ddPCR is one of the most accurate and applicable tools currently available for examining genetic alterations (6). ddPCR is used to detect rare mutations, quantify copy number variations and evaluate microRNA, and the technique can be extended to clinical applications due to its relatively simple workflow. However, it can only be used to screen for known mutations.

BEAMing is relatively sensitive and inexpensive when assessing a limited number of potential mutations (7). However, although it is fairly accurate, similar to ddPCR, BEAMing can only screen for known mutations. Furthermore, due to the complex workflow, dedicated instrumentation and the high cost of each sample, implementation of this technique in routine clinical settings is limited. Using BEAMing may be effective for detecting RAS mutations in blood (8).

The main features of TAm-Seq include a high sequencing flux, reduced sequencing time and cost, and the ability to simultaneously sequence millions of DNA molecules, thereby enabling the analysis of the transcripts and genomes of a species in detail. Forshew et al (9) used this method to investigate the tumor protein p53 gene in patients with advanced ovarian cancer and proved the presence of metastatic mutations in multiple primary tumors; in this study, the detection rate of TAm-Seq was >2%, and its sensitivity and specificity were ~97%.

CAPP-Seq can identify multiple mutations in patients with the same type of cancer and improve the assessment of tumor heterogeneity, thereby providing more comprehensive diagnostic information. Newman et al (10) detected ctDNA by CAPP-Seq in 50% of a patient population with stage I non-small cell lung cancer and 100% of patients with the same type of cancer at stages II–IV. The study identified that CAPP-Seq could detect tumor burdens prior to medical imaging, thus indicating that this technique is valuable in monitoring residual disease. CAPP-Seq can detect all major mutation types, including single nucleotide variants, insertions, rearrangements and copy number changes (11).

Complete rare tumor genome characterization shows great potential in assisting clinical decision-making and identifying unreserved treatment regimens, rare mutations and invisible oncogenes. However, several challenges limit the application of WGS in the clinical setting, including quality assurance, ethical issues and a lack of trained clinicians; it is also costly and time-consuming (12). Kim et al (13) performed WGS in ~250 untreated and 20 recurrent glioblastoma samples, and discovered that a change in the p53 pathway is a major molecular predictor of malignant glioma mutation. The results of the study thus suggested that the genetic changes in primary tumors may affect the subsequent evolution of tumor cells and the emergence of subclonal heterogeneity.

WES can be used to sequence the coding region of the human genome, in order to detect common or rare disease-related abnormalities. Lecomte et al (14) used WES to determine familial pancreatic cancer, and detected partner and localizer of BRCA2 as a susceptibility gene. The results of this study indicated that WES shows promising clinical utility in locating potential oncogenes and tumor suppressor genes. For example, it may provide a reference for the molecular diagnosis of tumors. Compared with traditional sequencing technology, its low cost and high yield are major advantages. However, WES currently remains in the early stages of development.

WGBS-Seq offers single cytosine measurement resolution and high accuracy. Due to these advantages, the technique has become the gold standard in DNA methylation analysis (15). WGBS-Seq has made important contributions to the discovery of partially methylated domains in cancer cells (16). However, the method presents limitations in its operation. In particular, DNA may exist at varying degrees of degradation and the method may exhibit reduced sensitivity during detection. Table I lists the advantages and disadvantages of the various detection methods.

An in vitro study revealed that live tumor cells could release DNA continuously, thus illustrating the feasibility of ctDNA in early-stage cancer (17). Schmiegel et al (18) recently reported that the RAS mutation status in plasma and tissue is consistent in patients with CRC. Underhill et al (19) demonstrated that the integrity of ctDNA could indicate cancer cell death, allowing the use of ctDNA fragment length as a reliable early detection marker. However, other colorectal diseases exhibit similar integrity, and whether this level of integrity could predict early cancer remains unknown. Therefore, further studies on this technique are required. The level of ctDNA in peripheral blood is closely associated with tumor initiation and progression, and patients at later stages of the disease are considered to carry more fragments than those at earlier stages (14). Furthermore, the release rate of ctDNA is associated with the location, size and vascular distribution of tumors (20). Blood tests for extensive population screening require strict specificity. Gao et al (21) indicated that ctDNA is not superior to protein biomarkers (carcinoembryonic antigen, CEA) in terms of sensitivity and specificity. As the most widely used serum markers in CRC, CEA presents no specific diagnostic value, as false-positive and false-negative results may affect early diagnosis. Therefore, using a combination of ctDNA and CEA may be a useful method for the diagnosis of early-stage CRC.

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, cetuximab and panitumumab, are effective treatments for advanced CRC. These therapies function by directly blocking the EGFR pathway and enhancing the activity of chemotherapy drugs (22). The National Comprehensive Cancer Network (NCCN) 2016 guidelines state that the Kirsten rat sarcoma virus oncogene homolog (KRAS) sequence must be tested prior to administering cetuximab in CRC treatment, and only patients with KRAS wild-type tumors respond with clinical efficacy. The NCCN and the European Society for Medical Oncology suggest avoiding cetuximab and panitumumab in patients with B-raf proto-oncogene (BRAF) mutant cancer. However, even patients with KRAS wild-type tumors could have no response to anti-EGFR therapy, particularly in the event of BRAF and phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α mutations, the overexpression of human epidermal growth factor receptor 2, MET proto-oncogene and KRAS, and the absence of phosphatase and tensin homolog expression. Tumor heterogeneity may be one of the most important reasons for the observed resistance (23). Relying on tissue biopsy to monitor resistance is unrealistic. These issues indicate the important role of ctDNA in assessing the genomic and subclone mutations repeatedly during treatment. ctDNA could also be used to monitor the clonal evolution and drug resistance of CRC in patients. ctDNA analysis could identify a second resistance mechanism not captured by single lesion biopsy (24), predicting the timing and causes of the treatment failure. Monitoring KRAS mutations in ctDNA can provide clinical guidance for the determination of treatment for patients with CRC (25). These findings indicate that ctDNA has great potential for acquired resistance monitoring.

Patients with locally advanced rectal cancer generally receive neoadjuvant chemotherapy followed by radical surgery. Sensitive monitoring of neoadjuvant chemoradiotherapy (nCRT) or post-surgical recurrence is important for patients with CRC, as early detection of recurrence is associated with increased survival times (26). Early-stage recurrence is mainly due to incomplete resectional treatments or the existence of unknown metastasis (27). A relapse rate of 50–60% was previously observed in patients with stage III cancer following complete resectional treatments (28). In general, ~80% of the recurrence occurred in the first 2 years after surgery (29,30). Carpinetti et al (31) compared ctDNA levels with clinical, radiological and pathological responses to nCRT, and identified that ctDNA could be used to detect disease recurrence and monitor treatment responses to nCRT, preceding an increase in CEA levels and radiological diagnosis. Diehl et al (32) detected independent gene mutations in patients with CRC and identified 16 cases of postoperative ctDNA in patients with recurrence and 4 cases of non-detectable ctDNA in patients with no lesions. A previous study revealed that, compared with conventional follow-up, ctDNA monitoring could identify the recurrence of CRC progression 10 months earlier than radiological reports (29), as tumor progression is accompanied by an accumulation of mutations (27). Tie et al (33) performed large-scale assays to determine the feasibility of using ctDNA in detecting residual disease among patients who had not been treated with adjuvant chemotherapy; ctDNA was detected postoperatively in 14 (7.9%) of 178 patients, 11 (79%) of whom exhibited recurrence at a median follow-up time of 27 months, whereas recurrence occurred in only 16 (9.8%) of the remaining 164 patients with negative ctDNA. While these findings suggest that ctDNA detection may provide direct evidence of residual disease, Habr-Gama et al (26) came to contrasting conclusions and suggested that whether ctDNA levels are proportional to systemic tumor burden is not clear since no independent method is yet available to detect this burden. Clinical applications of ctDNA detection in patients with CRC are summarized in Table II. Besides CRC, ctDNA detection is widely used in the detection of numerous other types of cancer (Table III). Although ctDNA has great prospects in monitoring the responses of patients post-surgery, a number of obstacles remain, including early risk prediction, real-time tracking of tumor progression and mutation detection of treatment resistance (34).

DNAme is an important epigenetic modification that refers to a reversible and heritable approach for regulating genomic functions; it mainly occurs in 5′-cytosine-guanine-3′ (CpG) dinucleotide sites in the G/C nucleotide-intensive sequences (CpG islands) (35). With further understanding of DNA methylation mechanisms and advances in methylation chip technology, DNAme has demonstrated great value in early tumor screening, prognostic evaluation and chemosensitivity prediction (36–38). Detection of tumor-specific DNAme alterations in ctDNA may assist in monitoring the tumor burden and treatment responses of patients with breast or hepatocellular cancer (39). Differentially methylated regions of ctDNA were tested in healthy subjects and patients with colon cancer, and it was demonstrated that the DNAme detection method exhibits high sensitivity and specificity for CRC (40).

CTCs refer to various types of tumor cells with the antigenicity and genetic characteristics of a specific tumor type, found in the peripheral blood (41). The CellSearch^® CTC test (Menarini Silicon Biosystems, Inc., Huntington Valley, PA, USA) is the only Food and Drug Administration-approved in vitro CTC diagnostic tool with positive counts associated with overall survival (OS) and progression-free survival (PFS) in patients with CRC (42). Lu et al (43) revealed that patients with detectable CTCs following adjuvant chemotherapy have higher recurrence rates than those without. Krebs et al (44) also suggested that patients with elevated CTC blood levels (≥3 cells per 7.5 ml) are more likely to benefit from intensive chemotherapy regimens than those with lower CTC levels, indicating that CTC levels may aid in guiding the adjustment of the treatment protocols of patients with CRC. In addition, CTCs are advantageous in other areas, for example, while complex chromosomal rearrangements, including translocation, cannot be easily detected using ctDNA, it is possible with CTC detection (45). The genetic and phenotypic profiles of CTCs are usually different from those of primary tumors; therefore, these biomarkers may be used to determine the most effective targeted therapy.

Patients with CRC have higher total cfDNA levels (which include ctDNA) than healthy individuals (46,47), implying that cfDNA levels may distinguish patients with cancer from those without. The level of degradation of cfDNA may be a sensitive indicator of therapeutic effect and disease progression (48,49), similar to the ctDNA. Lin et al (50) confirmed that patients with lower cfDNA levels have improved 5-year OS rates compared with those with higher levels. Kitahara et al (51) evaluated the levels and integrity (as determined by the ratio of long/short fragments) of cfDNA in plasma samples collected from 93 patients with CRC prior to immunotherapy and drew similar conclusions that cfDNA integrity could be a predictive biomarker of immunotherapy efficacy. Higher cfDNA integrity tends to indicate excessive tumor necrosis factors and a high likelihood of tumor rupture; it also predicts immunosuppressive resistance and shorter PFS time in patients with CRC (49).