[Early Tumor Screening] ctDNA Methylation Sequencing: The Most Potential Liquid Biopsy Technology Path

Cancer is a disaster for every person and family.
It is a huge expenditure on public health projects for the country.
According to the analysis report on the prevalence of malignant tumors in China released by the National Cancer Center in 2019, The number of cases continues to rise, and China’s annual medical expenditures are more than 220 billion yuan
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In the face of this situation, only early screening of tumors and killing them at the source of tumor occurrence and development can reverse this situation
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Early tumor screening has been repeatedly emphasized by the state, and a series of documents issued by it also demonstrate the country’s determination to tackle cancer medical problems
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In July 2019, the "Healthy China Action (2019-2030)" specifically proposed the "Cancer Prevention Action"
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Set hard action targets: by 2022 and 2030, the overall 5-year survival rate of cancer shall not be less than 43.
3% and 46.
6% respectively; the awareness rate of core cancer prevention knowledge shall not be less than 70% and 80% respectively; key areas with high incidence The rate of early diagnosis of cancer has reached 55% and above and continues to increase; it is basically realized that people at high risk of cancer participate in regular anti-cancer physical examinations
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In September of the same year, 10 departments including the National Health Commission jointly formulated the "Healthy China Action-Cancer Prevention and Control Implementation Plan (2019-2022)"
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By 2022, the cancer prevention and control system will be further improved, the comprehensive prevention and control of risk factors will make progress in stages, the level of cancer screening, early diagnosis and treatment, and standardized diagnosis and treatment will be significantly improved, the rising trend of cancer incidence and mortality will be curbed, and the overall cancer will be 5 years old.
The survival rate is 3 percentage points higher than in 2015, and the disease burden of patients is effectively controlled
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On May 24, 2021, the General Office of the State Council issued the "Key Tasks for Deepening the Reform of the Medical and Health System in 2021", proposing to expand the coverage of high-incidence cancer screening, and launch a pilot project for the construction of county-level cancer screening and early diagnosis and treatment centers
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Tumor screening is particularly important
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At present, early tumor screening is relatively unfamiliar to most people
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Due to China’s population of 1.
4 billion, medical resources appear to be very scarce.
At the same time, due to the limitations of traditional cancer screening methods, such as low accuracy of serological tumor markers, high false positive and false negative rates; endoscopy As an invasive examination method, the subject’s compliance is low; a series of problems such as the low popularity of imaging examinations such as B-ultrasound and CT have caused the current status of tumor screening in China to be unsatisfactory, and a new tumor is urgently needed.
Screening methods fill in the gaps
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With the rapid development of molecular biology and more and more extensive clinical applications, as well as the continuous development of liquid biopsy technology, the application of liquid biopsy to early tumor screening has become the most potential early screening technology path
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Liquid biopsy refers to the detection of biomarkers through the collection of blood samples and other non-solid biological tissues, and then the whole body tumor analysis
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At present, the biomarkers targeted by liquid biopsy can be divided into three categories: 1.
Complete cells in the blood, namely circulating tumor cells (CTC); 2.
Collect exosomes (EV) released by tumor cells; 3.
Analyze free tumor DNA or RNA, such as circulating tumor DNA (ctDNA), circulating cell-free RNA (cfRNA), miRNA and the like
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Comparison of analytes in solid and liquid biopsy samples From the perspective of tumorigenesis and metastasis, tumor cells release exosomes, ctDNA, cfRNA, etc.
into the blood through passive secretion (necrosis or apoptosis) and active secretion, which can be detected at an ultra-early stage Tumor signal
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The formation of CTC cells is mainly when tumor metastases.
The tumor epithelial cells transform into cells with a mesenchymal phenotype through the EMT effect, their adhesion ability is weakened, and they lose connection with the basement membrane and fall off from the primary tumor to enter the bloodstream.
System
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Therefore, the content of CTC in early tumors is low, even in patients with tumor metastasis, there are only 1-10 CTCs per 100 ml of whole blood, which is difficult to enrich
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At the same time, because ctDNA methylation can trace the clinical value of tumor tissue source, we believe that liquid biopsy technology represented by ctDNA has great potential for development in the field of early tumor screening
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Tumor metastasis and blood tumor component ctDNA gene mutation detection: signal amplification is a pain point.
It is known that mutation detection PCR is sufficient to track tumors.
It is necessary to distinguish the difference between ctDNA and normal cfDNA.
At present, it is mainly through gene mutation, methylation modification status, Abnormal copy number and nucleosome position are used as the basis for distinction
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Gene mutation is one of the root causes of cancer, so it is also the first target to be locked by detection and analysis
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According to the literature data of Nature Communications, EGFR (44.
2%) mutation in non-small cell lung cancer, RB1 (63.
4%) mutation in small cell lung cancer, APC (44.
4%) in colorectal cancer, KRAS (33.
8%) and PIK3CA (11.
2%) ) Mutations have shown the potential of using ctDNA mutations for tumor screening, and ctDNA mutations have also been shown to have a strong correlation with the frequency of driver genes detected by tissue biopsy
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However, the main difficulty of early tumor screening with ctDNA mutation detection is that the sensitivity is difficult to improve
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First of all, the abundance of ctDNA in blood is low, 10ml whole blood contains 4ml plasma on average, 6000GE
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According to Nature Genetics data, the detection sensitivity limit of VAF (variant allele frequency) recognition of tumor signal detection is 0.
01% (that is, it can detect a DNA fragment in 12,000 copies)
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The content of ctDNA in the blood is related to the size of the tumor.
Although it is possible to increase the concentration of ctDNA through close sampling (such as collecting stool for detection of colorectal cancer shedding markers), ctDNA is a unique marker without technological breakthroughs or effective sequencing and noise reduction methods.
Will be submerged in noise, under normal circumstances (collecting blood) it is difficult to identify tumors with a diameter of 12.
5mm (weight 1g) or less
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At the same time, because ctDNA is highly fragmented, has a short half-life, and the content of ctDNA varies greatly from person to person, excellent enrichment and sequencing depth are required to effectively capture early cancer signals related to gene mutations
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In addition, the technical challenges of using ctDNA mutations for early tumor screening include clonal hematopoiesis and early cancer location
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According to the cfDNA test results of 14,972 peripheral blood samples from 12377 patients in Nature Communications, 14% of the samples detected clonal hematopoietic (CH) mutations involving 15 mutated genes (the most common ones are DNMT3A, TP53, and TET2).
The frequency of CH mutations in melanoma, bladder cancer, uterine cancer and prostate cancer is greater than 20%
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At the same time, the frequency of CH mutations increases with age, which creates obstacles to the recognition of ctDNA mutations
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In terms of sequencing, ctDNA sequencing technologies mainly include PCR and NGS methods, while BEAMing, ARMSPCR and Tam-Seq are improved on the basis of PCR and NGS technologies
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At present, the detection of single cancer species often only needs to determine the known gene mutations of some targets.
At this time, PCR is sufficient for its short detection time, low cost, and high sensitivity
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The advantage of the NGS method is mainly high-throughput, which can sequence unknown sequences, but the cost is relatively high, and it may have advantages in identifying unknown gene mutations in the future
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ctDNA detection method (partial) comparison with ctDNA methylation sequencing: The most potential liquid biopsy technology path DNA methylation is an apparent regulatory modification that can participate in the regulation of protein synthesis without changing the base sequence
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This is mainly because the hypermethylation of the CpG island changes the conformation of the DNA region and affects the interaction between the protein and DNA and thus affects the transcription efficiency, while the methylation of the promoter region silences gene expression
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Compared with the mutation pattern of ctDNA at specific sites, the methylation of ctDNA in cancer often occurs in thousands of CpG sites, which is easier to detect and evaluate
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At the same time, according to the research published by Professor Zhang Kun (one of the founders of Kunyuan Biology) in Nature Genetics in 2017, methylation patterns can reflect the epigenetic origin of specific cancers and are used to reveal the unknown primary cancers.
Organization of origin
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Therefore, the detection of ctDNA methylation is stronger than the detection of gene mutations in both sensitivity and location of cancer
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One of the traditional DNA methylation sequencing is to capture and sequence methylated DNA binding protein (MBD) or methylated antibody (MeDIP) to obtain a methylation signal with a resolution of 100-1000bp in the whole genome, but it cannot be obtained.
Single-base resolution signal; Second, simplified genome methylation sequencing (RRBS) uses restriction enzymes to enrich the CpG region to obtain partial genome single-base resolution signals; whole genome methylation sequencing (WGBS) uses Bisulfite (BS) converts the unmethylated C to U, and then the methylation modification signal can be obtained by simply sequencing
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Although WGBS takes into account the advantages of whole-genome sequencing and acquisition of single-base signals, BS transformation is an extreme environment, which causes extremely high damage to DNA.
It is prone to mixing single and double strands, nicks, and notch damage, which can cause about 90% of DNA.
The template is lost, so the initial volume of its library construction is very high, and the effective data volume is low (in order to obtain a sequencing depth of 30X, a sequencing data volume of up to 230Gb is required, and the redundancy rate is more than half)
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Although WGBS sequencing can be optimized through a series of primer design, library construction optimization and other methods, there are still major shortcomings in clinical application
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 Considering the verification technology of ctDNA methylation markers, on the one hand, methylation detection has achieved good commercialization on the targeted sequencing platform (PCR and hybridization capture) driven by the advancement of targeted capture technology.
On the other hand, The BS-free sequencing technology for methylation is gradually achieving breakthroughs
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We believe that target selection for methylation detection and sequencing technology are still one of the barriers for early cancer screening companies.
Future technological breakthroughs are expected to improve sequencing accuracy and more accurately identify methylation signals
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Comparison of Methylation Sequencing Methods From the current commercialized products based on DNA methylation biomarkers, some methylation targets such as SEPT9 and MGMT have become star targets
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For example, after the FDA approved EpiproColon, an early screening product for colorectal cancer from Epigenomics, the domestic NMPA subsequently approved Borcheng (introduced Epigenomics patents), SEPT9 methylation detection kits for real organisms and Transjing Life, all for auxiliary diagnostic purposes
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South Korea's Genomictree company is suitable for colorectal cancer.
After EarlyTect, a product that detects SDC2 methylation, was approved by KFDA in 2014, the domestic SDC2 methylation test of Kolimin Biologics and Aide Biologics was subsequently approved by NMPA (also for auxiliary diagnostic purposes)
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It can be seen that the methylation detection products of currently approved targets overseas are easier to obtain certification and promote commercialization in China, but the methylation detection products of single target are more difficult to obtain approval for early screening purposes, and only serve as the gold standard.
Auxiliary diagnostic means of examination
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With the continuous development and improvement of liquid biopsy technologies such as ctDNA methylation sequencing, as a non-invasive and painless screening method, it will be extremely competitive in the field of early tumor screening in the future
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Article reference from: Soochow Securities "Tumor early screening, leveraging the market of 100 billion yuan, setting off the revolution of the times", the official website of the State Council, the official website of the National Health Commission