Circulating Tumor DNA-A Novel Biomarker of Tumor Progression and Its Favorable Detection Techniques

Abstract

Cancer is the second leading cause of death in the world and seriously affects the quality of life of patients. The diagnostic techniques for tumors mainly include tumor biomarker detection, instrumental examination, and tissue biopsy. In recent years, liquid technology represented by circulating tumor DNA (ctDNA) has gradually replaced traditional technology with its advantages of being non-invasive and accurate, its high specificity, and its high sensitivity. ctDNA may carry throughout the circulatory system through tumor cell necrosis, apoptosis, circulating exosome secretion, etc., carrying the characteristic changes in tumors, such as mutation, methylation, microsatellite instability, gene rearrangement, etc. In this paper, ctDNA mutation and methylation, as the objects to describe the preparation process before ctDNA analysis, and the detection methods of two gene-level changes, including a series of enrichment detection techniques derived from PCR, sequencing-based detection techniques, and comprehensive detection techniques, are combined with new materials. In addition, the role of ctDNA in various stages of cancer development is summarized, such as early screening, diagnosis, molecular typing, prognosis prediction, recurrence monitoring, and drug guidance. In summary, ctDNA is an ideal biomarker involved in the whole process of tumor development.

Keywords: cancer; ctDNA; detection technology; medication guide; prognosis.

Figure 1

Overview of ctDNA analysis.

Figure 2

Detection technology of ctDNA mutation. (A) Sensitivity of COLD-PCR (a) and ddPCR (b) for G12V-mutated DNA of metastatic colorectal cancer patients [59]. Aa, fast COLD-PCR sequence profiles of G12 V mutated DNA serially diluted with wild-type DNA (1 = 12.5%, 2 = 6.25%, 3 = 3.12%, 4 = 1.56%, 5 = 0.78%, 6 = 0.39%, 7 = 0.2%, 8 = 0.1% of mutated DNA). The antisense sequence is shown. Ab, sensitivity of the ddPCR G12S assay in discriminating different proportions of mutated alleles on serial dilutions starting from 50% up to 0.1% of the mutated allele. The respective percentages of fractional abundance obtained for each point are circled in red. (B) Melting curves and melting temperatures (Tm) of the wild-type ESR1 and ESR1 Y537S mutation [61]. (C) Sensitivity of enhanced-ice-COLD-PCR assay for ESR1 [60]. (D) Schedule of BEAMing [63]. (E) Comparison of BEAMing and NGS in detecting IDH mutation in glioma patients [65]. (F) The sensitivity of LAB-ddPCR for the detection of ctDNA T790M mutation was further improved compared to ddPCR and ARSM-PCR [68].

Figure 3

Technologies of ctDNA mutation based on enzyme and nanomaterial. (A–G) Technology based on CRISPR-Cas system. (A) method combining traditional PCR and CRISPR-Cas9 proposed by Wang [71]. (Aa) the introduced method mainly includes four steps: Preparation of templates, digestion of WT fragments by the Cas9 enzyme, PCR amplification, and Sanger sequencing or next-generation sequencing. (Ab) the PCR results of various template ratios (1/10, 1/100, 1/1000, and 1/10,000), respectively, after enrichment. The templates were mixed using mutant type DNA harboring a 15-bp deletion (c.2235_2249del) and wild-type DNA at various ratios. (Ac,Ad) the result for EGFR-exon19 15-bp deletion mutant (c.2235_2249del) and the wild-type at different ratios using Cas9/sgRNA digestion plus PCR amplification or without Cas9/sgRNA digestion and the fold increase. (B): CUT-PCR [72]. (Ba) schematic of the CUT-PCR enrichment process. (Bb) for the KRAS (c.35G4T) mutation, targeted deep sequencing after CUT-PCR was treated (red bars) or not (gray bars) were conducted for the plasmid mixtures in which mutant plasmids were originally mixed with wild-type plasmids at a ratio of from 100% to 0.01%. (C): Entropy-driven strand displacement reaction [73]. (Ca,Cb) schematic of the principle of the CRISPR/Cas9-triggered ESDR based on a 3D GR/AuPtPd nanoflower biosensor. (Cc) gel electrophoresis of the synthesized sgRNA (left) and DNA samples (right) after Cas9/sgRNA cleavage. (Cd) reproducibility of the electrochemical biosensor in different target concentrations (D): The combination method of graphene oxide screen printed electrode (GPHOXE) and dCas9 proteins and sgRNA [74]. (Da) schematic of CRISPR-dCas9 powered impedimetric biosensor. (Db) calibration curve, x-axis represents the ctDNA concentration, y-axis represents the impedance results (ohm). (E): A biosensor that was composed of a triple helix molecular switch (THMS) for recognition, ribonuclease HII, signal transduction probe (STP), capture probe fixed on the electrode, and deoxynucleotidyl transferase [76]. (Ea) schematic illustration of the dual enzyme assisted multiple amplification electrochemical biosensor. (Eb) (EbA) DPV responses for the detection of target ctDNA at concentrations of 0, 0.01 fM, 0.1 fM, 1 fM, 0.01 pM, 0.1 pM, and 1 pM (from a to g) with dual enzyme assisted multiple amplification, (EbB) Linear relationship between IMB and logarithm of target ctDNA with dual enzyme assisted multiple amplification, (EbC) DPV curves for the detection of ctDNA at concentrations of 0, 1 fM, 0.01 pM, 0.1 pM, 1 pM, and 0.01 nM (from a to f) without RNase HII-assisted target recycling amplification, (EbD) Linear relationship between IMB and logarithm of target ctDNA without RNase HII-assisted target recycling amplification. Error bars represent standard deviations of three parallel experiments. (F): A specific nucleic acid microfluidic capture device based on DNA nanomaterials [81]. Model for the flow simulation in the P-mesh microfluidic capture device. (Fa) Velocity. (Fb) Pressure. (Fc) MFI percentage of Cy5-labeled padlock probe attached on the PVDF membrane. MFI percentage of 1 μM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. MFI percentage of 1pM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. Differences between the two groups of samples were tested by t test. The level of significance was *** < 0.001. (G): A ctDNA ultrasensitive detection method dependent on the targeted recognition of the modified DNA probe on the gold-coated nanomaterials and the target fragment [82]. (Ga) Schematic illustration of DNA-mediated reduction of potassium ferricyanide (K3[Fe(CN)6]) by methylene blue (MB). (Gb) hybridization-induced change in the SWVs after exposing the sensor to different concentrations of complementary ctDNA target (101 nucleotides) wherein the probe DNA hybridized to the 3′ end (red data points) and the middle (black data points) of the ctDNA. (Gc) effect of hybridization time after exposing the sensor to 20 nM complementary ctDNA target (101 nucleotides) on the SWV current change. (H): A multiplexed ligation ctDNA single nucleotide nanopore detection method [83]. (Ha,Hb). the mutated sample exhibited clearly distinguishable optical spikes both in the red and green channels corresponding to passages of the target DNA molecules through the nanopore.

Figure 3

Technologies of ctDNA mutation based on enzyme and nanomaterial. (A–G) Technology based on CRISPR-Cas system. (A) method combining traditional PCR and CRISPR-Cas9 proposed by Wang [71]. (Aa) the introduced method mainly includes four steps: Preparation of templates, digestion of WT fragments by the Cas9 enzyme, PCR amplification, and Sanger sequencing or next-generation sequencing. (Ab) the PCR results of various template ratios (1/10, 1/100, 1/1000, and 1/10,000), respectively, after enrichment. The templates were mixed using mutant type DNA harboring a 15-bp deletion (c.2235_2249del) and wild-type DNA at various ratios. (Ac,Ad) the result for EGFR-exon19 15-bp deletion mutant (c.2235_2249del) and the wild-type at different ratios using Cas9/sgRNA digestion plus PCR amplification or without Cas9/sgRNA digestion and the fold increase. (B): CUT-PCR [72]. (Ba) schematic of the CUT-PCR enrichment process. (Bb) for the KRAS (c.35G4T) mutation, targeted deep sequencing after CUT-PCR was treated (red bars) or not (gray bars) were conducted for the plasmid mixtures in which mutant plasmids were originally mixed with wild-type plasmids at a ratio of from 100% to 0.01%. (C): Entropy-driven strand displacement reaction [73]. (Ca,Cb) schematic of the principle of the CRISPR/Cas9-triggered ESDR based on a 3D GR/AuPtPd nanoflower biosensor. (Cc) gel electrophoresis of the synthesized sgRNA (left) and DNA samples (right) after Cas9/sgRNA cleavage. (Cd) reproducibility of the electrochemical biosensor in different target concentrations (D): The combination method of graphene oxide screen printed electrode (GPHOXE) and dCas9 proteins and sgRNA [74]. (Da) schematic of CRISPR-dCas9 powered impedimetric biosensor. (Db) calibration curve, x-axis represents the ctDNA concentration, y-axis represents the impedance results (ohm). (E): A biosensor that was composed of a triple helix molecular switch (THMS) for recognition, ribonuclease HII, signal transduction probe (STP), capture probe fixed on the electrode, and deoxynucleotidyl transferase [76]. (Ea) schematic illustration of the dual enzyme assisted multiple amplification electrochemical biosensor. (Eb) (EbA) DPV responses for the detection of target ctDNA at concentrations of 0, 0.01 fM, 0.1 fM, 1 fM, 0.01 pM, 0.1 pM, and 1 pM (from a to g) with dual enzyme assisted multiple amplification, (EbB) Linear relationship between IMB and logarithm of target ctDNA with dual enzyme assisted multiple amplification, (EbC) DPV curves for the detection of ctDNA at concentrations of 0, 1 fM, 0.01 pM, 0.1 pM, 1 pM, and 0.01 nM (from a to f) without RNase HII-assisted target recycling amplification, (EbD) Linear relationship between IMB and logarithm of target ctDNA without RNase HII-assisted target recycling amplification. Error bars represent standard deviations of three parallel experiments. (F): A specific nucleic acid microfluidic capture device based on DNA nanomaterials [81]. Model for the flow simulation in the P-mesh microfluidic capture device. (Fa) Velocity. (Fb) Pressure. (Fc) MFI percentage of Cy5-labeled padlock probe attached on the PVDF membrane. MFI percentage of 1 μM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. MFI percentage of 1pM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. Differences between the two groups of samples were tested by t test. The level of significance was *** < 0.001. (G): A ctDNA ultrasensitive detection method dependent on the targeted recognition of the modified DNA probe on the gold-coated nanomaterials and the target fragment [82]. (Ga) Schematic illustration of DNA-mediated reduction of potassium ferricyanide (K3[Fe(CN)6]) by methylene blue (MB). (Gb) hybridization-induced change in the SWVs after exposing the sensor to different concentrations of complementary ctDNA target (101 nucleotides) wherein the probe DNA hybridized to the 3′ end (red data points) and the middle (black data points) of the ctDNA. (Gc) effect of hybridization time after exposing the sensor to 20 nM complementary ctDNA target (101 nucleotides) on the SWV current change. (H): A multiplexed ligation ctDNA single nucleotide nanopore detection method [83]. (Ha,Hb). the mutated sample exhibited clearly distinguishable optical spikes both in the red and green channels corresponding to passages of the target DNA molecules through the nanopore.

Figure 3

Technologies of ctDNA mutation based on enzyme and nanomaterial. (A–G) Technology based on CRISPR-Cas system. (A) method combining traditional PCR and CRISPR-Cas9 proposed by Wang [71]. (Aa) the introduced method mainly includes four steps: Preparation of templates, digestion of WT fragments by the Cas9 enzyme, PCR amplification, and Sanger sequencing or next-generation sequencing. (Ab) the PCR results of various template ratios (1/10, 1/100, 1/1000, and 1/10,000), respectively, after enrichment. The templates were mixed using mutant type DNA harboring a 15-bp deletion (c.2235_2249del) and wild-type DNA at various ratios. (Ac,Ad) the result for EGFR-exon19 15-bp deletion mutant (c.2235_2249del) and the wild-type at different ratios using Cas9/sgRNA digestion plus PCR amplification or without Cas9/sgRNA digestion and the fold increase. (B): CUT-PCR [72]. (Ba) schematic of the CUT-PCR enrichment process. (Bb) for the KRAS (c.35G4T) mutation, targeted deep sequencing after CUT-PCR was treated (red bars) or not (gray bars) were conducted for the plasmid mixtures in which mutant plasmids were originally mixed with wild-type plasmids at a ratio of from 100% to 0.01%. (C): Entropy-driven strand displacement reaction [73]. (Ca,Cb) schematic of the principle of the CRISPR/Cas9-triggered ESDR based on a 3D GR/AuPtPd nanoflower biosensor. (Cc) gel electrophoresis of the synthesized sgRNA (left) and DNA samples (right) after Cas9/sgRNA cleavage. (Cd) reproducibility of the electrochemical biosensor in different target concentrations (D): The combination method of graphene oxide screen printed electrode (GPHOXE) and dCas9 proteins and sgRNA [74]. (Da) schematic of CRISPR-dCas9 powered impedimetric biosensor. (Db) calibration curve, x-axis represents the ctDNA concentration, y-axis represents the impedance results (ohm). (E): A biosensor that was composed of a triple helix molecular switch (THMS) for recognition, ribonuclease HII, signal transduction probe (STP), capture probe fixed on the electrode, and deoxynucleotidyl transferase [76]. (Ea) schematic illustration of the dual enzyme assisted multiple amplification electrochemical biosensor. (Eb) (EbA) DPV responses for the detection of target ctDNA at concentrations of 0, 0.01 fM, 0.1 fM, 1 fM, 0.01 pM, 0.1 pM, and 1 pM (from a to g) with dual enzyme assisted multiple amplification, (EbB) Linear relationship between IMB and logarithm of target ctDNA with dual enzyme assisted multiple amplification, (EbC) DPV curves for the detection of ctDNA at concentrations of 0, 1 fM, 0.01 pM, 0.1 pM, 1 pM, and 0.01 nM (from a to f) without RNase HII-assisted target recycling amplification, (EbD) Linear relationship between IMB and logarithm of target ctDNA without RNase HII-assisted target recycling amplification. Error bars represent standard deviations of three parallel experiments. (F): A specific nucleic acid microfluidic capture device based on DNA nanomaterials [81]. Model for the flow simulation in the P-mesh microfluidic capture device. (Fa) Velocity. (Fb) Pressure. (Fc) MFI percentage of Cy5-labeled padlock probe attached on the PVDF membrane. MFI percentage of 1 μM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. MFI percentage of 1pM Cy5-labeled ssDNA captured by the P-mesh microfluidic capture device after storage over 6 months. Differences between the two groups of samples were tested by t test. The level of significance was *** < 0.001. (G): A ctDNA ultrasensitive detection method dependent on the targeted recognition of the modified DNA probe on the gold-coated nanomaterials and the target fragment [82]. (Ga) Schematic illustration of DNA-mediated reduction of potassium ferricyanide (K3[Fe(CN)6]) by methylene blue (MB). (Gb) hybridization-induced change in the SWVs after exposing the sensor to different concentrations of complementary ctDNA target (101 nucleotides) wherein the probe DNA hybridized to the 3′ end (red data points) and the middle (black data points) of the ctDNA. (Gc) effect of hybridization time after exposing the sensor to 20 nM complementary ctDNA target (101 nucleotides) on the SWV current change. (H): A multiplexed ligation ctDNA single nucleotide nanopore detection method [83]. (Ha,Hb). the mutated sample exhibited clearly distinguishable optical spikes both in the red and green channels corresponding to passages of the target DNA molecules through the nanopore.

Figure 4

Novel ctDNA methylation technologies. (A): Discrimination of Rare EpiAlleles by Melt developed by Thomas. et al. [87]. (Aa) DREAMing: analysis of epigenetic heterogeneity at single-copy sensitivity and single-CpG-site resolution. DNA is extracted from a liquid biopsy and undergoes bisulfite treatment (BST). (Ab,Ac) DREAMing primers optimized for high sensitivity. (Ab) for the p14ARF locus at various genomic DNA methylated total epiallelic fractions. (Ac) for the BRCA1 locus at various genomic DNA methylated total epiallelic fractions. Both assays exhibit sensitivities that provide detection of epiallelic fractions of 0.01% or lower. (B): A method based on pyrosequencing and digital microfluidics [89]. (Ba) schematic representation of the DNA methylation analysis based on DMF. (Bb) histogram of the signal intensities of T/C at various methylation levels for the first methylation site. (Bc) Linear relationship for various levels of input methylation levels for the first methylation site. Data are presented as mean ± SD from triplicate samples. (C): DISMIR based on ultra-low-depth WGBS data [88]. (Ca) Overview of DISMIR. (Cb) results of DISMIR and other methods on HCC diagnosis. (D): Dual-recognition-based determination grounded on peptide nucleic acid (PNA) and terminal protection of small-molecule-linked DNA (TPSMLD) [90]. (Da) the mechanism of the dual-recognition fluorescence biosensor for E542K-ds-ctDNA. (Db) fluorescence spectra of the dual-recognition fluorescence biosensor upon the addition of increasing concentration of E542K-dsctDNA. (Dc) calibration curve for E542K-ds-ctDNA detection. (Dd) bar chart of the fluorescent intensities in the presence of different DNA sequences.

Figure 4

Novel ctDNA methylation technologies. (A): Discrimination of Rare EpiAlleles by Melt developed by Thomas. et al. [87]. (Aa) DREAMing: analysis of epigenetic heterogeneity at single-copy sensitivity and single-CpG-site resolution. DNA is extracted from a liquid biopsy and undergoes bisulfite treatment (BST). (Ab,Ac) DREAMing primers optimized for high sensitivity. (Ab) for the p14ARF locus at various genomic DNA methylated total epiallelic fractions. (Ac) for the BRCA1 locus at various genomic DNA methylated total epiallelic fractions. Both assays exhibit sensitivities that provide detection of epiallelic fractions of 0.01% or lower. (B): A method based on pyrosequencing and digital microfluidics [89]. (Ba) schematic representation of the DNA methylation analysis based on DMF. (Bb) histogram of the signal intensities of T/C at various methylation levels for the first methylation site. (Bc) Linear relationship for various levels of input methylation levels for the first methylation site. Data are presented as mean ± SD from triplicate samples. (C): DISMIR based on ultra-low-depth WGBS data [88]. (Ca) Overview of DISMIR. (Cb) results of DISMIR and other methods on HCC diagnosis. (D): Dual-recognition-based determination grounded on peptide nucleic acid (PNA) and terminal protection of small-molecule-linked DNA (TPSMLD) [90]. (Da) the mechanism of the dual-recognition fluorescence biosensor for E542K-ds-ctDNA. (Db) fluorescence spectra of the dual-recognition fluorescence biosensor upon the addition of increasing concentration of E542K-dsctDNA. (Dc) calibration curve for E542K-ds-ctDNA detection. (Dd) bar chart of the fluorescent intensities in the presence of different DNA sequences.