CN111968702B - Malignant tumor early screening system based on circulating tumor DNA - Google Patents
Malignant tumor early screening system based on circulating tumor DNA Download PDFInfo
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Abstract
The invention discloses a malignant tumor early screening system based on circulating tumor DNA, which simultaneously utilizes mutation existing in sample circulating free DNA and leucocyte genome DNA, combines malignant tumor mutation capture library and malignant tumor related pathological mutation site comparison, eliminates non-tumor specific gene mutation, and can extract specific molecular markers released into a blood circulating system by malignant tumor cells from the gene mutation of the sample, namely circulating tumor DNA mutation sites. The invention can screen malignant tumor in early stage based on the variation of human body circulating tumor DNA, and improves the sensitivity and specificity of screening.
Description
Technical Field
The invention belongs to the technical field of biotechnology and medical detection, and particularly relates to early screening and risk assessment of malignant tumors by detecting circulating tumor DNA (ctDNA) molecular variation in human peripheral blood.
Background
Cancer is medically collectively called malignant tumor, is a complex serious disease severely threatening human health, and is the second leading cause of death in humans worldwide, next to cardiovascular and cerebrovascular diseases. Malignant tumor has the characteristics of hidden onset, long onset time, rapid progress, limited screening and treatment means and the like, and when patients perceive the corresponding symptoms, the malignant tumor often develops to middle and late stages, and clinical intervention and treatment are difficult. Mortality of malignant tumors is closely related to the period of clinical diagnosis, and the earlier the clinical diagnosis is, the higher the survival rate of patients and the lower the death rate are. It is counted that the five-year survival rate of most patients with advanced malignant tumors is less than 20%, and if diagnosis and treatment and intervention can be performed at an early stage, the five-year survival rate of most patients with advanced malignant tumors can be improved to more than 70%. At present, the prevention and treatment pressure of malignant tumors is huge, the types of the malignant tumors with high incidence rate in different countries and regions are often different, sex difference exists, and early detection, early diagnosis and early treatment of the malignant tumors are key links for reducing the death rate of the malignant tumors.
At present, the early malignant tumor screening means commonly used in clinic mainly comprise imaging examination and serum tumor protein marker detection. However, imaging screening technologies including PET-CT, nuclear magnetic resonance, and endoscopy have the characteristics of high operation difficulty, insufficient sensitivity and specificity, certain requirements on the size of malignant tumors, high cost, and the like, and particularly have the requirements on the size of tumors, so that most malignant tumors are in middle and late stages when passing imaging examination, and the imaging screening technologies have limitations in early screening of malignant tumors and cannot effectively perform early screening of malignant tumors. The serum tumor protein marker detection has the characteristic of simple operation, can be used for large-scale screening of malignant tumors, but has insufficient sensitivity and specificity, and is mainly characterized in that: the occurrence of certain other diseases can also obviously influence the content of the corresponding tumor protein markers, so that the screening efficiency of malignant tumors is further influenced, meanwhile, the existing serum tumor protein markers are often screened only for a certain or a certain type of malignant tumors, and the situation and risk of malignant tumors of people are difficult to comprehensively reflect.
Malignant tumors occur due to unrestricted proliferation and differentiation of cells caused by genetic variation, and are characterized by abnormal cell growth and abnormal differentiation caused by genetic mutation, while most malignant tumors occur involving complex interactions of genes with environmental factors including dietary nutrition, environmental pollutants, drugs, radiation, pathogenic microorganism infection, and the like. Malignant tumor driving genes are genes that play a decisive role in the development of malignant tumors, for example, activation of protooncogenes and inactivation of cancer suppressor genes in humans are key to the development of cancerous cells.
Changes from gene levels to clinically visible malignant tumors typically last years or even longer. In the early growth stage of malignant tumor, the malignant tumor can release its specific DNA fragments into human blood circulation system through apoptosis, necrosis, active release and other mechanisms, and these tumor specific DNA fragments free in peripheral blood are circulating tumor DNA (ctDNA), which usually contains point mutation (mutation of single base), deletion, insertion, rearrangement, copy number abnormality, methylation and other variants, and these variants can be used as specific malignant tumor molecular markers, wherein the maximum number of variants is mutation of single base. Therefore, detection of ctDNA in blood by biopsy technique in early stage of malignant tumor occurrence has a certain meaning for early screening of malignant tumor. However, early screening of malignant tumors based on circulating tumor DNA has some technical disadvantages and ultimately affects the sensitivity and specificity of early screening of malignant tumors: first, the existing detection method for the circulating tumor DNA is not strict in screening the circulating tumor DNA, the driving genes and the genetic variation sites adopted by different detection methods are large in difference, and the crowd difference is less considered. Secondly, the sequencing depth adopted when the second generation sequencing technology is utilized is low, so that trace circulating tumor DNA variation in blood can not be effectively detected; and lack effective sequencing error cancellation or compensation measures. Third, most of the circulating free DNA (cfDNA) variations in blood result from clonal hematopoiesis, resulting in a significant background noise in ctDNA detection when bioinformatic analysis is performed.
Disclosure of Invention
The invention aims to provide a malignant tumor early-stage screening system based on circulating tumor DNA, which can be used for carrying out early-stage screening on human malignant tumors based on the mutation condition of human circulating tumor DNA and improving the sensitivity and specificity of screening.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The early malignant tumor screening system comprises a circulating free DNA mutation analysis module, a leucocyte genome DNA mutation analysis module, a mutation site screening module and a malignant tumor screening and risk assessment module;
the circulating free DNA mutation analysis module is used for carrying out statistical analysis on the mutation of the corresponding target region of the sample circulating free DNA according to the sequencing result of the target region capture library, so as to obtain mutation sites in the sample circulating free DNA and serve as candidate mutation sites;
The leucocyte genome DNA mutation analysis module is used for carrying out statistical analysis on the mutation situation of the corresponding target region of the sample leucocyte genome DNA according to the sequencing result of the target region capture library, so as to obtain mutation sites in the sample leucocyte genome DNA and serve as candidate mutation sites;
The mutation site screening module is used for extracting the mutation sites of the circulating tumor DNA with the specificity of the malignant tumor cells from the candidate mutation sites of the circulating free DNA of the sample according to the sequencing depth, the mutation frequency, the mutation band number of the single mutation site and the mutation source by carrying out threshold comparison and sequence comparison on the candidate mutation sites in the circulating free DNA of the sample and the genome DNA of the leucocytes;
The malignant tumor screening and risk assessment module is used for judging early malignant tumor disease or malignant tumor risk increase according to the extraction result of the circulating tumor DNA mutation site with malignant tumor cell specificity (namely the extraction result of the mutation site screening module) and the relation between the extracted mutation sites and the malignant tumor clinical diagnosis result.
Preferably, the target region is selected from a malignant tumor driving gene mutation site.
Preferably, the malignant tumor is selected from one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, thyroid cancer, lymphoma, bladder cancer, renal cancer, endometrial cancer, nasopharyngeal cancer, prostate cancer, ovarian cancer, glioma, gall bladder cancer, leukemia, melanoma, oral cancer, laryngeal cancer, testicular cancer, osteosarcoma.
Preferably, the sample is selected from human peripheral blood, and the sample collection amount is 7.5-10 mL/human.
Preferably, in the mutation site screening module, all candidate mutation sites (mutation sites in the circulating free DNA and the leukocyte genomic DNA of the sample as determined by the sequencing and statistical analysis described above) below the sequencing depth threshold, the mutation frequency threshold and the mutation band number threshold of the single mutation site are excluded, i.e., these sites are not taken as circulating tumor DNA mutation sites with malignant cell specificity.
Preferably, the sequencing depth threshold is 10000× -40000×, the mutation frequency threshold is 3 mill-1%, and the mutation band number threshold of single mutation site is not less than 10.
Preferably, in the mutation site screening module, for any candidate mutation site that remains (not excluded) after the above threshold comparison, if the candidate mutation site is determined to be present in both the circulating free DNA and the leukocyte genomic DNA of the sample by the comparison, the candidate mutation site is excluded, i.e., these sites are not taken as circulating tumor DNA mutation sites with malignant cell specificity. So far, the mutation site screening module takes the remaining candidate mutation sites which are not excluded as the extraction result of the mutation sites of the circulating tumor DNA with the specificity of the malignant tumor cells and outputs the extraction result to the malignant tumor screening and risk assessment module.
Preferably, in the malignancy screening and risk assessment module, if the circulating tumor DNA mutation site with malignancy cell specificity is not extracted or the extracted circulating tumor DNA mutation site is irrelevant to clinical diagnosis of malignancy, determining that the sample is not from an early-stage malignancy patient or that the risk of the individual corresponding to the sample suffering from malignancy is the same as the general population (population prevalence); if the mutation sites of the circulating tumor DNA with the specificity of malignant tumor cells are extracted, and the extracted mutation sites are relevant to clinical diagnosis of malignant tumor, judging that the sample is from early malignant tumor patients or individuals corresponding to the sample are at higher risk of suffering from malignant tumor than the general population.
The beneficial effects of the invention are as follows:
The invention simultaneously utilizes mutation site information existing in circulating free DNA and leucocyte genome DNA of a sample, combines malignant mutation capture library and malignant tumor related pathological mutation site comparison, eliminates non-tumor specific gene mutation, and can extract specific molecular markers (namely circulating tumor DNA mutation sites) released into a blood circulation system by malignant tumor cells from the gene mutation of the sample, thereby improving sensitivity and specificity of early screening of malignant tumors. The invention can identify the released molecular variation signs at the early stage of malignant tumor occurrence, can discover and prompt malignant tumor risks earlier, and provides valuable time for early intervention of malignant tumor.
Furthermore, the average sequencing depth of the second generation sequencing technology is improved to more than 10000 multiplied by the invention, the quality of sequencing data is strictly controlled, and the mutation detection capability is improved.
Furthermore, the mutation detection error caused by other factors such as sequencing equipment is eliminated by setting the mutation frequency threshold and the mutation band number threshold.
Furthermore, the sample detected by the invention is a blood sample, is convenient to collect and has repeatability, and can provide dynamic change of internal circulation tumor DNA through collection and analysis at different time points, thereby indirectly reflecting the development trend of malignant tumor or adopting the intervention effect after intervention, and the invention has the characteristics of safety, minimally invasive, real time and the like.
Furthermore, the invention can improve the level of early screening and risk assessment of malignant tumors for specific people by constructing a malignant tumor type set.
Detailed Description
The present invention will be described in further detail with reference to examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
Construction of a local database
The local database stores the following known data:
1.1 Malignant tumor driving gene set)
The malignant tumor driving gene may be a protooncogene, an oncogene inhibitor or a malignant tumor-associated signal pathway gene;
1.2 Malignant tumor driver gene mutation site set)
Recording the position of the malignant tumor mutation site, the change condition of genes and genome bases, the change condition of amino acid caused by mutation and the occurrence frequency of site variation in various malignant tumors;
1.3 Malignant tumor driver gene mutation site targeting capture probe set
The attorney synthesis targeting capture probe is used for constructing a library;
1.4 Set of malignancy types)
According to national cancer statistics published by the national cancer center in 2019, population-specific malignancy types related to early screening of malignant tumors are experimentally screened, and the method specifically comprises the following steps: lung cancer, stomach cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, thyroid cancer, lymphoma, bladder cancer, kidney cancer, endometrial cancer, nasopharyngeal cancer, prostate cancer, ovarian cancer, glioma, gall bladder cancer, leukemia, melanoma, oral cancer, laryngeal cancer, testicular cancer, and osteosarcoma;
The malignant tumor driving genes and mutation site information related in the database come from biological information libraries such as the national center for biotechnology information NCBI(National Center for Biotechnology Information)、TCGA(The Cancer Genome Atlas)、COSMIC(Catalogue of Somatic Mutations in Cancer), and NCI (National Cancer Institute); the database has data updating and supplementing functions.
(II) early stage screening procedure for malignant tumor
1) 10ML of peripheral venous blood of a human subject is collected by using a standard STRECK CELL-Free DNA blood collection tube containing EDTA anticoagulant and special cell stabilizer.
2) Serum and leukocytes were isolated from the peripheral blood of the human subjects.
Specifically, the blood sample can be processed according to Shenzhen market standardized guiding technical document SZDB/Z186-2016 (human blood sample collection, processing, transportation and storage Specification for high-throughput sequencing research) to obtain serum and leucocyte respectively.
3) Cyclic free DNA (cfDNA) extraction and sequencing
3.1 CfDNA extraction of the isolated serum using standard human blood sample cfDNA extraction kit.
3.2 Sample quality inspection flow: the extracted cfDNA is detected by using the Nanodrop gel electrophoresis technology, the quality of nucleic acid (including purity, concentration and analysis of integrity or degradation degree) is evaluated, and SOP is formulated (the total cfDNA is required to be more than 30ng, and the ratio of absorbance values at 260nm and 280nm is about 1.8).
3.3 After the extracted cfDNA is detected to be qualified, library construction is performed by adopting a standard Illumina library construction flow.
3.4 Library quality inspection procedure: according to the current quality requirements and standards of library construction of target region capture, the constructed library is subjected to quality inspection by an Agilent2100 biological analyzer and a 7500 type real-time fluorescence quantitative PCR (polymerase chain reaction) instrument of ABI company, and library quality inspection SOP (the total amount of the library is required to be more than 30ng, the concentration is more than 20 ng/mu L, and the main peak of a 2100 peak graph is 310 bp+/-15 bp) is established.
3.5 Sequencing flow: on-machine sequencing is performed on an NGS platform of Illumina company, the average sequencing depth of cfDNA is 10000× (which cannot be lower than the index), and the sequencing quality q30% is not less than 80%.
3.6 Bioinformatics analysis flow: and (3) performing quality control and basic biological information analysis on the machine-down data, removing the result that the quality control cannot pass, and finally collecting the information such as malignant tumor driving genes matched with probes, corresponding mutation sites, sequencing depth, mutation frequency, mutation type, mutation site single-base sequencing quality, corresponding mutation band number and the like in the circulating free DNA of the tested person. Most of the extracted circulating free DNA of the tested person is a sequence which is not mutated, the DNA with mutation released by malignant tumor into blood only occupies a small part of all cfDNA, most cfDNA is released by apoptosis or necrosis of other normal cells into blood, the corresponding sections of the cfDNA do not carry tumor related mutation, and the mutation frequency of the cfDNA of the tested person and the mutation band number of the corresponding sites can be obtained through sequencing.
4) Extraction and sequencing of genomic DNA from leukocytes
4.1 The isolated leukocytes were subjected to genomic DNA extraction using standard human genomic DNA extraction kits.
4.2 Sample quality inspection flow: genomic DNA quality was checked using Nanodrop gel electrophoresis techniques in combination with OD values to assess nucleic acid quality (including purity, concentration, and integrity or degradation analysis).
4.3 For the qualified leukocyte genomic DNA, library construction was performed using a standard Illumina library construction procedure.
4.4 Library quality inspection procedure: and (3) according to the current requirements and standards of the quality of the target area capturing and library construction, carrying out quality inspection on the constructed library by adopting an Agilent2100 biological analyzer and a 7500 type real-time fluorescent quantitative PCR (polymerase chain reaction) instrument of ABI (Abies).
4.5 Sequencing flow: by utilizing on-machine sequencing on an NGS platform of Illumina company, the average sequencing depth of the leukocyte genome DNA is 10000× (which cannot be lower than the index), and the sequencing quality q30 is more than or equal to 80%.
4.6 Bioinformatics analysis flow: and (3) performing quality control and basic biological information analysis on the on-press data, removing the result that the quality control cannot pass, and finally collecting the malignant tumor driving genes matched with the probes in the genome DNA of the tested human leucocytes, and the corresponding mutation sites, sequencing depth, mutation frequency, mutation types, single-base sequencing quality of the mutation sites, the number of corresponding mutation bands and the like. The extracted leucocyte genome DNA of the tested human is mostly an undenatured sequence, some leucocyte genome DNA can be mutated due to the cloning hematopoiesis, the mutated DNA (namely, DNA with mutation) sequence only occupies a small part of the genome DNA sequence of all leucocytes, the abundance of the mutation carried by the subcloning of the hematopoietic stem cells is low (90 percent cloning hematopoiesis abundance is less than 1 percent), the mutation has obvious difference with germ line mutation from germ cells, but the detection result of ctDNA can be obviously influenced, and the mutation frequency and the corresponding mutation band number of the leucocyte genome DNA of the tested human can be obtained through sequencing.
5) On the basis of library construction, sequencing and basic bioinformatic analysis of the corresponding DNAs extracted from the serum and the white blood cells of the human subject according to the above steps, the malignant tumor driving gene mutation in the circulating free DNA and the white blood cell genomic DNA determined after the sequencing can be further analyzed by using a computer bioinformatic processing program, and whether the human subject is an early malignant tumor patient or is at risk of malignant tumor is determined according to the analysis result; the method comprises the following specific steps:
5.1 Setting the sequencing depth of 10000 multiplied by the sequencing depth threshold value, and removing malignant tumor driving gene mutation with the sequencing depth of less than 10000 multiplied by the circulating free DNA in the genome DNA of the leucocytes.
5.2 Setting mutation frequency of three thousandths as a mutation frequency threshold value, and removing malignant tumor driving gene mutation with mutation frequency lower than three thousandths in leukocyte genome DNA and circulating free DNA.
5.3 Setting the threshold value of the number of mutation bands of a single mutation site to 10, and removing malignant tumor driving gene mutations with the number of mutation bands lower than 10 in the genomic DNA of leucocytes and circulating episomal DNA.
5.4 After step5.3, the results of the sequencing of the genomic DNA of the human leucocytes and of the circulating free DNA (mutation sites) to be tested are compared with each other and the mutations detected by the sequencing of the genomic DNA of leucocytes are filtered out, which are not considered to be released into the blood by the tumor.
5.5 After step 5.1 to step 5.4 are completed, the mutation of the malignant tumor driving gene stored in the local database is also present in the sequencing result of the circulating free DNA of the tested person, and the pathological or possibly pathological gene loci are compared with the database such as clinvar, cosmic, tcga, so that the risk of the tested person for early stage malignant tumor patients or malignant tumor patients is judged to be increased, and meanwhile, the higher the number of mutation loci which are present after the treatment of the steps is, the risk of the tested person for malignant tumor patients is also increased. After completing steps 5.1 to 5.4, the test person is judged to have no malignancy or risk of malignancy if there is no malignancy-driven gene mutation stored in the local database in the sequencing result of the circulating free DNA of the test person.
(III) early malignant screening and risk assessment examples
Example 1
Mutation detection and analysis of circulating free DNA and leukocyte genomic DNA in blood samples of 162 stage i or stage ii malignancy patients (malignancy species meeting the set of types set by the above local database, specifically lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, and breast cancer) and 156 controls (healthy and non-malignant diseases) were performed using the above-described early malignancy screening procedure.
Blood samples from 156 control samples and 162 malignancy patients were collected from hospitalized and outpatient patients in third and second class hospitals in western security city of shanxi, 10 months in 2017.
Of the 162 malignant tumor patient samples, 133 malignant tumor patients were judged as a result of the analysis, i.e., the sensitivity to screening of malignant tumor patients was 82.1%. The analysis result shows that the number of cases of no malignant tumor or risk of malignant tumor is 29 as the general population, namely 17.9% of false negative results exist.
Of 156 control samples, the analysis result judges that the risk of suffering from malignant tumor is 148 samples equivalent to the general population, namely, the specificity of screening malignant tumor patients can reach 94.8%. The analysis results determined 8 samples with malignancy or increased risk of malignancy, i.e., about 5.2% false positive results were present.
Example 1 shows that the early malignant tumor screening technology based on the circulating tumor DNA (ctDNA) has higher sensitivity and specificity than the conventional clinical malignant tumor screening method (B ultrasonic, X-ray and serum tumor protein markers) and the conventional screening method based on the circulating tumor DNA.
In addition, when the mutation frequency threshold and the mutation band number threshold were not considered to be set in the processing of the sequencing result, the false negative result ratio of the 162 cases of samples of malignant tumor patients was 10.5%, and the false positive result ratio of the 156 cases of samples of controls was 46.7%.
Example 2
Mutation detection and analysis of circulating free DNA and leukocyte genomic DNA in blood samples of 75 healthy and non-malignant disease patients were performed using the above-described early malignant screening procedure.
75 Healthy and non-malignant disease patients are random populations, and come from a certain health physical examination organization in western An city of Shaanxi, and the blood sample collection time is 2017 and 11 months.
Of the 75 samples, the samples judged to have malignancy or an increased risk of having malignancy were 3. The 3 individuals are subjected to early malignant tumor screening (including conventional serum tumor protein markers and imaging screening) in third class first hospitals, and the results are negative, but after one year, one of the individuals is diagnosed as early lung cancer by third class first hospitals.
Example 2 shows that the early malignant screening technology based on circulating tumor DNA (ctDNA) can prompt malignant risk earlier before the existing screening technology means is used for diagnosing malignant tumor, namely, the malignant sign is found earlier than the traditional malignant screening technology.
In a word, the invention has a plurality of advantages compared with the existing tumor early screening technology, and has higher sensitivity and specificity compared with the conventional tumor protein marker screening technology; compared with B ultrasonic and CT based on imaging, the invention has no radiation to human body, and has higher sensitivity and specificity; compared with screening technologies such as gastroscope and rectoscope, the invention does not cause pain to the subject and has higher acceptance of the subject.
Claims (5)
1. An early malignant tumor screening system based on circulating tumor DNA, which is characterized in that: the early malignant tumor screening system comprises a circulating free DNA mutation analysis module, a leucocyte genome DNA mutation analysis module, a mutation site screening module and a malignant tumor screening and risk assessment module;
The circulating free DNA mutation analysis module is used for analyzing the mutation condition of the corresponding target region of the sample circulating free DNA according to the sequencing result of the target region capture library, so as to obtain candidate mutation sites in the sample circulating free DNA;
the leucocyte genome DNA mutation analysis module is used for analyzing the mutation condition of the corresponding target region of the sample leucocyte genome DNA according to the sequencing result of the target region capture library, so as to obtain candidate mutation sites in the sample leucocyte genome DNA;
The mutation site screening module is used for extracting the mutation sites of the circulating tumor DNA with the specificity of the malignant tumor cells from the candidate mutation sites of the circulating free DNA of the sample according to the sequencing depth, the mutation frequency, the mutation band number of the single mutation site and the mutation source by carrying out threshold comparison and sequence comparison on the candidate mutation sites in the circulating free DNA of the sample and the genome DNA of the leucocytes;
the malignant tumor screening and risk assessment module is used for judging early malignant tumor disease conditions or malignant tumor risk increase conditions according to the extraction result of the circulating tumor DNA mutation sites with malignant tumor cell specificity and the relation between the extracted circulating tumor DNA mutation sites and malignant tumor clinical diagnosis results;
In the mutation site screening module, candidate mutation sites lower than a sequencing depth threshold value, a mutation frequency threshold value and a mutation band number threshold value of single mutation sites are eliminated;
the sequencing depth threshold is 10000 x-40000 x, the mutation frequency threshold is 3 per mill-1%, and the mutation strip number threshold of single mutation site is more than or equal to 10;
in the mutation site screening module, for any candidate mutation site which is reserved after threshold comparison, if the candidate mutation site exists in circulating free DNA and leukocyte genome DNA of a sample at the same time, the candidate mutation site is eliminated.
2. The early malignant screening system based on circulating tumor DNA of claim 1, wherein: the target region is selected from a malignant tumor driving gene mutation site.
3. The early malignant screening system based on circulating tumor DNA according to claim 1 or 2, characterized in that: the malignant tumor is selected from one or more of lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, thyroid cancer, lymphoma, bladder cancer, renal cancer, endometrial cancer, nasopharyngeal cancer, prostatic cancer, ovarian cancer, glioma, gallbladder cancer, leukemia, melanoma, oral cancer, laryngeal cancer, testicular cancer and osteosarcoma.
4. The early malignant screening system based on circulating tumor DNA of claim 1, wherein: the sample is selected from human peripheral blood, and the sample collection amount is 7.5-10 mL/human.
5. The early malignant screening system based on circulating tumor DNA of claim 1, wherein: in the malignancy screening and risk assessment module, if a circulating tumor DNA mutation site with malignancy cell specificity is not extracted or the extracted circulating tumor DNA mutation site is irrelevant to clinical diagnosis of malignancy, judging that a sample is not from an early-stage malignancy patient or an individual corresponding to the sample has the same risk of malignancy as a general population; if the circulating tumor DNA mutation site with the specificity of the malignant tumor cells is extracted, and the extracted circulating tumor DNA mutation site is related to clinical diagnosis of the malignant tumor, judging that the sample is from an early malignant tumor patient or an individual corresponding to the sample is higher in risk of suffering from the malignant tumor than the general population.
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