Methylation biomarker combination for detecting tumor burden of metastatic prostate cancer and use

Abstract

Provided are a methylation biomarker combination for detecting the tumor burden of metastatic prostate cancer and the use. The methylation biomarker combination is selected from any combination of at least two of 171 methylation biomarkers. The methylation biomarker combination is used for preparing a detection kit, and the detection kit is used in the detection and diagnosis of the tumor burden of prostate cancer. By means of utilizing the high correlation between DNA methylation molecular markers and prostate cancer, a detection kit is prepared by means of combining the methylation biomarkers, which can be used for detecting the tumor burden of metastatic prostate cancer. The detection of the tumor burden of metastatic prostate cancer is realized in a non-invasive manner, and the sensitivity, specificity and accuracy of the detection of the tumor burden of metastatic prostate cancer are increased. Moreover, compared with existing detection methods, the method has significant progressive advantages in non-invasive diagnosis, convenience and rapidness, and greatly improved practicability, and also has great feasibility for clinical promotion.

Description

Methylation biomarker combination and application for detecting tumor burden in metastatic prostate cancer Technical Field

[0001] The present invention relates to the technical field of biomarker combination applications, and in particular to a methylation biomarker combination and application for detecting metastatic prostate cancer tumor load. Background Art

[0002] Prostate cancer is the second most common cancer and the fifth leading cause of cancer-related death in men. The incidence rate of prostate cancer in developed countries is 37.5 per 100,000 people, while in developing countries it is 11.3 per 100,000 people. The mortality rate is 8.1 per 100,000 people in developed countries and 5.9 per 100,000 people in developing countries. However, according to the latest national cancer report released by the China Cancer Center, the incidence rate of prostate cancer in my country has shown a clear upward trend in recent years, ranking sixth among male tumors. This is particularly true in major Chinese cities such as Shanghai, where the incidence rate is even higher, ranking fourth among male malignant tumors, surpassing liver cancer. Currently, approximately 10 million men are diagnosed with prostate cancer. Prostate cancer causes more than 400,000 deaths worldwide each year, and by 2040, the mortality rate is expected to reach over 800,000 per year.

[0003] The increasing number of patients not only places a heavy economic burden on society and families, but also brings more pain to patients and a decline in their quality of life. The clinical staging of newly diagnosed prostate cancer patients in China is very different from that in developed Western countries. Taking the United States as an example, among newly diagnosed prostate cancer patients, clinically localized cases account for 76%, local lymph node metastasis accounts for 13%, and distant metastasis accounts for only 6% (the remaining 5% are cases of unknown stage). Data from a multi-center study in China show that only one-third of newly diagnosed prostate cancer patients have clinically localized prostate cancer, and most patients are already in the middle or late stages at the time of initial diagnosis, resulting in the overall prognosis of prostate cancer patients in China being far worse than that in developed Western countries. Currently, the proportion of patients in my country who are initially diagnosed with high-risk or metastatic disease is around 50%. Although this is less than 70% in 2015, it is still far from the level of European and American countries.

[0004] The CHAARTED study defined high tumor burden as visceral metastasis, or bone metastasis lesions ≥ 4, of which at least 1 was outside the spine or pelvis. Low tumor burden was defined as no visceral metastasis and ≤ 3 bone metastasis lesions. The number of metastatic lesions and tumor burden in patients with metastatic prostate cancer are related to treatment prognosis. For example, surgery-based primary lesion treatment is expected to improve the prognosis of patients with low tumor burden and has better safety, while surgical treatment of patients with high tumor burden has little benefit. The treatment strategies for patients with different tumor burdens are different, and the indications for different treatment regimens are also different. Therefore, for patients with metastatic prostate cancer, the guidelines recommend stratifying patients according to low tumor burden and high tumor burden, and clinical treatment also needs to be stratified.

[0005] Circulating cell-free DNA (cfDNA) is small, free DNA fragments in peripheral blood, primarily derived from tumor-related necrosis or physiological apoptosis. It contains genetic information such as somatic mutations and DNA methylation. Detection of cfDNA, circulating tumor cells (CTCs), or exosomes in the blood is known as liquid biopsy. Compared with traditional tissue biopsies, it offers numerous advantages, including rapidity, low risk, low cost, and low invasiveness. To date, numerous studies have demonstrated that cancer-associated DNA mutations or methylation modifications can be detected in cfDNA from cancer patients. In 2017, Wyatt's team demonstrated that all somatic mutations found in prostate cancer metastatic tissue were also present in matched cfDNA samples. A growing number of clinical studies indicate that cfDNA can be used as a biomarker for the diagnosis and prognosis of prostate cancer patients.

[0006] The current methods for examining prostate cancer tumor burden mainly include bone scanning (ECT) and targeted prostate-specific membrane antigen (PSMA) PET / CT. Prostate cancer usually first metastasizes to the bones. When diagnostic examinations are required, technetium-99 radionuclide bone scanning is still the standard method for evaluating bone metastases. Although bone scans have high sensitivity, they lack specificity. In fractures, injuries, or local inflammation or degeneration, because of the osteogenesis process in bone repair, there will also be signal concentration, which can also cause false positives on bone scans. Therefore, patients with positive or unclear bone scans usually need further examinations. In recent years, the application of PSMA PET / CT imaging in the diagnosis and treatment of prostate cancer has gradually increased. PSMA PET / CT is more accurate and comprehensive than conventional imaging methods in detecting occult lymph node and bone metastases. Commonly used 68Ga-labeled PSMA-based imaging agents are primarily excreted through the urinary system, making it easy to miss small metastatic lesions or local recurrences around the bladder. Furthermore, approximately 10% of patients have low PSMA expression in their tumor lesions, resulting in negative PSMA PET imaging. PSMA is not only highly expressed in prostate cancer cells; it is also expressed to some extent on the endothelial membranes of neovascular cells in many solid tumors, such as lung, kidney, colon, thyroid, and brain tumors, and can be detected by PSMA PET / CT. Furthermore, nonspecific uptake of PSMA PET imaging agents can be observed in various benign lesions, such as bronchiectasis with infection, sarcoidosis, Paget's disease, and ganglia. PSMA PET / CT is expensive, and false positives and negatives plague the interpretation of imaging results. Its detection efficacy requires further research to explore and confirm.

[0007] Authoritative guidelines both domestically and internationally stratify prostate cancer treatment based on high and low tumor burden. Prostate cancer testing requires not only detecting metastasis but also tumor burden. Previous test kits rarely assess prostate tumor burden, while imaging tests are limited by false negatives and false positives. Therefore, the development and application of methylation biomarker combinations for detecting metastatic prostate cancer is crucial to address these challenges.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a methylation biomarker combination and application for detecting the tumor burden of metastatic prostate cancer. By utilizing the high correlation between DNA methylation molecular markers and prostate cancer, a detection kit is prepared by combining methylation biomarkers, which can be used for the detection of metastatic prostate cancer. The detection of metastatic prostate cancer tumor burden is achieved in a non-invasive manner, thereby improving the sensitivity, specificity and accuracy of the detection of metastatic prostate cancer, thereby addressing the above-mentioned deficiencies in the technology.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] A methylation biomarker combination for detecting metastatic prostate cancer tumor burden, selected from any combination of at least two of the following methylation biomarkers:

[0012] chr1:1137001-1137301、chr1:145573620-145573920、chr1:155620501-155620801、chr1:184006347-184006405、chr1:205631898-205632198、chr1:230208001-230208301、chr1:29564367-29564667、chr1:58234801-58235101、chr1:87767701-87768001、chr1:91089001-91089301、chr1:147487202-147487501、chr1:147532802-147533101、chr1:181059009-181059309、chr1:181059309-181059609、chr1:94146733-94146927、chr10:133506601-133506901、chr10:2095501-2095801、chr10:482262-482562、chr10:77158920-77159220、chr10:91330501-91330801、chr10:17270986-17271286、chr10:73156539-73156552、chr10:79397203-79397502、chr11:2867401-2867701、chr11:45672001-45672301、chr11:64875601-64875901、chr11:836101-836401、chr11:86900101-86900401、chr11:17373393-17373693、chr12:125076301-125076601、chr12:125139301-125139601、chr12:133353801-133354101、chr12:87835801-87836101、chr12:104851940-104852240、chr12:49393272-49393353、chr14:90849279-90849579、chr14:91579987-91580287、chr15:102303001-102303301、chr15:<h2 style=";text-align:left;direction:ltr">39872388-39872688、chr15:102286047-102286347、chr15:45479869- 45480127、chr15: 72667882-72668182、chr16: 1730317-1730617、chr1 6:5337901-5338201、chr16:83691601-83691901、chr17:80072101-80 072401、chr17:31619207-31619225、chr18:150901-151201、chr18:762 73801-76274101, chr19:16680412-16680712, chr19:29613901-29614201, chr19:38978952-38979252, chr19:38996401-38996701, chr19:49 703623-49703923, chr19:49703923-49704223, chr19:50944201-50944501, chr19:51303278-51303578, chr19:39282525-39282825, chr2:17 0552044-170552344、chr2: 170552344-170552375、chr2: 177023775-1 77024075、chr2:20068960-20069094、chr2:2275801-2276101、chr2:2 40033301-240033601, chr2: 240033601-240033901, chr2: 47703601-47703901, chr2: 88690201-88690501, chr2: 99796148-99796448, chr20: 29804820-29805120、chr20:56247772-56248072、chr20:4803590-480 3717、chr21:11169301-11169601、chr21:27041008-27041308、chr21:4 5078301-45078601, chr21: 28219250-28219313, chr22: 39855601-39855901, chr22: 46969801-46970101, chr22: 51177001-51177301, chr22:<h2 style=";text-align:left;direction:ltr">24199607-24199629、chr3:145173301-145173601、chr3:184243114-18 4243414、chr3:193119901-193120201、chr3:197113944-197114244、ch r3: 79606801-79607101, chr3: 93744201-93744501, chr3: 98381701-98382001, chr3: 50274237-50274536, chr4: 1039501-1039801, chr4: 15172 6801-151727101、chr4:44450164-44450464、chr4:9241061-9241361、c hr4:9245629-9245929、chr4:185937927-185937986、chr5:111409501-1 11409801, chr5: 149985301-149985601, chr5: 171576901-171577201, chr5: 175119901-175120201, chr5: 178398013-178398313, chr5: 1784083 06-178408606、chr5:63373501-63373801、chr5:79331327-79331526、c hr5:94956093-94956392、chr6:131404801-131405101、chr6:16127348 7-161273787、chr6:105584593-105584757、chr6:107810663-10781082 1、chr6: 131383975-131383979、chr6: 26189401-26189431、chr7: 144835 801-144836101, chr7:154592101-154592401, chr7:155135101-155135401, chr7:157485729-157486029, chr7:20823318-20823618, chr7:5623 0801-56231101, chr7: 56357341-56357641, chr7: 7605460-7605760, chr7: 7606060-7606112, chr7: 7612688-7612988, chr7: 7612988-7613288chr7: 7613288-7613588, chr7: 134144180-134144289, chr7: 23513704-23514004, chr7: 23514004-2 3514120, chr8: 110882401-110882701, chr8: 126445404-126445704, chr8: 131455029-131455329, ch r8: 55002982-55003282, chr8: 65711134-65711434, chr8: 7830601-7830901, chr8: 86566135-86566 435, chr8: 95182801-95183101, chr8: 99954843-99955143, chr8: 144853418-144853717, chr9: 13052 9101-130529401, chr9: 137378101-137378401, chr9: 44868901-44869201, chr9: 97317483-9731778 3. chr9: 102585769-102586068, chr9: 27529891-27530023, chr9: 37036916-37037215, chr9: 9395500 5-93955305, chrX:100672978-100673278, chrX:114468337-114468637, chrX:115005001-115005301, chrX:32723401-32723701, chrY:13489201-13489501 and another 35 methylation markers derived from Chinese patent application No. 202211120273.0.

[0013] As a preferred embodiment of the present invention, the methylation biomarker combination is used to prepare a detection kit.

[0014] As a preferred embodiment of the present invention, the detection kit is used to detect prostate cancer tumor load.

[0015] The method for detecting a methylation biomarker combination includes the above-mentioned methylation biomarker combination and a detection kit prepared therefrom, and the detection steps are as follows:

[0016] Step 1: extracting cfDNA from a blood sample to be tested; the blood sample is plasma, serum or blood;

[0017] Step 2: Treat the extracted cfDNA with bisulfite to obtain a converted cfDNA sample;

[0018] Step 3: Construct a methylated DNA library for the converted cfDNA sample;

[0019] Step 4: Construction of a targeted hybrid capture methylation library for prostate cancer;

[0020] Step 5: High-throughput sequencing to obtain probe-captured data. The sequencing can be performed by any sequencing method, including but not limited to the dideoxy chain termination method. Preferred high-throughput sequencing methods include but are not limited to second-generation sequencing technology or single-molecule sequencing technology.

[0021] Step 6: Methylation data analysis: confirm the final metastatic prostate cancer score and metastatic tumor burden score based on the methylation markers and algorithm model.

[0022] As a preferred embodiment of the present invention, during the methylation data analysis in step 6, conventional bioinformatics analysis and processing is performed on the raw data from the sequencer.

[0023] Methods for detecting DNA methylation are well known in the art, and the detection methods used are reagents used in pyrosequencing, bisulfite conversion sequencing, methylation chip method, fluorescent quantitative qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, methylation-sensitive restriction endonuclease analysis method, HPLC method, MassArray, methylation-specific PCR, or combinations thereof. In one or more embodiments, detection includes detecting any strand at a gene or locus.

[0024] In the above technical solution, the technical effects and advantages provided by the present invention are:

[0025] By utilizing the high correlation between DNA methylation molecular markers and prostate cancer, and by combining methylation biomarkers to make a detection kit, it can be used to detect the tumor burden of metastatic prostate cancer. The detection of metastatic prostate cancer tumor burden can be achieved in a non-invasive manner, which improves the sensitivity, specificity and accuracy of the detection of metastatic prostate cancer. Compared with existing detection methods, it has the obvious advantages of non-invasive diagnosis and convenience and speed, greatly improves practicality, and has high feasibility for clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0027] FIG1 is a ROC diagram of cfDNA methylation applied to the diagnosis of metastatic prostate cancer in Example 2 of the present invention;

[0028] FIG2 is a graph showing the predicted scores for metastatic prostate cancer in Example 2 of the present invention;

[0029] FIG3 is an ROC curve generated by comparing high tumor burden and low tumor burden in Example 2 of the present invention;

[0030] FIG4 is an algorithm model for the metastatic prostate cancer score and the prostate cancer metastasis load score in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to techniques and conditions described in literature in the field or according to product specifications, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Unless otherwise noted, the materials, reagents, and instruments used in the following examples were all commercially available.

[0033] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] In one aspect, disclosed herein are methods for disease diagnosis based on sequencing data (e.g., nucleic acids) from blood samples. cfDNA is used as a primary example throughout the application, but it should not limit the scope of the invention in any way.

[0035] The present invention provides a methylation biomarker combination for detecting metastatic prostate cancer, selected from any combination of at least two of the following methylation biomarkers:

[0036] chr1:1137001-1137301、chr1:145573620-145573920、chr1:155620501-155620801、chr1:184006347-184006405、chr1:205631898-205632198、chr1:230208001-230208301、chr1:29564367-29564667、chr1:58234801-58235101、chr1:87767701-87768001、chr1:91089001-91089301、chr1:147487202-147487501、chr1:147532802-147533101、chr1:181059009-181059309、chr1:181059309-181059609、chr1:94146733-94146927、chr10:133506601-133506901、chr10:2095501-2095801、chr10:482262-482562、chr10:77158920-77159220、chr10:91330501-91330801、chr10:17270986-17271286、chr10:73156539-73156552、chr10:79397203-79397502、chr11:2867401-2867701、chr11:45672001-45672301、chr11:64875601-64875901、chr11:836101-836401、chr11:86900101-86900401、chr11:17373393-17373693、chr12:125076301-125076601、chr12:125139301-125139601、chr12:133353801-133354101、chr12:87835801-87836101、chr12:104851940-104852240、chr12:49393272-49393353、chr14:90849279-90849579、chr14:91579987-91580287、chr15:102303001-102303301、chr15:39872388-39872688、chr15:102286047-102286347、chr15:45479869-45480127、chr15:72667882-72668182、chr16:<h2 style=";text-align:left;direction:ltr">1730317-1730617、chr16:5337901-5338201、chr16:83691601-836919 01、chr17:80072101-80072401、chr17:31619207-31619225、chr18:15 0901-151201, chr18: 76273801-76274101, chr19: 16680412-16680712, chr19: 29613901-29614201, chr19: 38978952-38979252, chr19: 38996 401-38996701, chr19:49703623-49703923, chr19:49703923-49704223, chr19:50944201-50944501, chr19:51303278-51303578, chr19:3928 2525-39282825、chr2:170552044-170552344、chr2:170552344-17055 2375、chr2:177023775-177024075、chr2:20068960-20069094、chr2:22 75801-2276101、chr2:240033301-240033601、chr2:240033601-24003 3901、chr2:47703601-47703901、chr2:88690201-88690501、chr2:997 96148-99796448, chr20: 29804820-29805120, chr20: 56247772-56248072, chr20: 4803590-4803717, chr21: 11169301-11169601, chr21: 2704 1008-27041308, chr21:45078301-45078601, chr21:28219250-28219313, chr22:39855601-39855901, chr22:46969801-46970101, chr22:511 77001-51177301, chr22: 24199607-24199629, chr3: 145173301-145173601, chr3: 184243114-184243414, chr3: 193119901-193120201, chr3:<h2 style=";text-align:left;direction:ltr">197113944-197114244、chr3:79606801-79607101、chr3:93744201-937 44501、chr3:98381701-98382001、chr3:50274237-50274536、chr4:103 9501-1039801、chr4:151726801-151727101、chr4:44450164-44450464 、chr4:9241061-9241361、chr4:9245629-9245929、chr4:185937927-185 937986, chr5: 111409501-111409801, chr5: 149985301-149985601, chr5: 171576901-171577201, chr5: 175119901-175120201, chr5: 17839801 3-178398313、chr5:178408306-178408606、chr5:63373501-63373801、 chr5:79331327-79331526、chr5:94956093-94956392、chr6:131404801- 131405101、chr6:161273487-161273787、chr6:105584593-105584757、 chr6:107810663-107810821、chr6:131383975-131383979、chr6:26189 401-26189431, chr7:144835801-144836101, chr7:154592101-154592401, chr7:155135101-155135401, chr7:157485729-157486029, chr7:208 23318-20823618, chr7: 56230801-56231101, chr7: 56357341-56357641, chr7: 7605460-7605760, chr7: 7606060-7606112, chr7: 7612688-76129 88, chr7: 7612988-7613288, chr7: 7613288-7613588, chr7: 134144180-134144289, chr7: 23513704-23514004, chr7: 23514004-23514120, chr8:110882401-110882701、chr8:126445404-126445704、chr8:131455029-131455329、chr8:55002982-55003282、chr8:65711134-65711434、chr8:7830601-7830901、chr8:86566135-86566435、chr8:95182801-95183101、chr8:99954843-99955143、chr8:144853418-144853717、chr9:130529101-130529401、chr9:137378101-137378401、chr9:44868901-44869201、chr9:97317483-97317783、chr9:102585769-102586068、chr9:27529891-27530023、chr9:37036916-37037215、chr9:93955005-93955305、chrX:100672978-100673278、chrX:114468337-114468637、chrX:115005001-115005301、chrX:32723401-32723701、chrY:13489201-13489501。

[0037] and another 35 methylation markers derived from Chinese patent application No. 202211120273.0 (chr1: 205532103-205532401, chr10: 2565003-2565301, chr11: 134253603-134253901, chr11: 46316648-46316946, chr11: 830025-830323, chr12: 113779503-113779801, chr12: 1311 74103-131174401, chr12: 124955703-124956001, chr12: 77272466-77272764, chr15: 96906603-96906901, chr15: 45406119-45406417, chr15: 33219303-33219601, chr16: 74440716-74441014, chr17: 79652103-79652401, chr19: 55994103-55994401, chr19: 49636737-49637035, chr2: 163323603-1633 23901, chr2: 103445403-103445701, chr2: 111875389-111875687, chr20: 21493409-21493707, chr20: 21497940-21498238, chr22: 50623397-5062 3695, chr22: 50988003-50988301, chr22: 42307184-42307431, chr4: 9557 9714-95580012, chr5: 148252503-148252801, chr5: 170744972-1707452 70. chr5: 172070703-172071001, chr6: 26189103-26189401, chr7: 237660 3-2376901, chr7: 5006703-5007001, chr7: 21765303-21765601, chr8: 105 86552-10586850, chr8: 23583988-23584286, chr9: 26547903-26548201).

[0038] Wherein, the methylation marker is a test sample of a subject's blood, such as plasma, serum or blood. The detection reagent of the combination marker may include primers and / or probes for detecting the methylation level of each gene in the combination marker.

[0039] The receiver operating characteristic (ROC) curve is a curve plotted based on a series of different binary classification methods (cutoff values ​​or decision thresholds), with sensitivity (true positive rate) on the vertical axis and 1-specificity (false positive rate) on the horizontal axis. The area under the ROC curve is an important indicator of test accuracy; the larger the area under the ROC curve, the greater the diagnostic value of the test.

[0040] Example 1

[0041] Regarding the detection method for detecting differential methylation markers of ctDNA in metastatic prostate cancer, this example provides specific detection steps:

[0042] 1. Extraction of plasma cfDNA;

[0043] Specifically: When extracting plasma cfDNA, the specific operating steps are carried out according to the operating instructions of QIAGEN's QIAamp Circulating Nucleic Acid Kit.

[0044] 2. Bisulfite conversion;

[0045] Specifically, 20 ng of cfDNA was added for bisulfite conversion, which deaminated the unmethylated cytosine in the DNA and converted it to uracil, while the methylated cytosine remained unchanged, thereby obtaining bisulfite-converted DNA. The specific operations of the above conversion process were carried out according to the instructions of Zymo Research's EZ DNA Methylation-Gold Kit.

[0046] 3. Methylation library construction;

[0047] The specific steps include:

[0048] 3.1, Sex change;

[0049] 3.1.1. Place 15 μl of the transformed sample in a PCR instrument and follow the following procedure:

[0050] 95℃ for 2 minutes

[0051] Heated cover 105℃

[0052] 3.1.2. After the PCR reaction is completed, remove the sample immediately and place it directly on ice for more than 2 minutes before proceeding to the next step.

[0053] 3.2, T7 connector connection;

[0054] 3.2.1. Prepare the reaction solution according to the following system;

[0055] 3.2.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0056] 3.3, second strand synthesis;

[0057] 3.3.1. Prepare the reaction solution according to the following system

[0058] 3.3.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0059] 3.4, first purification;

[0060] 3.4.1. After centrifugation of the reaction products from the previous step, add 101 μl of Agencourt AMPure Beads (previously equilibrated to room temperature) to each sample and mix thoroughly by pipetting.

[0061] 3.4.2. Let stand at room temperature for 5 minutes, transfer to a magnetic rack and let stand for 5 minutes, then carefully discard the supernatant.

[0062] 3.4.3 Place the PCR tube on the magnetic rack, add 200 μl of 80% ethanol, let it stand for 30 seconds, and then aspirate the ethanol.

[0063] 3.4.4. Repeat step 3.4.3 once;

[0064] 3.4.5. Open the lid and dry the magnetic beads for 2-3 minutes. After the alcohol has completely evaporated, place the PCR tube on a magnetic rack. Add 15 μl of Low EDTA TE, pipette to mix, and let stand at room temperature for 5 minutes.

[0065] 3.4.6. Place the PCR tube on a magnetic rack and let it stand at room temperature for 5 minutes. Pipette 15ul of supernatant into a new PCR tube.

[0066] 3.5, T5 connector connection;

[0067] 3.5.1. Prepare the reaction solution according to the following system

[0068] 3.5.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0069] 25℃ 15min

[0070] 4℃ hold

[0071] 3.6, Second purification;

[0072] 3.6.1. After centrifugation of the reaction products from the previous step, add 36 μl of Agencourt AMPure Beads (previously equilibrated to room temperature) to each sample and mix thoroughly by pipetting.

[0073] 3.6.2. Let stand at room temperature for 5 minutes, transfer to a magnetic rack and let stand for 5 minutes, then carefully discard the supernatant.

[0074] 3.6.3. Place the PCR tube on the magnetic rack, add 200 μl of 80% ethanol, let stand for 30 seconds, and then aspirate the ethanol.

[0075] 3.6.4. Repeat step 3.6.3 once;

[0076] 3.6.5. Open the lid and dry the magnetic beads for 2-3 minutes. After the alcohol has completely evaporated, place the PCR tube on a magnetic rack. Add 20 μl of Low EDTA TE, pipette to mix thoroughly, and let stand at room temperature for 5 minutes.

[0077] 3.6.6. Place the PCR tube on a magnetic rack and let it stand at room temperature for 5 minutes. Pipette 20ul of supernatant into a new PCR tube.

[0078] 3.7, Indexing PCR;

[0079] 3.7.1. Prepare the reaction solution according to the following system

[0080] 3.7.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0081] 3.8, third purification;

[0082] 3.8.1. After centrifugation of the reaction products from the previous step, add 40 μl of Agencourt AMPure Beads (previously equilibrated to room temperature) to each sample and mix thoroughly by pipetting.

[0083] 3.8.2. Let stand at room temperature for 5 minutes, transfer to a magnetic rack and let stand for 5 minutes, then carefully discard the supernatant.

[0084] 3.8.3. Place the PCR tube on the magnetic rack, add 200 μl of 80% ethanol, let stand for 30 seconds, and then aspirate the ethanol.

[0085] 3.8.4. Repeat step 3.8.3 once;

[0086] 3.8.5. Open the lid and dry the magnetic beads for 2-3 minutes. After the alcohol has completely evaporated, place the PCR tube on a magnetic rack. Add 20 μl of Low EDTA TE, pipette to mix, and let it stand at room temperature for 5 minutes.

[0087] 3.8.6. Place the PCR tube on a magnetic rack and let it stand at room temperature for 5 minutes. Pipette 20ul of supernatant into a new PCR tube.

[0088] 4. Methylation rapid hybridization assay;

[0089] The specific steps include:

[0090] 4.1. Calculate the amount of different DNA libraries and mix them evenly in a centrifuge tube. The amount of each library should not exceed 500ng, and the total amount should not exceed 4ug.

[0091] 4.2. Add the following pre-hybridization reagents to the mixed sample and mix well. The table is as follows

[0092] 4.3. Dry the mixed pre-hybridization reagents at room temperature in a vacuum concentrator;

[0093] 4.4. Add 20 μl of Fast Hybridization Mix to the dried sample and mix gently with your fingertips.

[0094] 4.5. Add 30ul Hybridization Enhancer to the surface of the above reagents;

[0095] 4.6. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0096] 4.7. Equilibrate the streptavidin magnetic beads to room temperature in advance and transfer 100 μl of the beads to a 1.5 ml centrifuge tube.

[0097] 4.8. Add 200 μl of binding buffer and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 minute or until the solution is clear, and discard the supernatant.

[0098] 4.9. Repeat step 4.8 twice, for a total of three washes.

[0099] 4.10. After the final wash, add 200ul of binding buffer, mix thoroughly, and transfer all the hybridization solution in the PCR instrument to the balanced magnetic beads.

[0100] 4.11. Mix the magnetic beads with hybridization solution thoroughly on a mixer at room temperature for 30 minutes.

[0101] 4.12. Remove the centrifuge tube from the mixer, centrifuge quickly, place it on a magnetic rack for 1 minute, and remove the supernatant.

[0102] 4.13. Add 200 μl of preheated Wash Buffer 1, mix well, incubate at 65°C for 5 min, transfer to a magnetic rack and let stand for 1 min, then carefully discard the supernatant.

[0103] 4.14. Repeat step 4.13 once.

[0104] 4.15. Add 200 μl of preheated Wash Buffer 2, mix well, incubate at 48°C for 5 min, transfer to a magnetic rack and let stand for 1 min, then carefully discard the supernatant.

[0105] 4.16. Repeat step 4.15 twice, for a total of three times.

[0106] 4.17. Add 45 μl of water, mix well, and incubate the solution on ice.

[0107] 4.18. Prepare the reaction solution according to the following system

[0108] 4.19. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0109] 4.20. Add 90 μl of DNA purification magnetic beads (allowed to equilibrate to room temperature in advance) to the amplified product and mix well.

[0110] 4.21. Let stand at room temperature for 5 minutes, transfer to a magnetic rack and let stand for 5 minutes, then carefully discard the supernatant.

[0111] 4.22. Place the PCR tube on the magnetic rack, add 200 μl of 80% ethanol, let it stand for 30 seconds, and then aspirate the ethanol.

[0112] 4.23. Repeat step 4.22 once.

[0113] 4.24. Open the lid and dry the magnetic beads for 2-3 minutes. After the alcohol has completely evaporated, place the PCR tube on a magnetic rack, add 32 μl of water, pipette to mix, and let it stand at room temperature for 5 minutes.

[0114] 4.25. Place the PCR tube on a magnetic rack and let it stand at room temperature for 5 minutes. Pipette 30ul of supernatant into a new PCR tube.

[0115] 5. Use Illumina's sequencer to sequence the sample after hybridization capture to obtain the sequencing results.

[0116] 6. Perform routine bioinformatics analysis on the raw data from the sequencer to obtain methylation data for the target region, differentially methylated region signals, and specific methylated fragments. Using the established algorithm model (Figure 4), calculate the metastatic prostate cancer score and the prostate cancer metastasis burden score. The algorithm calculation process is as follows:

[0117] 6.1. Based on the targeted methylation region designed in this patent, the methylation ratio value of the region and the methylation fragment information of the region are obtained respectively. After the methylation ratio level is calculated by Bismark software for CpG sites, all CpG methylation sites of all fragments in the region are weighted averaged according to the modified regional methylation site calculation method. The methylation fragment information is constructed by summarizing the base information of all sequenced fragments of the targeted methylation region, removing the low-quality region (sequencing read length 5' end 25bp), and then summarizing the base information of all fragments of the sequence ATCG and methylated C.

[0118] 6.2. For the regional methylation fragment information, the tissue LSTM model and Transformer model trained based on the established metastatic prostate cancer methylation fragment database are used to one-hot encode the methylation fragments and then use the tissue model to calculate the tissue methylation score of the sample.

[0119] 6.3. For the regional methylation ratio value, the blood methylation score of the sample was calculated based on the established metastatic prostate cancer blood methylation database and the established models, including the random forest model (RF), XGBoost model, and generalized linear model (GLM).

[0120] 6.4. Based on the tissue methylation score and blood methylation score of the sample, the prostate cancer tumor metastasis score and metastasis burden score of the sample are calculated using the generalized linear model trained with the metastatic prostate cancer signature and metastatic tumor burden signature.

[0121] Example 2

[0122] In this example, plasma samples from 70 patients with localized prostate cancer, 20 patients with low tumor burden of metastatic prostate cancer, and 23 patients with high tumor burden of metastatic prostate cancer were tested. The specific detection kit, experimental method, and data judgment and processing were consistent with those described in Example 1.

[0123] The differences in methylation levels among different groups were used to screen out biomarkers related to metastatic prostate cancer, and a total of 171 methylation markers were finally screened out.

[0124] The results are shown in Figure 1. At 96.1% specificity, the overall sensitivity for detecting metastatic prostate cancer and non-metastatic prostate cancer was 95.2%, and the overall AUC was 0.987. A scatter plot showing significant separation of the discrimination scores between the metastatic prostate cancer patient group and the non-metastatic prostate cancer patient group (see Figure 2). The results of differentiating between metastatic prostate cancer tumor burden are shown in Figure 3. At 90% specificity, the overall sensitivity for detecting high tumor burden and low tumor burden was 91.3%, and the overall AUC was 0.972.

[0125] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art may, without departing from the spirit and scope of the present invention, make various modifications and additions to the described embodiments in various ways, and such modifications and additions should also be considered within the scope of protection of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A methylation biomarker panel for detecting the tumor burden of metastatic prostate cancer, characterized in that: It is any combination of at least two selected from the following methylation biomarkers: chr1: 1137001 - 1137301, chr1: 145573620 - 145573920, chr1: 155620501 - 155620801, chr1: 184006347 - 184006405, chr1: 205631898 - 205632198, chr1: 230208001 - 230208301, chr1: 29564367 - 29564667, chr1: 58234801 - 58235101, chr1: 87767701 - 87768001, chr1: 91089001 - 91089301, chr1: 147487202 - 147487501, chr1: 147532802 - 147533101, chr1: 181059009 - 181059309, chr1: 181059309 - 181059609, chr1: 94146733 - 94146927, chr10: 133506601 - 133506901, chr10: 2095501 - 2095801, chr10: 482262 - 482562, chr10: 77158920 - 77159220, chr10: 91330501 - 91330801, chr10: 17270986 - 17271286, chr10: 73156539 - 73156552, chr10: 79397203 - 79397502, chr11: 2867401 - 2867701, chr11: 45672001 - 45672301, chr11: 64875601 - 64875901, chr11: 836101 - 836401, chr11: 86900101 - 86900401, chr11: 17373393 - 17373693, chr12: 125076301 - 125076601, chr12: 125139301 - 125139601, chr12: 133353801 - 133354101, chr12: 87835801 - 87836101, chr12:104851940 - 104852240, chr12:49393272 - 49393353, chr14:90849279 - 90849579, chr14:91579987 - 91580287, chr15:102303001 - 102303301, chr15:39872388 - 39872688, chr15:102286047 - 102286347, chr15:45479869 - 45480127, chr15:72667882 - 72668182, chr16:1730317 - 1730617, chr16:5337901 - 5338201, chr16:83691601 - 83691901, chr17:80072101 - 80072401, chr17:31619207 - 31619225, chr18:150901 - 151201, chr18:76273801 - 76274101, chr19:16680412 - 16680712, chr19:29613901 - 29614201, chr19:38978952 - 38979252, chr19:38996401 - 38996701, chr19:49703623 - 49703923, chr19:49703923 - 49704223, chr19:50944201 - 50944501, chr19:51303278 - 51303578, chr19:39282525 - 39282825, chr2:170552044 - 170552344, chr2:170552344 - 170552375, chr2:177023775 - 177024075, chr2:20068960 - 20069094, chr2:2275801 - 2276101, chr2:240033301 - 240033601, chr2:240033601 - 240033901, chr2:47703601 - 47703901, chr2:88690201 - 88690501, chr2:99796148 - 99796448, chr20:29804820 - 29805120, chr20:56247772 - 56248072, chr20: 4803590 - 4803717, chr21: 11169301 - 11169601, chr21: 27041008 - 27041308, chr21: 45078301 - 45078601, chr21: 28219250 - 28219313, chr22: 39855601 - 39855901, chr22: 46969801 - 46970101, chr22: 51177001 - 51177301, chr22: 24199607 - 24199629, chr3: 145173301 - 145173601, chr3: 184243114 - 184243414, chr3: 193119901 - 193120201, chr3: 197113944 - 197114244, chr3: 79606801 - 79607101, chr3: 93744201 - 93744501, chr3: 98381701 - 98382001, chr3: 50274237 - 50274536, chr4: 1039501 - 1039801, chr4: 151726801 - 151727101, chr4: 44450164 - 44450464, chr4: 9241061 - 9241361, chr4: 9245629 - 9245929, chr4: 185937927 - 185937986, chr5: 111409501 - 111409801, chr5: 149985301 - 149985601, chr5: 171576901 - 171577201, chr5: 175119901 - 175120201, chr5: 178398013 - 178398313, chr5: 178408306 - 178408606, chr5: 63373501 - 63373801, chr5: 79331327 - 79331526, chr5: 94956093 - 94956392, chr6: 131404801 - 131405101, chr6: 161273487 - 161273787, chr6: 105584593 - 105584757, chr6: 107810663 - 107810821, chr6: 131383975 - 131383979, chr6: 26189401 - 26189431, chr7: 144835801 - 144836101, chr7:154592101 - 154592401, chr7:155135101 - 155135401, chr7:157485729 - 157486029, chr7:20823318 - 20823618, chr7:56230801 - 56231101, chr7:56357341 - 56357641, chr7:7605460 - 7605760, chr7:7606060 - 7606112, chr7:7612688 - 7612988, chr7:7612988 - 7613288, chr7:7613288 - 7613588, chr7:134144180 - 134144289, chr7:23513704 - 23514004, chr7:23514004 - 23514120, chr8:110882401 - 110882701, chr8:126445404 - 126445704, chr8:131455029 - 131455329, chr8:55002982 - 55003282, chr8:65711134 - 65711434, chr8:7830601 - 7830901, chr8:86566135 - 86566435, chr8:95182801 - 95183101, chr8:99954843 - 99955143, chr8:144853418 - 144853717, chr9:130529101 - 130529401, chr9:137378101 - 137378401, chr9:44868901 - 44869201, chr9:97317483 - 97317783, chr9:102585769 - 102586068, chr9:27529891 - 27530023, chr9:37036916 - 37037215, chr9:93955005 - 93955305, chrX:100672978 - 100673278, chrX:114468337 - 114468637, chrX:115005001 - 115005301, chrX:32723401 - 32723701, chrY:13489201 - 13489501; and another 35 methylation markers derived from Chinese Patent Application No. 202211120273.

0.

2. The methylation biomarker combination for detecting the tumor burden of metastatic prostate cancer according to claim 1, wherein The methylation biomarker combination is used for preparing a detection kit.

3. The methylation biomarker combination for detecting the tumor burden of metastatic prostate cancer according to claim 2, wherein The detection kit is applied to detect the tumor burden of prostate cancer.

4. The methylation biomarker combination for detecting the tumor burden of metastatic prostate cancer according to any one of claims 1-3, characterized in that, The detection method of the methylation biomarker combination is as follows: Step 1: Extract cfDNA from the blood sample to be tested; Step 2: Perform bisulfite treatment on the extracted cfDNA to obtain the converted cfDNA sample; Step 3: Construct a methylation DNA library for the converted cfDNA sample; Step 4: Construct a targeted hybridization capture methylation and library for prostate cancer; Step 5: Perform high-throughput sequencing to obtain the data captured by the probes; Step 6: Analyze methylation data, and confirm the final metastatic prostate cancer signal and metastatic tumor burden signal according to methylation markers and algorithm models.

5. The methylation biomarker combination for detecting metastatic prostate cancer tumor burden according to claim 4, characterized in that: During the methylation data analysis in Step 6, perform routine bioinformatics analysis and processing on the raw data output from the sequencer.

6. The methylation biomarker combination for detecting metastatic prostate cancer tumor burden according to claim 4, wherein: The blood sample to be tested described in Step 1 is plasma, serum or blood.

7. The methylation biomarker panel for detecting metastatic prostate cancer tumor burden according to claim 4, wherein: In Step 5, use the sequencing library for high-throughput sequencing, and the sequencing can be performed by any sequencing method, including second-generation sequencing technology or single-molecule sequencing technology.

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