**6. Future directions**

Despite growing enthusiasm, ctDNA in CRC remains largely unavailable for clinical application outside of the trial setting. Recently, there has been a surge of research to further investigate the utility of more sensitive and accurate technologies for ctDNA detection and analysis, and to further elucidate its clinical implementation and significance in the various settings of CRC management.

**65**

*6.3.1 CTC*

*Current Utility and Future Applications of ctDNA in Colorectal Cancer*

failure rate of 0.02%) due to its ability to eliminate false positives [91].

tion and by applying custom solution hybrid capture enrichment [93].

Improved sensitivity techniques with the use of targeted-sequencing methods have been developed by several groups [9, 91]. For example, Lanman et al. validated the analytical and clinical use of a novel, ultra-high specific, digital sequencing technique (Guardant360) consisting of 54 clinically actionable cancer genes [91]. In 165 consecutively matched plasma and tumour tissue samples from patients with advanced cancer, this study demonstrated significantly improved sensitivity for Guardant360 in the plasma-derived cfDNA compared to that of tumour tissue. It also demonstrated the clinical success rate of the assay in 1000 consecutive plasma samples in the clinic (assay

Other investigators have combined the use of DNA fragment sequencing by using molecular barcodes with relevant bioinformatics filtering steps to enhance sensitivity and specificity [30, 69, 92, 93]. In a study using cfDNA from mCRC patients, Mansukhani et al. showed that false positive mutation calls could be reduced by 98.6% when incorporating novel molecular barcodes for error correc-

Several studies have explored the use of DNA methylation markers that may have a role in CRC screening and diagnosis, and which in some cases may have similar sensitivity and specificity to the aforementioned *Septin 9* methylation assay (for example, *APC*, *MGMT*, *RASSF2A*, *Wif-1*, *ALX4*, *NEUROG1*) [94–99]. More compelling is the evidence suggesting that the use of a combination of DNA methylation markers—a multigene methylation signature—may enhance sensitivity and specificity compared with single biomarker detection [94, 96]. Such an assay, utilising the methylation of both *BCAT1* and *IKZF1*, has shown promising results in

**6.3 Using CTC, extracellular vesicles, and microRNA as adjuncts biomarkers**

This chapter has highlighted the recognisable potential for a paradigm shift with the use of ctDNA for the molecular diagnosis and monitoring of CRC, as well as its multiple drawbacks when used in isolation. Notably, ctDNA is largely unable to evaluate biomarkers other than genomic aberrations. An alternative approach is the use of tools such as CTC, extracellular vesicles, and circulating microRNAs

A significant limitation of utilising CTC as a biomarker in CRC, particularly in early disease, is that they are difficult to detect in the blood due to a large proportion being captured in the liver prior to entering the general systemic circulation [101–103]. Furthermore, there have been a number of heterogenous studies, systematic reviews and meta-analyses which demonstrate conflicting results for the role of CTC as a prognostic biomarker [104–106]. This makes interpretation very challenging. Nonetheless, it is worth noting that detectable ctDNA and CTC as biomarkers are distinct entities and, in isolation, neither can be regarded as optimal surrogates of the multiclonal malignant state in an individual CRC patient. As discussed earlier, ctDNA is likely to be released by apoptotic or necrotic tumour cells, rather than highly proliferative cells, within a multiclonal tumour [103]. However, we do not know whether all clones have the same apoptotic potential, and therefore

(miRNA), in conjunction to ctDNA, to overcome these limitations.

*DOI: http://dx.doi.org/10.5772/intechopen.82316*

**6.1 Advancing ctDNA detection accuracy**

**6.2 Detecting aberrant DNA methylation**

this setting as previously discussed [51, 52, 64, 100].
