Abstract
Background
The field of liquid biopsies in oncology is rapidly expanding, with the application of cell-free circulating tumour DNA (ctDNA) showing promise in this era of precision medicine. Compared with traditional clinical and radiographic tumour monitoring methods, the analysis of ctDNA provides a minimally-invasive and technically feasible approach to assess temporal and spatial molecular evolutions of the tumour landscape. The constantly advancing technological platforms available for ctDNA extraction and analysis allow greater analytical sensitivities than ever before. The potential translational impact of ctDNA as a blood-based biomarker for the identification, characterization and monitoring of cancer has been demonstrated in numerous proof-of-concept studies, with ctDNA analysis beginning to be applied clinically across multiple facets of oncology.
Conclusions
In this review we discuss the biology, recent advancements, technical considerations and clinical implications of ctDNA in the context of cancer, and highlight important challenges and future directions for the integration of ctDNA into standardised patient care.
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1 Introduction
It is now well-accepted that cancer is fundamentally a genetic disease, where the sequential accumulation of somatic mutations and epigenetic changes disrupts the delicate homeostasis of cellular functioning. Advances in technological platforms such as next-generation sequencing (NGS) have allowed in-depth interrogations of such genetic aberrations and have, subsequently, led to significant advancements in our understanding of the key regulators of oncogenesis. As a consequence, we have entered the era of ‘precision medicine’, whereby therapeutic strategies may be tailored to patients based on their tumour molecular landscapes. With this comes the necessity to collect tumour samples for genomic analysis. Tissue biopsy remains the current gold-standard method for sampling a patient’s tumour for genotypic analysis [1, 2]. There are, however, multiple shortcomings to this method. Such invasive and costly sampling methods are associated with a high degree of complications. For example, 17% of lung cancer patients undergoing intra-thoracic image-guided biopsy suffer from an adverse event [3]. Additionally, owing to spatial and temporal tumour heterogeneity, a single-site biopsy may inadequately represent the total genetic landscape, and therapeutically actionable mutations or potentially resistant clones may thus be missed [1]. There is also a risk that a biopsy fails to obtain a sufficient number of cancerous cells to carry out adequate downstream analyses [4]. Moreover, current tissue preservation methods such as formalin fixation can introduce significant artefacts that may confound molecular testing [5]. In solution to these drawbacks, minimally-invasive biopsy methods have risen in popularity over recent years alongside the rise in molecular tumour profiling. Termed ‘liquid biopsies’, these methods include collecting blood or other bodily fluids from which to isolate various circulating components. In particular, cell-free DNA (cfDNA) holds promise as a biopsy surrogate, and has the potential to become a rapid, cost-effective and non-invasive means to assess the tumour landscape (Table 1).
2 Cell-free DNA biology
Cell-free DNA, double-stranded, highly fragmented extracellular DNA was first described in blood plasma in 1948 [25]. Subsequently, it has been detected in urine, saliva, peritoneal fluid, pleural fluid, uterine lavage fluid and cerebrospinal fluid [26,27,28,29,30], where it is presumably released from cells via both active secretion and apoptosis [31, 32]. Healthy individuals harbour between 1 and 10 nanograms of cfDNA in each millilitre of blood, with the major contributors being hematopoietic cells [30, 33,34,35]. Further studies are, however, required in order to definitively determine the threshold of ‘normal’ cfDNA concentrations. Whilst cfDNA has a short half-life of approximately 16 minutes, associations with various lipids and proteins can increase its longevity up to 10-fold [30, 36, 37]. cfDNA clearance from the body primarily involves nuclease action, followed by renal excretion [37, 38].
2.1 Biology and origin of tumour-derived cell-free DNA
Whilst a number of non-malignant causes of elevated cfDNA exist due to increases in cellular death and turnover (infection, autoimmune disease, trauma), it wasn’t until 1977 that the first connection between cfDNA and cancer was uncovered, when it was noted that circulating levels were increased in various types of cancers [39, 40]. This initial study by Leon et al. also provided a first indication that cfDNA levels may be proportional to tumour staging [41]. The presence of tumour-specific DNA among the pool of extracellular nucleic acids in the circulation was not confirmed until sensitive, mutant-specific PCR technologies were introduced nearly 15 years later [40, 42]. These early observations hinted at the possibility of using cfDNA to analyse tumour genomes in a minimally-invasive manner. Further technological advances such as the advent of next-generation sequencing (NGS) have brought these initial ideas to fruition.
In a malignant state, levels of cfDNA can increase to 50-1000 ng/ml blood, with tumour DNA comprising 3-90% of the total [43]. Increased levels are presumably due to a high turnover rate of tumour cells and an inefficient clearing of dead and dying cells [40]. The principal mechanisms through which tumour-derived cfDNA (herein referred to as ctDNA) is released in both solid and haematological malignancies are still under dispute, but multiple properties suggest cell death as the major contributor. The most common size of double-stranded ctDNA fragments is 160-180bp, as determined by sequencing-based approaches in humans and xenograft mouse models [30, 44, 45]. This size corresponds to the length of DNA occupying a nucleosome, the structural units of the eukaryotic chromosome [40]. Additionally, prominent but smaller fragment populations of ~330 and ~500 bp can be seen, corresponding to a linear progression of di- and tri-nucleosomal fragments [46] (Fig. 1). These observations implicate caspase-activated DNase, an endonuclease active in cellular apoptosis which cleaves chromatin DNA in an inter-nucleosomal pattern [46]. Another suggested method of ctDNA release is active secretion, in which newly-synthesised fragments are released in a lipid-protein complex [47]. This homeostatic mechanism, which was demonstrated early on by Abolhassani et al., can also produce DNA fragments of comparable lengths to those arising from apoptosis [48, 49]. Some studies have suggested that a large proportion of ctDNA is derived from active cellular secretion, such as the one published by Bronkhorst et al. who identified ctDNA fragments of novel length predominantly being released by viable, non-apoptotic osteosarcoma cells from [50]. There is also a case for necrotic release of tumour DNA, especially when considering the hypoxic environment of solid lesions. Necrotic cell death leads to the release of rapidly-cleaved, predominantly large (> 10,000 bp) nucleic acid fragments [43, 51]. A recent study using an in vivo inducible necrotic liver injury model demonstrated that large DNA fragments greater than 10,000 bp can be identified in the circulation which can significantly contribute to the pool of cfDNA fragments [43]. Current extraction methods, however, poorly recover larger ctDNA fragments and, therefore, the contribution of tumour cell necrosis to the ctDNA population is still incompletely understood [34, 52]. It is currently accepted that a combination of all three mechanisms may lead to ctDNA deposition, although further research is required to determine the relative contribution each makes to the total ctDNA levels in the context of different fluid compartments. Such knowledge may inform choices for cfDNA extraction methods in order to enrich for ctDNA.
Origins of circulating tumour DNA in the peripheral circulation. Capillary electrophoresis indicates that the bulk of ctDNA is present as mono-, di-, and tri-nucleosomal fragments, consistent with DNA release via cellular apoptosis
3 Technical considerations for ctDNA isolation and analysis
There are a number of factors that need to be considered when isolating patient ctDNA for analysis, and a thorough investigation of each may assist in the selection of standardized operating procedures to help control for preanalytical variables common during cfDNA processing. Whilst ctDNA has been identified in multiple fluid compartments, the most common source for analysis is blood (Box 1).
3.1 Plasma versus serum
ctDNA is, by nature, diluted in cfDNA present due to normal cellular turnover. Therefore, further dilution of the ctDNA by inadvertent lysis of leukocytes and other cells after sample collection is undesirable, as it can severely hinder the ability to detect low frequency mutations [58]. Past studies have favoured ctDNA collection from serum, as the total cfDNA yield is consistently greater compared to that of plasma [59, 60]. However, in recent years it has been demonstrated that this difference is due to the higher level of leukocyte lysis during blood coagulation [60]. For this reason, plasma has become the desired medium from which ctDNA may be extracted, and has since been shown to contain higher proportions of tumour-derived nucleic acids [45].
3.2 Collection tube and anti-coagulant
Between venepuncture and sample processing, the concentration of cfDNA will increase over time due to cellular lysis [61, 62]. To minimise genomic DNA contamination of ctDNA, prompt separation of plasma from the cellular components of blood via centrifugation is essential. The maximum time that a sample can be left unprocessed before it becomes compromised is still under debate, and ranges from 2 to 24 hours [63,64,65,66,67]. The type of blood collection tube should be taken into consideration when assessing the available infrastructure, or lack thereof, that may form a barrier to timely plasma isolation after venepuncture. The most common plasma collection tubes are ethylenediaminetetraacetic acid (EDTA) and cell-free DNA™ tubes (Streck), both of which contain the K3EDTA anti-coagulant, with the latter containing an additional preservative to reduce cellular DNA release [68]. Comparative studies in the fields of obstetrics and oncology have demonstrated the equivalence of both collection tube types if plasma isolation occurs within 6 hours of venepuncture [69]. Beyond this, cell-free DNA™ tubes outperform conventional EDTA tubes at preventing significant genomic DNA contamination [69,70,71], and have been shown to preserve the quality of cfDNA for five days at room temperature [72]. Other blood collection tubes include those containing citrate or heparin anti-coagulants, but K3EDTA surmounts both when considering cfDNA preservation and downstream analysis [65, 73].
3.3 Extraction and storage
Circulating DNA levels can be extremely variable and, therefore, all possible measures should be taken to maximise yield and minimise degradation in order to allow for sensitive detection of mutant alleles [74, 75]. Consequently, careful selection of a nucleic acid isolation kit suitable for low cfDNA is critical. There are currently numerous kits principally designed for the purpose of extracting cfDNA (Table 2), each with is own merits and limitations. After extraction, cfDNA can be stored between -20 and -80°C for extended periods of time, but multiple freeze-thaw cycles will invariably cause cfDNA fragmentation and should be avoided unless the DNA is stored in a low-salt elution buffer [61, 66]. Additionally, due to the hydrophilic nature of double-stranded helical DNA, use of plastic-ware containing hydrophobic polymers such as polypropylene can lead to reduced yields [61]. In contrast, polyallomer or specially-treated polypropylene plastic-ware has been shown to be more suitable for DNA storage and for maximising nucleic acid recovery during pre-analytical stages [84, 85].
3.4 Analysis platform
Similar to solid tumour biopsy analysis, the genomic interrogation of ctDNA ranges from single point mutation to whole-genome investigation. At present, there are numerous methods used to interrogate ctDNA, which can broadly be separated into candidate-gene approaches (for assessing 1-10 loci) and deep-sequencing approaches (Table 3). Whilst deep-sequencing methods provide the ability to assess a high number of loci without any prior knowledge of molecular alterations, and also affords a good platform for discovery of novel genes for precision medicine, it is expensive and requires comprehensive informatics analysis. Due to the low complexity and highly-damaged nature of ctDNA, whole genome studies are relatively scarce, although low-depth whole-genome sequencing has been used to identify somatic copy-number alterations in prostate cancer and neuroblastoma [86, 87]. As such, targeted sequencing approaches have become the preferred method when analysing ctDNA via deep sequencing [88, 89]. Candidate gene analysis approaches such as digital PCR have a comparable or increased sensitivity, are more cost-effective, and no bioinformatic analysis is required. However, such approaches only enable monitoring of known mutations, and multiplexing capabilities are low. It is likely that high-throughput, deep-sequencing methodologies will continue to be used for research purposes, and resulting data should inform a candidate-gene analysis approach that can easily be implemented in clinical practice where specific genomic expertise may be lacking. An assessment of the merits and limitations of current technologies used for ctDNA analysis is provided in Table 3.
3.4.1 Increasing analytical sensitivity
At present, both workflow approaches are lacking in the sensitivity required for ctDNA analysis to become imbedded in clinical practice. False-positives still occur [90], and it is suspected that many false-negatives arise due to insufficient detection of ctDNA in the larger pool of cfDNA, especially in the case of primary, low burden tumours. The addition of unique molecular identifiers (UMIs) in next generation sequencing approaches such as Safe-SeqS are expected to increase the sensitivity of deep-sequencing approaches for ctDNA detection. This molecular barcoding technique assigns unique sequences to each original DNA fragment, allowing the formation of UMI families following sequencing and the generation of corrected consensus reads without PCR or sequencing errors [91]. This allows utilisation of PCR replicates to reduce background errors and, therefore, increase true variant detection at lower allelic frequencies, which is useful when trying to identify ctDNA in a larger pool of normal cfDNA. Likewise, CAPP-seq uses a similar method, but each strand of the DNA template molecules is assigned a unique identifier allowing even pre-PCR errors to be removed, which further increases the sensitivity of the assay [92, 93]. Utilisation of targeted sequencing over whole genome or whole exome sequencing will also improve the sensitivity of NGS assays, as it allows increased coverage of specific regions of interest and, therefore, increases the likelihood of picking up a variant at low allelic frequency within the total pool of cfDNA. However, it should be noted that targeted methods reduce the discovery of novel cancer-associated loci.
Other methods that can be adapted to increase analytical sensitivity include in silico enrichment of shorter fragments from the cfDNA pool, as ctDNA fragments have been shown to be, in general, shorter than other cfDNA fragments [44, 94]. Additionally, when detecting a smaller tumour burden such as in early-stage malignancy, MRD or disease recurrence, a larger-volume sample from patients should be considered. Future studies should be aimed at assessing the best sample source and the utility of taking multiple samples concurrently (i.e., plasma and urine) to improve ctDNA yield.
4 Clinical applications of ctDNA
The clinical applications of circulating DNA in a cancer setting are numerous and varied. These applications include detection and localisation of neoplastic growth, prediction of tumour staging and prognosis, tumour genotyping and identification of potential targeted therapeutics, monitoring treatment efficacy and resistance, as well as identification of early-stage recurrence.
4.1 For early detection of disease
Early identification and intervention of cancer growth has the potential to improve therapy response and overall survival rates. ctDNA can be detected in patient plasma before conventional screening methods identify a cancerous lesion, as shown in one particular prospective study that found that KRAS2 and P53 mutations in otherwise ‘healthy’ individuals were associated with a higher risk of developing clinically detectable bladder cancer within 6 years (OR, 4.25 and 1.81, respectively) [95]. Whether this study represents the identification of a biomarker for cancer development, or a true identification of malignancy in pre-symptomatic individuals is still unclear. Nevertheless, it does represent a proof of concept for the potential incorporation of ctDNA into future screening programs. A deficit of this and similar studies is the interrogation of only 1 or 2 cancer-associated loci, making it likely that some true-positive patients may be overlooked for further investigative procedures in the absence of a ‘hallmark’ mutation [96, 97]. Moreover, a small number of healthy individuals harbour cancer-associated genomic alterations [98, 99], suggesting that analysis of a limited gene set is insufficient to confirm the presence or absence of ctDNA.
Assessment of the mutational and/or copy number status of a panel of pan-cancer-associated genes could potentially provide a more reliable method of identifying early-stage disease by increasing the sensitivity for detecting ctDNA. A multi-gene targeted NGS panel was recently utilised by Nair et al. on ctDNA isolated from uterine lavage fluid of women undergoing hysteroscopy for endometrial cancer screening. Ultra-deep sequencing revealed that all 7 women who received a positive early-stage diagnosis by classical histopathological assessment also had ctDNA harbouring significant cancer-associated gene mutations [26]. On a much larger scale, Cohen et al. reported the advent of CancerSEEK, a blood test with the ability to detect eight common cancer types across 1005 newly-diagnosed patients. CancerSEEK combines data collected from amplicon-based sequencing of cfDNA at 61 pan-cancer loci with levels of 8 circulating protein markers [100]. Of these eight cancer types, five currently have no effective screening test. With an assay specificity of over 99% and a sensitivity ranging from 69-98% (depending on cancer type), the possibility of using such liquid biopsies for early cancer detection is gaining momentum.
Still, whilst these early proof-of-concept studies are promising, a number of technical and biomedical challenges need to be overcome before a ctDNA screening assay becomes clinically attainable. Assay sensitivity requires ongoing improvement to detect early-stage cancer with high confidence and a low frequency of false-negative determinations. Currently, deep sequencing techniques meet this challenge better than low-throughput assays such as digital PCR. Additionally, the type and amount of specimen used (e.g. urine versus plasma) is also an important consideration when trying to identify small quantities of tumour DNA in an asymptomatic individual [26, 101]. Additionally, current patient follow-up tests may be of little use to ctDNA positive patients in which no neoplasm is detectable by current methods, except to pinpoint patients in need of close surveillance.
4.2 For cancers of unknown primary
Malignant cancers in which the disseminated site(s) are identified but not the primary location are associated with an extremely poor prognosis (mean survival < 12 months) due to a lack of tailored therapeutic strategies [102]. Epigenetic patterns of ctDNA are often highly stable and unique to particular cell or tissue types even in a cancer setting, and thus may facilitate the identification of the primary mass in CUPs (cancers of unknown primary). Recently, Lehmann-Werman et al. interrogated methylome databases to generate a list of methylation patterns than can be used to distinguish between different cell and tissue types. Consequently, through bisulfite sequencing, they demonstrated that methylation markers specific for exocrine pancreatic cells corresponded to those found in ctDNA of patients with pancreatic cancer [33]. This notion provides preliminary evidence that the ctDNA epigenome can be used to inform on cell-of-origin. Additionally, patterns of nucleosomal spacing of ctDNA fragments can also be used to infer cell types contributing to a pathological state. Snyder et al. assessed nucleosomal positioning in various solid tumours via deep sequencing and reported that the nucleosomal footprint of the tumour cells corresponds to ‘nucleosome maps’ derived from cancer cell lines [103]. In addition to epigenetic patterns, information on microsatellite instability has also been used to provide a primary source of diagnosis, as was the case in two trophoblastic tumours without a definitive histopathological diagnosis [104]. These proof-of-concept studies have revealed the potential utility of ctDNA for the localisation of a primary neoplastic site, and future studies should include larger cohort sizes and CUP patients to fully elucidate the clinical utility of ctDNA in providing a tissue-specific signal.
4.3 For tumour genotyping
Increasingly, there has been a transition away from anatomical and histological classification of tumours to a system based on molecular classification. This paradigm shift has allowed the introduction of precision medicine – customisation of healthcare and therapies based on genetic and molecular landscapes. The analysis of ctDNA has provided an avenue by which genetic and molecular signatures can be detected in a minimally invasive fashion. However, in order for ctDNA to become clinically relevant and play an integral part in day-to-day practice, there must be demonstration of a high concordance between mutational profiles found in ctDNAs and those found in solid biopsy samples. Studies in NSCLC have so far had the most success in this regard, with a recent trial showing the ability to detect epidermal growth factor receptor (EGFR) mutations in patient cfDNA with a concordance of 94.3% to matched solid biopsies. Additionally, 12 patients with a previously undefined mutational status due to technical difficulties with obtaining a tissue biopsy were identified as mutation-positive, and were subsequently treated with the EGFR inhibitor gefitinib [105]. There have been similar studies performed in a variety of other malignancies, including colon cancer and melanoma, were KRAS and BRAF mutations were interrogated, respectively [106,107,108,109]. Within these studies, variable concordance rates, sensitivities and specificities were reported, likely, in part, due to the assay types and technological platforms used. It should also be noted that rates of concordance between solid and liquid biopsies may not be an ideal method of assay validation, as ctDNA is expected to report on the genetic profile of all malignant populations [110], whereas a single tissue biopsy is unlikely to represent the full molecular landscape of a cancer.
4.4 For tumour staging and prognosis
Clinicopathological characteristics such as tumour stage and burden have been found to be significantly associated with both relative amounts and absolute concentrations of ctDNA [111,112,113]. Specifically, a 0.04% increase in plasma ctDNA levels (measured via TP53 mutant allele fraction) has been reported for every additional 1 cm3 of high grade serous ovarian tumour, as measured by computed tomography scans [114]. Such a statistic cannot be directly applied to other tumour types, due to the inherent differences in rates of cellular turnover, location and vascularity. Nevertheless, a liquid biopsy to determine tumour burden has clear advantages over conventional imaging and biomarker analyses as its short half-life allows real-time reporting [37]. In fact, in metastatic breast cancer, ctDNA has a greater correlation with tumour size when compared to both circulating tumour cells (CTCs) and the conventional protein biomarker CA 15-3 (Cancer Antigen 15-3) [115]. Studies measuring ctDNA levels to investigate tumour size almost always occur in a high burden setting, with the clinical utility of using ctDNA to report on precise tumour volumes in a minimal disease setting still being largely unclear.
The link between ctDNA levels and tumour burden suggests that cfDNA has prognostic ability. Indeed, many studies have observed a relationship between either total cfDNA or ctDNA levels and tumour staging (based on tumour size and extent of metastatic spread) [30, 74, 116, 117]. One particular study in 640 patients with various solid tumours found mutant fragments within plasma ctDNA increasing significantly between tumour stages (I through IV). The study also reported a significant association between mutant allele count and survival probability, with a two-fold rise reducing the 2-year survival probability from 25% to 10% [74]. A recent meta-analysis assessing a total of 1,170 non-small cell lung cancer (NSCLC) patients also concluded that a high cfDNA concentration was associated with a worse outcome in stage III-IV disease [118]. Other studies have reported similar findings [116, 119, 120] although, again, focus has been on tumour types with a high mutational burden and a distinct set of ‘hallmark’ alterations such as colorectal cancer. Larger gene panels are required to increase the sensitivity for detecting ctDNA in genetically heterogeneous tumours, or total cfDNA concentrations considered instead, although this comes with its own challenges due to fluctuations that may occur independently of tumour cell release [39, 40]. Additional challenges for using ctDNA for tumour staging arise from the high amount of inter-patient variability of plasma levels within the same stage and type of disease [74]. This is presumably due to differences in tumour vascularisation and extent of metastatic spread and, therefore, tumour burden. Such inter-individual differences need to be considered when using ctDNA as a staging tool, and perhaps a two-armed approach combining ctDNA with conventional methods may have the greatest utility.
4.5 For guiding treatment selection
The demonstration of reliable concordances between cfDNA and solid biopsies in regards to mutational profiling has given rise to studies investigating whether liquid biopsies can guide the selection of targeted therapies. Although retrospective in nature, multiple studies have showcased the ability of ctDNA to predict outcomes to targeted therapy. One such study in NSCLC reported that patients positive for the T790M EFGR mutation in plasma have outcomes that are equivalent to patients deemed positive by a tissue-based assay when treated with osimertinib, a third-generation EGFR inhibitor (overall response rates of 63% and 62%, respectively) [121]. Likewise, a study assessing BRAF mutations in melanoma ctDNA as a predictive marker of response to dabrafenib across four clinical trials concluded that it may be a useful marker for targeted therapy, although there was a small subset of plasma mutant-negative patients who experienced a therapeutic response [109]. Regardless, the robust data supporting ctDNA profiling in NSCLC has influenced major management guidelines allowing plasma biopsy as a viable alternative for molecular profiling before treatment in cases in which an initial tissue biopsy is non-diagnostic, and repeated biopsy is not feasible [4]. As a result, first-line targeted therapies in EGFR-mutated NSCLC based on liquid biopsy are now a reality due to approval by major regulatory bodies in the United States and Europe [122]. Other cancer types are not far behind, with numerous clinical trials currently underway assessing the predictive utility of ctDNA for approved targeted therapies.
In addition, total ctDNA levels may also be useful for guiding therapy. A recent study assessing plasma ctDNA in 21,807 advanced cancer patients revealed a relationship between ctDNA levels and the total mutational burden of a tumour, which may illuminate patients responsive to immunotherapy [123]. However, this link was only identified in breast cancer, colorectal cancer and NSCLC, and may be less applicable to cancer types with lower ctDNA fractions (e.g. glioma).
4.6 For monitoring treatment efficacy
Quantification and analysis of ctDNA represents a novel and timely method for measuring response (or lack thereof) to therapy. Currently, a combination of patient symptomology, biochemical markers and radiographic evaluation is used to objectively evaluate a patient’s response to treatment. These methods are not without significant drawbacks, however. Definitive symptoms can often lag behind overt disease progression, and tumour markers have been shown to lack specificity. For example, serum prostate-specific antigen (PSA) is widely used for disease monitoring in advanced prostate cancer despite progression regularly occurring in the absence of any detectable changes in PSA [124, 125]. Furthermore, the extended half-lives of tumour markers such as PSA allow them to persist in the circulation at levels that may not reflect treatment outcome, thereby hampering dynamic treatment decisions [126, 127].
In comparison, the unique properties of ctDNA, in particular its short half-life, make it an attractive real-time indicator of treatment efficacy and may lead to the identification of response earlier than clinical detection. Studies monitoring patients throughout therapy have reported that the dynamic changes in ctDNA throughout treatment correlates with response [128,129,130]. One such prospective study in women with metastatic breast cancer concluded that ctDNA is superior to other circulating biomarkers such as CA 15-3 in sensitivity and that, due to its dynamic range, it shows a greater correlation to changes in tumour burden [129]. Similar results have been reported in NSCLC, where reductions in ctDNA concentrations can predate favourable imaging findings by 4-6 weeks [131].
A recent study on metastatic melanoma suggested that clinical responses to anti-PD-1 immunotherapy may be preceded by reductions in ctDNA within 2-4 weeks of treatment initiation, although the cohort of 5 patients needs to be expanded [130]. It is likely that a drop in ctDNA is preceded by an initial peak in circulating ctDNA levels due to an initial increase in tumour cell death. Indeed, an earlier study on metastatic melanoma reported that an early peak in ctDNA level within the first week was indicative of a favourable response to T-cell transfer immunotherapy [132]. If confirmed, these data may be used to shape guidelines as to when ctDNA sampling should take place in order to assess early treatment responses to immunotherapy.
4.7 For identifying treatment resistance
It is well-appreciated that targeted therapies exert selective pressure on sensitive populations of neoplastic cells, ultimately leading to a molecular evolution of a patient’s tumour and the development of treatment resistance [133]. If, during treatment, relative changes in mutations can be observed early, then clinicians may be one step ahead, preparing for a second line of therapy to immediately combat the rise of novel resistant clones. Serial analysis of ctDNA represents an avenue through which clonal evolution can be closely monitored for the manifestation of resistance-conferring mutations that invariably develop during the treatment course [134,135,136] (Fig. 2). The ability of ctDNA to report on the genetic landscape of multiple tumour sites will also ensure that novel metastases arising from sub-clones are not ignored [110, 137]. Most evidence outlining the utility of ctDNA in monitoring treatment resistance currently comes from NSCLC, with the T790M mutation conferring resistance to EGFR tyrosine kinase inhibitors being reliably detected in the ctDNA of patients 16 to 49 weeks before clinical or radiological progression is observed [138,139,140]. There is also emerging evidence that ctDNA can be used to investigate therapeutic resistance in other malignancies, including advanced prostate cancer, where various somatic aberrations have been linked to resistance to the androgen-axis targeting therapeutic compounds enzalutamide and abiraterone acetate [75, 141, 142].
Monitoring of tumour clonal evolution via serial liquid biopsies. Targeted therapeutics puts cancer cells under selective pressure, changing the genetic landscape and, thereby, altering treatment efficacy. Detection of such clonal evolution in circulating tumour DNA can help to guide the selection of ensuing therapies
The preferred method of interrogating the ctDNA mutational landscape for resistance-conferring mutations is via a candidate gene approach such as digital droplet PCR, presumably due to its superior sensitivity (Table 3). However, the application of ctDNA to the identification of treatment resistance warrants deep sequencing studies interrogating multiple targets in order to identify all potential resistance-conferring clones. Whole exome or large-panel sequencing of cfDNA are currently limited by their lower sensitivity and higher costs, with patient-specific mutation panels presenting a more desirable alternative [143]. This, however, also presents the risk of missing de novo events.
4.8 For monitoring MRD
A central question after any anti-tumour therapy (surgery or otherwise) is whether the treatment has left behind any residual cancer cells or microscopic metastatic deposits that are responsible for relapse. Minimal residual disease (MRD) is a prominent concept in haematological malignancies, however the ability to detect small populations of residual tumour cells in solid tumours is still underdeveloped. A highly sensitive and accurate assay capable of detecting the presence of ctDNA can potentially be used to identify patients in need of further adjuvant therapies whilst saving low-risk patients from unnecessary and expensive treatments. It has been estimated that approximately 44 million malignant cells are required for significant plasma ctDNA detection, a number below the limit of resolution of current radiological techniques [144]. The ability to detect ctDNA even in the absence of any other clinical evidence of cancer makes it a prime candidate for measuring early-stage disease recurrence.
A recent prospective correlative biomarker study in 230 early-stage colon cancer patients reported that only 10% of patients with no detectable post-operative ctDNA suffered radiological relapse within three years whilst 100% of ctDNA-positive patients suffered disease recurrence [145]. As a result, post-operative ctDNA detection has been incorporated into a prospective clinical trial of stage II colon cancer patients receiving adjuvant chemotherapy. Such a high predictive value is presumably due to the personalised Safe-SeqS assays developed based on the molecular characteristics of the patient’s tumour resected during surgery, and the large volume of plasma collected at each time point (10 ml). Whilst this study clearly shows the validity of a patient-specific assay to detect MRD, results of similar studies in other cancer types are less clear, such as a separate study in multiple myeloma patients which found plasma ctDNA to be a poor predictor of disease recurrence, although one potential limitation of this study is a lack of sensitivity due to under sampling of patient serum (1 ml) [146]. It should also be noted that the design of a personalised assay for monitoring MRD based on somatic mutations in a resected sample may reduce assay sensitivity due to a potential lack of representation of heterogeneous tumours, and the emergence of novel sub-clones post-surgery.
5 Concluding remarks and future directions
Liquid biopsies are likely to play an increasing role in the implementation of precision medicine and the personalised treatment of various cancers. At present, many proof-of-concept studies have paved the way for the field to move from exploratory studies to implementation in clinical trials where ctDNA profiling guides patient management decisions. Currently, over 200 clinical trials investigating the prognostic and/or predictive biomarker utility of ctDNA across various cancer types are underway or actively recruiting (Fig. 3). However, there remain multiple issues delaying the widespread incorporation of ctDNA into routine patient care. Future focus should be on establishing optimal techniques for sample collection, cfDNA isolation (to improve yield of all relevant fragment sizes) and analysis. In addition, further studies are required to better understand the biological characteristics of ctDNA, such as mechanisms of release and clearance, and a proper characterisation of normal reference ranges. It is also important to understand how non-malignant conditions (e.g. inflammation, illness) contribute to the total cfDNA pool, as this may be informative for the optimal time for liquid biopsy collection.
Current clinical trials utilising cell-free DNA in cancer settings. Trials are either recruiting or underway, as detailed on clinicaltrials.gov. Search performed on 29/06/2018
As with any biomarker, a critical step to incorporation into clinical care is validation of a highly sensitive, universally agreed-upon platform. At present, multi-gene approaches capable of identifying several variant types (e.g. SNVs, CNVs) such as hybrid-capture based deep sequencing appear to be the most promising for biomarker discovery and development, and may soon match candidate-driven approaches in terms of sensitivity. Such assays should be developed with rigorous attention to analytical validity, accompanied by precise reporting on methods in accordance to best practice guidelines [147, 148]. Only by first addressing analytical validity one can begin to broach to the idea of clinical validation in prospective clinical cohorts.
On the challenge of clinical validation, there is an imperative that investigators continue to design biomarker-driven, prospective clinical trials, where patients receive specific treatment(s) on the basis of a predefined marker of interest [149, 150]. This focused approach, where the ctDNA biomarker will define the management plan, enriches study cohorts for patients likely to benefit, potentially streamlining drug development and accelerating drug approval [151]. It is important that ctDNA biomarker testing in such trials is restricted to clinical testing laboratories with a Clinical Laboratory Improvement Amendments (CLIA) certification, preferably with ample prior experience with the assay is question. Finally, robust evidence demonstrating definitively improved clinical outcomes or superior diagnostic capability compared to any current gold-standard is crucial before we can confidently transfer a ctDNA biomarker assay into routine clinical practice.
A future with ctDNA as a validated biomarker is on the horizon. As concordance of detectable driver alterations between ctDNA and solid biopsies improves, it will only be a matter of time before such a minimally-invasive liquid biopsy becomes a vital component of clinical practice and precision medicine.
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Fettke, H., Kwan, E.M. & Azad, A.A. Cell-free DNA in cancer: current insights. Cell Oncol. 42, 13–28 (2019). https://doi.org/10.1007/s13402-018-0413-5
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DOI: https://doi.org/10.1007/s13402-018-0413-5