Cancer Genetics and Epigenetics, 2025, Vol.13, No.6, 287-299 http://medscipublisher.com/index.php/cge 290 sequencing (NGS) (Henriksen et al., 2023; Chen et al., 2025). dPCR techniques like ddPCR have high detection sensitivity and quick results, making them highly suitable for detecting known hot spot mutations or a small number of tumor-specific gene changes. They can detect variant allele frequencies as low as 0.01% (VAF), but the number of mutations they can detect at one time is limited, which is their drawback (Chung et al., 2024; Shim et al., 2025). The detection method using NGS can sequence a large number of cancer-related genes, and even complete whole exome or whole genome sequencing, thereby achieving extensive genetic analysis (Henriksen et al., 2023). Tumor individualized NGS testing represented by Signatera first sequences the patient's tumor tissue to identify its specific gene mutations, and then conducts deep sequencing on plasma samples. This method is based on the genetic variation of the patient themselves and helps to reduce false positives caused by clonal hematopoiesis or technical interference, thereby significantly improving the sensitivity and accuracy of minimal residual lesion detection (Hashimoto et al., 2025). In recent years, individualized detection panels based on whole genome sequencing have been further developed and their sensitivity has been further improved. They can even detect ctDNA less than 0.001%, which is also applicable to tumor types with usually low ctDNA release (Chen et al., 2025). In practical applications, the choice of which detection platform to use should be determined based on clinical needs, required sensitivity, and the type of mutation to be tracked (Shim et al., 2025). 3.3 Factors affecting detection sensitivity and specificity The sensitivity and accuracy of ctDNA testing are influenced by a variety of technical and biological factors, among which the amount of blood collected is particularly crucial. The more plasma there is, the more DNA can be extracted and the easier it is to detect extremely small amounts of ctDNA, which is especially suitable for MRD detection. Approximately 4 mL of plasma can be obtained from about 10 mL of blood, containing about 12,000 diploid genomes. Theoretically, about 0.01% of mutations can be detected. The detection rate can be further improved and missed detections reduced by increasing the sample size or collecting blood multiple times (Gu et al., 2025). To detect low-frequency mutations, especially when using individualized NGS detection panels, ultra-deep sequencing of more than 100,000 times is usually required. Meanwhile, individualized panels should be reasonably designed and optimized to select relatively stable tumor-specific targets, improve detection efficiency and reduce the minimum detectable level, so as to make MRD detection more sensitive (Hashimoto et al., 2025). The accuracy of the detection is also related to the analytical method. It depends on whether true tumor-related variations can be accurately identified from non-tumor sources such as background interference, technical errors or clonal hematopoiesis. "Tumor information detection" that tracks the specific mutations of patients' tumors is more accurate than detection that does not target specific tumors. Some factors before testing, such as sample processing methods, processing time and storage conditions, may all affect the results. Only by unifying standards can the reliability of the results be guaranteed. In addition, biological factors such as tumor location, metastatic site, and individual ctDNA shedding characteristics can also affect the detection rate of ctDNA. For cases like lung metastasis and peritoneal metastasis, the detection difficulty is even greater (Andersen et al., 2024). To enable ctDNA to play a greater role in the monitoring and treatment of colorectal cancer, efforts have been made to improve detection protocols, optimize panel designs, and standardize clinical operation procedures. 4 Application of ctDNA in Postoperative Recurrence Monitoring 4.1 Early recurrence prediction based on MRD detection After a patient undergoes radical surgery, the detection of circulating tumor DNA (ctDNA) in the blood can directly and in real time determine whether there is minimal residual disease (MRD), which has completely changed the way the risk of recurrence of colorectal cancer (CRC) is evaluated. Many prospective studies and comprehensive analyses have shown that regardless of the stage of colorectal cancer, patients with positive ctDNA after surgery have a much higher risk of recurrence than those with negative CTDNA, with the risk ratio of recurrence-free survival (RFS) ranging from 7 to 18 (Abidoye et al., 2025). Regardless of the risks indicated by traditional clinical pathological examinations, the predictive value of ctDNA holds true, which also indicates that it can uniquely reflect whether there are residual cancer cells in the body-and these cancer cells often cannot avoid
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