Application of DNA replication late region in pan-cancer diagnosis

Abstract

The present application relates to the field of molecular diagnosis technology, and specifically provides an application of a late DNA replication region in pan-cancer diagnosis. The present application uses a variety of omics data to find that the late DNA replication region presents DNA hypomethylation and high mutation frequency in tumors, and presents a higher fragment length ratio and unique terminal motif characteristics in tumor cfDNA. This phenomenon exists in multiple cancers and is a pan-cancer marker, based on which a pan-cancer diagnostic method and application is established.

Description

[0001] This patent application claims the priority of the prior Chinese application date of March 7, 2023, with application number CN202310225516.5. Technical Field

[0002] The present application relates to the field of molecular diagnosis technology, and in particular to the application of a late DNA replication region as a pan-cancer diagnostic marker. Background Art

[0003] Since early detection of cancer patients can be intervened in advance, it can significantly reduce the mortality rate of patients, improve their quality of life and prolong their survival time. With the development and maturity of liquid biopsy technology, the cancer early screening market is expected to enter a high-speed growth stage from the early exploration stage. Leading domestic and foreign companies represented by Grail and Burning Rock have deployed in the field of pan-cancer early screening. Although the detection technology and screening site areas of each company are different, they are all based on the comprehensive consideration of the intensity and abundance of tumor signals. Among them, the research team of Grail, a leading pan-cancer early screening company, found in the first sub-study that whole gene methylation sequencing (WGBS) is better than targeted sequencing to detect mutations and whole genome sequencing (WGS), so the research team used targeted methylation sequencing analysis methods for early detection of tumor patients. The development of pan-cancer early screening products has the characteristics of high technical barriers, large capital investment and long development cycle. In particular, the quality of early biomarker screening affects the quality of later project effects, so early screening companies have done a lot of research work in the biomarker screening stage.

[0004] Circulating tumor DNA (ctDNA) is DNA from tumor cells released into the plasma. Its content in free DNA (cfDNA) is very low, and in early-stage tumor patients it is less than 1%. This makes the tumor signal extremely weak and difficult to detect, so how to select the molecular genetic characteristics of ctDNA and the range of its characteristics on the genome is crucial. For ctDNA mutation characteristics, the detection limit that can be achieved by high-depth sequencing detection methods is between 0.1-0.5%, which cannot meet the requirements for early detection of cancer, especially those cancers that release very little ctDNA into the blood, such as lung cancer. As another focus of early screening, the methylation characteristics of ctDNA are favored by various companies.

[0005] Most of the traditional screening of ctDNA methylation biomarkers is based on the TCGA public database. Even if some projects use whole-genome methylation sequencing (WGBS) of a single cancer type to screen out some areas, it is difficult to design better methylation sites or regions from the whole genome level due to the small number of tumor samples. Therefore, each early screening research team is looking for biomarkers with high signal intensity and signal abundance to reach the detection limit of lower pan-cancer types. Recently, many literature reports have used changes in ctDNA DNA methylation levels or differences in fragment characteristics as features for tumor prediction and diagnosis, and have achieved good results.

[0006] In view of this, this application is filed. Summary of the invention

[0007] To solve the above problems, this application found through WGBS sequencing of tumor tissues and para-cancer samples of 9 cancers (lung cancer, colon cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, lymphoma) that the interval of DNA replication in the late stage of tumor tissue is enriched with DNA hypomethylation, and this phenomenon is common in these 9 cancers. At the same time, these regions are enriched in multiple features such as mutations, pattern sequences, and fragment length ratios, which expand the signal intensity and abundance and make up for the shortcomings of ctDNA in cfDNA signals. Therefore, the late DNA replication region of the genome is enriched as a pan-cancer candidate region, and multiple omics signals such as the genome and epigenome are enriched, including DNA hypomethylation, high mutation frequency, fragment length ratio, end pattern sequence (endmotif) (abundance difference), breakpoint pattern sequence (break-point motif) (abundance difference), etc. This region is used as a biomarker for pan-cancer early screening. It can be used to integrate multi-omics tumor signals such as DNA methylation, fragment characteristics, and mutations to perform early screening for pan-cancer species. The signal intensity and abundance of the detection and the performance of the product are superior to existing technologies and products.

[0008] Therefore, this application has at least the following purposes:

[0009] The first purpose of this application is to seek a new use for pan-cancer diagnosis;

[0010] The second purpose of this application is to seek a new product suitable for pan-cancer diagnosis;

[0011] The third purpose of this application is to seek a new method for pan-cancer diagnosis.

[0012] To achieve the above objectives, this application proposes the following specific technical solutions:

[0013] The present application first provides an application of a late region of DNA replication as a marker in the diagnosis or prediction of pan-cancer species, or an application of a detection agent or component for obtaining the level of a late region of DNA replication in a sample in the preparation of a product for the diagnosis or prediction of pan-cancer species.

[0014] In some aspects, the product includes, but is not limited to, a kit form, a system device form, a computer readable medium, or a computer system form.

[0015] In some aspects, the late region of DNA replication serves as an independent indicator for pan-cancer diagnosis or prediction.

[0016] In some aspects, the level of the late region of DNA replication includes one or more of the DNA methylation level, mutation frequency level, and fragment characteristic level in the late region of DNA replication; preferably, the methylation level is a low methylation level, and the mutation frequency level is a high mutation frequency level; more preferably, the fragment characteristic level includes one or more of the fragment short-to-length ratio level, the terminal pattern sequence abundance difference level, and the breakpoint pattern sequence abundance difference level.

[0017] Furthermore, the acquisition is carried out through sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0018] Furthermore, the acquisition method includes but is not limited to any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0019] In some aspects, the product also includes a detection agent or component for obtaining the levels of other pan-cancer diagnostic markers for combined detection.

[0020] In some aspects, the product also includes sample processing reagents, such as nucleic acid extraction reagents.

[0021] In some aspects, the sample comprises one or more of serum, plasma, whole blood, tissue, cells, body fluid, urine, semen, cerebrospinal fluid, saliva, pleural effusion, ascites, feces or synovial fluid; preferably, the sample is serum, plasma, whole blood or tissue.

[0022] In some aspects, the use comprises the steps of:

[0023] (i) obtaining the level of the late region of DNA replication in the sample of the subject;

[0024] (ii) comparing the level of the late region of DNA replication with that of a control sample, wherein a significant difference in the level of the late region of DNA replication between the subject sample and the control sample is an indication that the subject suffers from pan-cancer; or, comparing with a set threshold absolute amount, wherein the level of the subject sample is higher than the threshold absolute amount is an indication that the subject suffers from pan-cancer.

[0025] In some aspects, the diagnosis includes but is not limited to pan-cancer screening, early diagnosis, auxiliary diagnosis, diagnosis, prediction / judgment of disease severity, assessment of disease activity, monitoring of treatment, etc.; preferably, early diagnosis.

[0026] The present application also provides a product for diagnosing or predicting pan-cancer, including a detection agent or component for obtaining the level of the late region of DNA replication in a sample.

[0027] In some aspects, the product includes, but is not limited to, a product in the form of a kit, a system device, a computer readable medium, or a computer system.

[0028] In some aspects, the late region of DNA replication can be independently used as an indicator for pan-cancer diagnosis or prediction.

[0029] In some aspects, the level of the late region of DNA replication includes one or more of the DNA methylation level, mutation frequency level, and fragment characteristic level in the late region of DNA replication; preferably, the methylation level is a low methylation level, and the mutation frequency level is a high mutation frequency level; more preferably, the fragment characteristic level includes one or more of the fragment short-to-length ratio level, the terminal pattern sequence abundance difference level, and the breakpoint pattern sequence abundance difference level.

[0030] Furthermore, the acquisition is carried out through sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0031] Furthermore, the acquisition method includes but is not limited to any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0032] In some aspects, the product also includes a detection agent or component for obtaining the levels of other pan-cancer diagnostic markers for combined detection.

[0033] In some aspects, the product also includes sample processing reagents, such as nucleic acid extraction reagents.

[0034] In some aspects, the sample comprises one or more of serum, plasma, whole blood, tissue, cells, body fluid, urine, semen, cerebrospinal fluid, saliva, pleural effusion, ascites, feces or synovial fluid; preferably, the sample is serum, plasma, whole blood or tissue.

[0035] In some aspects, the diagnosis includes but is not limited to pan-cancer screening, early diagnosis, auxiliary diagnosis, diagnosis, prediction / judgment of disease severity, assessment of disease activity, monitoring of treatment, etc.; preferably, early diagnosis.

[0036] The present application also provides a method for diagnosing pan-cancer in vivo or in vitro, comprising the step of obtaining the level of the late region of DNA replication in a sample of a subject.

[0037] In some aspects, the method comprises the steps of:

[0038] (i) obtaining the level of the late region of DNA replication in the sample of the subject;

[0039] (ii) comparing the level of the late region of DNA replication with that of a control sample, wherein a significant difference in the level of the late region of DNA replication between the subject sample and the control sample is an indication that the subject suffers from pan-cancer; or, comparing with a set threshold absolute amount, wherein the level of the subject sample is higher than the threshold absolute amount is an indication that the subject suffers from pan-cancer.

[0040] In some aspects, the late region of DNA replication can be independently used as an indicator for pan-cancer diagnosis.

[0041] In some aspects, the level of the late region of DNA replication includes one or more of the DNA methylation level, mutation frequency level, and fragment characteristic level in the late region of DNA replication; preferably, the methylation level is a low methylation level, and the mutation frequency level is a high mutation frequency level; more preferably, the fragment characteristic level includes one or more of the fragment short-to-length ratio level, the terminal pattern sequence abundance difference level, and the breakpoint pattern sequence abundance difference level.

[0042] Furthermore, the acquisition is carried out through sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0043] Furthermore, the acquisition method includes but is not limited to any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0044] In some aspects, the product also includes a detection agent or component for obtaining the levels of other pan-cancer diagnostic markers for combined detection.

[0045] In some aspects, the product also includes sample processing reagents, such as nucleic acid extraction reagents.

[0046] In some aspects, the sample comprises one or more of serum, plasma, whole blood, tissue, cells, body fluid, urine, semen, cerebrospinal fluid, saliva, pleural effusion, ascites, feces or synovial fluid; preferably, the sample is serum, plasma, whole blood or tissue.

[0047] In some aspects, the diagnosis includes but is not limited to pan-cancer screening, early diagnosis, auxiliary diagnosis, diagnosis, prediction / judgment of disease severity, assessment of disease activity, monitoring of treatment, etc.; preferably, early diagnosis.

[0048] In some aspects, the subject mentioned above is preferably a human.

[0049] The present application also provides a method for detecting a marker in a subject having or suspected of having cancer in vivo or in vitro, the method comprising determining or detecting the level of the late region of DNA replication in a sample from the subject.

[0050] In some aspects, the method can be used for disease diagnosis or non-disease diagnosis.

[0051] The present application may also provide a method for evaluating or screening pan-cancer diagnostic markers, comprising the steps of performing correlation or consistency analysis between potential biomarkers and late regions of DNA replication, and verifying the diagnostic value of the potential markers through the conclusions of the correlation or consistency.

[0052] The present application also provides a method for evaluating or screening pan-cancer therapeutic agents in vivo or in vitro, the method comprising the step of evaluating the level of the late region of DNA replication in a sample treated with the therapeutic agent.

[0053] The present application also provides a marker for diagnosing pan-cancer, including a late region of DNA replication.

[0054] Beneficial technical effects of this application:

[0055] 1) This application found that the DNA replication period is positively correlated with tumor methylation, and that low methylation is present in the late stage of DNA replication, which is significantly different from normal cfDNA methylation. This phenomenon exists in multiple cancer types and is a pan-cancer marker. Based on this, a pan-cancer diagnostic method and application is established.

[0056] 2) The late region of DNA replication is enriched with multi-omics features, such as low methylation, mutation frequency, motif specificity, and short fragments. This application can further improve the classification of pan-cancer early screening products by integrating multi-omics features, and the performance is significantly better than existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0058] Figure 1 , Flow chart of DNA replication late region screening in this application;

[0059] Figure 2 , 9 The relationship between the whole genome methylation level and different stages of DNA replication in each cancer type;

[0060] Figure 3 , Methylation levels of paracancerous tissues and tumor tissues of lung and liver at different stages of DNA replication;

[0061] Figure 4 , Pan-cancer mutation frequency diagrams at early, late, exon, and whole genome;

[0062] Figure 5 , the distribution of cfDNA fragment length in healthy people, lung cancer and liver cancer;

[0063] Figure 6 , the distribution of End-motif terminal features of cfDNA in healthy people, lung cancer and liver cancer in early and late stages;

[0064] Figure 7 , the distribution of break-point motif features of cfDNA in healthy individuals, lung cancer and liver cancer in early and late stages;

[0065] Figure 8 , pan-cancer cross-validation of methylation markers in the late stage of DNA replication, that is, using eight cancer types to predict the ninth cancer type;

[0066] Fig. 9 , cfDNA characteristic curve (ROC) of the pan-cancer DNA methylation panel in subjects with lung cancer, pancreatic cancer, and esophageal cancer. DETAILED DESCRIPTION

[0067] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application, and the examples are part of the present application, rather than all of the examples. Based on the examples in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0068] Definition of some terms

[0069] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0070] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0071] The terms "approximately" and "substantially" in this application represent an accuracy interval that can be understood by a person skilled in the art and still guarantee the technical effect of the feature in question. The term usually means ±10%, preferably ±5%, of the deviation from the indicated value. Indefinite or definite articles such as "a" or "an", "the" used when referring to a singular noun include the plural form of the noun.

[0072] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0073] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0074] Diagnostic Uses

[0075] This application screened the late DNA replication region that can be used as a pan-cancer methylation marker by integrating and analyzing the Repli-seq sequencing data of 15 cell lines in the UCSC database and the whole genome methylation sequencing (WGBS) data of tumors and adjacent cells of 9 cancer types (lung cancer, colon cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, lymphoma). This region is enriched with multi-omics tumor signals, including DNA hypomethylation, mutation frequency (high frequency), fragment length ratio (FSR), end motif (abundance difference), breakpoint motif (abundance difference). Using this region as a biomarker for pan-cancer early screening, it can be used to integrate multi-omics tumor signals such as DNA methylation, fragment characteristics, and mutations, and perform early screening for pan-cancer species. The signal intensity and abundance of the detection and the performance of the product are better than existing technologies and products.

[0076] Therefore, various aspects of the present disclosure provide a method for diagnosing pan-cancer using a late region of DNA replication as a marker, and a method for preparing a product for diagnosing pan-cancer using a detection agent for detecting a late region of DNA replication in a sample.

[0077] The "DNA replication late region" described in this application refers to the replication termination region of human DNA. Generally, human chromosomes have many replication origins, and DNA replicates from the replication origin to form a replication fork, which extends to the left and right. Two adjacent replication forks will meet on the chromosome and terminate DNA replication. These encounter and termination intervals are DNA replication late regions.

[0078] In some embodiments, the DNA replication late region level described in the present application includes one or more of the DNA methylation level, mutation frequency level, and fragment characteristic level of another DNA replication late region; preferably, the methylation level is a low methylation level, and the mutation frequency level is a high mutation frequency level; more preferably, the fragment characteristic level includes one or more of the fragment short-to-length ratio level, the terminal pattern sequence abundance difference level, and the breakpoint pattern sequence abundance difference level. It can be understood that the above-mentioned multiple indicators can be used for the prediction or diagnosis of pan-cancer species independently or in combination, as demonstrated in the embodiments of the present application.

[0079] The "methylation level" mentioned in this application refers to the methylation level of cytosine at the CpG site on DNA catalyzed by DNA methylase; the "DNA hypomethylation level" refers to the hypomethylation level of cytosine at the CpG site on DNA not catalyzed by DNA methylase.

[0080] The "frequency mutation level" mentioned in this application refers to the frequency level of point mutations in a certain region on the DNA of tumor patients; the "high frequency mutation level" refers to a significant increase in the number of point mutations in a certain region on the DNA of tumor patients, which is significantly higher than the average level of the genome.

[0081] The "fragment features" described in this application refer to the sequence features of cfDNA, including the fragment length ratio (FSR) feature of the sequence, the end motif (EDM) feature, and the breakpoint motif (BPM) feature. Among them, the fragment length ratio refers to the ratio of short sequence to long sequence fragments, usually the short sequence length is 90-150bp, and the long sequence length is 180-220bp. The end pattern sequence (EDM) refers to the 4bp sequence from the 5' to 3' direction of the positive and negative chains of the reference genome at the start and end positions of the comparison after the inserted fragment is aligned to the reference genome, and 256 types of EDM can be obtained. The breakpoint pattern sequence (BPM) refers to the 3bp on the left and right of the 5' to 3' direction of the positive and negative chains of the reference genome at the start and end positions of the comparison after the inserted fragment is aligned to the reference genome, respectively, for a total of 6bp sequences, and 4096 types of BPM can be obtained.

[0082] As used herein, the term "diagnosis" refers to the process of identifying pan-cancer species through their signs, symptoms, and especially the results of various diagnostic processes (including detecting the level of the late region of DNA replication in a biological sample (such as serum) obtained from an individual). Moreover, the term "diagnosis" as used herein includes cancer screening, early diagnosis, auxiliary diagnosis, diagnosis (determining whether cancer exists), prediction or judgment of the severity of cancer, assessment of disease activity, monitoring treatment, such as monitoring cancer deterioration or recurrence during treatment, evaluating the therapeutic efficacy of cancer, and the selection of a given treatment regimen, etc. Without limitation, in some specific embodiments, the present application has fully demonstrated that the level of the late region of DNA replication can be used to determine the presence or absence of cancer in a subject, and can then be used for screening, early diagnosis, auxiliary diagnosis or diagnosis, etc.

[0083] As used herein, the terms "sample", "specimen", "test sample", "subject sample", etc. include various sample types obtained from patients, individuals or subjects and can be used for diagnosis or monitoring determination. Samples can be obtained from healthy subjects or cancer patients. In addition, the sample obtained from the patient can be divided into parts, and only a part can be used for diagnosis. In addition, the sample or a part thereof can be stored under the condition of maintaining the sample for subsequent analysis. This definition specifically includes blood and other liquid samples of biological origin (including but not limited to tissues, cells, body fluids, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid, etc.). In a specific embodiment, the sample includes a blood sample. In a specific embodiment, the sample includes a serum sample. This definition also includes samples that are manipulated in any way after the sample is obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells in culture, cell supernatants, tissue samples, organs, etc. Samples may also include fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or studies by immunohistochemistry.

[0084] The terms "individual," "subject," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0085] The term "ROC" or "ROC curve" as used herein may refer to a receptor operating characteristic curve. A ROC curve may be a graphical representation of the performance of a binary classifier system. For any given method, a ROC curve may be generated by plotting sensitivity for specificity under a variety of threshold settings. In addition, as long as at least one of the three parameters (e.g., sensitivity, specificity, and threshold settings) is provided, and the ROC curve may determine the value or expected value of any unknown parameter. Unknown parameters may be determined using a curve fit for the ROC curve.

[0086] As used herein, the term "AUC" or "ROC-AUC" generally refers to the area under the receiver operating characteristic curve. This metric takes into account the sensitivity and specificity of the method and can provide a measure of the diagnostic utility of the method. Typically, ROC-AUC is in the range of 0.5 to 1.0, where values ​​closer to 0.5 indicate that the method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and values ​​closer to 1.0 indicate that the method has greater diagnostic utility (e.g., higher sensitivity and / or specificity). See, e.g., Pepe et al., "Limitations of the Odds Ratio in Gauging the Performance of a Diagnostic, Prognostic, or Screening Marker", Am. J. Epidemiol 2004, 159 (9): 882-890, which is incorporated herein by reference in its entirety. Other methods of characterizing diagnostic utility using likelihood functions, odds ratios, information theory, predictive values, calibration (including goodness of fit), and reclassification measures are summarized in Cook, "Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction), Circulation 2007, 115:928-935, which is incorporated herein by reference in its entirety.

[0087] The detection agent or component for obtaining the level of the late region of DNA replication in the sample can be understood to include a detection reagent for directly obtaining the level of the late region of DNA replication in the sample, or a component for indirectly obtaining the level of the late region of DNA replication in the sample (such as a computer program directly obtaining an indicator of the level of the late region of DNA replication in a sample that has been detected).

[0088] According to some embodiments of the present application, the marker level index can be obtained in any way such as nucleic acid level. The detection method is not limited, but any method that can be used to directly or indirectly evaluate the nucleic acid in the late region of DNA replication is suitable for the present application. It can be understood that the nucleic acid level can include DNA level or RNA level. There are many ways to detect DNA or RNA levels in this field, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in the present application. In some specific embodiments of the present application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0089] According to the experimental data of the present application, the level of the late region of DNA replication can be used as an independent indicator for pan-cancer diagnosis, and effective pan-cancer diagnosis can be achieved based solely on the level of the late region of DNA replication.

[0090] It is understandable that in order to further enhance the diagnostic effect of pan-cancer, the biomarkers detected by the reagent can also be other biological materials that can be used as markers, such as nucleic acid fragments, proteins, metabolites, etc. with diagnostic effects that are well known to those skilled in the art. In this way, a combination of multiple biomarkers can be used, and they cooperate with each other to achieve a better diagnostic effect on pan-cancer patients, thereby achieving a more effective diagnostic evaluation. Therefore, in addition to being an independent indication for pan-cancer diagnosis, the late DNA replication region can also be detected in combination with other existing known diagnostic markers to improve diagnostic efficacy.

[0091] In some embodiments of the present application, the test sample described in the present application can be selected from tissues, cells, body fluids serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid; in a preferred embodiment, the test sample is selected from any one of serum, plasma or whole blood.

[0092] Diagnostic Methods

[0093] The applicant found that certain levels of late regions of DNA replication in pan-cancer patients are higher than those in healthy people. Therefore, a more accurate prediction of pan-cancer diagnosis (especially early diagnosis) can be made based on the levels of late regions of DNA replication (such as low methylation levels).

[0094] The core of the diagnostic method includes the steps of detecting or determining the late region of DNA replication in the subject's sample;

[0095] In some specific embodiments, the method comprises the following steps:

[0096] (i) detecting or determining the level of the late region of DNA replication in the test sample;

[0097] (ii) comparing the level of the late region of DNA replication with that of a control sample; wherein a significant difference in the level of the late region of DNA replication between the test sample and the control sample is an indication that the subject suffers from pan-cancer; or, comparing with a set threshold absolute amount; wherein the level of the subject sample being higher than the threshold absolute amount is an indication that the subject suffers from pan-cancer.

[0098] It is understandable that the control sample can be selected according to actual needs. For example, in diagnosis, the control sample is a normal population sample, while when used for prognosis assessment, it can be a control sample of different prognostic conditions.

[0099] In some specific embodiments, a set value for the expression level of the late region of DNA replication can be given, and the set value can be determined based on the expression level of the late region of DNA replication of normal samples of normal people and / or non-pan-cancer patients. For example, the average value of the expression level of the late region of DNA replication of normal samples of an appropriate number of samples is selected, or a reasonable multiple is set based on the average value, such as 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the expression level of the late region of DNA replication of the subject is higher than the set value, it is judged as cancer. It can be understood that the set value determined based on the average value or the multiple of the average value needs to have a good classification significance, and the known samples can be tested by using a commonly used statistical test method through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard. Among them, the level of the late region of DNA replication refers to the value of this biomarker for the subject by direct measurement or further indirect deduction based on direct measurement, which is usually at least partially derived from the abundance or concentration of the biomarker in the sample of the subject. Among them, the indirectly derived value includes the value derived by applying a function to the measured value of this biomarker. Directly measured values ​​include, but are not limited to, values ​​of biomarkers determined by at least one of sequencing, hybridization, mass spectrometry, immunoassay, immunofluorescence, flow cytometry, and the like.

[0100] product

[0101] According to the core diagnostic use of this application, the process for detecting the level of the late region of DNA replication can be configured into a corresponding product form for the diagnosis or prediction of pan-cancer species. The product includes reagents or components for detecting biomarkers in the late region of DNA replication. It is understood that such product forms are diverse, including but not limited to kit forms, system devices, computer-readable media or computer system forms.

[0102] Kit Format

[0103] In some embodiments of the present application, a kit for detecting or analyzing the level of the late region of DNA replication to predict cancer is also disclosed herein. Such a kit may include reagents for detecting the level of one or more markers and instructions for predicting cancer based on the detected level.

[0104] The kit may include a set of reagents for generating a data set by at least one assay. The set of reagents is capable of detecting the level of the late region of quantitative DNA replication. The set of reagents may also further detect one or more other marker levels, wherein the reagents are detected at the nucleic acid level.

[0105] It is understood that there are many nucleic acid detection methods in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0106] Thus, for example, in some embodiments, the reagent may be a sequencing reagent, such as a first-generation Sanger sequencing reagent, a second-generation sequencing ("NGS") reagent, a third-generation sequencing reagent, etc. In other embodiments, the reagent is a PCR primer reagent, wherein the primer refers to a primer that can specifically amplify a gene in the late region of DNA replication, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting point for synthesizing primer extension products. In other embodiments, the reagent is a probe reagent, wherein the probe refers to a probe that can specifically identify a gene in the late region of DNA replication or a transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other portion of another molecule, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a specific sequence in the late region of DNA replication as a target nucleotide) through complementary base pairing.

[0107] In some embodiments, such a kit may include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing one of the individual elements to be used in the method. The kit of the present application may include a container as described above and one or more other containers containing materials required from the standpoint of a commercial end user, including buffers, diluents, filters, and package inserts with instructions for use.

[0108] In some embodiments, the kit further includes a sample processing reagent, which may include at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0109] In some embodiments, the product further comprises at least one of a standard, a calibrator, a reference substance, and a buffer. The reference substance is a control substance for testing the effectiveness of the experiment and serves as a control for judging the results. The buffer can be any solution known in the art that can provide appropriate buffer conditions during the detection process.

[0110] System Devices

[0111] In some embodiments of the present application, a system for detecting or analyzing the level of a late region of DNA replication to predict cancer is also disclosed herein. Such a system may include a set of reagents for detecting the level of one or more markers; a device configured to receive a mixture of one or more reagents and a test sample obtained from a subject to measure the level of the late region of DNA replication; and a computer system that is coupled to the device in communication to obtain the measured level and determine a score that predicts the likelihood of a patient suffering from cancer.

[0112] The device is configured to detect the DNA replication late region level in the mixture of reagent and test sample. For example, the device can determine the DNA replication late region level by the assay method of nucleic acid detection. The mixture of reagent and test sample can be provided to the device by various containers, and the examples of these containers include holes, bottles or pipes of orifice plates (for example, 96 orifice plates). Because of this, the device can have an opening (for example, a slot, a cavity, an opening, a sliding tray), and the opening can receive the container that holds the reagent test sample mixture and read to generate the quantitative expression value of a soluble medium.

[0113] The computer system communicates with the device to receive the quantitative expression value of the soluble mediator. The computer system analyzes the quantitative expression value by applying a prediction model and determines the likelihood of a pan-cancer activity event in the subject.

[0114] Computer system or computer readable medium

[0115] In some embodiments of the present application, the method or use of the present application can be performed on a computer, including detecting or analyzing the level of the late region of DNA replication by a computer to predict cancer.

[0116] For example, the construction and execution of the prediction model for generating a score can be implemented in hardware or software or a combination of the two. In one embodiment, a readable storage medium is provided, such as a medium including a data storage material encoded with machine-readable data, when a machine programmed with instructions utilizing the data is used, the data storage material can display any data set of the prediction model of the present application and execution and results. Such data can be used for a variety of purposes, such as cancer patient monitoring, diagnosis, treatment considerations, etc. The embodiment of the above method can be implemented in a computer program executed on a programmable computer, the computer including a processor, a data storage system, a graphics adapter, a network adapter, at least one input device and at least one output device, etc. The display is coupled to a graphics adapter. Program code is applied to input data to perform the above functions and generate output information. Output information is applied to one or more output devices in a known manner. The computer can be, for example, a personal computer, a microcomputer, or a workstation of traditional design.

[0117] Each program can be implemented in a high-level program or object-oriented programming language to communicate with a computer system. However, if desired, the program can be implemented in an assembly or machine language. In any case, the language can be a compiled language or an interpreted language. Each such computer program is preferably stored in a storage medium or device (e.g., ROM or disk) readable by a general or special programmable computer, for configuring and operating a computer to perform the program described herein when the computer reads the storage medium or device. The system can also be considered to be implemented in the form of a computer-readable storage medium configured with a computer program, wherein the storage medium so configured causes the computer to operate in a specific and predefined manner to perform the functions described herein.

[0118] Feature patterns and their databases can be provided in various media to facilitate their use. "Medium" refers to a manufactured article containing feature pattern information of the present application. The database of the present application can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electronic storage media such as RAM and ROM; and hybrids of these categories, such as magnetic / optical storage media. Those skilled in the art can easily understand how any currently known computer-readable medium can be used to produce a record containing current database information. "Record" refers to the process of storing information on a computer-readable medium using any such method known in the art. Based on the method used to access the stored information, any convenient data storage structure can be selected. A variety of data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0119] Other aspects

[0120] It can be understood that based on the belief that the level of the late region of DNA replication can be used as a pan-cancer diagnostic marker, technical personnel in this field can evaluate or screen other potential pan-cancer diagnostic markers based on this. Usually, the potential biomarkers are evaluated with the late region of DNA replication through correlation. On the basis of reaching the conclusion of positive correlation, it helps to illustrate the value of the potential diagnostic marker.

[0121] The present application may also relate to a method for detecting markers in a subject having or suspected of having pan-cancer in vivo or in vitro, the method comprising determining or detecting the level of a late region of DNA replication in a biological sample from the subject, and the method may include uses for non-disease diagnosis.

[0122] A method for evaluating or screening pan-cancer therapeutic agents in vivo or in vitro, the method comprising the step of evaluating the level of the late region of DNA replication in a sample treated with the therapeutic agent, analyzing the improvement of the disease after treatment with the therapeutic agent by evaluating the level of the late region of DNA replication, and further evaluating the effect of the therapeutic agent.

[0123] The above contents are only used to exemplify the present application and are not used to limit the present application. The corresponding specific embodiments are further described as follows.

[0124] Example 1 Screening of late DNA replication regions

[0125] This embodiment adopts Figure 1 The process screened out markers that can be used for pan-cancer diagnosis or prediction, specifically:

[0126] 1) Collect tumor tissues and adjacent cancer samples from 9 cancer types, including lung cancer, colon cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, and lymphoma. Perform tissue section microscopy on the samples and filter out samples with a low tumor percentage (<15%). 50 tumor tissues and 50 adjacent cancer samples were obtained for each cancer type.

[0127] 2) DNA was extracted from the samples, treated with bisulfite reagent, and the library was constructed using the SWIFT methylation library construction kit and sequenced on the Novo-seq 6000. The methylation sequencing data was aligned to the human hg19 reference genome using BWA-meth, and PCR duplications and reads with low alignment quality were removed.

[0128] 3) Use ichorCNA to estimate the tumor proportion of the whole genome methylation sequencing data of tumor samples, and eliminate samples with low tumor proportion (<15%).

[0129] 4) Correction of methylation levels of CpG sites in tumor samples:

[0130] a. Use ichorCNA to assess the tumor proportion of each tumor sample and calculate the main clone and subclone proportions of the tumor sample.

[0131] b. Calculate the average methylation level of CpG sites in all adjacent para-cancer samples.

[0132] c. According to the methylation level of CpG sites in each tumor sample, the methylation level of CpG sites in adjacent samples, and the proportion of major clones and subclones, the corrected methylation level (M) of the CpG sites in the tumor sample was calculated according to the following method. adj ).

[0133]

[0134] Among them, M raw represents the original CpG methylation level, M norm Indicates the average methylation level of the CpG site in all adjacent paracancerous samples. x Represents the percentage of tumor cells calculated by ichorCNA.

[0135] 5) Screening of late regions of whole genome duplication:

[0136] The sequencing data of Repli technology of 15 cell lines were downloaded from the UCSC database. The data of different periods of DNA replication were sorted out, and the early and late regions of DNA replication were divided according to the signal value intensity obtained by Repli-seq in each period. Specifically, the data of different replication periods of the whole genome DNA sequence of each cell line were integrated according to 1000bp segments, and the early and late regions of DNA replication were defined by comparing the difference in active signal values ​​of each segment in the early DNA replication cell line and the late DNA replication cell line; combined with the above-corrected CpG site methylation level, the correlation between the average methylation level and the differential signal value in each segment was calculated, and the region with correlation>0, which showed a positive correlation, was screened. A total of 30M whole genome DNA replication late regions ( Figure 1 ).

[0137] 6) Relationship between genome-wide methylation levels and different stages of DNA replication in 9 cancer types

[0138] Based on the screened 30M whole genome DNA replication late region, a genomic region (chr2: 115,000,000-130,000,000) derived from the 30M whole genome DNA replication late region was taken as an example ( Figure 2), describing the correlation between the signal values ​​at different DNA replication stages and the distribution of DNA methylation levels of 9 major cancer types. The genome of this example region covers a base length of approximately 15,000,000bp, accounting for about 5% of the total length of the human genome, and based on the above statistics of this application, this genomic region contains more than one thousand late DNA replication regions, as shown in the following table (due to the large number, the intermediate values ​​have been hidden), so the analysis based on this genomic region can have statistical and deductive significance.

[0139]

[0140] Figure 2 In the figure, A is the distribution of early and late signal values ​​of DNA replication obtained from sequencing data of Repli-seq technology, the peak is the early stage of DNA replication, and the trough between the two peaks is the late stage of DNA replication; B is the distribution of early and late regional methylation levels in lung cancer tissue and adjacent tissue of lung cancer; C is the distribution of early and late regional methylation levels in colorectal cancer tissue and adjacent tissue of colorectal cancer; D is the distribution of early and late regional methylation levels in gastric cancer tissue and adjacent tissue of gastric cancer; E is the distribution of early and late regional methylation levels in breast cancer tissue and adjacent tissue of breast cancer The distribution of methylation levels in the early and late regions of liver cancer tissue and adjacent tissues of liver cancer; the distribution of methylation levels in the early and late regions of esophageal cancer tissue and adjacent tissues of esophageal cancer; the distribution of methylation levels in the early and late regions of pancreatic cancer tissue and adjacent tissues of pancreatic cancer; the distribution of methylation levels in the early and late regions of ovarian cancer tissue and adjacent tissues of ovarian cancer; the distribution of methylation levels in the early and late regions of lymphoma tissue and adjacent tissues of lymphoma; and the distribution of methylation levels in the early and late regions of leukocytes. The red in the BJ figure is cancer tissue, and the blue is adjacent tissue. (Note: For the definition or division of methylation levels, please refer to the definition in the content of the invention).

[0141] It can be seen that in the sample area, in the early stage of DNA replication, i.e., the peak area of ​​Figure A, all 9 cancer types show a significant high methylation level distribution (red), while in the late stage of DNA replication, i.e., the trough area of ​​Figure A, all 9 cancer types show a significant low methylation level distribution (red). Throughout the entire area, the pan-cancer methylation level and the signal value of the DNA replication period show a positive correlation (see Figure 2 and Figure 3 ).

[0142] Taking lung cancer and liver cancer as representative cancers, the methylation levels of 136M early DNA replication and 30M late DNA replication regions in the whole genome were statistically compared in all the early and late regions of DNA replication screened in the whole genome, showing that lung and liver tumor tissues were in a high methylation state in the early DNA replication region ( Figure 3Red), the late DNA replication region is in a low methylation state ( Figure 3 Blue), the adjacent tissues are both highly methylated during the two DNA replication periods. Therefore, the late DNA replication region with the largest difference in methylation levels between tumor tissue and adjacent tissues was selected as the candidate interval for pan-cancer detection ( Figure 3 (above).

[0143] Example 2: Evaluation of differences in mutation frequency among pan-cancer species in the late DNA replication region

[0144] This application also evaluates the enrichment of mutation frequencies in the screening regions during the late DNA replication period across cancer species.

[0145] 1) Download the somatic mutation data of 15 cancer types sequenced by PCGA, including bile duct cancer, bone tumor, breast cancer, central nervous system tumor, esophageal cancer, head and neck tumor, kidney cancer, liver cancer, lymphoma, myeloid tumor, ovarian cancer, pancreatic cancer, prostate cancer, skin tumor and stomach cancer.

[0146] 2) Based on the 136M whole genome DNA replication early region and 30M whole genome DNA replication late region, as well as the human genome exon region and human whole genome region screened in Example 1 above, they were segmented according to 1 kb.

[0147] 3) Based on the genome-wide somatic mutation information of the 15 cancer types obtained in 1) above, the average number of somatic mutations per 1kb in the 4 genomic regions in 2) above was counted to compare and evaluate the characteristics of mutation enrichment in the late DNA replication region of the 30M whole genome in pan-cancer types. The results are as follows: Figure 4 shown.

[0148] Figure 4 It can be seen that among 15 different types of cancer, the number of mutations (per 1kb) in the late region of 30M whole genome DNA replication (orange) is significantly higher than that in the early region of whole genome DNA replication (blue) and the whole genome exon region (gray). This phenomenon is more obvious in skin cancer, esophageal cancer and liver cancer.

[0149] Therefore, the above mutation frequency characteristics in the late region of 30M whole genome DNA replication are significantly enriched in tumor signals in pan-cancer species. The mutation frequency characteristics based on the late region of 30M DNA replication can also be used as candidate features for pan-cancer early screening signals.

[0150] Example 3: Evaluation of the differences in fragment features of pan-cancer species in the late DNA replication region

[0151] In this example, low-pass whole genome DNA sequencing (LP-WGS) was performed on cfDNA of three cancer types, namely lung cancer, liver cancer and colon cancer, and the enrichment of fragment features in the late region of 30M whole genome DNA replication of the three cancer types was evaluated. The specific steps are as follows:

[0152] 1) For lung cancer, liver cancer, colorectal cancer and healthy people, 10 cfDNA samples were collected and tested with LP-WG S. The sequencing data were quality controlled, compared with the human reference genome and deduplicated to obtain the bam format results for subsequent statistics.

[0153] 2) Extract the cfDNA fragment feature information of each sample:

[0154] a. Fragment length ratio: For the bam results of each sample tested by LP-WGS, the number of cfDNA fragments with a length of 90-150 bp and the number of cfDNA fragments with a length of 180-220 bp were counted respectively, and the ratio of the two was the length ratio of the cfDNA fragments.

[0155] b. End-motif (EDM): For the bam results of each sample tested by LP-WGS, a total of 256 EDMs were obtained based on the 4bp sequences from 5′ to 3′ of the positive and negative strands of the reference genome at the start and end positions of the cfDNA fragment alignment. The ratio of the number of cfDNA fragments supporting each EDM to the total number of cfDNA fragments detected in the sample, i.e., the abundance of the EDM, was calculated.

[0156] c. Breakpoint motif (BPM): For the bam results of each sample tested by LP-WGS, 3 bp on each side of the reference genome from 5′ to 3′ in the positive and negative strands at the start and end positions of the cfDNA fragment insertion alignment, a total of 6 bp sequences, a total of 4096 BPMs were obtained. The ratio of the number of cfDNA fragments supporting each BPM to the total number of cfDNA fragments detected in the sample was statistically analyzed, that is, the abundance of the BPM.

[0157] 3) The distribution of the three cfDNA fragment features in 2) above in the 136M whole genome DNA replication early region and the 30M whole genome DNA replication late region obtained by screening in Example 1 was counted for comparison and evaluation of the enrichment effect of the 30M whole genome DNA replication late region on the tumor cfDNA fragment features. The results are as follows Figure 5-7 shown.

[0158] The results are as follows Figure 5-7 As shown:

[0159] 1) Fragment length ratio

[0160] like Figure 5 As shown in the figure, the cfDNA fragment length distribution of healthy people and patients with lung cancer and liver cancer was compared, and it was found that the cfDNA fragment length distribution in the late region of 30M whole genome DNA replication was different between healthy people and cancer patients (lung cancer and liver cancer) ( Figure 5 ). It is mainly manifested as the fragment length in the 30M DNA replication late stage region of lung cancer and liver cancer patients ( Figure 5 , orange) than healthy people ( Figure 5 , blue) is more to the left, that is, the fragment lengths of patients with lung cancer and liver cancer are shorter (or the proportion of short fragments is higher), that is, they have a higher level of short-to-long fragment ratio.

[0161] 2) End-motif terminal feature abundance difference level

[0162] like Figure 6 As shown in the figure, Figure A and Figure B refer to two groups of EDM features, H represents the abundance distribution of EDM in the whole genome of healthy people, Tlung represents the abundance distribution of EDM in the whole genome of lung cancer, Tliver represents the abundance distribution of EDM in the whole genome of liver cancer, lung-E represents the abundance distribution of EDM in the early DNA replication region of lung cancer samples, lung-L represents the abundance distribution of EDM in the late 30M DNA replication region of lung cancer samples, liver-E represents the abundance distribution of EDM in the early DNA replication region of liver cancer samples, and liver-L represents the abundance distribution of EDM in the late 30M DNA replication region of liver cancer samples. T test was performed on the abundance of EDM in healthy samples and tumor samples, and the top 10 EDMs with the largest differences were obtained for analysis. Significant differences in the abundance of EDM in the early DNA replication region and the late DNA replication region were found in both lung cancer and liver cancer cfDNA.

[0163] 3) Break-point motif feature abundance difference level

[0164] like Figure 7As shown, Figure A and Figure B refer to two groups of BPM characteristics, H represents the abundance distribution of BPM in the whole genome of healthy people, Tlung represents the abundance distribution of BPM in the whole genome of lung cancer, Tliver represents the abundance distribution of BPM in the whole genome of liver cancer, lung-E represents the abundance distribution of BPM in the early DNA replication region of lung cancer samples, lung-L represents the abundance distribution of BPM in the late 30M DNA replication region of lung cancer samples, liver-E represents the abundance distribution of BPM in the early DNA replication region of liver cancer samples, and liver-L represents the abundance distribution of BPM in the late 30M DNA replication region of liver cancer samples. T test was performed on the abundance of BPM in healthy samples and tumor samples, and the top 10 BPMs with the largest differences were obtained for analysis. Significant differences in the abundance of BPM in the early DNA replication region and the late DNA replication region were found in both lung cancer and liver cancer cfDNA.

[0165] Therefore, the above three cfDNA fragment features in the late region of 30M whole genome DNA replication are significantly enriched in tumor signals in different cancer types (lung cancer, liver cancer, and colorectal cancer). The three cfDNA fragment features based on the late region of 30M DNA replication can also be used as candidate features for pan-cancer early screening signals.

[0166] Example 4: Evaluation of diagnostic prediction performance

[0167] 1) Cross-validation evaluation of methylation signatures of tissue samples

[0168] Based on the whole genome methylation sequencing results of tumor tissues and para-cancer samples of 9 cancers in Example 1, all tumor samples and para-cancer samples were merged respectively, and the methylation level difference of CpG sites between tumor tissues and corresponding para-cancer and leukocytes in the 30M whole genome DNA replication late region obtained in Example 1 was screened>0.2, and if 100bp contained 3 or more CpG difference sites, they were merged into one interval. The methylation intervals in the late DNA replication region screened out in 8 cancer tissues using the above method were counted, the methylation level of each methylation interval was counted, and a logistic regression classification model was constructed, and the methylation level of the late DNA replication region corresponding to the remaining ninth cancer was used for verification, similar to the 9-fold cross-validation, and the ROC (Receiver Operating Characteristic) curve and its area under the curve AUC (Area Under the Curve) were used to evaluate the effectiveness and stability of methylation features in the late DNA replication region in pan-cancer tumor classification and diagnosis.

[0169] like Figure 8As shown, the methylation interval in the late stage of 30M whole genome DNA replication can well distinguish tumors and adjacent tissues of 9 cancer types, among which 8 cancer types have an AUC of more than 0.96, and pancreatic cancer is slightly lower but also reaches 0.76.

[0170] Therefore, the above cross-validation results show that the methylation level in the late 30M whole genome DNA replication period can be effectively applied to pan-cancer tumor classification and diagnosis.

[0171] 2) cfDNA sample verification

[0172] In order to further verify the various characteristics of the late DNA replication period, this example designs a pan-cancer methylation probe capture panel based on the genomic methylation intervals in the late DNA replication region of the 30M whole genome screened in Example 1 above. At the same time, a series of clinical samples, including cfDNA of lung cancer (205 high-risk people vs. 194 lung cancer patients), pancreatic cancer (114 high-risk people vs. 120 pancreatic cancer patients) and esophageal cancer (98 high-risk people vs. 92 esophageal cancer patients) samples, were converted by methylase method, and the methylation panel in the late DNA replication region was used for capture sequencing. After sequencing data quality control, human reference genome alignment, and deduplication, the methylation level of each sample was calculated in the above methylation interval. A logistic regression classification model was constructed based on the methylation level of each cancer type and the corresponding high-risk population, and the ROC curve and its area under the curve AUC were used to evaluate the effectiveness of tumor classification and diagnosis of methylation characteristics in the late DNA replication region in cfDNA of different cancer types. Fig. 9 As shown in the figure, the area under the curve (AUC) values ​​of cfDNA in different cancer types are: lung cancer AUC = 0.937; pancreatic cancer AUC = 0.896; esophageal cancer AUC = 0.953 ( Fig. 9 ,red).

[0173] In addition, this example further integrates the mutation frequency in the late region of DNA replication, the short-to-length ratio of cfDNA fragments, the end feature (EDM) and the breakpoint feature (BPM) in the above-mentioned Examples 2 and 3 to evaluate the effectiveness of tumor classification and diagnosis in cfDNA. Fig. 9 As shown in the figure, the tumor classification and diagnostic performance in cfDNA can be further improved, and the area under the curve (AUC) values ​​are: lung cancer AUC = 0.945; pancreatic cancer AUC = 0.904; esophageal cancer AUC = 0.966 ( Fig. 9 ,blue).

[0174] It can be seen that the late DNA replication regions screened by the method of the present application have good tumor diagnosis and prediction performance in different omics characteristics of pan-cancer cfDNA samples (including methylation, mutation frequency, cfDNA fragment length ratio, EDM, BPM).

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a detection agent or component for obtaining the level of a late region of DNA replication in a sample in the preparation of a pan-cancer diagnostic product; the level of the late region of DNA replication is the methylation level of DNA in the late region of DNA replication; the methylation level is a low methylation level; the steps for obtaining the late region of DNA replication are as follows: collating data from different periods of DNA replication, and dividing the early and late regions of DNA replication according to the signal value intensity obtained by Repli-seq in each period; combining the corrected CpG site methylation levels, calculating the correlation between the average methylation level and the differential signal value in each segment, screening the regions with correlation > 0, and obtaining the late region of DNA replication in the whole genome; the pan-cancer is lung cancer, colon cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer or lymphoma.

2. The use according to claim 1, characterized in that: The acquisition is carried out by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

3. The use according to any one of claims 1-2, characterized in that: The sample includes one or more of serum, plasma, whole blood, tissue, cells, urine, semen, cerebrospinal fluid, saliva, pleural effusion, ascites or feces.

4. The use according to any one of claims 1-2, characterized in that: The application comprises the following steps: (i) obtaining the level of the late region of DNA replication in the subject's sample; (ii) comparing the level of the late region of DNA replication with that of a control sample, wherein a significant difference in the level of the late region of DNA replication between the subject sample and the control sample is an indication that the subject suffers from pan-cancer; or, comparing with a set threshold absolute amount, wherein the level of the subject sample is higher than the threshold absolute amount is an indication that the subject suffers from pan-cancer.

Images