**4. CtDNA in detection of residual disease in early stage CRC**

Although surgical resection can cure a high percentage of patients with CRC, tumour recurrence occurs in approximately 30–50% of all patients who undergo curative resection. The majority of these recurrences take place during the first 2 years after surgery and 90% recur within 5 years [53, 54]. Recommendation for adjuvant cytotoxic therapy is based on clinicopathological risk, although it may not be necessary in many patients and toxicity is substantial. Thus, biomarkers that would aid in identifying patients at high risk of recurrence and who would benefit from adjuvant therapies is of utmost importance. Carcinoembryonic antigen (CEA), a blood proteinbased tumour marker, is currently used for monitoring CRC treatment and can also be detected at elevated levels in pancreatic, gastric, lung and breast cancers, as well as a number of benign conditions. Whilst CEA is upregulated in the majority of advanced

**61**

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

CRC, the sensitivity for recurrence detection has been shown to be unacceptably low,

These preliminary studies support the need for large prospective trials evaluating pre- and post-operative ctDNA-based biomarkers to help predict recurrence and evaluate prognosis. However, it is not yet known whether ctDNA represents the molecular diversity of disease or whether only selective clones (for example, highly apoptotic clones) are secreted into the bloodstream. Furthermore, it is yet to be established whether early detection of recurrence can indeed improve survival

For the majority of patients with mCRC, the mainstay of treatment consists of palliative-intent systemic therapy with median overall survival (OS) approaching 24 months. Three classes of cytotoxic agents (fluoropyrimidine, irinotecan and oxaliplatin) and two classes of molecularly-targeted agents (monoclonal antibodies targeting vascular endothelial growth factor [VEGF], and the epidermal growth factor receptor [EGFR]) are currently approved for use in mCRC, although the optimal sequencing and scheduling of these treatments are debated. To optimise their therapeutic ratio and minimise toxicity, effective and accurate means of assessing treatment response are needed. In the following section, we summarise the evidence on the use of ctDNA in mCRC management. These include prognostication, monitoring tumour burden and predicting treatment efficacy, guiding targeted treatment selection, and detecting anti-EGFR therapy resistance.

**5.1 CtDNA in mCRC: prognostic value and monitoring tumour burden**

The association between the presence or high levels of ctDNA and adverse survival outcomes in mCRC has been demonstrated in several studies [66–69]. For example, in a landmark study, Bettegowda et al. observed a steady decrease in 2-year survival rate as ctDNA concentration increased [69]. Moreover, a systematic review exploring the prognostic role of ctDNA in CRC (mostly mCRC) found that most studies, although not all, demonstrated a negative correlation between ctDNA

It is well known that in CRC, there is high genomic concordance between the primary tumour and its metastases [57]. Therefore, a promising strategy to detect minimal residual disease or even relapsed disease, could be to use ctDNA to track and quantify key genomic aberrations (*APC*, *KRAS*, *BRAF* and *TP53*), which are recognised as playing a role in early CRC, and may persist in metastatic disease [18, 58]. Several studies have shown that peri-operative ctDNA detection is associated with higher rate of recurrence and in some cases, poorer overall survival; albeit with varying detection methodology, sensitivity and specificity [59–63]. Additionally, the aforementioned methylation markers *BCAT1 and IKZF1* have also shown promise in a study of patients with resected stage I–IV CRC, where post-operative positivity for *BCAT1/IKZF1* methylation was more sensitive (68%) for recurrence detection than CEA (32%, p < 0.05) and its odds of recurrence given a positive test (14.4, 95% CI: 5–39) was twice that of CEA (6.9, 95% CI: 2–22) [64]. However, they fail to detect advanced adenomas despite their frequent presence in cancer and adenoma tissue [65]. It would be reasonable to speculate that the release of any DNA from neoplasia seems to be a function of progression along the oncogenesis pathway and it is not a simple reflection of whether or not

approximately 30% [55, 56], supporting the need for alternative markers.

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

the change is present in tissue.

outcomes, if treatment is followed soon after.

**5. CtDNA in metastatic CRC (mCRC)**

and disease-free survival and OS [70].

### *Current Utility and Future Applications of ctDNA in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82316*

*Advances in the Molecular Understanding of Colorectal Cancer*

undetectable levels in the population tested.

of CRC with specificity of 92% [51, 52].

that observed in matched adenocarcinoma and high-grade intra-epithelial neoplasia tissues (45%) [43]. Although this is a small study, the results suggest that either this particular assay is not sufficiently sensitive, or that ctDNA was found at low or

The detection of epigenetic alterations characterised by aberrant DNA methylations is an alternative approach to mutational ctDNA analysis. Aberrant DNA methylation leads to transcription silencing of tumour suppressor genes, occurs early in CRC carcinogenesis, and may be more commonly seen and consistent in cancer patients compared with somatic mutations. Indeed, DNA methylation profiles in plasma have been used in biomarker development to identify emergence of early CRC by several groups [44]. One of the methylated promotors that has gained a lot of interest is the *Septin 9* gene promoter. Methylation in the *Septin 9* promoter demonstrated high sensitivity in preclinical studies and a small clinical cohort [45, 46], however a large prospective screening study demonstrated a sensitivity profile of only 48% [47]. In addition, the sensitivity to detect advanced adenomas was low (11%) [47]. Despite this, in 2016, the FDA approved the use of the Epi pro-Colon, a plasma-derived *Septin 9* methylation assay, for the screening of CRC. This decision occurred in the setting of encouraging results from a meta-analysis comparing the pooled sensitivity of methylated *Septin 9* with FOBT as a screening tool [48], and the improved sensitivity and specificity results of a modified version of the Epi proColon assay (2.0 version) [49, 50]. Recently, promising results have been reported in the utilisation of two methylation markers in the screening context—*branched-chain amino acid transaminase 1* (*BCAT1*) and *ikaros family zinc finger protein 1* (*IKZF1*)—where methylation of either gene identified close to 70%

To date, ctDNA is yet to demonstrate clinical utility in the CRC screening setting. Challenges lie in minimising false positive readings, whilst developing a test sensitive enough to detect small amounts of ctDNA. For example, normal physiological ageing is associated with the development of somatic mutations in the absence of malignant disease, and false positive readings may also be seen in patients with chronic inflammatory disease. False positive results can lead to unnecessary follow-up procedures and anxiety. Studies examining a large number of healthy control individuals will be essential to evaluate the specificity of potential screening assays. Yet another challenge with ctDNA-based screening is the identification of the underlying tissue of origin. Because the same gene mutations drive multiple tumour types, ctDNA tests based on genomic analysis alone gener-

ally cannot identify the anatomical location of the primary tumour.

**4. CtDNA in detection of residual disease in early stage CRC**

Although surgical resection can cure a high percentage of patients with CRC, tumour recurrence occurs in approximately 30–50% of all patients who undergo curative resection. The majority of these recurrences take place during the first 2 years after surgery and 90% recur within 5 years [53, 54]. Recommendation for adjuvant cytotoxic therapy is based on clinicopathological risk, although it may not be necessary in many patients and toxicity is substantial. Thus, biomarkers that would aid in identifying patients at high risk of recurrence and who would benefit from adjuvant therapies is of utmost importance. Carcinoembryonic antigen (CEA), a blood proteinbased tumour marker, is currently used for monitoring CRC treatment and can also be detected at elevated levels in pancreatic, gastric, lung and breast cancers, as well as a number of benign conditions. Whilst CEA is upregulated in the majority of advanced

**60**

CRC, the sensitivity for recurrence detection has been shown to be unacceptably low, approximately 30% [55, 56], supporting the need for alternative markers.

It is well known that in CRC, there is high genomic concordance between the primary tumour and its metastases [57]. Therefore, a promising strategy to detect minimal residual disease or even relapsed disease, could be to use ctDNA to track and quantify key genomic aberrations (*APC*, *KRAS*, *BRAF* and *TP53*), which are recognised as playing a role in early CRC, and may persist in metastatic disease [18, 58]. Several studies have shown that peri-operative ctDNA detection is associated with higher rate of recurrence and in some cases, poorer overall survival; albeit with varying detection methodology, sensitivity and specificity [59–63]. Additionally, the aforementioned methylation markers *BCAT1 and IKZF1* have also shown promise in a study of patients with resected stage I–IV CRC, where post-operative positivity for *BCAT1/IKZF1* methylation was more sensitive (68%) for recurrence detection than CEA (32%, p < 0.05) and its odds of recurrence given a positive test (14.4, 95% CI: 5–39) was twice that of CEA (6.9, 95% CI: 2–22) [64]. However, they fail to detect advanced adenomas despite their frequent presence in cancer and adenoma tissue [65]. It would be reasonable to speculate that the release of any DNA from neoplasia seems to be a function of progression along the oncogenesis pathway and it is not a simple reflection of whether or not the change is present in tissue.

These preliminary studies support the need for large prospective trials evaluating pre- and post-operative ctDNA-based biomarkers to help predict recurrence and evaluate prognosis. However, it is not yet known whether ctDNA represents the molecular diversity of disease or whether only selective clones (for example, highly apoptotic clones) are secreted into the bloodstream. Furthermore, it is yet to be established whether early detection of recurrence can indeed improve survival outcomes, if treatment is followed soon after.
