Circulating Tumor Cells and Circulating Tumor DNA Detection Technology

Circulating Tumor Cells and Circulating Tumor DNA Detection Technology. Malignant tumors are the most important disease threatening human health, and early detection of tumors is the most effective way to fight tumors.

Studies have found that tumor cells or tumor DNA fragments are released into body fluids in the early stages of tumor occurrence. Therefore, detection of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in body fluids is useful for early diagnosis of tumors.

CTCs were first proposed by Nowell [1] and defined by Nowell as tumor cells that originate from primary tumors or metastatic tumors, gain the ability to break away from the basement membrane and enter blood vessels through the invading tissue matrix. They are primary tumors and metastatic tumors.

The bridge between them plays an important role in tumor diagnosis, treatment effect evaluation, and recurrence and metastasis monitoring [2]. ctDNA is an episomal gene fragment released by tumor cells, which is between 20 and 200 bp in length and carries the characteristics of tumor cell gene mutations.

The various detection technologies of CTCs and ctDNA have developed rapidly. Because of their advantages of non-invasiveness, multiple sampling, high specificity, short metabolic cycle, etc., they can reflect tumor changes in real time, and are gradually used in the medical field, but they exist in clinical applications. The problem requires careful analysis.

The development of CTCs detection technology

The amount of CTCs in the blood circulation is very small, and the detection is easily interfered by blood cells. Therefore, it is necessary to enrich the CTCs before the detection to obtain higher purity CTCs. There are currently two main enrichment methods: immunology and biophysics-based enrichment technology; CTCs detection methods have as many as 40 kinds, mainly including CTCs counting method, immunofluorescence method, gene detection method, etc. 

(1) Enrichment technology of CTCs

1. Enrichment technology based on immunology:

Immunological technology is mainly immunomagnetic bead enrichment method, which is divided into positive enrichment method and negative enrichment method [3]. The positive enrichment method is to directly isolate the target cells from the cell mixture. It is mainly suitable for epithelial cell tumors (breast cancer, prostate cancer, colon cancer, lung cancer, etc.). The most representative method is the CellSearch CTCs detection method. Criteria: Cell EpCAM+/CK+/CD45- characteristic markers of CTCs, the relationship between the number of CTCs and patient prognosis, etc. The negative enrichment method uses magnetic beads to bind to background cells (such as white blood cells), and then uses anti-CD45 or anti-CD61 antibodies to remove polymerized cells and platelets in the blood to obtain CTCs target cells [4]. The negative enrichment method is more suitable for research. Specimens without selective markers or difficult to remove certain conjugates.

2. Detection technology based on biophysics:

The detection technology based on biophysical characteristics mainly uses CTCs cell volume, density, deformability, electrophoresis characteristics and other biophysical characteristics to distinguish CTCs cells from other cells such as leukocytes. It does not need to rely on biomarkers and overcomes the immune magnetic beads. Some flaws of the law [5].

Separation method based on cell volume: Generally speaking, the cell volume of CTCs is larger than that of white blood cells [6], and the cytoplasm is twice that of white blood cells [7]. The heterogeneity of the volume of CTCs may be due to cell apoptosis or cells in different cell metabolic cycles, which can be used to isolate CTCs and identify or stage tumors through detection.

Separation method based on cell variability: The variability of CTCs cells is greater than that of non-tumor cells, and their characteristics are conducive to separation. The commonly used density gradient centrifugation method is based on the density and variability of red blood cells, white blood cells, and tumor cells, and the corresponding cells are obtained by centrifugation. Density gradient centrifugation is generally used as a preliminary method for the separation of CTCs.

Based on the microfiltration membrane separation technology: The principle of the microfiltration membrane method is based on the large cell volume of CTCs and the high hardness of the cell membrane. This method was first used by Seal for the separation of CTCs [8], and its representative technologies are ISET® (Rarecells Diagnostics) and ScreenCell® (ScreenCell). ISET® is widely used in clinical research, mainly for tumor cell metastasis, non-small cell lung cancer, etc. [9]. ScreenCell® is mainly used for research on cell mechanism, cell culture and cell molecular testing. With the CellSieve™ microfilter, an average of 56 CTCs can be found in the blood of each metastatic tumor patient (10 ml) [10], and tumor-related macrophages are even found [11].

3. CTCs culture method:

The culture method can isolate and culture CTCs with tumor characteristics from malignant tumors such as lung cancer, cervical cancer, gastric cancer, bladder cancer, breast cancer, etc. [12], and then perform cell test. The results have strong specificity, but the culture takes a long time. (14~28 d), it is not conducive to a large number of clinical development.

(2) Detection method of CTCs

1. CTCs counting method:

The CTCs counting method is a basic detection method that uses specific molecular markers on the cell membrane surface to separate and count, and the results can show the prognosis [13].

2. CTCs immunofluorescence labeling detection method:

The immunofluorescence labeling detection method mainly uses different fluorescent markers to label different cells. The most commonly used detection method is the CellSeach® system [14], which is a general standard for the detection of CTCs. The automatic fluorescence microscope is used to detect CK+/DAPI+/CD45- count. This method has high specificity and is easy to standardize, but due to the lack of specific antigen, it is easy to cause false negatives.

3. Genetic testing method:

Including fluorescence quantitative PCR, CTCs gene chip technology, CTCs gene sequencing and microfluidic technology detection.

Fluorescence quantitative PCR: Fluorescence quantitative PCR is to first lyse the enriched CTCs, and use PCR-related techniques to detect free RNA in the blood to confirm CTCs. One CTCs can be detected in (1-10)×106 normal cells, but it is easy Non-specific RNA products appear, causing false positives. Reverse transcription PCR is more specific than PCR. It can detect tumor-specific RNA levels and has high sensitivity. However, reverse transcription PCR is prone to sample contamination, and some inflammatory reactions can also secrete CTCs, resulting in false positive test results. Real-time quantitative fluorescent PCR and real-time quantitative probe PCR are new methods that have emerged in recent years. Specific probes and real-time quantitative detection can be used to jointly detect CTCs, which improves the efficiency and sensitivity of amplification and has higher clinical application value.

CTCs gene chip technology: The detection technology developed on the principle of microfluidic control technology is mainly based on the principle of fluidics combined with antigen-antibody reaction to capture CTCs in the sample. This technology has successfully detected a large number of CTCs in the peripheral blood of patients with lung cancer, pancreatic cancer, and colon cancer; after 2010, a second-generation chip technology that can be stained on a chip and characterized by a fishbone-like matrix has been developed [ 15], shorten the time of antigen-antibody reaction and greatly increase the detection rate of CTCs, but the application of gene chip technology in clinical application still needs more practice and verification.

CTCs gene sequencing: The sequenced information may contain mutation information that is not in the primary tumor. Comparing the genetic changes between the primary tumor and CTCs can also help to discover the relationship between gene mutations and disease progression and treatment. It is a malignant tumor. To provide help.

Microfluidic technology detection: It is a technology that uses microfluidic chips to control tiny fluids at the micron and sub-micron levels. It can simulate the interaction of cells and has the advantages of non-contact, high precision, tissue culture, nutrient supply, and waste removal. As well as high throughput, small reaction volume, and small sample size, it has the characteristics of parallel analysis of multiple items on a small amount of sample, so that the reaction, pretreatment and detection processes in the experiment are integrated into a microfluidic system Completed, it has great development potential and application value [16,17]. However, because microfluidic products are usually not used in clinical laboratories, their quality control, repeatability, linearity and precision are still lacking, and clinical testing still needs more related research.

Development of ctDNA detection technology

In 1989, a study found that some of the free DNA in the plasma of tumor patients came from tumor cells [18]. The content of ctDNA in peripheral blood is very small, the fragments are small, and it is easy to combine with plasma proteins. The quality and quantity of DNA isolated from different tumors vary greatly. In recent years, more and more high-sensitivity and high-specificity technologies have been applied to the detection of ctDNA, making ctDNA widely used in tumor-related research. At present, there are many types of ctDNA detection technologies, mainly including PCR technology and next-generation sequencing (NGS)-based detection technologies. 

(1) ctDNA detection technology based on PCR

1. Digital PCR technology:

Digital PCR realizes “single-molecule template PCR amplification” and uses a positive reactor to determine the copy number of target cell ctDNA. The droplet digital PCR can even detect the lower limit of detection of 0.001% [19]. The disadvantage of this detection method is that it cannot detect unknown mutations and is not suitable for detection of high-concentration DNA samples. Therefore, the current digital PCR technology is mostly used in scientific research and has not been widely used in clinical practice.

2. BEAMing technology:

BEAMing technology is a combination of digital PCR and flow cytometry, using specific PCR primers to combine with the corresponding magnetic beads and amplify, and then use flow cytometry to detect fluorescent labels to determine mutations, such as breast cancer patients can be detected Phosphatidylinositol 3-kinase catalytic subunit α mutation, epidermal growth factor receptor mutation in non-small cell lung cancer, etc. This detection method is highly sensitive, but it is currently difficult to detect mutations in smaller tumors, nor can it be used to discover new mutations [20,21].

(2) NGS-based ctDNA detection technology

1. Marker amplified deep sequencing:

The label-amplified deep sequencing has high throughput and short sequencing time, which can detect hundreds of thousands to millions of DNA molecules at a time. The main principle is to design primers for the target region for 15 cycles of pre-amplification, then selectively amplify the amplicon region with mutations by single-plex PCR, and finally add sequencing adapters and specific tags at both ends of the amplicon The sequence was sequenced single-ended. This technology can detect gene mutations in cancer patients, and is also conducive to the guidance of personalized medicine. Compared with whole genome sequencing, it is more economical and effective, but there are certain false positives.

2. Deep sequencing tumor personalized analysis method:

The deep sequencing method of individualized tumor analysis is a capture sequencing technology that uses sources such as tumor gene mutation databases to find the sequence corresponding to the target cell, and then performs targeted capture of the sample before performing in-depth testing. This technology is mainly used to detect the ctDNA of non-small-cell lung carcinoma (NSCLC). The sensitivity of ctDNA in NSCLC above stage II is 100%, and the sensitivity of stage I is 50%. The specificity is 96% [22].

3. Barcode sequencing technology:

Barcode sequencing technology has high fidelity, mainly through aptamer connection to increase the barcode sequence, so as to perform deep sequencing, and then use linear amplification to eliminate errors in PCR, which can be used to identify individual molecules. This technology reduces the probability of errors in PCR amplification, but the detection throughput is low, and only one nucleosome factor can be studied at a time. More research is needed to improve its detection throughput to be widely used in clinical practice.

4. Whole exome sequencing technology:

Whole exome sequencing (WES) is a high-throughput sequencing analysis method that uses sequence capture technology to capture and enrich the exon region DNA of the whole genome. Exome sequencing has obvious advantages over other sequencing technologies in terms of screening range and detection rate. Compared with genome resequencing, the cost is lower. It also has greater advantages for studying SNPs and Indels of known genes, such as comparison Tissue biopsy of neuroblastoma, somatic mutations of ctDNA and changes in gene copy number, to explore the tumor heterogeneity of neuroblastoma [23]. Its disadvantage is that it can only obtain the information of the variation inside or at the boundary of the exon region, and cannot detect the large structural variation in the genome.

5. Targeted sequencing:

Targeted sequencing, also known as target region sequencing, is the use of PCR or probe hybridization to capture and enrich the target genomic region, perform high-throughput sequencing, detect genetic mutation sites, and obtain mutation information in the designated target region. Compared with the traditional first-generation sequencing, whole-genome sequencing, and whole-exome sequencing, the target area sequencing can obtain deeper coverage and higher data accuracy, and improve the detection efficiency of the target area.

Conclusion and outlook

Compared with traditional tissue biopsy or imaging techniques, CTCs and ctDNA have significant advantages. The specimen source can be blood or other body fluids, and non-invasive detection can be performed. At the same time, finding CTCs or ctDNA in the blood or body fluids has more direct evidence for the diagnosis of tumors, and can be used to guide treatment and prognosis evaluation. However, the detection of CTCs or ctDNA has certain limitations, and its content in blood or body fluids is very low, especially ctDNA. Secondly, due to the heterogeneity of tumor cells, the technical requirements for CTCs and ctDNA are very high. How to extract high-purity CTCs and ctDNA is also a challenge for the clinical application of these two methods. In addition, not all tumors will have CTCs or ctDNA into the blood circulation or other body fluids, so there are also certain false negatives.

When CTCs and ctDNA are used in clinical diagnosis and treatment, the following issues need to be paid attention to:

(1) Standardization of specimen processing procedures before testing. Different detection methods use different types of cytoprotective agents, and the storage time after collection, the centrifugation process, and the extraction and purification of ctDNA are different, and these differences may affect the results.

(2) Standardization of testing procedures. Different detection methods or platforms have different sample volumes, detection throughput, and detection targets for CTCs and ctDNA, and the operating procedures are also very different, resulting in poor comparability of results between different companies, so standardized detection procedures are required. Make the test results comparable.

(3) The government improves relevant policies and regulatory mechanisms. At present, the clinical application of CTCs and ctDNA is subject to certain restrictions. In addition to the high testing technology and platform requirements, their prices are also attracting attention.

The sensitivity and specificity of different methods or platforms are very different, and the clinical diagnostic performance is also different, which causes certain difficulties in the comparability and repeatability of the results. How to choose a method that is more suitable for their own research or laboratory feasible among the complicated detection methods is also a problem that medical workers need to consider.

In addition to mastering the various principles, advantages and disadvantages of current existing detection technologies, the method to solve the bottleneck of detection technology is more important to stimulate more research ideas through methodological comparison, and to further improve or perfect related detection technologies. CTCs and ctDNA have been widely used in clinical practice.

(source:internet, reference only)

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