Circulating cell-free DNA and its clinical utility in cancer

Amanda Salviano-Silva; Cecile L. Maire; Katrin Lamszus; Franz L. Ricklefs

doi:10.1515/labmed-2022-0047

Article Open Access

Circulating cell-free DNA and its clinical utility in cancer

Amanda Salviano-Silva , Cecile L. Maire , Katrin Lamszus and Franz L. Ricklefs

Published/Copyright: July 19, 2022

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From the journal Journal of Laboratory Medicine Volume 46 Issue 4

Abstract

Liquid biopsies are a valuable non-invasive biomarker source for the diagnosis, prognosis and monitoring of cancer patients. The detection of circulating cell-free DNA (cfDNA) derived from tumor cells (ctDNA) has emerged as a promising clinical approach, as their levels are elevated in many cancers and contains tumor-related mutations and specific methylation patterns. ctDNA can be released from tumor cells into the bloodstream, either linked to extracellular vesicles (EV-DNA) or in an EV-free form when associated with nucleosomes and other proteins, or even as a component of macromolecular structures such as neutrophil extracellular traps (NET DNA). These different types of cfDNA can mirror cancer progression and predict patient outcome. This review presents the recent benefits of cfDNA in cancer, distinguishing between EV-DNA and EV-free DNA, and highlights their clinical utility.

Keywords: cell-free DNA; extracellular vesicles; liquid biopsy

Introduction

Finding minimally invasive techniques to detect, characterize and monitor cancer has long been a central goal in oncology research. In the past decade, there have been major breakthroughs in our ability to examine tumor-derived material in the circulation (blood/plasma) and other biofluids, including cerebrospinal fluid (CSF), saliva, and urine [1]. This has been possible due to the development of sensitive assays, capable of detecting the so-called needle in a haystack which refers to rare cancer-specific analytes hidden within the abundance of analytes of healthy cells. Current analytes used for liquid biopsy approaches include circulating tumor cells (CTCs), cell-free DNA (cfDNA), extracellular vesicles (EVs), tumor-educated platelets and other entities (i.e. proteins, mRNA, miRNAs, lncRNAs, metabolites, lipids). Each analyte has its advantages and disadvantages that must be considered when it is used to answer specific clinical questions. For example, cfDNA is an easily accessible and widely available analyte, yet its half-life is less than 1–2 h [2, 3], whereas EVs and CTCs can protect their cargo from degradation but require elaborated isolation techniques, resulting in relatively low quantities.

Most published studies used cfDNA as analyte for the detection of tumor-specific mutations via liquid biopsy [4], due to the relative easeness of DNA isolation and the availability of sequencing technologies to assess cancer-specific alterations. Circulating DNA is usually fragmented into ∼167 bp sequences, and samples from individuals with a high tumor burden show a trend toward even shorter sizes (<150 bp) [5]. It is assumed that the fragmented DNA suffices to detect tumor-specific mutations, however, the protocols that are used to isolate cfDNA from the blood of cancer patients usually do not involve an EV-removal step resulting in a mix of bona fide circulating DNA and EV-associated DNA [6]. While conflicting results regarding the proportion of EV-DNA within cfDNA have been reported (41–93%) [7, 8], EV-DNA has the advantage of being much larger (up to >10 kb) [9, 10]. It is thus unclear whether the circulating EV-DNA or fragmented cfDNA are the main source of the detected tumor-specific mutations and which analyte is better suited for identifying genome-wide mutations. Moreover, cfDNA sequencing can yield false positive results and detect mutations that are not present in the original tumor. Discordant results are sometimes due to clonal hematopoiesis, but also technical issues that compromise the specificity of mutation detection [4]. In this review, we summarize the main information contained in literature concerning circulating cfDNAs and their potential utility as tumor biomarkers in liquid biopsies.

Cell free DNA

Liquid biopsy from plasma, CSF or urine is a source of easily accessible cfDNA that due to advances in sequencing techniques is now used as biomarker in routine prenatal diagnostic screening [11], as well as in tumor classification, tumor heterogeneity assessment and therapy monitoring [12]. cfDNA combines genomic, mitochondrial and microbial DNA, double and single stranded degraded fragments from all kind of shredded or apoptotic cells. Despite the low amount and poor DNA quality, essential inputs can be inferred from bulk cfDNA and tumor-derived ctDNA to identify tumor specific genomic alterations and infer tumor subclasses, emergence of specific clones and treatment evolution. The confirmation that plasma ctDNA whole genome as well as whole exome sequencing is a valid method to identify unknown point mutations and copy number alterations from metastatic prostate and breast cancer [13] opened the door for ctDNA analysis as a diagnostic tool. Recently, WGS on ctDNA extracted from CSF of pediatric patients with medulloblastoma, was proven to be an essential asset to identify residual disease and for survival outcome prediction [14]. Targeted sequencing, an often more affordable approach, can also be used on plasma ctDNA to detect residual disease and patient outcome in gastric cancer [15]. In neurooncology, ctDNA analysis was until recently still extremely limited and ineffective due do the limiting amount of ctDNA from brain tumors that can be detected in the plasma and the low percentage of point mutations found in general in glioblastoma (GBM). However, the use of personalized sequencing panels designed from matched tumor combined with machine learning models was successful for glioma-ctDNA in plasma, urine, and CSF [16]. Whole genome bi-sulfite sequencing recently became a more promising technique to interrogate genome-wide methylation patterns as well as querying copy number variations and SNV indels. Such methylation profiling performed on ctDNA proved that methylation patterns are informative enough to detect over 50 different types of cancer in plasma [17]. Methylated DNA immunoprecipitation sequencing (MeDIP-seq), which provides a good coverage of the low CpG density genomic regions [18], has also been used successfully on ctDNA to identify differentially methylated regions which turn out to be sufficient to classify different types of brain tumors [19]. Genome wide DNA methylation profiling on ctDNA could also be achieved through CpG based arrays despite the plasma ctDNA degradation and can yield valuable information [20] as methylation profiling can be used to classify CNS tumors and assign them to specific subclasses with different susceptibility to treatment [21]. Additionally, specific mutations in the TERT promoter (C228T, C250T) have been recently identified to be present in more than 70% of GBM which opened the door for the use of digital droplet PCR to detect TERT mutations in GBM plasma ctDNA [22].

Whole genome sequencing and methylation profiling of cell free DNA led to new opportunities in early diagnosis as well as therapy monitoring and patient survival prediction, however, it is still unclear if the cfDNA used in most of these publications is linked with EVs or organized into DNA macromolecular complexes (Figure 1).

Figure 1:

Main cfDNA types.

Tumor cfDNA can reach the circulation being carried by extracellular vesicles (EV-DNA) (middle) or in an EV-free way, either by association with nucleosomes, as consequence of cell apoptosis (left), or as macromolecular complexes released from neutrophils (NET-DNA) (right).

DNA associated with extracellular vesicles (EV-DNA)

Extracellular vesicles (EVs) play an important role in intercellular communication between different tumor cells and also between tumor and normal somatic cells [23, 24]. EVs comprise a heterogenous population, with differences in biogenesis, content, and size. These parameters allow a differentiation of EVs in either small-EVs (S-EVs–including exosomes and small microvesicles) and large-EVs (L-EVs–including oncosomes and apoptotic bodies). S-EVs are ∼100 nm membrane vesicles, generally released into the extracellular space by multivesicular bodies that fuse with the plasma membrane. In contrast, L-EVs are usually bigger and detach from the plasma membrane, carrying unique surface markers. Both EV entities carry complex biological information, consisting of soluble and transmembrane proteins, RNAs and miRNAs, DNA, and lipids. EVs are taken up by recipient cells and release their bioactive contents, which can significantly alter the phenotype of the recipient cell [24]. Moreover, EVs are released from tumor cells into the bloodstream, and their cargo are protected from fragmentation and degradation by the membranous envelope, making circulating tumor EVs an optimal biomarker source for obtaining diagnostic information about the tumor and monitoring therapeutic progress by “liquid biopsy” [24].

Despite intensive research on EVs as potential sources of biomarkers and their role in physiological functions, the associated DNA remains largely unexplored. Most studies have so far focused almost exclusively on EV proteins, lipids and RNA cargo, and their composition. That EVs possess DNA is now widely accepted, but it remains uncertain where this DNA comes from, where it is located in relation to EVs, and what characteristics it possesses.

L-EVs detach from the plasma membrane and their seemingly non-specific loading mechanism can explain that their DNA content is reflective of the cell of origin seen at least evident for apoptotic bodies [25]. S-EVs, on the other hand, use more complex loading mechanisms using the ESCRT complex. However, it is not clear whether DNA can also be loaded into S-EVs via this loading mechanism. On the other hand, it has been shown that micronuclei, small constrictions of the nucleus as a result of DNA damage, can release DNA into the cytosol which then colocalizes with tetraspanin markers, which are established EV markers [26, 27]. These results support the early assumption that EVs are a waste removal system of cells to purify cytosolic DNA and maintain cellular homeostasis to avoid cellular senescence and apoptosis [28, 29].

Another topic that continues to be much debated is the localization of DNA in EVs. Most studies looking at L-EVs with or without comparison to S-EVs proved the presence of large gDNA (>2 million bp) in their vesicular lumen [30], [31], [32]. DNA associated with small EVs seems to be predominantly located to the outer membrane, yet a small proportion remains in the intraluminal space. Notably, although multiple groups provided strong evidence of DNA in EVs [9, 10, 28, 30, 32], [33], [34], [35], [36], a recent report questioned these findings and suggested that DNA is mainly associated with non-vesicular components [37].

The true localization of the DNA in EVs is clearly complex, and no consensus has yet been found. These differences are a result of the diversity of EV isolation and DNA characterization methods as well as the heterogeneity of EV DNA itself [38], [39], [40], [41]. Therefore, a consensus on EV DNA isolation and characterization methods must first be reached before an understanding of the true nature of EV-DNA localization can be obtained. In GBM patients’ tumor-specific DNA, especially mutant copies of IDH gene have been detected in EVs from glioma cell cultures or liquid biopsies [42], [43], [44], [45], [46], [47], [48]. While these results highlight the diagnostic potential of EVs, the significance of mutations in a few selected genes affected by recurrent hotspot mutations such as IDH1 or EGFR is limited to only a subset of patients with these alterations. More comprehensive profiling is needed to classify tumors with unknown genetic alterations and to monitor changes in genetic or epigenetic tumor makeup during the course of treatment and disease progression. Studies in other cancers have shown that high-molecular-weight double-stranded DNA from all chromosomes is present in EVs and can reflect the genome-wide mutational status of parental tumor cells [9, 10, 28, 30, 32]. Additionally, mutant KRAS alleles were found in late stages of pancreatic cancer progression to be associated not only to EVs, but also to histones in the circulation [49], suggesting both EV- and EV-free associated DNA as interesting sources for cancer biomarkers. The investigation of EV-DNA as a tumor biomarker and their role in therapeutic approaches has been extensively reviewed in the literature [50], [51], [52].

EV-free cfDNA (EF-DNA)

When not associated to EVs, cfDNA can be associated to many soluble proteins, including immunoglobulins [53], albumin [54], histones/nucleosomes [55] and lipoprotein-RNA complexes [56]. Such DNA/protein bindings are formed by electrostatic interactions and protect the extracellular DNA from degradation by nucleases.

Nucleosomes are stable complexes consisting of double stranded DNA arranged around a histone octamer. These nuclear structures can be found in the bloodstream as a result of cell apoptosis, carrying fragments of DNA with a characteristic size of approximately 150–200 bp, [55, 57]. The expression and structure of extracellular nucleosome DNA correlates with the cell of origin [57], is altered in various cancers [58], [59], [60] and carries tumor-derived mutations [49]. In addition, ctDNA is smaller and more fragmented than DNA from healthy cells [16, 57, 60], [61], [62].

CfDNA associated with nucleosomes can also exist in macromolecular structures called extracellular traps (ETs) [63]. The main types of ETs studied are neutrophil extracellular traps (NETs), which are released by activated neutrophils as a result of cell death (NETosis) or DNA extrusion without cell lysis. These structures are large fibers of nuclear or mitochondrial DNA, combined with histones and cytotoxic enzymes, such as myeloperoxidase (MPO) and neutrophil elastase (NE) [63]. NETs are mainly involved in pathogen recognition and inflammation initiation, and under pathological conditions they account for a significant proportion of EV-free cfDNA, as their increased production can be harmful to tissues [64], [65], [66]. Considering that inflammation is a hallmark of cancer and neutrophil infiltration is a common finding in tumor microenvironment, NETs have been increasingly investigated in the crosstalk between neutrophils and tumor cells [65, 67]. NETs are induced by several cancers, in response to reactive oxygen species (ROS) production, hypoxic conditions, proteases, interleukins, cytokines, and tumor-derived exosomes [68], supporting tumor growth and progression by different mechanisms. Citrullinated histone 3 (H3cit), MPO and NE, which are the main NET markers, contribute to platelets activation and cancer-associated thrombosis [69], [70], [71], [72], [73], [74], [75]. NETs are also involved in ECM disruption and cancer metastasis [68], as well as in exacerbated inflammation in advanced cancers [67].

Lung cancer studies have shown that NET DNA can trap CTCs, promoting their adhesion in distant organs and leading to metastasis. This metastatic adhesion has been reversed by NET disruption in vitro and in vivo [76, 77], and is mediated in part by Integrin ß1 (78,79). Furthermore, integrin α3β1 signaling pathway can be activated by NET-induced proteolytic laminin remodeling during lung inflammation, leading to the awakening of dormant cancer cells and aggressive metastasis [78]. In sharp contrast, an antitumor effect of NETs has been observed in melanoma cells, where integrin-mediated NET adhesion can inhibit tumor cell migration [79]. Conflicting data have also been observed in ovarian cancer, where a higher proporation of NETs may be associated with a favorable outcome [80] or a deleterious effect promoting metastasis [81]. NETs production is stimulated by breast cancer cells and promotes cancer proliferation and dissemination [82], [83], [84]. NETs are also increased in different digestive malignancies [68], contributing to progression, hypercoagulation and metastasis in gastric [85, 86], pancreatic [87] and colorectal cancers [88], [89], [90].

High levels of free fatty acids stimulate NET formation in nonalcoholic steatohepatitis patients, increasing inflammation, leading to the progression to hepatocellular carcinoma (HCC) [91]. Increased NET levels are also observed in HCC patients to promote metastasis [92, 93], also by inducing inflammatory response [94], or even as a consequence of surgical stress [95]. Moreover, NETs are also elevated and associated with thrombosis in non-solid tumors, such as in myeloproliferative neoplasms (MPN) [96] and acute promyelocytic leukemia (APL) [97].

Furthermore, in a genome wide association study (GWAS) performed with chronic diseases patients, the plasma levels of NETs were associated with the presence of genetic variants located in genes predicted to be involved in cancer pathways, such as OR10H1 and KHDRBS1 [98]. This suggests the possibility of genetic markers for NET levels with prognostic value in cancer, although no studies were performed in this specific field until the moment.

New diagnostic approaches with circulating cell free DNA

The detection of ctDNA in liquid biopsies is a non-invasive method which allows a real-time monitoring of cancer patients, being attractive for diagnostic and prognostic purposes. Therefore, many efforts have been made in the last decades to integrate the use of cfDNA and other circulating biomolecules into clinical routine. In this context, numerous studies analyze cfDNA associated with EVs, as the nucleic acids are protected and less fragmented when carried by these nanoparticles, and therefore highly representative of their original cells. Therefore, it is not surprising that the number of studies with EV DNA is increased in different types of cancer [50], [51], [52], compared to NET DNA.

Although most studies using NETs have been conducted in inflammatory and autoimmune disorders, interest in these macromolecular DNA structures has increased in cancer studies due to their influence on the tumor microenvironment and their potential utility as clinical markers. As aforementioned, NET DNA is elevated in several cancers and has been associated with poor prognosis, whereas NET disruption with DNase I has been associated with better outcomes [67, 71]. Interestingly, the NET degradation with DNase I has been shown in vivo to decrease the anti-PD-1 blockade resistance and attenuate the tumor growth. In this context, DNase I administration is suggested to be combined with immune checkpoint inhibitors for colorectal cancer patients [99, 100]. Other proposed NET inhibitors include prostaglandin 2 [101, 102] and inhibitors of peptidylarginine deiminase [103].

Clinical trials are currently being conducted with cfDNA in cancer patients [104]. Among them, RB1 mutations carried by EVs are investigated in childhood cancer retinoblastoma (ClinicalTrials.gov identifier: NCT04164134), while EVs carrying T790M derived from non-small cell lung cancer cells are being used to evaluate the efficacy of a potential antitumor treatment (NCT03228277). EV-DNA is also currently researched in early diagnosis of lung (NCT04529915) and thyroid cancers (NCT04742608). NETs have also been investigated in clinical trials, such as for detection of occult cancers in patients with venous thromboembolism (NCT03781531), and also for the prognosis of patients with high risk for thromboembolism, as the case of hepatocellular carcinoma (NCT05040347), myeloproliferative neoplasms (NCT04177576), and pancreatic, gastric and colon cancers (NCT04294589). In breast cancer, clinical trials are currently being conducted to evaluate NETs side effects as response to Tamoxifen administration (NCT05056857). Further clinical trials have been conducted, although there is still a long way to go to incorporate EV- and NET-DNA samples into routine clinical practice.

Conclusions

Detection of ctDNA in liquid biopsies is a promising tool for monitoring patients and predicting tumor status, in real time and in a non-invasive way. EV-DNA, NET-DNA and other types of cfDNA have been increasingly studied and some of them are already presented in the literature as potential clinical biomarkers for tumor progression and prognosis. However, there are still many challenges regarding their low sensitivity in detection. In addition, some important questions regarding cfDNA remain to be addressed. For example, the proportion of ctDNA in total cfDNA is still unclear, and it is largely unknown how this proportion changes during treatment. There is therefore a great need to characterize different DNA analytes and directly compare their suitability for detecting tumor-specific mutations in cancer patients.

Corresponding author: Franz L. Ricklefs, Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany, Phone: +49-40-7410-0, fax: +49-40-7410-55590, E-mail: f.ricklefs@uke.de

Research funding: None declared.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Authors state no conflict of interest.
Informed consent: Not applicable.
Ethical approval: Not applicable.

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Received: 2022-04-06

Accepted: 2022-06-09

Published Online: 2022-07-19

Published in Print: 2022-08-26

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/labmed-2022-0047

Keywords for this article

cell-free DNA; extracellular vesicles; liquid biopsy

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