**3. Conclusion**

**2.3. cfDNA genetic variations**

40 Liquid Biopsy

*2.3.2. Mutation of cfDNA*

*2.3.3. SNP of cfDNA*

associated with traditional [63, 64].

**2.4. cfDNA methylation as epigenetic biomarker**

*2.3.1. Copy-number variations of cfDNA*

Cell-free DNA generates from apoptosis or necrosis cells and contains the same genetic variations with intra-tissues. cfDNA is widely used as a genetic biomarker for disease diagnosis and monitoring by detection of the copy-number variations, SNPs, and mutation occurred in cfDNA.

Copy-number variations (CNV) are always associated with the occurrence of complex disorders. The CNV of urine cfDNA in advanced prostate cancer patients is significantly associated with tumor burden, and the CNV change after stage-specific therapies reflected disease progression status and overall survival [51]. Copy-number variations of HLA-DRB5 in 135 systemic lupus erythematosus (SLE) patients were higher than that in 219 healthy controls and were associated with the risk of SLE. The copy-number at 6p21.32 is aberrant in the majority of SLE patients [52]. In the plasma of neuroblastoma patients, the copy-number alterations of cfDNA displayed concordant high patterns and can be used as a cost-effective, noninvasive,

Mutation is a widespread phenomenon in biology, the effect of which is permanent alteration of nucleotide sequence. Mutations play a vital role in both normal and abnormal biological processes, such as evolution and cancer. Many studies display that the mutation detection in cfDNA will enable noninvasive tumor diagnosis and monitoring with higher sensitivity and specificity in advance [54, 55]. Many patients with advanced lung cancers that are resistant to AZD9291 therapy carried *EGFR C797S* mutation in cfDNA [56]. EGFR mutations in cfDNA were significantly associated with overall survival (OS), progression-free survival (PFS), and response to therapy in the EURTAC trial. The EGFR L858R mutations in cfDNA proved to be a novel prognostic marker [57]. In melanoma, BRAF mutation in cfDNA can be detected earlier than primary lesion [58]. KRAS mutation in cfDNA for pancreatic ductal adenocarcinoma (PDAC) provided a new diagnostic marker and could optimize therapeutic strategies for patients [59].

Single-nucleotide polymorphism is a variation in a single nucleotide occurring in the genome at a specific position. Detection of SNP in circulating cell-free DNA has been widely used in prenatal screening. The detection of SNP located in SRY gene or TSPY gene in Y chromosome proved to be a highly accurate and clinically applicable noninvasive prenatal diagnosis (NIPD) marker for fetal gender determination [60–62]. The study reported first trimester contingent screening used nuchal translucency and cell-free DNA, the latter has higher detection rate that is up to 98% for trisomy 21, but noninvasive prenatal testing will not be cost-effective

Epigenetic modifications are heritable molecular events that affect gene expression without changing DNA sequences, including DNA methylation, histone modification, and so on.

rapid, robust, and sensitive biomarker for neuroblastoma prognosis [53].

At present, cfDNA has been used as an independent marker for prenatal screening and also has great applicable value in the disease prognosis and monitoring, particularly in cancer. When the concentration of cfDNA is above a baseline 30 ng/ml and is closed to hundreds or even thousands ng/ml, or/and when the size of cfDNA is obviously short and displays ladder pattern, or/and when vital genetic and epigenetic mutations are reported in cfDNA, patients should be recommended for further examination. The appearance of cfDNA conforms to the current trend of precision medicine in the disease and achieves accurate diagnosis and precise treatment. However, there are many challenges in the real clinic applications. Firstly, the detected method is not uniform and the standardization process is lacking [72]. Secondly, the level of cfDNA can be too low, so the detection technology needs to be improved to increase the sensitivity and specificity [73, 74].

The study of cfDNA is still in its infancy, and a lot of in-depth research is needed to further confirm its clinical application value.
