In this study, TP53 mutation and MD/TD in circulating exoDNA were detected to evaluate their diagnostic and prognostic value in HCC. Exosomes are secreted by a wide range of sources, including tumor cells, which is why exosome DNA is easier to detect than cfDNA. This study demonstrated that exosomes and purified DNA can be extracted from serum volumes as low as 250μL for the detection of TP53 mutations.
Our results showed that TP53 mutations were detected in 82.5% of patients, which is immensely higher than the previous detection rate for tumor-specific mutations in cfDNA. We previously detected TERT, TP53 and CTNNB1 mutations in cfDNA of HCC patients, with a sensitivity of only 19.5%. Other studies have shown that the different stages of cfDNA at between 30.8% and 57.9%. In addition, a meta-analysis showed that cfDNA lacks robustness in the diagnosis of HCC. This finding may be explained by the single source of cfDNA and the small number of circulating gene fragments produced from tumor tissues, with the total number of circulating DNA fragments <1.0%[21, 22], and it makes traditional technology difficult to detect. No studies thus far have described the detection of TP53 mutations in circulating exoDNA in patients with HCC. Therefore, we used ddPCR to detect TP53 mutations in circulating exoDNA of HCC patients and explored the possibility of circulating exoDNA as a new non-invasive liquid biopsy method in the diagnosis and prognosis of HCC. Krimmel et al reported that the detection of TP53 mutations was related to age. However, our study did not find an association between age and TP53 mutation status.
Our results showed that patients with high MD/TD were more likely to show MVI. HCC patients with vascular invasion have a poor prognosis, and our study confirmed that patients with high MD/TD were more likely to relapse compared with patients with low MD/TD (P<0.001). Wang et al. reported that circulating tumor DNA correlates with MVI and predicts tumor recurrence of HCC. These results are consistent with our findings. We speculate that the association between MD/TD and vascular invasion may be because MVI makes it easier for nucleic acid fragments from the tumor to enter the circulation. Our findings suggest that HCC patients with high MD/TD are more likely to show MVI and a poor prognosis. Therefore, MD/TD may be a candidate factor to predict the occurrence of MVI in HCC patients and thus to assess patient prognosis.
Like most tumors, HCC tumors show heterogeneity[25, 26]. After the multiple stages of cell division and proliferation during tumor development, tumor cells undergo various genetic changes, resulting in aberrant growth rate, invasion ability, sensitivity to drugs, and other activities. This may also contribute to the limitations of traditional biopsy. Notably, the detection of tumor-specific mutations in circulating exoDNA overcomes obstacles associated with tumor heterogeneity. Detection of mutations in exoDNA is therefore a promising non-invasive liquid biopsy approach for early diagnosis, efficacy assessment, follow-up monitoring, and prognosis assessment in HCC patients.
Targeted sequencing of circulating exoDNA has been applied to a variety of tumors. Allenson et al. detected KRAS mutations in 66.7% (22/33), 80% (12/15) and 85% (17/20) of patients with localized, locally advanced and metastatic pancreatic cancer, respectively, with rates similar to our sensitivity detection results of 82.5%. In addition, Yang et al. demonstrated the value of circulating exoDNA as a rapid and low-cost method for identification of cancer-driving mutations in circulating exoDNA such as KRAS and TP53 mutations in pancreatic cancer patients and healthy individuals, respectively. In contrast to these studies, we did not detect TP53 gene mutations in circulating exoDNA in healthy individuals, which also led to the lack of specificity in our experimental results. Uchiyama et al. reported that due to low exosome DNA concentrations, these samples may be in a "gray area" for detecting, which may be a potential limitation of circulating exoDNA as a liquid biopsy. Moreover, compared with other reports, we detected the TP53 mutation with a coverage area of only 87 bp, which made the detection of circulating exoDNA less effective as a non-invasive liquid biopsy.
To increase the value of circulating exoDNA detection in HCC application, we are currently planning a prospective study that will cover almost all HCC-specific mutations. We will also improve the exoDNA extraction technology to improve the sensitivity of ddPCR, so as to improve the detection of low exosome DNA concentrations. We anticipate that this approach will provide beneficial information for the molecular evaluation of personalized HCC therapy. The detection of circulating exoDNA may enable further development of precision medical technologies to realize early diagnosis of HCC and to customize personalized treatment strategies.