Challenges and proposed solutions to the adoption of cell free DNA in screening, detecting and prognosticating colorectal cancer.

Comprehensive genome profiling cfDNA tests for CRC - Guardant360 ® CDx and FoundationOne ® Liquid CDx - were recently approved by the FDA for the detection of genomic changes in cancer-associated genes. Both assays are recommended as companion diagnostics for guidance of therapeutic decision making in multiple cancers. In particular, Guardant360 ® CDx has been shown to accurately identify 28 of 29 (96%) of pre-treatment plasma of CRC patients as bearing an amplification of ERBB2 , which as previously stated in this review, is a common driver mutation in normal colonic epithelium. This might potentially allow clinicians to identify patients at a higher risk of malignant colonic transformation. This would also allow the prediction of the patients’ response to HER2-targeted therapy[ 84 ]. Another test that could have prognostication utility in CRC patients is the commercial Oncomine ™ cfDNA assay which constructs 48 amplicons covering key hotspot mutations of 14 genes. In a pre-planned analysis of the VALENTINO trial using Oncomine ™ , Manca et al [ 85 ] studied the ctDNA VAF as a prognostic marker in patients with wild-type RAS metastatic CRC treated with an anti-EGFR-based treatment (folinic acid, fluorouracil, and oxaliplatin). They noted that higher VAF was found in patients with liver metastases, and that patients with high VAF had shorter overall survival (OS) compared to those with low VAF (21.8 months vs 36.5 months). The prognostication value of VAF hence exceeded that of baseline CEA by being significantly correlated with OS ( P = 0.003), hence confirming its reliability for this purpose. CfDNA based assays are also emerging as promising noninvasive approaches for detection of CRC recurrence post-resection and evaluating treatment response. The DYNAMIC trial highlighted that the ctDNA-based detection approach significantly reduced the use of adjuvant chemotherapy without increasing the risk of CRC recurrence in patients with stage II CRC[ 5 ]. Various NGS assays for surveillance of MRD post CRC resection have been developed over the past decade. One such example is Signatera ™ (Natera), a personalized, tumour-informed, multiplex PCR-based NGS assay for ctDNA detection currently commercially available in the United States. A large observational surveillance trial by Reinert et al [ 86 ] evaluated CEA levels, computed tomography imaging, and ctDNA in patients with stage I to III CRC. Patients underwent post-resection surveillance by Signatera ™ after adjuvant chemotherapy as well as by radiographic imaging. They found that Signatera ™ identified disease recurrence at a median of 8.7 months before radiographic imaging. More notably, while patients were awaiting radiologic detection, their ctDNA levels increased 5-fold, indicating that tumour burden increased markedly during the 8.7 months of lead time. Guardant Reveal ™ is another NGS assay that utilizes ctDNA detection as a surveillance strategy. Unlike Signatera ™ , it is a tumour-uninformed assay which analyses epigenetic signatures related to anomalous DNA methylation in addition to the detection of CRC-specific somatic alterations employed by most MRD assays. A prospective cohort study by Parikh et al [ 87 ] found that the augmentation of MRD detection by the integration of epigenomic signatures increased sensitivity by 36% compared to somatic alterations alone. In most CRC patients, ctDNA was detected from both genetic and epigenetic alterations, however a significant proportion were detected as positive by either genetic or epigenetic alterations, demonstrating that combining these two modalities may improve sensitivity for MRD detection. Other than its utility as a postoperative surveillance tool, the Guardant Reveal™ assay is also being tested in prospective clinical trials to assess its efficacy as a guide for adjuvant therapy.

Genetic mutations such as single-nucleotide variations (SNVs) and copy number variations (CNVs) have been used as diagnostic biomarkers for cfDNA-based modalities[ 28 ]. Modalities which detect genetic mutations ( i.e. , SNVs and CNVs) include quantitative polymerase chain reaction (PCR), targeted sequencing, whole-genome sequencing (WGS), and whole-exome sequencing. However, these mutation-based diagnostic modalities might not be adequately sensitive for patients with precancerous lesions or early-stage cancer given the lower number of recurrent mutations. A possibly superior approach may be through the detection of large-scale epigenetic alterations instead as they are tissue and cancer-type specific, and therefore are not constrained by low cell numbers[ 29 ]. Though initially discovered in the blood, cfDNA fragments have now been found in all human body fluid types, such as pleural and peritoneal effusions, cerebrospinal fluids, urine, saliva, stool and seminal fluid. The advantages of using plasma as a source of cfDNA are as follows: It is easily obtainable hence allowing for longitudinal sampling at multiple timepoints, and tumour heterogeneity might be better captured than with tissue biopsy sampling. However, the low ctDNA to cfDNA ratio due to the predominance of clonal haematopoiesis makes using plasma cfDNA as a diagnostic modality difficult[ 30 ]. In contrast, the lower proportion of cfDNA originating from haematopoietic cells in non-plasma bodily fluids suggests a higher ctDNA fraction (ctDNA%) and higher variant allele frequencies (VAFs) in non-plasma sources compared to plasma[ 31 ]. This reduction in cfDNA levels can be beneficial when searching for low-frequency genetic alterations due to a reduction of the background noise created by clonal haematopoiesis. Furthermore, ctDNA from non-plasma sources might be more representative of the primary tumour, as shown in the pleural fluid of patients with advanced stage lung cancer[ 31 ] or in CSF from patients with leptomeningeal metastases[ 32 ]. Specifically in the context of CRC, stool-derived cfDNA can be particularly advantageous due to physical proximity to the colorectal neoplastic tissue. A multitarget stool DNA test (Cologuard ® ) showed a sensitivity of 92.3% and specificity of 86.6% for the detection of CRC[ 33 ]. Hence, depending on the specific tumour type, non-plasma cfDNA can be a more viable option for cancer detection.

In contrast, DNA methylation signatures exhibit significant differences between healthy individuals and those with malignant tumours[ 19 ]. Compared to searching for point mutations, characterizing plasma epigenetic changes has shown to improve detection sensitivity by studies exploring the utility of cfDNA methylation for cancer detection[ 48 ]. Moreover, cfDNA methylation has shown utility in locating the cancer’s TOO[ 8 ], otherwise known as tissue deconvolution. This is possible as different tissues have different DNA methylation signatures[ 49 , 50 ] and even between different cell types within the same tissue[ 51 ]. Analyzing differentially methylated positions or regions can even allow detection of patients with malignant tumours[ 52 - 54 ]. Since cfDNA has various release sources, the methylation level measured at each cytosine-phosphate-guanine dinucleotide site is essentially a mixed signal originating from multiple tissues, including those harbouring tumorigenic driver mutations[ 55 ]. Hence, pinpointing the TOO is possible by deconvolution of blended methylation signatures.

Despite the major strides of progress in the last decade regarding cfDNA assay for CRC detection, surveillance and prognostication, only a handful of tests have been approved for widespread clinical use due to multiple limitations, as summarized in Figure 3 . The challenges of using circulating tumour DNA for the detection and prognostication of colorectal cancer and proposed solutions. Figure was created with Biorender.com. Challenges preventing the adoption of cell free DNA to detect and prognosticate colorectal cancer include the exorbitant cost of deep sequencing which makes it infeasible for population-wide screening, inefficient circulating tumour DNA extraction from plasma or non-plasma sources, and the lack of technologies with sufficient sequencing depth. Potential solutions that can be explored are to channel more resources into the development of sequencing technologies with sufficient depth and sensitivity, and to formulate an effective and standardized method of circulating tumour DNA extraction. CRC: Colorectal cancer; ctDNA: Circulating tumour DNA. One of the main limitations is that ctDNA is not readily available in patients with early-stage tumours due to the low input volume and poor signal-to-noise ratio. Since ctDNA is not very different from normal cfDNA, specific extraction is challenging with no current standard for extraction. Extraction efficiency is pivotol for a successful ctDNA analysis, especially in the early stages of CRC when ctDNA load is low to begin with. However, there is currently no standardized protocol for ctDNA isolation. Most methods require centrifugation of plasma which is time-consuming, inefficient and drains resources. Most methods also lack extraction efficiency as they are unable to detect for low molecular weight DNA which is typical of most cfDNA fragments[ 88 ]. Hence, the entire process of ctDNA isolation is not only labour-intensive but also costly, calling for the need of a standardized and efficient purification protocol. Additionally, somatic mosaicism in blood plasma remains an immense challenge for accurate cfDNA analysis[ 89 ]. Clonal hematopoiesis is a common age-related process involving the expansion of a clonal population of hematopoietic stem cells[ 90 ]. However, the detection of these non-tumorigenic clonal hematopoietic mutations is a common source of background signal for cfDNA-based assays[ 89 ]. Hence, the detection of CRC-specific mutations from plasma-derived cfDNA remains largely infeasible. In order to address the issue of poor signal-to-noise ratio, we need detection technologies with higher sensitivity and specificity to detect the < 1.0% of ctDNA in total cfDNA to allow for earlier intervention. However, the cost of high-sensitivity sequencing is generally more costly and is hence infeasible for population-wide screening. It might be advantageous to explore the use of new cutting edge sequencing technologies such as Beads, Emulsion, Amplification, and Magnetics, clustered regularly interspaced short palindromic repeats-mediated, Ultrasensitive detection of Target DNA-PCR-PCR, or CAncer Personalised Profiling by Deep Sequencing for the augmentation of cfDNA analysis to increase sensitivity and specificity[ 91 ]. As the consistency of mutation profiles between paired plasma and tumour tissue samples increases, it is becoming clear that cfDNA-based liquid biopsy for CRC detection, surveillance and prognostication has immense potential for clinical and precision medicine. However, two main challenges still stand in the way. The first challenge is the poor signal-to-noise ratio amidst a background of somatic non-tumorigenic mutations, which is further compounded by a lack of technologies with sufficient sequencing depth. The only way to circumvent this issue would be to develop technologies capable of deep sequencing to pick up mutations more sensitively. However, even if such technologies are developed, the cost of high-sensitivity detection is likely to be expensive and infeasible for population-wide screening. The second challenge is the low quantity of plasma ctDNA found in healthy, precancerous and even early-stage CRC patients. This significantly affects the sequencing performance of some, if not all, ctDNA assays. It might be possible to circumvent this issue by harvesting ctDNA from non-plasma sources like the stool or peritoneal fluid, which might be more representative of the primary tumour. However, obtaining peritoneal fluid is unrealistic for population-wide screening due to the invasive nature of a paracentesis. On the other hand, obtaining stool as a non-plasma source of ctDNA is more feasible and is already being used clinically. However, due to the gastrointestinal microbiome, only 0.01% of the total DNA content of stool is human-derived. This leaves us to face the first challenge again: The issue of poor signal-to-noise ratio and the lack of technologies with sufficiently deep sequencing. Hence, there is no conceivable way to utilize ctDNA assay as a mainstream modality of CRC detection and prognostication if the above two challenges are not addressed. If they are circumvented, however, the potential of liquid biopsy for the detection, surveillance and prognostication of CRC would be limitless.

The potential for cfDNA to revolutionize the screening, detection and prognostication of patients with CRC rests on overcoming the challenges detailed in this manuscript. Given the potential for improved screening, as well as improved OS in patients with CRC, efforts need to be focused on overcoming these challenges with expediency.

Existing literature suggests that cfDNA is released into the circulation via two different routes: Passive release mechanisms and active release mechanisms. Passive release mechanisms include processes like apoptosis, necrosis, and breakage of CTCs. Active release mechanisms involve cfDNA release from extracellular vesicles (EVs). Apoptosis is a type of programmed cell death carried out by caspases occurring in both physiological and pathological conditions. This process is triggered by a complex signalling cascade and involving morphological changes like cell shrinkage, pyknosis, plasma and chromatin condensation. Membrane blebbing leads to the release of apoptotic bodies from cells. These apoptotic bodies are subsequently engulfed by phagocytic cells and their components recycled[ 17 , 18 ]. It is believed that the large majority of cfDNA originates from apoptotic processes. Due to the multiple cancer hallmarks and cell-death mechanisms involved in tumorigenesis, an origin of cfDNA from apoptosis alone is unlikely[ 19 ]. Besides apoptosis, necrosis has been mentioned as a potential source of cfDNA in cancer patients. Cells undergoing necrosis exhibit organelle dysfunction and degradation of the plasma membrane, exposing its intracellular DNA to degradative agents such as nucleases and leading to the release of DNA into the extracellular space. CfDNA molecules are then fragmented by the various nucleases. Other passive mechanisms include cfDNA release from CTCs and chromosome fragments due to chromosomal instability. Other cell death mechanisms that have also been hypothesised to contribute to cfDNA load include necroptosis, oncosis, pyroptosis and ferroptosis[ 20 - 22 ]. Despite the abundant literature suggesting that cfDNA is mainly associated with apoptotic and necrotic processes, one recent study showed that cfDNA concentration had no correlation with apoptosis and necrosis. Active release mechanisms via exosomes constitute an alternative mechanism in which cfDNA may be found in the blood. The study found that breast cancer cells in the G1 phase released cfDNA via exosomes, and that the majority of cfDNA from breast cancer cells was released via active cellular secretion processes[ 23 ]. Current literature mentions active release mechanisms via exosomes, apoptotic blebs, shedding vesicles, and microparticles[ 24 , 25 ], however, the majority of active secretion of cfDNA occurs via EVs, which are spherical lipid-bound particles acting as mediators of physiological and biological processes[ 26 ] such as homeostasis[ 27 ]. Tumour-derived EVs are known to promote tumour invasion, metastasis and tumour migration as they can transfer tumour traits by entering other cells[ 18 ].

Mutation-based diagnostic modalities involve utilizing CNVs[ 34 , 35 ] and SNVs[ 36 - 38 ] as discriminative molecular features to reliably assess tumour-derived cfDNA. Current tumour fraction prediction methods based on CNVs rely on WGS with higher-coverage of more than 100-fold sequence coverage. Cutting edge algorithms, ichorCNA14[ 33 ] and ACE23[ 39 ], were initially developed from low-coverage WGS to generate an estimate of tumour fraction in cfDNA. However, both fail to provide an accurate estimate of tumour fractions due to high levels of aneuploidy and chromosomal instability[ 40 , 41 ]. Moreover, CNVs and SNVs are challenging to detect given the low number of mutated ctDNA fragments in early-stage cancer or certain tumour types[ 19 ]. While next-generation sequencing (NGS) technology enables high degrees of target multiplexing, the current depth of NGS sequencing is not deep enough to reliably search for mutations in a background of non-tumour-derived cfDNA[ 12 , 42 ]. The proportion of cfDNA fragments which harbour tumorigenic mutations is too low[ 43 ] which makes it difficult to search for bona fide variations amidst background signal from sequence changes introduced in library preparation. Extensive efforts have been made to improve the signal-to-noise ratio for more sensitive mutation detections, however these new methods rely on high-throughput sequencing and only analyze specific parts of the genome[ 37 , 42 ]. Such methods have limited efficacy for detecting cancer, especially at early stages, due to the low number of tumour genome equivalents in cfDNA[ 44 , 45 ]. The specificity of mutation detection is also hindered by the presence of somatic mutations in normal, non-malignant tissues[ 46 , 47 ]. Additionally, mutation-based screening modalities are largely incapable of localising the tissue of origin (TOO) of the tumour as the same driver mutation can be shared by many different cancers[ 37 ].

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, with a lifetime risk in average individuals of approximately 4% to 5%[ 1 ]. Novel approaches in screening and detection of CRC are needed to reduce overall mortality. Current approaches in the detection of CRC include modalities such as endoscopy and faecal occult blood tests. Liquid biopsy-based approaches are a promising avenue for non-invasive cancer detection, prognostication and surveillance. Liquid biopsy is a molecular-biological diagnostic approach for detecting significant tumour-derived markers in bodily fluids without the need for invasive tissue biopsy. This includes circulating tumour cells (CTCs), cell-free DNA (cfDNA), mRNA, microRNA, exosomes, nucleosomes, and various glycoproteins and antigens[ 2 ]. Although each liquid biopsy analyte has unique advantages and disadvantages with regards to detection, this present review will focus on cfDNA, which are DNA fragments released into circulation during cellular apoptosis and necrosis[ 3 ]. This review focuses on cfDNA because it is an attractive candidate for non-invasive cancer detection. CfDNA carries information about cancer-specific genetic and epigenetic mutations[ 4 ]. CfDNA levels during treatment have been shown to correlate with oncologic outcome and provide direct evidence of minimal residual disease (MRD) in patients post-surgical resection[ 5 ]. Some studies report cfDNA to outperform imaging modalities like computer tomography in the detection of recurrent tumour[ 6 ]. Moreover, cancer patients’ blood has been shown to contain increased levels of cfDNA compared to that of healthy individuals[ 7 ]. The analysis of cfDNA methylation patterns has also shown to successfully detect a wide range of cancers with specificity and sensitivity performance approaching the standard for population-level screening[ 8 ]. However, few studies have explored the use of cfDNA analysis in healthy, presymptomatic individuals. This is a novel concept that is difficult to implement for a few reasons: Lack of knowledge regarding the molecular basis of tumour initiation, significantly lower cfDNA concentration in the plasma of healthy individuals compared to cancer patients[ 9 ], and lack of mutant ctDNA molecules present in the plasma of healthy individuals with zero tumour burden[ 10 ]. In this article, we will explore the role of cfDNA analysis across the oncogenic spectrum of CRC, beginning during tumour initiation at a point when the tissue is phenotypically normal, then proceeding into a precancerous polypoidal lesion, and finally at the point when cancer has been established. The clinical roles of cfDNA with regards to normal, precancerous and cancerous colonic epithelium are summarized in Figure 1 . The role of cell free DNA in patients with normal, precancerous or cancerous colon. Figure was created with Biorender.com. Cell free DNA can play different roles in different patient populations. In presymptomatic individuals, it can be used to screen for colorectal cancer (CRC) by detecting the presence of circulating tumour DNA in plasma or non-plasma samples. Even if patients are proven to be CRC-free, they can be evaluated for driver mutations which would predict their risk of developing CRC. In individuals with CRC however, cell free DNA can be used for the surveillance of minimal residual disease post-surgery, to prognosticate, or to inform therapeutic decision making. CRC: Colorectal cancer; cfDNA: Cell free DNA; ctDNA: Circulating tumour DNA.