Progress in cfDNA research and the need for standardization

Preface

Cell free DNA (cfDNA) is becoming increasingly prominent as a novel molecular marker for disease prediction and tumor prevention. However, there are still many obstacles before achieving its widespread clinical application, mainly the lack of standards related to cfDNA, resulting in the lack of comparability of current research reports and the lack of stable reproducibility of assay results, thus reducing the reliability of clinical application. This review focuses on the progress of cfDNA-related research in recent years, including sample sources, sample preservation, qualitative and quantitative assays and applications of cfDNA, and analyzes and compares the methods used in various aspects of cfDNA research and their advantages and disadvantages, analyzes and summarizes the problems that exist among them, and indicates the urgency of developing cfDNA-related standards.

Cell free DNA (cfDNA) is a fragment of DNA released into the serum or plasma by all tissues of the body, including normal functioning tissues and abnormal functioning tissues such as intestinal flora or cancer, as well as cells, bacteria, viruses, nucleic acid-protein complexes, etc. cfDNA is usually thought to be found in blood plasma, but has also been found in other body fluids such as bile, feces, urine and cerebrospinal fluid. Its types are mainly classified as circulating tumor DNA (ctDNA) and cell-free fetal DNA (cffDNA).

The first reports of elevated cfDNA concentrations were in studies of patients with autoimmune diseases and leukemia. In the following decades, numerous studies have shown that patients with cancer and some inflammatory conditions typically have higher levels of cfDNA compared to healthy subjects. Therefore, human health monitoring through cfDNA testing is of great importance. The first successful application of cfDNA technology in human health monitoring is the cfDNA-based fetal chromosome aneuploidy, non-invasive prenatal testing. In addition, cfDNA testing can be used for tumor prevention and treatment. Many adult tumor patients have to undergo 20-30 years of lesions before cancer eventually forms. A large part of the high mortality rate of cancer is due to the failure to detect tumor formation early enough to lead to later medical intervention. Changes in cfDNA content can be detected several months earlier in the early stage of cancer cell formation, and thus early cfDNA testing can be used for tumor prevention and treatment purposes. Compared to tumor biopsies, cfDNA has been shown to better represent the complete genetic profile of tumors, and thus cfDNA can be used as a marker for screening healthy and asymptomatic groups, early detection of disease, diagnosis and treatment, evaluation of treatment efficacy, and prevention and control of disease recurrence. cfDNA's role as a molecular marker in clinical applications of tumors has been detailed.

Many studies have exploited the differences between cfDNA base lengths for fetal prenatal noninvasive testing, tumor and transplantation therapy monitoring. cfDNA breaks naturally into small fragments with an average length of 167 bp through a potential mechanism of nucleosome-mediated protection of circulating nucleases, consistent with similar studies finding that more than 70% of plasma cfDNA fragments are less than 300 bp in length, with an average size of 170 bp results are consistent. To avoid in vivo complications, Bronkhorst et al. established an in vitro model by culturing cells in vitro and extracting cfDNA for property exploration, and together with the experimental results of Yu et al. showed that the band of cfDNA at 166 bp was prominent, while the size of cfDNA fragments released into the body was found to vary, and usually the size of these fragments was related to the nucleosomal DNA extension of These fragments are usually associated with the fold of nucleosomal DNA elongation, ranging from 150 bp to 1,000 bp, which may be due to the different pathways of cfDNA release into the circulatory system. There are numerous speculations about the pathway of cfDNA release from the body to the circulatory system, and excluding the partially disproven ones, there are three suspected sources: apoptosis, necrosis, and active cellular release. Whereas DNA fragments from necrotic cells are usually larger than 10,000 bp, suggesting that most cfDNA originates from apoptotic cells in both diseased and healthy individuals, most electrophoresis results of plasma or serum DNA also demonstrate a ladder pattern similar to that of apoptotic cells.

1. Sample Collection and Treatment

1.1 Sample collection

The sample sources of cfDNA involve in vivo and in vitro. The samples used for in vivo extraction are from a wide range of sources, including plasma, serum and whole blood. Fluorescence quantitative PCR (qPCR) measurements found that cfDNA levels in serum are 10-fold higher than in plasma, but plasma minimizes dilution of tumor-derived cfDNA, and mutation detection results for plasma-derived cfDNA are more similar to biopsy results. The mutation detection results of plasma-derived cfDNA are closer to those of biopsies. Thirty percent of the DNA in preserved plasma samples or extracted and isolated cfDNA is degraded each year. The blood collection tubes used during sample collection can have an impact on cfDNA; tubes with cell lysis capabilities should not be used, and blood collection tubes used for plasma preparation must be treated with anticoagulants in advance. In addition, the transport of samples can introduce changes that are difficult to control. Filtration, centrifugation time, centrifugation force, and centrifugation procedures can affect the extraction and quantification of cfDNA. Samples that cannot be processed immediately and need to be stored for a short period of time should be placed in BCT tubes and placed in a cold environment, while ensuring that the samples are not exposed to extreme temperatures during transport.

1.2 Sample pre-treatment

When the collected samples cannot be extracted in time, the time interval between collection and processing and the storage temperature should be controlled to avoid affecting the yield of cfDNA. Risberg et al. found that when whole blood samples were collected in K3EDTA tubes and left at room temperature for 0, 6, 24, 48, 96 hours and one week after extraction of cfDNA, there is no statistically significant differences were observed of the number of mutant molecules expressed as copies/ml of plasma. In addition, some pretreatment operations can improve the cfDNA yield. The results of Bronkhorst et al. showed that the addition of proteinase K increased the cfDNA yield, but snap-freezing of the samples did not increase the cfDNA yield. The highest yield of cfDNA was obtained by the manual method with sodium iodide treatment. During the extraction of cfDNA, it is important to avoid the use of manipulation methods and reagent supplies that are biased towards the collection of cfDNA, to prevent missing small fragments and to avoid contamination of cfDNA by degradation of genomic DNA.

2. cfDNA quantitative detection method

2.1 Total cfDNA content quantitative detection

Quantitative methods for cfDNA include two main levels: quantification of the total cfDNA content to assess extraction efficiency and yield, and quantification of variant genes in cfDNA to guide clinical applications. There are numerous methods for the quantification of total cfDNA content, including UV spectroscopy, fluorescent dyes, fluorescent magnetic particles, competitive binding radioimmunoassay (sensitivity range 25~1 000 µg/ L), radiolabeled DNA (sensitivity up to 1.6 µg/ L), DNA embedding method, microbeads, emulsion, amplification and magnetics (beam), but usually due to the extraction of the resulting cfDNA concentration is low, resulting in the lack of accuracy of such quantitative methods. In addition, gel staining can achieve quantitative and semi-quantitative cfDNA fragment size, but cannot accurately detect the presence of small amounts of cfDNA; microarray electrophoresis can determine cfDNA fragment size distribution and measure content, but quantitative results depend on DNA standards with accurate quantitative values and certain fragment lengths.

2.2 Quantification of cfDNA gene variants

Tumor cells release DNA into the serum or plasma through a variety of mechanisms that enable the detection of cancer-related genetic alterations in the blood, including point mutations, copy number changes, chromosomal rearrangements, and epigenetic aberrations. Earlier tests have shown that somatic alterations can be effectively detected from samples containing 5% to 10% of tumor cells using whole exome sequencing at standard depths (~150× coverage). High-throughput sequencing technologies allow for the detection of target variant genes in cfDNA, and in particular, the advent of second-generation sequencing (NGS) has improved the accuracy of mutant gene detection to a certain extent by enhancing the mutation signal in a lower percentage of wild-type DNA background. yang et al. developed an NGS-based method for cfDNA allele counting, called cfDNA barcoding, which can be used for noninvasive prenatal diagnosis of monogenic diseases. ichorCNA software was developed by Adalsteinsson et al. to quantify the content of tumor mutated genes in cfDNA from whole genome sequencing data with 0.1-fold coverage in the presence of prior unknown tumor mutations.

Based on the PCR principle, qPCR and dPCR can achieve quantitative analysis of total cfDNA content and target gene variants. qPCR quantifies by fitting standard curves with standards, and the primary problem is to identify reference genes, and there are various types of reference genes available, including GAPDH, β-bead protein, ERV and TERT genes, etc., while the results of β-bead protein quantification and the two GAPDH and ERV There are significant differences between the quantitative results of β-bead protein and the quantitative results of two target genes, GAPDH and ERV, and the statistical analysis method of choosing the joint system of multiple reference genes may be able to balance the influence of different reference genes on the quantitative results of cfDNA. The lack of uniform reference genes and accurate content of cfDNA standards has led to the limitation of qPCR technique in the quantitative analysis of cfDNA.

dPCR is useful in advanced tumor detection, but lacks sensitivity and reliability in early screening, for which Diehl et al. established a novel bead emulsion amplification magnetics (BEAMing) technique that can determine the error rate during PCR. the short oligonucleotide mass spectrometry (SOMA) method established by Laken et al. likewise helps to improve the sensitivity of cfDNA gene mutation detection. With the development of technology, ddPCR is increasingly used in research with its high sensitivity, but mainly for mutant gene detection where the information is known. Pre-screening methods such as denaturing high performance liquid chromatography (DHPLC), time temperature gradient electrophoresis (TTGE) and single strand conformation polymorphism (SSCP) are required when gene variant information is unknown. Many previous methods to screen for cancer-derived cfDNA have focused on targeting somatic single nucleotide variants (SSNVs) in repeat mutant cancer genes, but studies have found that the vast majority of metastatic cancers contain somatic copy number alterations (SCNAs) at the chromosomal arm length level, and SCNAs may be suitable for a broader range of applications. In addition, methylation assays aimed at tumor localization are being extensively developed, and methylation assays combined with hmC-CATCH technology have been shown to greatly improve the sensitivity of cfDNA detection.

Third-party validation of test parameters such as sensitivity, reliability, use of internal and external controls, and the need for regulatory guidance from food and drug regulatory authorities and health departments must be considered for the application of cfDNA assays in a true clinical setting.

3. Applications

When tumor biopsy is not feasible, screening of cfDNA and ctDNA is the only valuable marker for selecting patients suitable for kinase blockade targeted therapy. The most common mutated genes include KRAS, EGFR, PIK3CA, BRAF, TP53, HER2, etc.

Among the current topical studies on the application of cfDNA assays, most of the quantitative assays focus on dPCR techniques with high sensitivity, which allow systematic comparison of mutation abundance at different gene loci. However, the reference genes used for quantification in individual study reports are yet to be unified, and the lengths of the selected cfDNA fragments are different, which is not conducive to exploring the integrity of cfDNA compared with genomic DNA and cannot further explore the mechanism of cfDNA release in blood, which affects the clinical application of cfDNA as a detection marker.