**2.1 Pre-analytic considerations**

*Advances in the Molecular Understanding of Colorectal Cancer*

be better suited for serial surveillance, by reducing procedural time and potential harm. Blood sampling may also provide a more accurate representation of global tumoural heterogeneity, not limited to the site-specific characteristics detected through tissue biopsy [5]. The focus of this review will be directed towards circulating tumour DNA (ctDNA) found in blood samples, which at present, of all the liquid biopsy approaches, has had the greatest clinical impact. CtDNA is thought to be released by tumour cells, containing tumour-specific genetic and epigenetic alterations [6]; and has been found to correlate with tumour stage, burden of

Herein, we provide an overview of ctDNA technologies in use and highlight the emerging clinical applications of ctDNA in various CRC management settings (**Figure 1**). The future directions of this rapidly advancing field will also be

**2. ctDNA methodological approaches and technical considerations**

Circulating cell-free DNA (cfDNA) was first detected in 1948 by Mendel and Metais in the peripheral blood plasma of healthy and diseased individuals [8].

by physiological conditions such as exercise and acute inflammation [9]. In 1977, Leon et al. found that cfDNA was more elevated in cancer patients compared with healthy subjects, with higher levels correlating with higher burden of disease [10]. In 1989, Stroun et al. discovered that at least part of the plasma DNA in cancer patients originated specifically from cancer cells [11]. In the ensuing decades, knowledge and applications of tumour-derived cfDNA has rapidly evolved due to advances in molecular techniques, and also gave rise to the term, circulating tumour

in plasma and can be affected

disease and response to therapy in CRC [7].

*Emerging ctDNA applications in various CRC management settings.*

CfDNA levels can vary between 1 and 10 ng mL<sup>−</sup><sup>1</sup>

**56**

DNA (ctDNA).

explored.

**Figure 1.**

Numerous inherent challenges have affected the development of ctDNA preanalytic and analytic methods. These include variable fragmentation, low abundance in plasma or serum volumes, tumour heterogeneity, and low stability as a result of the aforementioned biological factors [16].

To minimise sample degradation and optimise stability, a number of preanalytical steps need to be carefully planned. Although there are currently no standardised methodology guidelines on ctDNA collection, storage and processing, the typical workflow is illustrated in **Figure 2**.
