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Circulating tumor DNA in blood: Future genomic biomarkers for cancer detection

Open AccessPublished:June 23, 2018DOI:https://doi.org/10.1016/j.exphem.2018.06.003

      Highlights

      • Tumor heterogeneity causes significant challenges in designing cancer therapies.
      • Cell free circulating tumor DNA (ctDNA) can monitor tumor burden, therapy response, and relapse.
      • Liquid biopsy can be a future marker used in the diagnosis of cancer.
      • Integrated digital error suppression shows promise in the detection of ctDNA in cancer.
      Cancer is characterized by Darwinian evolution and is a primary cause of mortality and morbidity around the globe. Over the preceding decade, the treatment of cancer has been markedly improved by many targeted therapies, but these treatments have given birth to new challenges and issues. Clonal evolution and tumor heterogeneity present a significant challenge in designing cancer therapies. Fortunately, these restrictions have been overcome by technological advancements allowing us to track both genetic and epigenetic aberrations. Cell-free circulating tumor DNA (ctDNA) analysis, or “liquid biopsy” from a blood sample, provides the opportunity to track the genetic landscape of cancerous lesions. This review focuses on ctDNA analysis as a noninvasive method and versatile biomarker for cancer treatment and technological advancements for ctDNA analysis. This method may able to cope with all the challenges associated with previous cancer therapies and has the potential to monitor minimal residual disease, tumor burden, and therapy response and provide rapid detection of relapse. However, there are many challenges that still need to be addressed. Future prognosis, diagnosis, and analysis of ctDNA require reproducibility and accuracy of results, which are not possible without the validation and optimization of procedures. Integrated digital error suppression has thus far shown promise in the detection of ctDNA in cancer.
      If complexity is defined, cancer is at the top hits. In 2012, 8.2 million reported deaths and 14 million new cases of cancer illustrate that it is the primary cause of mortality and morbidity around the globe. This number is expected to rise by 70% in the next 20 years, which is an alarming situation [
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ,

      Stewart B, Wild CP. World cancer report 2014. Health 2017.

      ,
      • Weir HK
      • Thompson TD
      • Soman A
      • Møller B
      • Leadbetter S
      The past, present, and future of cancer incidence in the United States: 1975 through 2020.
      ]. Approximately 90% of cancer-associated mortality is caused by metastasis, a process in which cells from the primary tumor mass move to a distant site, where they proliferate and form secondary tumors. This process involves a series of events in which cells from the primary tumor invade the surrounding tissues and move into the microvasculature of blood and lymph. After this, cells pass through the bloodstream to microvessels of distant tissues, exit the bloodstream, and survive in the microenvironment of tissues, resulting in cell proliferation and the formation of secondary tumors (Figure 1) [
      • Brooks SA
      • Lomax-Browne HJ
      • Carter TM
      • Kinch CE
      • Hall DM
      Molecular interactions in cancer cell metastasis.
      ].
      Fig 1
      Figure 1Illustration of cancer-transformed cells leading to clonal expansion and formation of cancerous subclones and invasion into the basement membrane, followed by intravasation and entry into the blood. Afterward, tumor cells extravasate to colonize at a distant site and start angiogenesis of metastatic clone, resulting in secondary tumors.
      Over the preceding decade, the treatment of cancer has been markedly improved by adjuvant systemic treatments, surgery, solid biopsy, and many other targeted therapies, but these treatments have resulted in many new issues and challenges. Some of the challenges faced by medical practitioners include potential morbidity of biopsies, lack of effective drugs against most genomic aberrations, cost, technical limitations of molecular tests, and reimbursement and regulatory hurdles. Apart from these, clonal evolution and tumor heterogeneity present a significant challenge in the design of cancer therapies [
      • Valastyan S
      • Weinberg RA
      Tumor metastasis: molecular insights and evolving paradigms.
      ].
      Different tumor cells vary in their gene expression, proliferation, metastatic potential, motility, metabolism, and cellular morphology [
      • Marusyk A
      • Polyak K
      Tumor heterogeneity: causes and consequences.
      ]. Intratumoral heterogeneity in primary tumors, with spatially separated chromosomal imbalances and somatic mutations, offers a serious challenge for cancer therapies because therapeutically associated lesions could be overlooked during the examination of tissue biopsy as a part of tumor mass [
      • Gerlinger M
      • Rowan AJ
      • Horswell S
      • et al.
      Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.
      ]. Although clonal evolution in primary metastasis is partly stimulated by intratumoral heterogeneity, under the selection pressure resulting from anticancer treatments, clonal evolution continues to occur even after metastasis has developed [
      • Aparicio S
      • Caldas C
      The implications of clonal genome evolution for cancer medicine.
      ]. Sequential monitoring of tumor genome through molecularly targeted cancer treatments such as tumor biopsy is costly, invasive, related to potential morbidity, and not sufficient to monitor tumor heterogeneity and clonal evolution. However, there is a dire need to develop methods for detecting tumor heterogeneity and clonal evolution because these are often not detected by conventional methods [
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ].
      Recent advancements in cancer therapies have resulted in a promising noninvasive method based on the detection of circulating cell-free tumor DNA (ctDNA) from the blood sample, commonly referred to as a liquid biopsy. This is a noninvasive, rapid, and cost effective method for cancer detection and can act as a potential biomarker for cancer treatment. Liquid biopsies have several advantages over conventional tissue biopsies for cancer screening (Table 1) [
      • McLarty J
      • Yeh C
      Circulating cell-free DNA: The blood biopsy in cancer management.
      ]. Liquid biopsy is dominant over tissue biopsy because a tumor comprising approximately 5000 malignant cells is sufficient for ctDNA detection; for comparison, computerized tomography requires a tumor size no less than 7–10 mm containing one billion malignant cells [
      • Sholl LM
      • Aisner DL
      • Allen TC
      • et al.
      Liquid biopsy in lung cancer: A perspective from members of the Pulmonary Pathology Society.
      ].
      Table 1Comparison between blood biopsy and tissue biopsy
      Key CharacteristicsBlood Biopsy>Tissue BiopsyReference
      InvasivenessNoYes
      • Sacher AG
      • Paweletz C
      • Dahlberg SE
      • et al.
      Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer.
      Sample Availability Throughout the Disease ProcessYesNo
      • Oxnard GR
      • Paweletz CP
      • Kuang Y
      • et al.
      Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.
      Sample Stability When Maintained Ex VivoYesStable when processed
      • Oxnard GR
      • Paweletz CP
      • Kuang Y
      • et al.
      Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.
      Utility for Longitudinally Disease MonitoringYesNo
      • Reck M
      • Kaiser R
      • Mellemgaard A
      • et al.
      Docetaxel plus nintedanib versus docetaxel plus placebo in patients with previously treated non-small-cell lung cancer (LUME-Lung 1): a phase 3, double-blind, randomised controlled trial.
      Global Molecular Status of PatientYesNo
      • Crowley E
      • Di Nicolantonio F
      • Loupakis F
      • Bardelli A
      Liquid biopsy: monitoring cancer-genetics in the blood.
      CostLowHigh
      • Yang JC
      • Wu YL
      • Schuler M
      • et al.
      Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials.
      Processing TimeShortLong (involvement of tissue sectioning, staining and pathologists)
      • Yang JC
      • Wu YL
      • Schuler M
      • et al.
      Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials.
      Rejection/Failure RateLowHigh (due to QNS or TNI)
      • Reck M
      • Kaiser R
      • Mellemgaard A
      • et al.
      Docetaxel plus nintedanib versus docetaxel plus placebo in patients with previously treated non-small-cell lung cancer (LUME-Lung 1): a phase 3, double-blind, randomised controlled trial.
      Starting Material for Multiple TestingSufficeScarce
      • Sacher AG
      • Paweletz C
      • Dahlberg SE
      • et al.
      Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer.

      Sources of tumor DNA: circulating tumor cells and ctDNA

      Tumor cell sheds two types of material in the blood that are susceptible to comprehensive molecular analysis: intact circulating tumor cells (CTCs) and ctDNA [
      • Haber DA
      • Velculescu VE
      Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA.
      ]. According to a previous study, CTCs are less sensitive in detecting tumor-associated genetic rearrangements than ctDNA because the number of CTCs is very low in the blood (Speicher & Pantel, 2014). ctDNA is isolated from the supernatant, whereas CTCs are isolated from the cell pellet, which, in addition to CTCs, also contain platelets, white blood cells, and other fragments, resulting in the dilution of CTCs (one cell per 109 normal blood cells) and making detection more difficult. Therefore, for CTCs, an enrichment step with advanced PCR approaches is required to detect cancer-related changes using highly sensitive techniques [
      • Alix-Panabieres C
      • Schwarzenbach H
      • Pantel K
      Circulating tumor cells and circulating tumor DNA.
      ].

      Origin and biology of ctDNA

      ctDNA is a fraction of extracellular nucleic acid (cell-free DNA) that is released into the bloodstream by tumor cells. It was first discovered by two French biochemists, Mandel and Metais, in 1948 in the serum of healthy individuals [
      • Riva F
      • Dronov OI
      • Khomenko DI
      • et al.
      Clinical applications of circulating tumor DNA and circulating tumor cells in pancreatic cancer.
      ]. It is reported that cell-free DNA enters into the blood circulation under normal physiological and pathological conditions. In normal physiological conditions, phagocytes destroy the apoptotic and necrotic cells, thereby fragmenting the DNA into nucleosomal units, but the level of ctDNA remains low. Tumor cells shed their DNA as a result of apoptosis, necrosis, autophagy, and a number of other mechanisms, but the exact mechanism of release remains unclear [
      • Jung K
      • Fleischhacker M
      • Rabien A
      Cell-free DNA in the blood as a solid tumor biomarker: A critical appraisal of the literature.
      ].
      Cell-free DNA fragments generated through apoptosis are between 180 and 200 base pairs, whereas necrosis releases fragments of large size as a result of incomplete and random digestion of genomic DNA. ctDNA has half-life ranges from 15 minutes to few hours and is rapidly cleared away by kidney, liver, and spleen. Various physiological filtering events (clearance, degradation, etc.) of the lymphatic system and blood determine the amount of ctDNA in the blood circulation [
      • Schwarzenbach H
      • Hoon DS
      • Pantel K
      Cell-free nucleic acids as biomarkers in cancer patients.
      ]. The level of ctDNA is much higher in cancer patients than in normal individuals, but the level varies widely among different patients, ranging from about 0.01% to more than 90%. These variations in the level of ctDNA among cancer patients are due to tumor burden, stage, vascularity, cellular turnover, and response to therapy [
      • Elshimali YI
      • Khaddour H
      • Sarkissyan M
      • Wu Y
      • Vadgama JV
      The clinical utilization of circulating cell-free DNA (CCFDNA) in blood of cancer patients.
      ]. ctDNA is characterized by the presence of cancer-related genetic and epigenetic mutations such as point mutations, rearranged genomic sequences, copy number variation (CNV), microsatellite instability (MSI), loss of heterozygosity (LOH), and DNA methylation. These characteristics are responsible for distinguishing them from remaining cell-free DNA fraction, thus allowing their use as potential biomarkers for determining disease state, progression, and recurrence [
      • Marzese DM
      • Hirose H
      • Hoon DS
      Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients.
      ].
      In cancer patients, ctDNA releases from all tumor deposits without being affected by intratumoral heterogeneity than a single specimen of tumor tissue. The quantity of detectable ctDNA fraction is lower in localized diseases but higher in metastatic diseases [
      • Riva F
      • Dronov OI
      • Khomenko DI
      • et al.
      Clinical applications of circulating tumor DNA and circulating tumor cells in pancreatic cancer.
      ]. For ctDNA analysis, ctDNA can be isolated both from plasma and serum. The quantity of cell-free DNA can be two to four times higher in serum than in plasma, but it is not recommended as a preferred source for ctDNA because of its contamination from cells during the clotting process. It is suggested to use plasma having lower concentrations of background wild-type DNA as a source of ctDNA [
      • Heitzer E
      • Ulz P
      • Geigl JB
      Circulating tumor DNA as a liquid biopsy for cancer.
      ].

      Technologies for ctDNA analysis

      The presence of a small fraction of tumor-specific DNA along, with a larger fraction of wild-type cell-free DNA, makes its isolation and detection difficult, emphasizing the need for highly sensitive techniques (Figure 2). Classical approaches for the analysis of cell-free DNA (cfDNA) were based on quantitative real-time polymerase chain reaction (qRT-PCR), fluorescence, and spectrophotometry [
      • Ignatiadis M
      • Dawson SJ
      Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?.
      ].
      Fig 2
      Figure 2Schematic representation illustrating the potential of liquid biopsy to monitor cancer. Cell-free DNA is released through multiple mechanisms (e.g., necrosis, apoptosis) from different healthy and tumorous tissues. ctDNA can be isolated from the plasma and various genetic mutations can be detected. Variable amounts of ctDNA can be detected using different techniques. Liquid biopsy may prove to be a diagnostic tool for cancer diagnosis, detection of minimal residual disease, therapy monitoring, and prognosis.

      qRT-PCR

      In qRT-PCR, the signal emitted from the fluorescently labeled probe during amplification of the target gene allows the estimation of the nucleic acid concentration. The concentration of cfDNA has been measured by amplification of several genes such as human telomerase reverse transcriptase and betaglobin [
      • Pinzani P
      • Salvianti F
      • Zaccara S
      • et al.
      Circulating cell-free DNA in plasma of melanoma patients: qualitative and quantitative considerations.
      ]. It is a reliable method for the measurement and detection of amplicon produced during each PCR cycle. The target sequence was hybridized with the oligonucleotide probe designed to have a quencher dye at the 3′end and a reporter fluorescent dye at the 5′ end, thereby eliminating the need for post-PCR processing [
      • Lee SJ
      • Li Z
      • Sherman B
      • Foster CS
      Serum levels of tumor necrosis factor-alpha and interleukin-6 in ocular cicatricial pemphigoid.
      ]. Initially, when probe binds to the PCR product, the fluorescence emitted by the reporter dye significantly decreases due to close proximity of quencher dye. The fluorescence signal is radiated only when probe is degraded into smaller fragments due to exonuclease activity of Thermus aquaticus (i.e., Taq) DNA polymerase enzyme following the fluorescence resonance energy transfer principle [
      • Arya M
      • Shergill IS
      • Williamson M
      • Gommersall L
      • Arya N
      • Patel HR
      Basic principles of real-time quantitative PCR.
      ]. Early-phase detection has distinct advantages over the traditional method because end-stage detection confers low sensitivity, poor precision, and minimal resolution and requires a manual procedure and the use of agarose gel electrophoresis for the identification of amplicons. In addition to that, it is time consuming and gives variable results based on size discrimination, which cannot be analyzed through agarose gel electrophoresis [
      • Niesters HG
      Quantitation of viral load using real-time amplification techniques.
      ].

      Methylation-specific PCR

      Methylation-specific PCR is a technique in which the amplification of the gene of interest through PCR is carried out to determine the status of CpG islands. The primers used in this technique are specific for methylated or non-methylated DNA sequence bisulfite modification. The combination of both real-time PCR and methylation-specific PCR helps in the analysis of the methylation pattern of different genes, as well as in the quantification of methylated cfDNA sequences [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].

      Digital PCR

      Another sensitive technique is digital PCR, which was developed to detect mutations that are present in low allele fraction. To increase the extent of conventional PCR, Vogelstein and Kinzler developed another sensitive technique called as digital PCR [
      • Vogelstein B
      • Kinzler KW
      Digital PCR.
      ]. In this approach, single molecules are isolated after making serial dilutions of DNA and are then examined for the presence of mutations [
      • Baker M
      Digital PCR hits its stride.
      ]. Amplification of PCR template (single copy) is achieved by running PCR at its optimum conditions. Hybridization of fluorescently labeled probes with the amplicons permits the detection of sequence-specific PCR product. Therefore, the number of both alleles in the sample (maternal and paternal) can be counted directly using digital PCR [
      • Pohl G
      • Shih Ie M
      Principle and applications of digital PCR.
      ]. Through this technique, alterations in PIK3CA mutations in breast cancer were screened in one study and yielded 93.3% sensitivity and 100% specificity. The technique of digital PCR is used in a number of different methods including droplet-based systems, microfluidic platforms, and beads, emulsions, amplification and magnetics (BEAMing) [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].

      Next-generation sequencing

      Next-generation sequencing (NGS) has emerged as a comprehensive technique for the analysis of ctDNA, allowing screening of mutations across wider genomic regions in many cancer types. Targeted deep-sequencing methods based on either PCR or capture-based techniques such as TAm-Seq, Safe-Seq, Ion AmpliSeq, and cancer personalized profiling by deep sequencing (CAPPSEQ) have also been developed and utilized to determine the sequence of specified genomic regions in plasma DNA. In addition, whole-exome analysis of plasma DNA has provided a new way to broadly characterize mutation profiles [
      • Ignatiadis M
      • Dawson SJ
      Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?.
      ]. NGS or massively parallel sequencing involves the amplification and sequencing of billions of DNA fragments simultaneously instead of serially amplifying and sequencing the individual clones. A number of different platforms are currently available for massively parallel sequencing, but the principle behind all of them is the same. The first step is library preparation for which DNA is fragmented into lengths of 500 bp, followed by ligation of adaptors [
      • Liu T
      • Yu L
      • Liu L
      • Li H
      • Li Y
      Comparative transcriptomes and EVO-DEVO studies depending on next generation sequencing.
      ]. After library preparation, the clones are amplified by either bridge amplification or emulsion PCR in order to produce multiple copies of individual clones [
      • Metzker ML.
      Sequencing technologies: The next generation.
      ]. The billions of clones generated are then sequenced simultaneously using different platforms. The reversible dye terminator sequencing method is most commonly used for NGS. The sequence of clusters generated is then aligned to a reference genome and the data generated are finally transformed into a BAM file for further detailed analysis. Massively parallel sequencing (MPS) is a fast, cost-effective, and high-throughput approach that can revolutionize the field of human genome analysis, particularly the diagnosis of cancer, by providing deeper insights into the hidden complexities of tumor profiles [
      • Hagemann IS
      • Cottrell CE
      • Lockwood CM
      Design of targeted, capture-based, next generation sequencing tests for precision cancer therapy.
      ].

      MPS

      The MPS approach has also been used for assaying the ctDNA isolated from primary and metastatic tumors. For this purpose, plasma samples were taken from a 66-year-old breast cancer patient. Fifteen mutations were discovered in primary and metastatic tumors, but two mutations were detected only in metastatic tumors. The heterogeneity of tumors has been attributed due to the all these variation that were detected in ctDNA [
      • De Mattos-Arruda L
      • Weigelt B
      • Cortes J
      • et al.
      Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle.
      ]. Early indications of disease progression can be obtained by ctDNA analysis rather than by radiologic and biochemical evaluations by scanning the mutant allele fractions of ctDNA during treatment. The sequencing analysis of ctDNA of metastatic cancer patients receiving treatment has showed that the fraction of mutant alleles rises with increase in therapy resistance [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].

      Personalized analysis of rearrangement ends

      For the detection of chromosomal rearrangements in ctDNA, a method called personalized analysis of rearrangement ends (PARE) was developed. This method provides considerable sensitivity and specificity for the use of ctDNA as biomarker. There are certain limitations of this technique, including that its sensitivity depends on the amount of sequence data, cost, false-positive results, and absence of information about the source of ctDNA. Through the advancements and improvements in the sensitivity of genomic approaches, NGS will play a significant role in the analysis of ctDNA [
      • Leary RJ
      • Sausen M
      • Kinde I
      • et al.
      Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing.
      ].

      CAPPSEQ

      CAPPSEQ is a recently developed technique for highly sensitive ctDNA quantification that overcomes a number of technical limitations related to the previous techniques. CAPPSEQ is a capture-based NGS technique for ctDNA detection covering multiple mutations classes, including insertions, deletions, copy number variations, and rearrangements. In this approach, deep sequencing of hundreds of captured genomic regions is carried out with the subsequent application of different bioinformatics tools for the assessment of the tumor genomic profiles. The detection limit of this method is 0.02% in cfDNA and ctDNA from patients in both the initial and advanced stages of cancer [
      • Newman AM
      • Bratman SV
      • To J
      • et al.
      An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
      ]. The overall cost of the technique per assay is $200–$400 and there will be further reduction in costs in coming years [
      • Bettegowda C
      • Sausen M
      • Leary RJ
      • et al.
      Detection of circulating tumor DNA in early- and late-stage human malignancies.
      ]. Through this technique, frequently mutated genes have been detected by using a probe panel and exploiting whole-exome sequencing data for lung cancer patients from the Cancer Genome Atlas. The probe panel was targeted at 521 exons and 13 introns from 139 recurrently mutated genes analyzing the ctDNA of stage II–IV non-small-cell lung cancer (NSCLC) patients. For the mutant allele fraction that occurs in a low percentage of patients, this method achieved a specificity of 96% [
      • Newman AM
      • Bratman SV
      • To J
      • et al.
      An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
      ].
      In another study, NGS of plasma and tumor DNA detected mutations in the genes p53, PIK3CA, PTEN, AKT1, IDH2, and SMAD4. Mutation allele frequency in plasma may be less than the detection limit of the method used. In addition, mutations were detected in the plasma and not in tumor DNA in another two samples [
      • Mohan S
      • Heitzer E
      • Ulz P
      • et al.
      Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.
      ]. These mutations were not identified by biopsy in tumor clones. Whole-genome sequencing of cfDNA has the potential to detect new mutations that cannot be detected by targeted sequencing, but is limited by its cost and the requirement for expertise to analyze the large amount of data generated. To avoid this problem, sequencing of exonic regions of the genes that are mostly mutated is recommended [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].

      BEAMing

      Another important technique is BEAMing, which was designed to detect mutations in ctDNA [
      • Richardson AL
      • Iglehart JD
      BEAMing up personalized medicine: mutation detection in blood.
      ]. This technique uses the primers having known tags sequences to amplify the DNA segment of interest. The DNA sequences are amplified again by emulsion PCR using primers targeted against the tags. After this, the DNA sequences are covalently attached to magnetic beads, followed by the addition of the labeled nucleotides to the sequences. Finally, with the help of flow cytometry, beads containing mutant allele are separated from those containing wild-type allele [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ]. This technique has the ability to identify and measure only one mutated DNA from 10,000 DNA molecules, providing increased sensitivity. This technique has also been used to identify APC, EGFR, and PIK3CA variations in CRC, lung cancer, and breast cancer, respectively [
      • Richardson AL
      • Iglehart JD
      BEAMing up personalized medicine: mutation detection in blood.
      ].

      Peptide nucleic acid clamp-mediated PCR

      Peptide nucleic acid (PNA) is a synthetic nucleic acid analog consisting of peptide backbone with N-(2-aminoethyl)-glycine units in place of phosphodiester backbone. PNA complementary to wild-type sequences function as clamp in PNA-mediated PCR reactions [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ]. The presence of a mismatch differentiates between a wild-type sequence and a mutated sequence. PNA-mediated PCR clamping is based on the competitive binding of PNA or PCR primer to the common target site. PNAs have more affinity for the wild-type sequences and their binding favors the impairment of sequence amplification because of the inability of DNA polymerase to recognize them as primers. In contrast to PNAs, the PCR primers specific for the target sequences only bind to the mutated sequences and are then further amplified [
      • Orum H
      • Nielsen PE
      • Egholm M
      • Berg RH
      • Buchardt O
      • Stanley C
      Single base pair mutation analysis by PNA directed PCR clamping.
      ]. The important parameters for proper functioning of clamping PCR are the concentration of both PNAs and DNA primer, thermal stability of the PNA and DNA for their respective target sites, and the kinetics of PNA and DNA hybridization [
      • Giesen U
      • Kleider W
      • Berding C
      • Geiger A
      • Orum H
      • Nielsen PE
      A formula for thermal stability (Tm) prediction of PNA/DNA duplexes.
      ]. This PCR approach facilitates the detection of single-base-pair gene variants for mutation screening and gene isolation. PNA-clamp-mediated PCR has been used for the detection of EGFR variants in NSCLC patients. Limitations of this method are sensitivity and time. KRAS codon 12 variants in pancreatic cancer patients were identified by performing PCR clamping, along with melting curve analysis, giving a sensitivity of about 1–5 × 105 [
      • Kim HR
      • Lee SY
      • Hyun DS
      • et al.
      Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping.
      ].
      Although the techniques of PCR and BEAMing have proven to be sensitive, they require prior information about variants in patients, whereas the technique of NGS does not, which helps in the analysis of different cancer-related genetic mutations in the blood. The detection of genetic alterations in the circulating tumor DNA through NGS will open up new avenues in the field of cancer therapy (Table 2) [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].
      Table 2Technologies for circulating tumor DNA (ctDNA) detection
      Principle of detectionMethodType of alterationAdvantagesLimitations
      PCR-basedNested real-time PCR

      ARMS/Scorpian PCR-SSCP

      Mutant allele-specific PCR

      Mass spectrometry
      Known point mutations such as KRAS, EGFR and PK3CA hotspot alterationsEase of use, lowest costLower sensitivity, only detect limited genetic loci
      Digital PCRBEAMing

      Droplet-based digital PCR

      Microfluidic digital PCR
      Known point mutations, genetic rearrangementsHigh sensitivityOnly detect limited genetic loci
      Targeted Deep SequencingSafeSeq

      TamSeq

      Ion-AmpliSeq

      CAPPSEQ
      Selected SNVs, CNVs and rearrangements across targeted regionsHigh sensitivity, relatively inexpensiveLess comprehensive than WES methods
      Whole-Exome SequencingDigital karyotyping

      PARE
      Genome-wide SNVs, CNVs and rearrangementsBroad applicationExpensive
      SNV = single-nucleotide variant.

      CancerSEEK

      Many scientists endeavored for a long time to establish a single test for earlier detection of various cancers, which is key to reducing cancer-linked mortality significantly. Recently, Cohen et al. succeeded in developing a noninvasive blood test, cancerSEEK, which successfully diagnosed the eight most common benign tumors in 1005 patients clinically detected with localized liver, stomach, ovary, pancreas, colorectum, esophagus, breast, and lung cancers. This test is based upon detection of circulating proteins and mutations in cell-free DNA and scored high sensitivity (69–98%) and specificity (>98%). This innovation opened up a new vista for cancer detection and developing screening tests for average-risk patients [
      • Jiang Y
      • Wang D
      Liquid biopsy in the OMICS era of tumor medicine.
      ].

      Potential applications of ctDNA in clinical oncology

      Liquid biopsies are a promising tool in the field of cancer care research because they allow researchers to trace the tumor-specific mutations and thus facilitate the development of targeted therapies for individual patients (Table 3). Periodic measurement of ctDNA levels in plasma/serum can also help to follow the progression of metastatic disease and monitor the efficacy of therapeutic treatments [
      • Heitzer E
      • Ulz P
      • Geigl JB
      Circulating tumor DNA as a liquid biopsy for cancer.
      ]. Therefore, ctDNA offers a convenient, repeatable, and noninvasive “liquid biopsy” to detect molecular markers that reflect tumor burden. These methylated biomarkers serve a prognostic and/or predictive function, which is useful for predicting outcome and monitoring treatment in various cancers [
      • Fackler MJ
      • Bujanda ZL
      • Umbricht C
      • et al.
      Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.
      ].
      Table 3Potential application of ctDNA in clinical oncology
      Cancer ScreeningLocalized CancerMetastatic CancerRefractory Cancer
      Early diagnosis and early interventionIdentifying specific genomic alterations to guide therapeutic selection, monitoring tumor burden and therapeutic responses, detecting minimal residual disease, assessing risk of dissemination and recurrenceEarly identification of relapse and treatment resistance, guidance of treatment selection, monitoring therapeutic responsesUnderstanding mechanism of resistance, determining new treatment
      Similar to the mutational spectrum of tumors, ctDNA harbors genetic and epigenetic alterations and these characteristic mutations in ctDNA ensure their usefulness as specific biomarkers in personalized diagnosis and management of metastatic disease. High-throughput analysis and ease of collection are the profound characteristics of ctDNA analysis. In contrast to CTCs, ctDNA genotyping has enormous clinical applications because it is rapid, economical, and reliable [
      • Ignatiadis M
      • Dawson SJ
      Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?.
      ], as shown in Figure 3.
      Fig 3
      Figure 3Schematic representation of ctDNA analysis and clinical implications using NGS. A specimen taken from either plasma or tumor tissue can be subjected to NGS. Then whole-exome sequencing or targeted sequences in ctDNA isolated from plasma can be performed. Genetic variations can be assessed for prospective clinical utilization and prognosis. Mutations can be identified in patient's plasma for ctDNA detection at the time of diagnosis or later after surgery.

      Tumor genotyping: detection of genetic and epigenetic alterations

      Because ctDNA represents the entire epitome of mutations present in primary and metastatic tumors, blood-based tumor genotyping assays using ctDNA will contribute greatly to the development of personalized treatment of various types of cancer (Figure 4). Some of these variations in tumor cells make cancer patients resistant to the effects of therapeutic drugs. Therefore, ctDNA analysis would allow us to improve the efficacy of treatment by testing the patients for drug-resistant mutations [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ].
      Fig 4
      Figure 4Prospective clinical applications of ctDNA analysis. Cancer cells release cell-free tumor DNA and tumor cells into circulation, which can be used for scrutiny, early identification of tumors, selection of auxiliary therapy, and to determine disease progression/resistance to proposed therapy.

      Detection of tumor-specific mutations and CNVs

      In the past 20 years, various mutations have been detected in ctDNA of individuals with different types of cancers. Whole-genome sequencing analysis of ctDNA has disclosed the presence of single nucleotide variants (SNVs) and CNVs in advanced-stage cancer patients. Identification of genetic and epigenetic alterations in ctDNA can help in the discovery of genes that can play a critical role in the development, progression, and therapeutic resistance of cancer [
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ,
      • Crowley E
      • Di Nicolantonio F
      • Loupakis F
      • Bardelli A
      Liquid biopsy: monitoring cancer-genetics in the blood.
      ]. Presently, tumor genotyping has allowed indications for melanoma (BRAF), non-small-cell lung cancer (EGFR and EML4–ALK mutations), including forthcoming applications of BRAF+ EGFR-directed therapies in colorectal cancer and PIK3CA-targeted treatments in breast cancer and other cancers [
      • Haber DA
      • Velculescu VE
      Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA.
      ,

      Skog JKO, Balaj L, Noerholm M, Breakefield XO. Cancer-related biological materials in microvesicles. Google Patents.

      ].

      Detection of microsatellite instability and LOH in ctDNA

      Microsatellite instability (MSI) such as LOH in tumor tissues was first reported by Nawroz in 1996 [
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ]. MSI can provide an increased detection of cancer diagnosis and progression. However, because the detection of MSI in cfDNA depends on small number of known mismatch repair genes or microsatellite loci, this problem makes this application difficult to use. In lung cancer and melanoma, patient survival and disease status were associated with MSI alterations [
      • Aarthy R
      • Mani S
      • Velusami S
      • Sundarsingh S
      • Rajkumar T
      Role of circulating cell-free DNA in cancers.
      ]. Several studies have shown that LOH can be correlated with disease status and it can also indicate tumor recurrence. For example, high LOH frequencies in breast cancer were associated with the aggressiveness of the disease and unfavorable prognosis [
      • Alix-Panabieres C
      • Pantel K
      Circulating tumor cells: liquid biopsy of cancer.
      ].

      Detection of tumor-associated DNA methylation in ctDNA

      Aberrant DNA methylation in noncoding gene sequences or in the promoter regions of genes is associated with tumor initiation, spreading, and progression and was first detected in liver, breast, and lung cancer patients in 1999 [
      • Mok T
      • Wu YL
      • Lee JS
      • et al.
      Detection and dynamic changes of EGFR mutations from circulating tumor DNA as a predictor of survival outcomes in NSCLC patients treated with first-line intercalated erlotinib and chemotherapy.
      ]. Examination of tumor-derived plasma DNA can provide an informative methylation profile on a genome-wide scale, which can help in the early detection of different types of cancers. Moreover, advanced disease and aggressive tumor biology is often associated with the presence of high amounts of ctDNA methylation. Therefore, these methylation-based biomarkers are a very useful tool for assessing cancer risk and prognosis in a clinical setting [
      • Elshimali YI
      • Khaddour H
      • Sarkissyan M
      • Wu Y
      • Vadgama JV
      The clinical utilization of circulating cell-free DNA (CCFDNA) in blood of cancer patients.
      ].

      Monitoring disease burden and treatment response

      In cancer patients, measuring the dynamic changes in the levels of tumor-derived cfDNA is of prognostic significance because it is useful for assessing tumor burden, disease progression, and therapeutic responses. Currently, radiological imaging (CT, PET, and MRI) and protein biomarkers (carcinoembryonic antigen, prostate-specific antigen, cancer antigen) are being widely used for determining disease burden and response to treatment. However, the low specificity and poor reliability of protein biomarkers and exposure of patients to ionizing radiation in medical imaging are some of the drawbacks of these techniques. ctDNA has a distinct advantage over these conventional methods by overcoming some of these shortcomings [
      • Bratman SV
      • Newman AM
      • Alizadeh AA
      • Diehn M
      Potential clinical utility of ultrasensitive circulating tumor DNA detection with CAPP-Seq.
      ,
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ].
      In lymphoma, high amounts of ctDNA levels were associated with aggressive disease and worse prognosis. Similarly, in breast cancer, metastasis and disease progression were correlated with increased cfDNA, nucleosomes, and protease (Caspase) activities [
      • Ellsworth RE
      • Blackburn HL
      • Shriver CD
      • Soon-Shiong P
      • Ellsworth DL
      Molecular heterogeneity in breast cancer: state of the science and implications for patient care.
      ]. In contrast to metastatic disease, patients with non-metastatic tumors had lower ctDNA levels in their blood. Conversely, a reduction in the amount of cfDNA indicated improved clinical condition and a better therapeutic response. Further, these ctDNA levels were lower in patients that were disease-free than in those expected to relapse. However, these levels remain unchanged or increased further in individuals who showed no response to treatment [
      • Qin Z
      • Li X
      • Zhang J
      • et al.
      Chemotherapy with or without estramustine for treatment of castration-resistant prostate cancer: A systematic review and meta-analysis.
      ]. Therefore, monitoring ctDNA during treatment provides highly sensitive, integrally specific, and comprehensive real-time information about disease status and treatment response.

      Monitoring minimal residual disease

      An appealing feature of ctDNA analysis to study disease progression is its potential to detect minimal residual disease and deduce the mechanism of relapse. In some cancers, patients with primary stage localized tumors are cured by surgery alone, but there are no effective means of identifying patients who have been cured and those who are at a risk of disease recurrence. ctDNA is a highly sensitive biomarker for residual disease monitoring and for identifying individuals at risk of relapse. This is possible by assessing tumor-specific mutations that offer an exquisitely profound way to monitor early tumor recurrence. This strategy will help in the development of effective therapies at a time when burden of resistant cells is still low [
      • Sacher AG
      • Paweletz C
      • Dahlberg SE
      • et al.
      Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer.
      ].

      Cancer screening: noninvasive/early diagnosis of cancer

      Finally, early detection of cancer is likely to be the most important aspect of a noninvasive blood-based diagnosis and patient survival. Conventional cancer-screening programs are often associated with false-positive results, which can lead to unnecessary invasive procedures that further add to the cost of health care systems. Diagnostic precision of screening procedures can be enhanced by detection of ctDNA in the blood serum/plasma, which reduces the false-positive results [
      • Haber DA
      • Velculescu VE
      Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA.
      ]. However, there are several factors that complicate the analysis of ctDNA in this context: primary tumors are small so the concentration of ctDNA is very low; some mutations in the circulating DNA may be the result of mosaicism and not be associated with tumors; and tumor mutations specific to a patient are unknown. CAPPSEQ could overcome some of these obstacles because of its high analytical sensitivity and specificity. Current advancements in technology would facilitate greater diagnostic accuracy and eventually make ctDNA-based cancer screening practicable [
      • Bratman SV
      • Newman AM
      • Alizadeh AA
      • Diehn M
      Potential clinical utility of ultrasensitive circulating tumor DNA detection with CAPP-Seq.
      ].

      Challenges in ctDNA analysis

      Diagnostic science has provided unique edge and lifesaving results using ctDNA as a biomarker, but many challenges need to be managed to make it applicable in clinical practice.

      Clinical study

      Extensive study has been done related to old age disease progression with high level of ctDNA, but studies in early age disease and low level of ctDNA detection are lacking. Moreover, during inflammation, the aggregation of normal genomic DNA in the bloodstream hinder ctDNA detection by diluting it [
      • Li ZS
      • Deng CZ
      • Yao K
      • et al.
      Bilateral pelvic lymph node dissection for Chinese patients with penile cancer: a multicenter collaboration study.
      ].Therefore, in some clinical trials, nonprogressive benign lesions might also be detected in ctDNA analysis [
      • Alix-Panabières C
      • Pantel K
      Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy.
      ].

      Optimization and standardization

      Before long, rapid optimization of clinical processes, standardization of different steps of analytical pathways and validation of clinical and analytical processes are the need of time [
      • Hofman V
      • Ilie M
      • Long E
      • et al.
      Detection of circulating tumor cells from lung cancer patients in the era of targeted therapy: promises, drawbacks and pitfalls.
      ]. Most significantly, well-designed clinical standards need to be maintained by clinical trials involving large groups of patients and control samples to confirm ctDNA as a promising biomarker [
      • Qin Z
      • Ljubimov VA
      • Zhou C
      • Tong Y
      • Liang J
      Cell-free circulating tumor DNA in cancer.
      ].

      Sensitivity of the techniques

      Detection of very low amount of ctDNA in the early stages of disease progression is challenging due to methodological constraints [
      • Diaz Jr LA
      • Williams RT
      • Wu J
      • et al.
      The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.
      ]. In the early stages of disease, a very low amount of ctDNA is released in the blood. As reported, tumors of about 1 cm diameter have a half billion cells and release only 0.01% of the genome into the circulation, thus representing only 17–20 tumor cells. Therefore, Sanger sequencing (the conventional technique) is not appropriate for the accurate detection of such low concentrations. NGS is a better technique for this situation and many other PCR-based techniques are also making appreciable contributions in this regard [
      • McLarty J
      • Yeh C
      Circulating cell-free DNA: The blood biopsy in cancer management.
      ].

      Intratumoral heterogeneity

      Tumors are heterogeneous in nature and have polyclonal expansion properties that cause hindrances in diagnosis and treatment strategies. Furthermore, extensive understanding is required to explore the complexity of intratumoral heterogeneity because it is unclear whether ctDNA is representative of all metastasized cells at different places or if it represents all DNA of subclones at different zones which can prop-up clinical progression of disease or therapeutic defiance [
      • McLarty J
      • Yeh C
      Circulating cell-free DNA: The blood biopsy in cancer management.
      ]. Recent studies shown that not only are genetic factors the substructure of intratumoral heterogeneity, but epigenetic factors are also expanding the level of its complexity [
      • Gerlinger M
      • Rowan AJ
      • Horswell S
      • et al.
      Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.
      ].

      Limitation of blood biopsy

      Because tumor DNA is fragmented and present in small quantities, current techniques for its isolation are not adequate because they require high sample volume as input and are costly and labor-intensive [
      • Hong B
      • Zu Y
      Detecting circulating tumor cells: current challenges and new trends.
      ]. In current techniques, much of the sample is lost during the washing, elution, and binding steps. Therefore, it is essential to construct new methodologies for the detection of ctDNA with high sensitivity and specificity [
      • McLarty J
      • Yeh C
      Circulating cell-free DNA: The blood biopsy in cancer management.
      ,
      • Hong B
      • Zu Y
      Detecting circulating tumor cells: current challenges and new trends.
      ].

      Future perspectives

      Considerable differences in qualitative and quantitative circulating tumor profile have caused much confusion. These differences are attributable to different methods of ctDNA measurement, lack of standards used to evaluate DNA, and variations in the results obtained using different techniques for sample processing. Future prognosis, diagnosis, and analysis of ctDNA require reproducibility and accuracy of results, which is not possible without the validation and optimization of procedures [
      • Pathak AK
      • Bhutani M
      • Kumar S
      • Mohan A
      • Guleria R
      Circulating cell-free DNA in plasma/serum of lung cancer patients as a potential screening and prognostic tool.
      ,
      • Diehl F
      • Schmidt K
      • Choti MA
      • et al.
      Circulating mutant DNA to assess tumor dynamics.
      ]. Despite the availability of considerable information regarding ctDNA biomarkers, the lack of recurrent genetic changes in tumor lesions causes researchers to divert their attention to enhancing the analytical and diagnostic sensitivity of genome-wide analysis and the identification of specific cancer signatures [
      • Heitzer E
      • Ulz P
      • Geigl JB
      Circulating tumor DNA as a liquid biopsy for cancer.
      ].
      In the clinical settings, the integration of ctDNA requires extensive study. Validation of simple blood tests will be promising in the detection of early tumors, lessening the need for surgical treatment. ctDNA as a potential intermediate biomarker will be used to study efficiency of intervention [
      • Pathak AK
      • Bhutani M
      • Kumar S
      • Mohan A
      • Guleria R
      Circulating cell-free DNA in plasma/serum of lung cancer patients as a potential screening and prognostic tool.
      ]. Optimization and standardization of both CTCs and ctDNA analysis with extensive study, advancements in technology, and laboratory trials will position them as a valid clinical test with striking results and ultimately will be helpful in the management of disease [
      • Ignatiadis M
      • Dawson SJ
      Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?.
      ].
      To obtain a reliable, effective, and targeted noninvasive treatment approach, comprehensive genome-wide analysis of tumor DNA, time, and cost are major hurdles. Cost is continuously being reduced as a result of ongoing technological advancement in NGS, yet time remains a major issue to be addressed. By overcoming all of these problems, the use of ctDNA and CTCs as biomarkers will become routine practice in laboratories for accessing early and late risks of cancers [
      • Ignatiadis M
      • Dawson SJ
      Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?.
      ]. Further developments can be made in this field by doing research in three following areas: origin and biological importance of ctDNA, well-designed clinical trials, and methodical advancements. Several methodological issues, such as sample preparation and storage, introduction of innovative techniques, and characterization of assay conditions, require attention for determining qualitative and quantitative changes of ctDNA [
      • Jung K
      • Fleischhacker M
      • Rabien A
      Cell-free DNA in the blood as a solid tumor biomarker: A critical appraisal of the literature.
      ].

      Apprehensions linked to liquid biopsies

      The fact that the use of liquid biopsies for early diagnosis of cancer may result in overdiagnosis is a concerning for clinicians and scientists. However, overdiagnosis should not be misinterpreted as being equivalent to false-positive results. Although diagnosed as actual abnormalities, we are unsure which cases will lead to cancer and which will be indolent and not become symptomatic. The problem is that cancer progression is not a linear process. Such overdiagnoses in screening cancers should be avoided as a potential source of psychological and emotional distress [
      • Bangma C
      • Roemeling S
      • Schröder F
      Overdiagnosis and overtreatment of early detected prostate cancer.
      ,
      • Salam M.
      Early detection of prostate cancer: Bangladesh perspective.
      ]. Overdiagnosis will inevitably led to overtreatment; most commonly, radiotherapy and chemotherapy are used for treatment and many patients suffering from benign tumors did not actually need any treatment. Significant rethinking has occurred over last 7 years for screening of breast and prostate cancers. There has been backlash when considering the side effects of cancer treatments, unless the number of patients needing treatment is higher than the number dying from interventions [
      • Alcover J
      • Filella X
      Identification of candidates for active surveillance: should we change the current paradigm?.
      ].

      Conclusion

      With the ever increasing number of cancer-related deaths in both developed and developing countries, effective diagnostic and therapeutic advances offer a real hope for both current and future generations of patients with cancer. ctDNA analysis, also termed liquid biopsy, is a noninvasive method that allow clinicians to assess cancer patients’ disease status and management in a much cheaper, faster, and more reliable manner. In addition to the conventional DNA detection techniques, including PCR, PARE, and BEAMing, recent advances in NGS bring new options for ctDNA study and examination. ctDNA is a promising diagnostic, prognostic, and predictive biomarker that provides clinicians with highly precise and useful information in the course of disease monitoring. ctDNA analysis also contributes to the field of precision medicine because it provides information on each patient's disease status in real time. However, several challenges still need to be addressed and various improvements have to be made before this technique becomes widely accepted in the clinical setting.

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