of China [81830070]
  2. Autonomous Project of State Key Laboratory of Cancer Biology, China [CBSKL2019ZZ06]
  3. National Science and Technology Major Projects of China [2018ZX10302205-002]
  4. Key Research and Development Program of Shaanxi Province, China [2018ZDXM-SF-061]

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Mitochondrial DNA mutations play a role in the development of cancer, leading to the use of circulating cell-free mtDNA as a potential tumor biomarker. A new approach for profiling ccf-mtDNA mutations was developed based on systematic optimization of sequencing methodology. The study also highlighted the importance of proper processing of plasma samples for accurate detection of tumor-specific mtDNA mutations in cancer patients.
Mitochondrial DNA (mtDNA) mutations are closely implicated in the pathogenesis of multiple cancers, making circulating cell-free mtDNA (ccf-mtDNA) as a potential non-invasive tumor biomarker. However, an effective approach to comprehensively profile ccf-mtDNA mutations is still lacking. In this study, we first characterized ccf-mtDNA by low-depth whole-genome sequencing (WGS) and found that plasma DNA samples exhibited a dramatic decrease in mtDNA copy number when compared with fresh tumor tissues. Further analysis revealed that plasma ccf-mtDNA had a biased distribution of fragment size with a peak around 90 bp. Based on these insights, we developed a robust captured-based mtDNA deep-sequencing approach that enables accurate and efficient detection of plasma ccf-mtDNA mutations by systematic optimization of probe quantity and length, hybridization temperature, and PCR amplification cycles. Moreover, we found that placement of isolated plasma for 6 h at both 4 degrees C and room temperature (RT) led to a dramatic decrease of ccf-mtDNA stability, highlighting the importance of proper plasma sample processing. We further showed that the optimized approach can successfully detect a substantial fraction of tumor-specific mtDNA mutations in plasma ccf-mtDNA specifically from hepatocellular carcinoma (HCC) patients but not from colorectal cancer (CRC) patients, suggesting the presence of a potential cancer-specific difference in the abundance of tumor-derived mtDNA in plasma.

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