Origin of circulating free DNA in patients with lung cancer

Abstract

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.

Extracellular Vesicle-Derived DNA vs. CfDNA as a Biomarker for the Detection of Colon Cancer

“…A higher number of copies of mutants in cfDNA can be ascribed to the higher concentration of DNA obtained from plasma. Few earlier studies have reported a higher number of copies of mutant allele in cfDNA obtained from the supernatant left after extraction of EV than EV-DNA [10,13].…”

“…Wan et al found EV-DNA to be superior compared to cfDNA in EGFR mutation detection in the early stage of non-small cell lung cancer. However, recent study on liquid biopsy of lung carcinoma patients found a higher mutant allele frequency of EGFR in cfDNA than EV-DNA [13]. Thus, it still remains under debate whether analysis of EV-DNA has any advantage over cfDNA in cancer detection.…”

Current achievements and future perspectives with liquid biopsy

“…Although ctDNA was reported to be derived from apoptotic or necrotic cancer cells as well as released from intact cells [14], it origin has not been clearly clarified. Recently, we reported a bimodal distribution of cfDNA long fragments of 5 kb and short fragments of 170 bp in patients with advanced lung cancer, and both fractions contained ctDNA [45,46]. The total amount of cfDNA is higher among patients with distant metastases, but this is more obvious with long fragment cfDNA.…”

“…On the basis of experiments with lung cancer cell lines, we found that long fragment cfDNA co-exists with extracellular vesicles (EVs). EVs are well known to mediate tumor progression by facilitating interaction between cancer cells and stromal cells [46]. Therefore, it is possible that long fragment cfDNA also contributes to tumor progression in concert with EVs.…”

“…According to the principle that nucleosome positions or patterns near transcription start sites may differ between cancer cells and normal cells, it was shown that cfDNA from cancer patients was more variable and shorter than that from healthy individuals. We recently found that short fragment cfDNA exists in the supernatant of human plasma samples after centrifugation at 100,000 g, and the supernatant contained two times as much ctDNA as in plasma isolated by the conventional procedure [46]. If we can more efficiently isolate ctDNA from the entirety of cfDNA on the basis of the evidence mentioned above, precise genomic analysis with liquid biopsy could become possible even in the early stages of various cancers.…”