Methylation marker combination for pan-cancer detection and its application

Abstract

The present disclosure provides a combination of methylation markers for detecting pan-cancers and its application. The methylation marker combination is used for joint detection of eight cancer screenings and organ tracing of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, and ovarian cancer. The methylation marker combination can significantly improve the detection rate of cancer patients while ensuring a low false positive rate, and accurately predict the source organ of the cancer signal, which helps to further improve the early screening rate of patients.

Description

Technical Field

[0001] The present disclosure belongs to the field of biological detection technology, and specifically relates to a methylation marker combination and application thereof for screening of various cancers. Background Art

[0002] Liver cancer is a common malignant tumor in my country, with an insidious onset, high malignancy and poor prognosis. About 70% of HCC patients in China are in the middle or late stage at the time of initial diagnosis, and the recurrence rate is as high as 70% 5 years after surgery. Currently, alpha-fetoprotein (AFP) is commonly used in clinical diagnosis in combination with imaging examinations. When AFP exceeds the normal range, it is confirmed by abdominal B-ultrasound or enhanced CT. According to relevant reports, the sensitivity of AFP for early liver cancer screening is only 30-40%, and the sensitivity of combined ultrasound examination is only 63%.

[0003] Lung cancer ranks first in cancer incidence and mortality in my country. Currently, about 75% of patients are already in the advanced stage of lung cancer when diagnosed, missing the best time for radical treatment. Therefore, early screening and diagnosis of lung cancer are very important and can significantly improve patient survival. Clinically, imaging examinations are commonly used to screen for lung cancer, mainly including chest X-ray, chest LDCT, magnetic resonance imaging (MRI), PET-CT, etc., but the screening ability for early cancer is still insufficient.

[0004] The number of new cases of colorectal cancer ranked second among malignant tumors in my country in 2022. Colonoscopy is the gold standard for the diagnosis of colorectal cancer, but it is painful and has poor patient compliance, which is not conducive to large-scale screening. In addition, the fecal occult blood test (FIT) can be used for detection, but its sensitivity is poor.

[0005] Gastric cancer and esophageal cancer are common digestive tract malignancies in my country. Due to changes in dietary structure, the incidence rate has increased year by year. The early symptoms of gastric cancer and esophageal cancer are not obvious, and the patient consultation rate is low. Once discovered, it is already in the late stage. The commonly used diagnostic methods in clinical practice are gastroscopy and endoscopy screening, which is difficult to screen on a large scale.

[0006] Breast cancer and ovarian cancer are the most common malignant tumors in Chinese women. Ultrasound, molybdenum target X-ray and magnetic resonance imaging (MRI) are currently commonly used to screen for breast cancer, but they rely on the doctor's operating level and have a high rate of missed diagnosis, making early screening difficult. B-ultrasound and MRI can be used for ovarian cancer screening, but since there are no specific symptoms in the early stage and the patient consultation rate is low, it is necessary to seek a simpler screening method that is suitable for a large population.

[0007] Pancreatic cancer is one of the most malignant tumors. Its incidence rate is increasing year by year, and its prognosis is poor. In addition, early pancreatic cancer is often asymptomatic and difficult to detect. There is a lack of standardized screening methods in clinical practice, and only imaging examinations can be used for diagnosis, but this has great limitations for early diagnosis. Therefore, for the screening and diagnosis of early cancer, it is necessary to find more effective detection methods that are more conducive to large-scale screening.

[0008] Single cancer screening methods are more suitable for auxiliary diagnosis in high-risk groups. Since some high-risk factors for cancer (such as alcoholism and smoking) are correlated with multiple cancers, when using single cancer screening methods to screen healthy people and high-risk groups, multiple tests may lead to cumulative false positive rates, increasing the medical burden. Multi-cancer screening can screen and assist in the diagnosis of multiple cancers in healthy people at the same time, especially for those with a family history, high risk of environmental exposure, or known genetic susceptibility factors, to improve screening efficiency and compliance. Therefore, it is of great significance to develop a simple, accurate, and non-invasive method for early detection of multiple cancers.

[0009] When tumor cells rupture or die, they release circulating tumor DNA (ctDNA), which carries the genetic information of tumor cells. Liquid biopsy provides a new idea for early cancer screening because of its non-invasive sampling, low invasiveness and high accuracy. Detecting ctDNA through liquid biopsy technology can detect cancer earlier and predict the possibility of cancer.

[0010] DNA methylation sequencing is a high-resolution, high-throughput technology used in cancer screening, diagnosis, and monitoring. However, the methylation markers used in current DNA methylation sequencing are limited in the types of cancer they detect, the methylation sites they cover are not comprehensive, the sensitivity is low, and the accuracy of tracing back to the source needs to be improved. There is an urgent need for methylation markers that can detect multiple cancer types, cover comprehensive sites, have high sensitivity, and can accurately trace back to the source. Summary of the invention

[0011] In order to solve at least one of the above problems, the present disclosure provides specific hypermethylation markers, including a combination of detection and tracing markers, through which the tumor signal and tracing signal of cancer cfDNA can be effectively identified.

[0012] According to one aspect of the present disclosure, a methylation marker combination for detecting pan-cancer is provided, wherein the methylation marker combination includes methylation markers for detecting cancer types, based on the sequence of the human reference genome Hg19, and the methylation markers for detecting cancer types include any one or more combinations of the methylation markers shown in Table 1.

[0013] In some embodiments, the methylation marker combination also includes an organ-tracing methylation marker, based on the sequence of the human reference genome Hg19, and the organ-tracing methylation marker includes any one or more combinations of the methylation markers shown in Table 2.

[0014] In some embodiments, the pan-cancer species includes solid tumors.

[0015] In some embodiments, the pan-cancer species includes any of a primary tumor or a recurrent tumor.

[0016] In some embodiments, the pan-cancer comprises any one or more of respiratory system cancers, digestive system cancers, or reproductive system cancers.

[0017] In some embodiments, the pan-cancer comprises any one or more of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer and ovarian cancer.

[0018] According to another aspect of the present disclosure, a primer pair is provided, wherein the primer pair uses the nucleotide sequence where the methylation marker combination is located as the target sequence and is used for specific amplification of the target sequence.

[0019] According to another aspect of the present disclosure, a probe is provided, wherein the probe is used to specifically capture the nucleotide sequence where the methylation marker combination is located.

[0020] According to yet another aspect of the present disclosure, a kit is provided, comprising the methylation marker combination, the primer pair and / or the probe.

[0021] In some embodiments, the kit further comprises a conversion reagent capable of converting methylated or unmethylated cytosine into uracil.

[0022] In some embodiments, the conversion reagent comprises bisulfite or Ten-Eleven Translocation Enzymes (TET enzymes).

[0023] According to another aspect of the present disclosure, a pan-cancer detection method is provided, comprising the step of using the methylation marker combination, the primer pair, the probe or the kit to detect a sample to be tested collected from a subject.

[0024] In some embodiments, the cancer detection methylation marker, the primer pair for the cancer detection methylation marker, or the probe that specifically captures the cancer detection methylation marker is used to detect the sample to be tested collected from the subject; and / or

[0025] The organ-tracing methylation marker, the primer pair targeting the organ-tracing methylation marker, and the probe that specifically captures the organ-tracing methylation marker are used to detect the sample to be tested collected from the subject.

[0026] In some embodiments, the sample to be tested includes a tissue sample or a body fluid sample.

[0027] In some embodiments, the body fluid sample includes: saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, tumor cells, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge and their processed products; and / or,

[0028] The tissue samples include tissues, paraffin sections and their processed products.

[0029] According to another aspect of the present disclosure, a storage medium is provided, on which a computer program is stored, and when the computer program is executed by a processor, the pan-cancer detection method is implemented.

[0030] According to another aspect of the present disclosure, provided is the use of the methylation marker, the primer pair, the probe, the kit, and the pan-cancer detection method in preparing a product for early cancer screening, diagnosis, dynamic monitoring, organ tracing, or prognosis assessment.

[0031] In some embodiments, the pan-cancer species includes solid tumors.

[0032] In some embodiments, the pan-cancer species includes primary tumors and / or recurrent tumors.

[0033] In some embodiments, the pan-cancer comprises any one or more of respiratory system cancers, digestive system cancers, or reproductive system cancers.

[0034] In some embodiments, the pan-cancer comprises any one or more of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer and ovarian cancer.

[0035] In some embodiments, the pan-cancer species includes stage I to IV cancers.

[0036] In some embodiments, the pan-cancer includes any one or more of stage I-IV liver cancer, stage I-IV lung cancer, stage I-IV colorectal cancer, stage I-IV gastric cancer, stage I-IV esophageal cancer, stage I-IV pancreatic cancer, stage I-IV breast cancer and stage I-IV ovarian cancer.

[0037] The methylation marker combination disclosed in the present invention, in addition to the 8 cancer hypermethylation markers, also includes organ-tracing methylation markers, which can be used for cancer detection and accurate tracing of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, ovarian cancer, etc. While ensuring a low false positive rate, the detection rate of cancer patients can be significantly improved, and the source organ of the cancer signal can be accurately predicted. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The ROC curve of the 5-fold cross-training set of the early screening model is shown.

[0039] Figure 2 The performance results of the 5-fold cross-training set of the traceability model are shown.

[0040] Figure 3 The traceability prediction performance results of early screening positive samples are shown. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0042] At present, the main methods of cancer screening are imaging examination and cancer marker detection, but there are problems such as poor patient compliance, poor specificity and sensitivity, and some cancers lack effective screening methods. Therefore, in order to further improve the detection rate of early screening of multiple cancers and accurately predict the location of cancer onset, the present disclosure provides a methylation marker combination that can jointly detect eight cancer screening and tracing of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, and ovarian cancer.

[0043] In some embodiments, the present disclosure provides a set of methylation marker combinations that can be used for risk assessment of eight types of cancer and prediction of cancer occurrence sites. By performing whole genome methylation sequencing and targeted methylation high-depth sequencing on tissue samples, plasma samples and non-cancer plasma samples of eight types of cancer in stages, 437 methylation markers related to cancer risk assessment and 838 methylation markers related to organ tracing prediction were finally screened out.

[0044] The combination of methylation markers for screening eight cancers contains all or part of 437 methylation markers related to cancer risk assessment (Table 1) and / or 838 methylation markers related to organ tracing prediction (Table 2). The physical positions of all methylation markers are determined based on the comparison of the human whole genome sequence (version number is hg19).

[0045] Table 1: Methylation markers for early screening

[0046]

[0047]

[0048]

[0049]

[0050] Table 2: Traceable methylation markers

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Finally, plasma samples from eight other cancer patients, as well as healthy plasma samples and benign cancer plasma sample data were used as independent validation sets to verify the performance of the early screening cancer risk assessment system and the organ tracing prediction system when using the above-mentioned combination of markers.

[0060] The verification results show that the 437 markers related to cancer detection and the 838 markers related to tracing provided by the present invention can effectively identify the tumor signals and tracing signals of cfDNA of eight types of cancer.

[0061] definition

[0062] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0063] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0064] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0065] The term "cfDNA molecule" or "cfDNA" as used herein includes a DNA molecule that naturally exists in an extracellular form in a subject (e.g., in blood, serum, plasma, or other body fluids such as lymph, cerebrospinal fluid, urine, or sputum). Although cfDNA is initially present in one or more cells in a large complex biological organism (e.g., mammal), cfDNA undergoes release from the cell into the fluid present in the organism and can therefore be obtained by obtaining a sample of the fluid without the need for an in vitro cell lysis step.

[0066] The term "CpG site" as used herein is an abbreviation of "-C-phospho-G-", also referred to as a "CG site", and refers to a region on a DNA sequence in which a base sequence appears as cytosine followed by guanine, with a phosphodiester bond linking cytosine (C) and guanine (G), with C at the 5' end and G at the 3' end. Cytosine in a CpG site can be methylated to 5-methylcytosine. In mammals, 70% to 80% of the cytosine in CpG sites is methylated.

[0067] The term "methylation" or "DNA methylation" as used herein refers to the addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'→3' direction of a nucleic acid sequence). In some embodiments, DNA methylation refers to the addition of a methyl group to adenine, such as in N6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of cytosine). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine to generate 5-methylcytosine (5mC). In some embodiments, methylation includes derivatives of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxycytosine ( 5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the 3rd carbon of cytosine). In some embodiments, 3C methylation includes adding a methyl group to the 3C position of cytosine to generate 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT or CpC sites. DNA methylation can change the activity of methylated DNA regions. For example, when the DNA in the promoter region is methylated, the transcription of the gene may be repressed. DNA methylation is essential for normal development, and abnormalities in methylation can disrupt epigenetic regulation. Disruption of epigenetic regulation, such as repression, can cause diseases, such as cancer. Promoter methylation in DNA can indicate cancer.

[0068] As used herein, the term "cancer" refers to a disease or disorder caused by the proliferation of cells that are transformed by carcinogenesis. "Cancer" shall include any one or more of a wide range of benign or malignant tumors, including those that are able to grow and metastasize through the human or animal body or parts thereof, for example, via the lymphatic system and / or bloodstream invasion. Although the present invention is particularly intended to diagnose or detect malignant tumors and solid cancers, as used herein, the term "tumor" includes benign and malignant tumors or solid masses. Cancer includes (but is not limited to) liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer and ovarian cancer.

[0069] As used herein, the term "sequencing" refers to the process of determining the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, a nucleic acid, such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturation temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), composite probe anchor polymerase sequencing (cPAS), and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., etc. For example, BGI DNBseq sequencing platforms such as BGISEQ-500, BGISEQ-50, MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-T20X2, or Illumina's HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, NovaSeq6000, etc.

[0070] The term "subject" as used herein refers to any animal, mammal or human. The subject has, may have or is suspected of having any one or more of the combined diseases. The subject may have cancer, the subject may show symptoms associated with cancer, the subject may not show symptoms associated with cancer, or the subject may not have been diagnosed with cancer. In some embodiments, the subject is a human.

[0071] As used herein, the term "computer-readable medium" (e.g., data storage, data storage, etc.) or "computer-readable storage medium" refers to any medium that participates in providing instructions to a processor for execution. Such media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include but are not limited to optical disks, solid-state disks, magnetic disks, such as storage devices. Examples of volatile media can include but are not limited to dynamic memory, such as memory.

[0072] Common forms of computer readable media include, for example, floppy disks, diskettes, hard disks, magnetic tape or any other magnetic medium, CD-ROMs, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, RAM, PROM and EPROM, FLASH-EPROM, any other memory chip or cassette, or any other tangible medium from which a computer can read.

[0073] In addition to computer-readable media, data can be provided as a signal on a transmission medium included in a communication device or system to provide one or more instruction sequences to a processor of a computer system for execution. For example, a communication device may include a transceiver with a signal indicating instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in this disclosure. Representative examples of data communication transmission connections may include, for example, telephone modem connections, wide area networks (WANs), local area networks (LANs), infrared data connections, NFC connections, etc.

[0074] The term "diagnosis" as used herein refers not only to "judgment", but also includes "determination", "differentiation", "identification", "inspection", "detection" and "prediction". It includes distinguishing / differentiation or assisting in distinguishing / differentiating cancer and benign lesions, judging / determining cancer and the degree of development of cancer, predicting the risk of cancer, treatment effect, prognosis and risk of recurrence. In some embodiments, the diagnosis includes distinguishing or assisting in distinguishing cancer and benign lesions, judging the degree of development of cancer, or predicting the risk of cancer, treatment effect, prognosis and risk of recurrence.

[0075] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure, but do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be noted that for those skilled in the art, several variations and improvements made on the basis of this public creative conception all belong to the protection scope of the present disclosure. The reagents used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0076] Example

[0077] The samples involved in the examples were obtained from the Sun Yat-sen University Cancer Center and were approved by the Ethics Committee of the Sun Yat-sen University Cancer Center.

[0078] The methylation testing process includes the following steps:

[0079] 1. Sample DNA Extraction

[0080] 1.1 Plasma sample extraction

[0081] For whole blood, plasma / blood cell separation should be performed promptly (EDTA anticoagulant tube, within 4 hours; Streck tube within 72 hours). The separation steps are as follows:

[0082] ① Centrifuge at 1600g for 10 minutes at 4°C. After centrifugation, divide the upper layer of plasma into multiple 1.5mL or 2.0mL centrifuge tubes. Be careful not to aspirate the white blood cells in the middle layer during the plasma aspiration process.

[0083] ② Centrifuge at 16000g for 10 min at 4°C to remove residual cells, transfer the supernatant into a new 1.5mL or 2.0mL centrifuge tube (be careful not to aspirate the white blood cells at the bottom of the tube) to obtain the required plasma.

[0084] Plasma according to MagMAX TM Cell-Free DNA Isolation Kit extraction reagent instructions for extracting plasma cfDNA.

[0085] 1.2 Tissue sample extraction

[0086] DNA was extracted from liver cancer tissues and paired adjacent liver cancer tissues using RNeasy Mini Kit (Qiagen), and 100 ng of gDNA was ultrasonically sheared. The sheared DNA was retained for subsequent library construction and sequencing experiments.

[0087] 2. Methylation library construction

[0088] Using Hieff The extracted DNA was used to construct a library using the Ultima Pro DNA Library Prep Kit for Illumina, which was then oxidized by TET enzyme and reduced by pyridine borane, followed by PCR amplification to construct a prelibrary.

[0089] 3. Hybridization and Sequencing

[0090] If whole genome methylation sequencing is performed, hybridization is not required. If targeted methylation sequencing is performed, the library needs to be hybridized and captured in advance, and then the Gene+seq sequencer and other sequencers with the same principle are used for sequencing. The sequencing experiment is performed according to the operating instructions provided by the manufacturer.

[0091] Example 1 Clinical tissue and plasma sample detection and cancer marker combination screening

[0092] In this example, a total of 733 tissue samples (detailed sample types are shown in Table 3) and 271 healthy plasma samples (sample information is shown in Table 3) were used to perform 30× and 60× GMseq whole genome methylation sequencing, respectively.

[0093] The wgbstools tool was used to divide the human genome into multiple regions based on the whole genome methylation sequencing results of all cancer tissues, which were used as marker regions for subsequent screening and analysis.

[0094] For methylation marker screening, we first adopt a strategy of cancer-by-cancer screening and then merging. For each type of cancer, we select matching cancer tissues and adjacent cancer tissues for differential analysis, and preliminarily screen out high-methylation regions in which the methylation level in cancer tissues is significantly higher than that in adjacent cancer tissues. Then, we compare the cancer tissue of a target cancer type and any other cancer-related tissue (including cancer tissue and adjacent cancer tissue). After multiple rounds of one-to-one comparisons, we preliminarily screen out specific high-methylation regions in which the methylation level in the cancer tissue of each target cancer type is significantly higher than that in the other cancer-related tissues. We filter out high-noise regions in healthy plasma samples, screen out marker regions related to early screening and tracing, and customize probes based on these screened regions.

[0095] Table 3: Information of tissue samples tested

[0096] Cancer Number of cancer tissue samples Number of adjacent cancer / normal tissue samples Liver cancer 50 12 Bowel cancer 37 23 Lung cancer 97 210 Ovarian cancer 119 20 Pancreatic cancer 36 18 Breast cancer 35 5 Gastric cancer 24 7 Esophageal cancer 32 8

[0097] Furthermore, the above customized probes were used to perform targeted methylation detection on 1,374 plasma samples, and the test results were divided into two parts: training set and validation set.

[0098] The first part of the data (training set) is used for plasma screening of markers and model training, including 520 cancer plasmas and 453 non-cancer plasmas (see Table 4 for details); plasma screening includes following the strategy of screening by cancer and then merging. For each cancer type, the distribution ratio of markers with higher methylation levels in all plasma samples of this cancer type relative to non-cancer plasma is evaluated, and markers with high distribution ratios in all plasma samples of this cancer type are screened as markers related to cancer risk assessment; in addition, following the strategy of screening by cancer and then merging, for each cancer type, the proportion of markers with higher methylation levels in all plasma samples of this cancer type relative to other cancer plasma is evaluated, and markers with high distribution ratios in all plasma samples of this cancer type are screened as traceability markers. Finally, 437 methylation markers related to cancer risk assessment (as shown in Table 1) and 838 methylation markers related to organ traceability prediction (as shown in Table 2) were screened.

[0099] The second part of the data (validation set) was used to independently verify the clinical predictive performance of the early screening model and the tracing model, which included 215 cancer plasmas and 186 non-cancer plasmas (see Table 4 for details).

[0100] Table 4: Clinical information of plasma samples tested

[0101]

[0102]

[0103] Example 2 Model Construction

[0104] 1. Construction of an early screening model combining eight cancer markers

[0105] The early screening model was constructed by using the 437 methylation markers related to cancer risk assessment screened in Example 1. In short, the 973 clinical plasma samples (including 520 cancer plasma samples and 453 non-cancer plasma samples) in the training set shown in Table 4 were used to construct the model using the LogisticRegression function in sklearn (V1.3.0), and the model parameters were continuously optimized by cross-validation to finally lock in the multi-cancer early screening model.

[0106] Figure 1 The ROC curve of the 5-fold cross-validation performance of the early screening model is shown, where the horizontal axis represents the false positive rate (specificity) and the vertical axis represents the true positive rate (sensitivity). Figure 1 As shown, the established early screening model has good judgment ability.

[0107] 2. Construction of the traceability model

[0108] The traceability model was constructed by using the methylation markers related to the 838 organ traceability predictions proposed in Example 1. In short, the 520 clinical cancer plasma samples shown in Table 4 were used, and the LogisticRegression function in sklearn (V1.3.0) was used to complete the construction of the traceability model using the "one-vs-rest" strategy. The organ category with the highest predicted probability was selected as the traceability prediction result of Top1, and the organ category with the second highest probability was selected as the traceability prediction result of Top2.

[0109] The prediction performance of the training set was preliminarily evaluated through 5-fold cross validation, in which only the tumor samples predicted as positive by the early screening model were counted. The results are as follows: Figure 2 As shown. Figure 2 It can be seen from the results that the traceability model disclosed in the present invention has achieved a high prediction accuracy.

[0110] Example 3 Performance verification of early screening traceability model

[0111] The main purpose of this embodiment is to verify the performance of the early screening and tracing model through 215 cancer blood samples (27 lung cancers, 30 liver cancers, 38 colorectal cancers, 27 pancreatic cancers, 36 esophageal cancers, 15 gastric cancers, 20 breast cancers, and 22 ovarian cancers) and 186 non-cancerous plasma samples in Embodiment 1 that have not been used in the previous embodiments.

[0112] First, early screening prediction and tracing prediction were performed using the pre-built model in Example 2. Through statistical analysis of the predicted results, the early screening model specificity in the independent validation set was 99.5%, the overall sensitivity of early screening was 74.0%, and the detailed sensitivity of each cancer type and stage is shown in Table 5. The organ tracing accuracy of the top 1 tumor sample with positive early screening was 83.6%, and the organ tracing accuracy of the top 2 was 93.1%. The details of the prediction of the organs with the highest probability of each cancer are shown in Table 5. Figure 3 The sensitivity of the early screening model and the overall performance of the tracing model are shown in Table 6.

[0113] Table 5: Early screening prediction performance of each cancer stage in the independent validation set in Example 5

[0114]

[0115]

[0116] Table 6: Sensitivity of early screening model and overall performance of tracing model

[0117]

[0118] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A methylation marker combination for detecting pan-cancer species, characterized in that: The methylation marker combination includes cancer detection methylation markers, which are based on the sequence of the human reference genome Hg19, and the cancer detection methylation markers are composed of differential methylation regions shown in Table 1; the pan-cancer type is a combination of any one or more of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer and ovarian cancer.

2. The methylation marker combination according to claim 1, characterized in that: The methylation marker combination also includes an organ-tracing methylation marker, which is based on the sequence of the human reference genome Hg19. The organ-tracing methylation marker is composed of the differential methylation marker regions shown in Table 2.

3. The methylation marker combination according to claim 1, characterized in that: The pan-cancer types include primary tumors and / or recurrent tumors.

4. A primer pair combination, characterized in that: The primer pair combination specifically amplifies the methylation marker combination according to any one of claims 1 to 3.

5. A probe set, characterized in that: The probe set is used to specifically capture the methylation marker combination according to any one of claims 1 to 3.

6. A kit, characterized in that: The kit comprises the primer pair combination according to claim 4 or the probe set according to claim 5.

7. Use of the primer pair combination of claim 4, the probe set of claim 5, and the kit of claim 6 in the preparation of a product for early cancer screening, cancer diagnosis, dynamic cancer monitoring, or cancer organ tracing of a sample to be tested, wherein the cancer is any one or more of liver cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, and ovarian cancer.

8. The use according to claim 7, characterized in that: The cancer includes stage I to IV cancer.

9. The use according to claim 7, characterized in that: The sample to be tested includes: a sample to be tested collected from a subject.

10. The use according to claim 7, characterized in that: The sample to be tested includes a tissue sample or a body fluid sample.

11. The use according to claim 10, characterized in that: The body fluid samples include: saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, tumor cell fluid, alveolar lavage fluid, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge and their processed products; and / or, The tissue samples include tissues, paraffin sections and their processed products.

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