Tumor-derived fragmented DNA in the bloodstream
Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. ctDNA should not be confused with cell-free DNA (cfDNA), a broader term which describes DNA that is freely circulating in the bloodstream, but is not necessarily of tumor origin. Because ctDNA may reflect the entire tumor genome, it has gained traction for its potential clinical utility; "liquid biopsies" in the form of blood draws may be taken at various time points to monitor tumor progression throughout the treatment regimen.
Recent studies have laid the foundation for inferring gene expression from cfDNA (and ctDNA), with EPIC-seq emerging as a notable advancement. This method has substantially raised the bar for the noninvasive inference of expression levels of individual genes, thereby augmenting the assay's applicability in disease characterization, histological classification, and monitoring treatment efficacy.
ctDNA originates directly from the tumor or from circulating tumor cells (CTCs), which describes viable, intact tumor cells that shed from primary tumors and enter the bloodstream or lymphatic system. The precise mechanism of ctDNA release is unclear. The biological processes postulated to be involved in ctDNA release include apoptosis and necrosis from dying cells, or active release from viable tumor cells. Studies in both human (healthy and cancer patients) and xenografted mice show that the size of fragmented cfDNA is predominantly 166bp long, which corresponds to the length of DNA wrapped around a nucleosome plus a linker. Fragmentation of this length might be indicative of apoptotic DNA fragmentation, suggesting that apoptosis may be the primary method of ctDNA release. The fragmentation of cfDNA is altered in the plasma of cancer patients.
In healthy tissue, infiltrating phagocytes are responsible for clearance of apoptotic or necrotic cellular debris, which includes cfDNA. ctDNA in healthy patients is only present at low levels but higher levels of ctDNA in cancer patients can be detected with increasing tumor sizes. This possibly occurs due to inefficient immune cell infiltration to tumor sites, which reduces effective clearance of ctDNA from the bloodstream. Comparison of mutations in ctDNA and DNA extracted from primary tumors of the same patients revealed the presence of identical cancer-relevant genetic changes. This led to the possibility of using ctDNA for earlier cancer detection and treatment follow up.
Methods.
Pre-analytical considerations.
When blood is collected in EDTA tubes and stored, the white blood cells begin to lyse and release genomic wild type DNA in to the sample in quantities typically many fold higher than the ctDNA is present in. This makes detection of mutations or other ctDNA biomarkers more difficult. The use of commercially available cell stabilisation tubes can prevent or delay the lysis of white cells thereby reducing the dilution effect of the ctDNA. Sherwood et al. demonstrated superior detection of "KRAS" mutations in matched samples collected in both EDTA K3 and Streck BCT tubes. The advantages of cell stabilisation tubes can be realised in situation where blood cannot be processed to plasma immediately.
Other procedures can also reduce the amount of "contaminating" wild type DNA and make detection of ctDNA more feasible:
Extraction of ctDNA.
The main appeal of ctDNA analysis is that it is extracted in a non-invasive manner through blood collection. Acquisition of cfDNA or ctDNA typically requires collection of approximately 3mL of blood into EDTA-coated tubes. The use of EDTA is important to reduce coagulation of blood. The plasma and serum fractions of blood can be separated through a centrifugation step. ctDNA or cfDNA can be subsequently extracted from these fractions. Although serum tends to have greater levels of cfDNA, this is primarily attributed to DNA from lymphocytes. High levels of contaminating cfDNA is sub-optimal because this can decrease the sensitivity of ctDNA detection. Therefore, the majority of studies use plasma for ctDNA isolation. Plasma is then processed again by centrifugation to remove residual intact blood cells. The supernatant is used for DNA extraction, which can be performed using commercially available kits.
Analysis of ctDNA.
The analysis of ctDNA after extraction requires the use of various amplification and sequencing methods. These methods can be separated into two main groups based on whether the goal is to interrogate all genes in an untargeted approach, or if the goal is to monitor specific genes and mutations in a targeted approach.
Untargeted approaches.
A whole genome or whole exome sequencing approaches may be necessary to discover new mutations in tumor DNA while monitoring disease burden or tracking drug resistance. Untargeted approaches are also useful in research to observe tumor heterogeneity or to discover new drug targets. However, while untargeted methods may be necessary in certain applications, it is more expensive and has lower resolution. This makes it difficult to detect rare mutations, or in situations where low ctDNA levels are present (such as minimal residual disease). Furthermore, there can be problems distinguishing between DNA from tumor cells and DNA from normal cells using a whole genome approach.
Whole genome or exome sequencing typically use high throughput DNA sequencing technologies. Limiting the sequencing to only the whole exome instead can decrease expense and increase speed, but at the cost of losing information about mutations in the non-coding regulatory regions of DNA. While simply looking at DNA polymorphisms through sequencing does not differentiate DNA from tumor or normal cells, this problem can be resolved by comparing against a control sample of normal DNA (for example, DNA obtained through a buccal swab.) Importantly, whole genome and whole exome sequencing are useful for initial mutation discovery. This provides information for the use of more sensitive targeted techniques, which can then be used for disease monitoring purposes.
Whole genome sequencing enables to recover the structural properties of cfDNA, the size of fragments and their fragmentation patterns. These unique patterns can be an important source of information to improve the detection of ctDNA or localize the tissue of origin of these fragments. Size-selection of short fragments ( ;150bp) with in vitro or in silico methods could improve the recovery of mutations and copy number aberrations.
Digital karyotyping.
This method was originally developed by the laboratory of Bert Vogelstein, Luis Diaz, and Victor Velculescu at Johns Hopkins University. Unlike normal karyotyping where a dye is used to stain chromosomal bands in order to visualize the chromosomes, digital karyotyping uses DNA sequences of loci throughout the genome in order to calculate copy number variation. Copy number variations are common in cancers and describe situations where loss of heterozygosity of a gene may lead to decreased function due to lower expression, or duplication of a gene, which leads to overexpression.
Personalized analysis of rearranged ends.
After the whole genome is sequenced using a high throughput sequencing method, such as Illumina HiSeq, personalized analysis of rearranged ends (PARE) is applied to the data to analyze chromosomal rearrangements and translocations. This technique was originally designed to analyze solid tumor DNA but was modified for ctDNA applications.
DNA methylation and hydroxymethylation.
Proper epigenetic marking is essential for normal gene expression and cell function and aberrant alterations in epigenetic patterns is a hallmark of cancer. A normal epigenetic status is maintained in a cell at least in part through DNA methylation. Measuring aberrant methylation patterns in ctDNA is possible due to stable methylation of regions of DNA referred to as “CpG islands”. Methylation of ctDNA can be detected through bisulfite treatment. Bisulfite treatment chemically converts unmethylated cytosines into a uracil while leaving methylated cytosines unmodified. DNA is subsequently sequenced, and any alterations to the DNA methylation pattern can be identified. DNA hydroxymethylation is a similarly associated mark that has been shown to be a predictive marker of healthy versus diseased conditions in cfDNA, including cancer.)
Targeted approaches.
In a targeted approach, sequencing of ctDNA can be directed towards a genetic panel constructed based on mutational hotspots for the cancer of interest. This is especially important for informing treatment in situations where mutations are identified in druggable targets. Personalizing targeted analysis of ctDNA to each patient is also possible by combining liquid biopsies with standard primary tissue biopsies. Whole genome or whole exome sequencing of the primary tumor biopsy allows for discovery of genetic mutations specific to a patient's tumor, and can be used for subsequent targeted sequencing of the patient's ctDNA. The highest sensitivity of ctDNA detection is accomplished through targeted sequencing of specific single nucleotide polymorphisms (SNPs). Commonly mutated genes, such as oncogenes, which typically have hotspot mutations, are good candidates for targeted sequencing approaches. Conversely, most tumor suppressor genes have a wide array of possible loss of function mutations throughout the gene, and as such are not suitable for targeted sequencing.
Targeted approaches have the advantage of amplifying ctDNA through polymerase chain reactions (PCR) or digital PCR. This is especially important when analyzing ctDNA not only because there are relatively low levels of DNA circulating in the bloodstream, but also because ctDNA makes up a small proportion of the total cell-free DNA extracted. Therefore, amplification of regions of interest can drastically improve sensitivity of ctDNA detection. However, amplification through PCR can introduce errors given the inherent error rate of DNA polymerases. Errors introduced during sequencing can also decrease the sensitivity of detecting ctDNA mutations.
Droplet digital polymerase chain reaction.
Droplet digital PCR (ddPCR) is derived from the digital polymerase chain reaction, originally named by Bert Vogelstein’s group at Johns Hopkins University. Droplet Digital PCR utilizes a droplet generator to partition single pieces of DNA into droplets using an oil/water emulsion. Then individual polymerase chain reactions occur in each droplet using selected primers against regions of ctDNA and proceeds to endpoint. The presence of the sequences of interest is measured by fluorescent probes, which bind to the amplified region. ddPCR allows for highly quantitative assessment of allele and mutant frequencies in ctDNA but is limited by the number of fluorescent probes that can be used in one assay (up to 5). The sensitivity of the assay can vary depending on the amount of DNA analyzed and is around 1 in 10,000. Specificity should be augmented through the use of either minor groove binding (MGB) modified probes or of an alternative such as locked nucleic acids (LNAs).
Beads, emulsification, amplification, and magnetics.
Beads, emulsification, amplification, and magnetics (BEAMing) is a technique that builds upon Droplet Digital PCR in order to identify mutations in ctDNA using flow cytometry. After ctDNA is extracted from blood, PCR is performed with primers designed to target the regions of interest. These primers also contain specific DNA sequences, or tags. The amplified DNA is mixed with streptavidin-coated magnetic beads and emulsified into droplets. Biotinylated primers designed to bind to the tags are used to amplify the DNA. Biotinylation allows the amplified DNA to bind to the magnetic beads, which are coated with streptavidin. After the PCR is complete, the DNA-bound beads are separated using a magnet. The DNA on the beads are then denatured and allowed to hybridize with fluorescent oligonucleotides specific to each DNA template. The resulting bead-DNA complexes are then analyzed using flow cytometry. This technique is able to capture allele and mutation frequencies due to coupling with ddPCR. However, unlike with ddPCR, a larger number of DNA sequences can be interrogated due to the flexibility of using fluorescently bound probes. Another advantage of this system is that the DNA isolated can also be used for downstream sequencing. Sensitivity is 1.6 in 104 to 4.3 in 105.
Cancer Personalized Profiling by deep Sequencing.
Cancer personalized profiling by deep sequencing (CAPP-Seq) was originally described by Ash Alizadeh and Maximilian Diehn's groups at Stanford University. This technique uses biotinylated oligonucleotide selector probes to target sequences of DNA relevant to ctDNA detection. Publicly available cancer databases were used to construct a library of probes against recurrent mutations in cancer by calculating their recurrence index. The protocol was optimized for the low DNA levels observed in ctDNA collection. Then the isolated DNA undergoes deep sequencing for increased sensitivity. This technique allows for the interrogation of hundreds of DNA regions. The ctDNA detection sensitivity of CAPP-Seq is reported to be 2.5 molecules in 1,000,000.
Tagged amplicon deep sequencing.
Tagged amplicon deep sequencing (TAM-Seq) allows targeted sequencing of entire genes to detect mutations in ctDNA. First a general amplification step is performed using primers that span the entire gene of interest in 150-200bp sections. Then, a microfluidics system is used to attached adaptors with a unique identifier to each amplicon to further amplify the DNA in parallel singleplex reactions. This technique was shown to successfully identify mutations scattered in the TP53