|
| Methods | Advantages | Limitations | Bioinformatics tools |
|
| ALU-based q-PCR | Acceptable specificity, diagnoses CRC patients from healthy individuals [121] (screening and prognostic value for CRC disease) | Low sensitivity for differential diagnosis of stage I/stage II CRC and adenomas [121] | REST, pyQPCR, PrimerPy, GenEx, MultiD PowerNest, qBASE+ [122] |
|
| KRAS mutation analysis | Exists in 40% CRC cases mostly in codons 12 [36], negative predictive factor for metastatic CRC to treat with anti-EGFR antibodies [123], and correlates with the prognosis of the disease [124] | Not declared | COSMIC, TCGA, GSEA, GO enrichment, KEGG, STRING, CPTAC, [125–127] |
|
NGS assay:
(1) Safe-SeqS
(2) TEC-Seq | Detects somatic mutations in CRC early stages [8], prognosis and prediction to anti-EGFR antibodies or drug selection [8] | Not declared | Ion Torrent, Illumina, Read Filtering and Trimming, Burrows–Wheeler transform algorithm, Torrent Mapping Alignment Program, SAMtools Genome Analysis Toolkit (GATK), and Picard [128] |
|
| BRAF mutation analysis | Differentiating CRC from precancerous or state and distinctive histologic subtype of CRC [129], correlates with poor prognosis of the disease [130], and predictive biomarker for targeted therapy and prognosis [95] | It exists in 5%–10% of CRC tumors and should be used with other markers [131] | CBioportal, oncomine [132] |
|
| APC mutation analysis | Exists in 85% of colorectal tumors and 60% of stage I/II CRC patients, differentiating CRC from precancerous or lesions [133], and a potential screening and predictive marker to immunotherapy [134] | The wide spread of the mutations over variable codons [134] | CBioportal, oncomine [132] |
|
| RAS mutation analysis | Excellent concordance with liver metastases in CRC patients who account for RAS mutations in exons 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146), predictive marker for monitoring the resistance to treatment with monoclonal antibodies (as monotherapy or combined with chemotherapy) [65] | Not declared | COSMIC, TCGA, GSEA, GO enrichment, KEGG, STRING, CPTAC, [125–127] |
|
| OncoBEAMTM RAS | One-step ultra-sensitive quantitative detection of plasma-derived KRAS and NRAS mutations for diagnosis, treatment, and immunotherapy monitoring of mCRC patients after surgery [84, 95], significant low threshold detection [67], kinetical assaying of the mutated haploid GE quantity [67], prognostic value of MAF indicator in mCRC patients [67] | Not declared | MPprimer, Ultiplex [135, 136] |
|
| IdyllaTM RAS | Accuracy sensitivity rate of ≤1% and ≤5 for KRAS mutations in exons 2, 3, and 4, respectively (potential assay for highly individualized anti-EGFR therapy and chemotherapy treatment decisions) [84] | Two-step assay of RAS mutational status with lower clinical sensitivity than OncoBEAM assay [84] | Biocartis Idylla™ System [84] |
|
| SSCP method | High sensitivity, specificity, for rapid diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) families based on hMLH1 and hMSH2 mutations with a good limit of detection (10%) [137–140] | The longest turnaround time [137–140] | GenBank, MUSCLE alignment program [141] |
|
| Direct sequencing | The gold standard, highly sensitive, and cost-effective, the quantitative measuring of an individual mutant allele in ctDNA for distinguishing CRC histological type [35, 142, 143] | Limited potency for low amount mutant sequences detection in full wild-type DNA sequence context [35, 142, 143] | SnackVar, SangeR [144, 145] |
|
| Long cell-free DNA fragment/β-globin ratio | Biomarker of early colorectal liver metastasis recurrence [146] (patient’s monitoring after CRC surgery) | β-globin gene must also be analyzed in cfDNA as an index of overall impair [146] | Twoddpcr [147, 148], REST [149] |
|
