A competition for cancer cells and human medical testing – How to use liquid biopsy to break through the testing dilemma?

Editor/ Alma Wu
Review/ Thomas Huang, Jane Lee

Liquid biopsy has been selected by MIT Technology Review as one of the top 10 breakthrough technologies in 2015. It can be used for Non-Invasive Prenatal Testing (NIPT), early screening for disease, companion diagnostics, dynamic monitoring, cancer relapsing prediction, and prognosis evaluation. This is done by collecting specific biomarkers in patient’s body fluid including blood, saliva, and urine etc. Compared to traditional invasive sampling, it is convenient, instantaneous and minimally invasive. Moreover, it can improve the acceptance of subjects. In recent years, related research and application have become more extensive, and the future of liquid biopsy in clinical application is arriving.

Liquid biopsy provides new directions for cancer diagnosis and monitoring

There are three major applications of liquid biopsy: Circulating tumor cells (CTCs), Cell free DNA (cfDNA), and Circulating exosomes or extracellular vesicles. Among them, the application of CTCs and cfDNA are much more mature. CTCs purification technology ( invented in 1990), and the clinical applications of cfDNA/ ctDNA has made a lot of progress since the new generation sequencing (NGS) came to the market in 2007-2008. It has also been a popular trend of clinical application of cancer in recent years. Traditional tumor tissue biopsy is collecting samples by aspiration biopsy or surgery, which is an invasive testing. For patients, the testing risk are not only high, the physical consumptions are also huge. Also the treatment evaluation could vary for different sampling locations. This could affect prognosis and other issues. Therefore, since the liquid biopsy only needs the patient’s body fluid as sample, which is non-invasive or minimally invasive; it would eliminate bodily injuries to the patients, improve the process, and reduce the sampling cost. This is a great alternative solution for patients who are not good candidates for tissue slicing ( whom are not suitable for surgery when the tumor is located in the brain, blood vessels or inaccessible regions of the body).

Clinically, liquid biopsy is mostly done by real-time PCR (RT-PCR) or digital droplet PCR (ddPCR). Both known as using the mutation sites to design probes and detect mutations in the sample as a reference for physician evaluation, diagnosis, and prescription. RT-PCR can monitor the results of the experiments on the computer instantly and significantly reduce the testing time, and ddPCR can accurately determine the number of DNA molecules in nucleic acids. Compared to other relatively quantitative tests, ddPCR can achieve absolutely quantitative accuracy. However, the design of the probe does not contain a map of all genetic variations, which may lack the less common clinically relevant mutational identities and limit the testing of other emerging biomarkers. By using NGS method of testing, the overall variation can be more comprehensive and complete.

It is important to choose the appropriate tool for detecting trace amounts of ctDNA!

In the 1940s, scientists discovered the presence of cfDNA from the blood. When normal cells are metabolized and ruptured in the body, all intracellular substances are released. DNA is also released from the cells into the blood and becomes cfDNA. In general, such free DNA is cleared by macrophages. However, when free DNA is generated at a rate greater than the clearance rate, it may persist in the blood, such as DNA from tumor cells. The cfDNA does contain circulating tumor DNA (ctDNA) released after tumor cell apoptosis. Therefore, it can track the presence or absence of cancer cells, and detect ctDNA mutations by detecting ctDNA in blood, as well as the reference for the diagnosis and prescription. In addition, the half-life of circulating DNA is short, so the composition of ctDNA will change with the progress of the tumor, and the disappearance, spread, and recurrence of the tumor can be presented instantly, accurately and sensitively.

Although the use of liquid biopsy to assist cancer diagnosis is a major breakthrough in medical testing, it still confronts many challenges, such as:

1. Tumor heterogeneity: Due to the high heterogeneity of cancer cells, the circulating DNA does not represent the overall cell characteristics and the appearance of the tumor. Therefore, the interpretation of the test results must be more cautious and conservative.

2. The technical purification and sensitivity issues: It is more difficult to apply to early tumor testing since amount ctDNA from early tumor is low. Unless the testing technology can be improved for its purification and sensitivity, early tumor detection remains challenging.

How to improve the testing accuracy? The key is to sample and QC correctly!

Since the amount of ctDNA is minimal, it accounts for only one per million cfDNA in the blood, so the sensitivity of purification and testing becomes the key during the testing process. The following session we will discuss how to quality control (QC) in four procedures of cfDNA diagnosis application from the application of cfDNA in the test and quality control (QC) to respectively: Sampling, Purification, Detection, Analysis:

1. Sampling / Preservation: Sampling of liquid biopsy has the advantage of convenience, instant, and minimally invasive. However, due to short half-life of cfDNA, the process of sampling, delivering and preservation may also lead to rupture of nuclear cells, the release of genomic DNA, and interference with the accuracy of subsequent tests. As a result, the use of appropriate preservation tubes can effectively reduce the degradation of cfDNA, maintain the integrity of the other cells and improve the preservation quality.

2. Purification: Because the amount of ctDNA is low; the purification requires more effort. In general, purified products on the market can be classified into magnetic beads or silica membrane according to a purification method. The principle of the magnetic beads is to provide a suitable environment for DNA which adsorbed onto the surface of the carboxylic acid modified macromolecular magnetic beads, in order to collect DNA and separate DNA from the magnetic beads by elution, which lead to the purpose of purification. The silica membrane uses a silicon-containing filter membrane as a specific adsorption material for nucleic acids through the adsorption capacity of the filter membrane to nucleic acids adjustment of salts and pH. The key difference between these two are the recovery. There is little difference in the purification of general DNA. However, because ctDNA is extremely low, the probability of collision with magnetic beads is low. And the silica membrane will be more advantageous since it acts like catching the fish with the net.

3. Detection: According to the detection requirements, the purified ctDNA can be detected with microarray, PCR, qPCR, ddPCR, NGS. Before the experiment, the QC of the sample plays a key role to ensure the accuracy of the detection. The mother’s blood may contain fetal DNA from the placenta. The blood of the stroke or heart disease patient may also carry DNA fragments. Therefore, besides determining basic concentration, using high-sensitivity capillary electrophoresis (CE), such as: Qsep series of BiOptic Inc., Advanced Analytical Fragment Analyzer or Agilent Bioanalyzer to confirm the size of the sample fragment (the general ctDNA size should between 160~ 200 bp), the possibility of genomic DNA or carrier RNA contamination can be excluded.

4. Analysis: We will apply to medical diagnosis and assistance after the data analysis.

As the saying goes, good tools are prerequisite to the success of a job. It is more dominant to choose the appropriate weapons and tools to fight against intangible enemies such as diseases. Liquid biopsy is an important step towards clinical precision medicine, and it also revolutionizes cancer medical treatment, which not only applies for companion diagnostics, dynamic monitoring, but also for cancer recurrence and prognosis. The disease is constantly changing, so the medical professionals are expecting more weapons to fight against the diseases.

Therefore, it is impossible to meet the medical demands by detecting the presence or absence of the diseases. Scientists are trying to link ctDNA to protein markers and attempt to trace the results of liquid biopsy back to the sites of cancer, or using liquid biopsy to identify whether cancers need to do the immediate treatment. Looking forward to the future, the meaningful liquid biopsy should be more accurate, comprehensive and sensitive than traditional testing. It is possible to detect early cancer patients in asymptomatic people, as well as confirm the sites of cancer and whether treatment is needed. Using the right tools and applying technology flexibly will bring more benefits to patients.

“This article is sponsored by BiOptic Inc. and INVISION BIOTECH”

Author

GeneOnline