Pre-analytical considerations When blood is collected in EDTA tubes and stored, the white blood cells begin to lyse and release genomic wild type<unk>  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<unk>  and make detection of ctDNA more feasible: * Never freeze a blood sample before extracting the plasma for ctDNA analysis * Process the sample to plasma within 2–4 hours (if collected in EDTA tube) * Never use heparinised tubes, heparin inhibits PCR by mimicking the helical structure of<unk>  * Perform a double centrifugation step (centrifuge the blood to extract plasma, then repeat on the plasma to remove from debris in the bottom of the tube) to remove more cellular debris prior to<unk>  extraction. * Plasma is better than serum for ctDNA recovery 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<unk>  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<unk>  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<unk>  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<unk>  from tumor cells and<unk>  from normal cells using a whole genome approach. Whole genome or exome sequencing typically use high throughput<unk>  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<unk> . While simply looking at<unk>  polymorphisms through sequencing does not differentiate<unk>  from tumor or normal cells, this problem can be resolved by comparing against a control sample of normal<unk>  (for example,<unk>  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 (4 to 4.3 in 105. Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) This method 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<unk>  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<unk>  levels observed in ctDNA collection. Then the isolated<unk>  undergoes deep sequencing for increased sensitivity. This technique allows for the interrogation of hundreds of<unk>  regions. The ctDNA detection sensitivity of CAPP-Seq is reported to be 2.5 molecules in 1,000,000. Tagged AMplicon deep Sequencing (TAM-Seq) 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<unk>  in parallel singleplex reactions. This technique was shown to successfully identify mutations scattered in the TP53 tumor suppressor gene in advanced ovarian cancer patients. The sensitivity of this technique is 1 in 50. Safe-Sequencing (Safe-Seq) This method was originally described by Bert Vogelstein and his group at Johns Hopkins University. Safe-Seq decreases the error rate of massively parallel sequencing in order to increase the sensitivity to rare mutants. It achieves this by addition of a unique identifier (UID) sequence to each<unk>  template. The<unk>  is then amplified using the added UIDs and sequenced. All<unk>  molecules with the same UID (a UID family) should have the same reported<unk>  sequence since they were amplified from one molecule. However, mutations can be introduced through amplification, or incorrect base assignments may be called in the sequencing and analysis steps. The presence of the UID allows these methodology errors to be separated from true mutations of the ctDNA. A mutation is considered a ‘supermutant’ if 95% of the sequenced reads are in agreement. The sensitivity of this approach is 9 in 1 million. Duplex sequencing This method is an improvement on the single UIDs added in the Safe-Seq technique. In duplex sequencing, randomized double-stranded<unk>  act as unique tags and are attached to an invariant spacer. Tags are attached to both ends of a<unk>  fragment (α and β tags), which results in two unique templates for PCR - one strand with an α tag on the 5’ end and a β tag on the 3’ end and the other strand with a β tag on the 5’ end and an α tag on the 3’ end. These<unk>  fragments are then amplified with primers against the invariant sequences of the tags. The amplified<unk>  is sequenced and analyzed.<unk>  with the duplex adaptors are compared and mutations are only accepted if there is a consensus between both strands. This method takes into account both errors from sequencing and errors from early stage PCR amplification. The sensitivity of the approach to discovering mutants is 1 in 10^7. Integrated Digital Error Suppression (iDES)-enhanced CAPP-Seq iDES improves CAPP-Seq analysis of ctDNA in order to decrease error and therefore increase sensitivity of detection. Reported in 2016, iDES combines CAPP-Seq with duplex barcoding sequencing technology and with a computational algorithm that removes stereotypical errors associated with the CAPP-Seq hybridization step. The method also integrates duplex sequencing where possible, and includes methods for more efficient duplex recovery from cell free<unk> . The sensitivity of this improved version of CAPP-Seq is 4 in 100,000 copies.
Circulating tumor DNA
Circulating tumor cell
Tumor metabolome
Cancer Likelihood in Plasma
Tumor mutational burden
Circulating mitochondrial DNA
Tumor marker
Tumor antigen