**2.2 Detection methods**

A variety of methods for detecting and characterising ctDNA have been reported. These can be broadly categorised as targeted and non-targeted approaches. Differing performance characteristics, strengths and disadvantages may also facilitate complementary roles of these approaches in molecular analysis. **Table 1** lists examples of described methods. Applying any of these approaches in

## **Figure 2.**

*Pre-analytical components in ctDNA analysis. (1) Collection of blood samples (usually 5–10 mL) via phlebotomy. Currently, there is no guidance on the comparative impact of the source of blood draw (for example, peripheral venepuncture or intravascular ports) on ctDNA analysis [5]; (2) samples should be collected in tubes containing anticoagulants compatible with polymerase chain reaction methods, such as ethylenediaminetetraacetic acid (EDTA) [9]; (3) centrifugation of blood to separate cells should be performed promptly. The exact optimal time to centrifugation is not known and may depend on storage conditions and the presence of stabilising agents [16]. Current evidence suggests that plasma is preferred to serum samples, as in the latter case, cfDNA released during white blood cell lysis may lead to a dilutional effect [9]. (4) Processed plasma is then generally stored frozen, often in aliquots; (5) CfDNA is extracted using commercially available kits. There are multiple DNA purification strategies and modifications, which may variably impact on DNA yield and purity [5].*

routine clinical practice in a credentialed laboratory would require considerable scaling up, standardisation, and optimisation of methodological efficiency and accuracy, while minimising cost [5].

Earlier approaches such as Sanger sequencing and conventional polymerase chain reaction (PCR)-based methods have limited sensitivity for ctDNA detection, particularly for rare alterations [6]. A number of digital PCR-based approaches were subsequently developed, capable of improved limits of detection (up to 0.001%), low frequency allele detection and nucleic acid quantitation. Commonly used digital PCR methods for ctDNA detection include BEAMing (beads, emulsion, amplification and magnetics) and droplet digital PCR (ddPCR). BEAMing which combines beads in emulsion and flow cytometry, was first described in 2003 [17] and facilitated serial tracking of mutant allele fractions in patients with CRC [9, 18]. This method and its variations are now widely applied in ctDNA analysis. DdPCR involves the massive partitioning of nucleic acids into thousands of droplets to enable highly sensitive and precise detection and quantitation of small concentrations of DNA [19, 20].

Next generation sequencing (NGS) or massively parallel sequencing are broad terms describing a range of high throughput methods capable of the simultaneous analysis of thousands to millions of DNA molecules, and also encompasses both targeted and non-targeted approaches. Targeted sequencing platforms such as safe sequencing system (Safe-SeqS) [21] and tagged-amplicon deep sequencing (TAm-Seq) [22] have the advantage of improved multiplex capability and evaluating a larger number of loci simultaneously in the genomic areas of interest [15]. However, targeted PCR-based or NGS methodologies mostly rely on prior knowledge of molecular changes and cannot identify variants located in areas that are not analysed.

In contrast, non-targeted genome or exome-wide sequencing allows discovery of *de novo* alterations as well as detection of structural changes including rearrangements, gene fusions and copy number alterations [23]. These may be advantageous in patients who do not have accessible tumour tissue for biopsy. Several techniques have been described and used in the ctDNA setting. The personalised analysis of rearranged ends (PARE) method uses paired-end sequencing, and was utilised in a proof-of-principle study to identify unselected genome-wide chromosomal alterations characteristic of tumour DNA in cfDNA in patients with CRC and


*PCR, polymerase-chain reaction; ARMS, amplification-refraction mutation system; Bi-PAP, bidirectional pyrophosphorolysis-activated polymerisation; BEAMing, beads, emulsion, amplification and magnetics; ddPCR, droplet digital PCR; Safe-Seq, safe-sequencing system; TAm-Seq, tagged-amplicon deep sequencing; WGS, wholegenome sequencing; WES, whole-exome sequencing; PARE, personalised analysis of rearranged ends.*

**59**

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

commonly seen in germline DNA to avoid false positives [15].

although substantial heterogeneity was noted across studies [39].

breast cancer, including copy number changes and rearrangements [24]. Another group demonstrated the feasibility and utility of exome-wide sequencing in ctDNA to identify mutations associated with acquired therapeutic resistance in a small cohort of patients with advanced cancer [25]. Current limitations of non-targeted approaches include lower sensitivity and relatively prohibitive costs impeding routine clinical implementation [24]. Furthermore, due to the relatively large amount of resulting sequencing data, substantial bioinformatic expertise and filters are required to decipher somatic tumoural alterations from the structural variants

Five-year survival for CRC patients is highly dependent on the timing of disease detection and tumour stage. CRC screening can achieve early disease detection and treatment, including that of pre-malignant dysplastic lesions, and has been shown to improve CRC-related mortality. However, 60–70% of patients are diagnosed at mid- to late stage CRC despite recent advances in screening methods [33]. Screening approaches used to test asymptomatic people for a presence of unsuspected disease, which have proven efficacy in CRC include endoscopic visualisation and faecal occult blood tests (FOBT). The former is invasive and expensive with associated morbidity, thus affecting patient compliance and acceptance. FOBT measure the presence of haemoglobin in faeces and can be categorised into guaiac-based (gFOBT) and the newer haemoglobin-antibody-based faecal immunochemical test (FIT). FIT is used more commonly in current practice and has largely superseded gFOBT, due to its superior sensitivity for colorectal bleeding, improved analytical characteristics and it is also less likely to be affected by dietary and medication factors [34–37]. In addition, FIT has better acceptance and participation which improves population participation [38]. In a meta-analysis of 19 studies, FIT was found to have high accuracy and specificity, and moderately high sensitivity,

This prompted the development of faecal-based tests targeting genetic and epigenetic alterations. Cologuard is the first commercially-approved faecal test which combines several technologies including molecular assays for aberrant *NDRG4* and *BMP3* methylation, *β-actin* (a reference gene for human DNA quantity), and *KRAS* mutations; and a haemoglobin immunoassay [40]. The haemoglobin component of the Cologuard test contributes to 80% of the cancer detection in the algorithm. A large randomised clinical trial comparing Cologuard and FIT screening showed that the sensitivity of Cologuard was superior to that of FIT in the detection of CRC and precancerous lesions [40]. However, the higher cost of Cologuard and its lower specificity compared with FIT has limited its adaptation as a screening tool [33]. CtDNA analysis may offer a more convenient screening approach compared with faecal-based tests. The malignant transformation pathway, from adenoma to carcinoma, is driven by mutations such as *APC*, *KRAS* and *TP53* [41]. However, somatic mutational profiles are highly variable between patients. For example, *KRAS* and *BRAF V600E* are seen in approximately 40 and 7% of patients with CRC respectively [42]. To date, the vast majority of cancer patients evaluated with mutation-based blood plasma assays have advanced-stage disease. A challenge in early stage disease is the often-minute fraction of ctDNA present in the total circulating DNA—may be as low as <0.01%—which may be below the limit of detection assays [18]. A study which enrolled 170 patients with positive FOBT investigated differences in *KRAS* mutation levels in plasma and tissue samples [43]. The rate of *KRAS* mutations in plasma (3%) was found to be lower compared with

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

**3. CtDNA in screening**

#### **Table 1.** *Methods of ctDNA detection.*

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

breast cancer, including copy number changes and rearrangements [24]. Another group demonstrated the feasibility and utility of exome-wide sequencing in ctDNA to identify mutations associated with acquired therapeutic resistance in a small cohort of patients with advanced cancer [25]. Current limitations of non-targeted approaches include lower sensitivity and relatively prohibitive costs impeding routine clinical implementation [24]. Furthermore, due to the relatively large amount of resulting sequencing data, substantial bioinformatic expertise and filters are required to decipher somatic tumoural alterations from the structural variants commonly seen in germline DNA to avoid false positives [15].
