DNA methylation markers tagme-8, 9, and 10 for broad-spectrum identification of cancer and use thereof

Abstract

Provided are DNA methylation markers TAGMe-8, 9, and 10 for broad-spectrum identification of cancer and use thereof. The provided markers exhibit significant methylation differences between cancer tissue and para-carcinoma tissue. Whether epigenetic modification markers of cancer are in abnormally high methylation states can be analyzed to analyze whether a person is at high risk of developing cancer, and the markers are suitable for analyzing different types of cancer.

Description

TAGMe-8, 9, 10, and their applications in broad-spectrum cancer identification DNA methylation markers Technical Field

[0001] This invention belongs to the fields of oncology and epigenetics; more specifically, this invention relates to novel DNA methylation markers TAGMe-8, 9, 10 that can be used for cancer identification and their uses. Background Technology

[0002] The mechanisms of cancer development and progression are complex, and humanity has yet to fully understand their patterns, seriously impacting life and health. With the development of molecular biology and the completion of the Human Genome Project, we have gained a deeper understanding of cancer at the genetic level. Under the influence of various factors (including genetic and environmental factors), the DNA in normal human cells is damaged, leading to mutations. The continuous accumulation of mutations eventually results in the activation of multiple oncogenes and the inactivation of tumor suppressor genes, causing cancer cells to grow and proliferate uncontrollably, invading adjacent normal tissues and metastasizing to distant tissues and organs. Invasion and metastasis are fundamental biological characteristics of malignant tumors and are also the main causes of death in cancer patients. Tumor metastasis includes local invasion, infiltration into adjacent blood vessels or lymphatic vessels, survival and transport within the circulatory system, extravasation from the lumen of the circulatory system to distant tissues, and the clonal formation of visible tumors in distant tissues. Tumor metastasis is a continuous process of complex interactions and mutual influences between tumor cells, host cells, and the tumor microenvironment. Multiple pathways, genes, and cytokines participate in the entire process of invasion and metastasis.

[0003] Research has found that in addition to gene mutations playing an important role in the occurrence and development of cancer, epigenomics is also closely related to the occurrence, development, or metastasis of malignant tumors.

[0004] Over 98% of CpG dinucleotides in the genome are scattered within transcriptionally dependent repetitive sequences with transposition potential. In normal cells, these CpGs are in a state of high methylation / transcriptional silencing, while in tumor cells, these CpGs undergo extensive demethylation, leading to transcriptional changes in repetitive sequences, transposon activation, increased genomic instability, and enhanced proto-oncogene transcription. The remaining approximately 2% of CpGs are densely distributed in smaller regions (CpG islands). CpG islands are present in or near approximately 40-50% of gene promoter regions, suggesting that DNA methylation may be involved in the transcriptional regulation of these genes. In tumor cells, these CpG islands, which are normally hypomethylated, become hypermethylated, leading to transcriptional inactivation of the genes. Studies have shown that tumor cell DNA methylation is characterized by overall hypomethylation but localized hypermethylation. Affected genes include DNA repair genes, cell cycle control genes, and tumor suppressor genes such as anti-apoptotic genes. Therefore, this abnormal DNA methylation pattern in tumor cells has become a new area of ​​research in tumor biomarkers. After genomic DNA is treated with bisulfite, PCR (methylation-specific PCR, MSP) can be performed to effectively determine the methylation status of specific sites on the tested DNA fragment. As a qualitative analysis method, MSP can detect single-digit abnormally methylated tumor cells in complex clinical samples containing 10,000 normal cells.

[0005] While early cancer screening based on DNA methylation molecular markers has gradually gained attention, very few programs are actually used clinically. This is partly due to issues with the sensitivity and specificity of DNA methylation markers, which is why cancer screening is often classified as a specialized test. Furthermore, most existing tumor markers are only effective for specific tumor types, with very few available for screening multiple cancers.

[0006] Therefore, finding molecular targets that can be used for the diagnosis, prognosis, and prediction of cancer development is of great significance for early cancer screening, clinical intervention, and guiding patient treatment. Summary of the Invention

[0007] The purpose of this invention is to provide novel DNA methylation markers TAGMe-8, 9, 10 that can be used for cancer identification and their applications.

[0008] In a first aspect of the present invention, a method for preparing a reagent for detecting cancer is provided, comprising:

[0009] (a) Providing an epigenetic modification marker or a nucleic acid derived therefrom, said epigenetic modification marker comprising:

[0010] (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site of SEQ ID NO: 1, 2, or 3; or

[0011] (2) The epigenetic modification markers or fragments that are sequentially complementary to those in (1);

[0012] The nucleic acid derived therefrom is an epigenetic modification marker corresponding to (1) or (2), in which the unmodified cytosine is converted to T or U, while the cytosine C at the modified CpG site remains unchanged.

[0013] (b) Using the epigenetic modification markers in (a) as targets, design a detection reagent that specifically detects the CpG site modification status of the targets.

[0014] As one implementation, the SEQ ID NO: 1, 2 or 3 may also include its sequence variants or homologous sequences.

[0015] In one implementation, the sequence variant or homologous sequence is a sequence having more than 80%, 85%, 90%, 92%, 95%, 96%, 98%, 99%, 99.5%, or 99.8% sequence identity compared to the sequence shown in SEQ ID NO: 1, 2, or 3. Accordingly, epigenetic modification markers derived from the transformation of the sequence variant or homologous sequence (conversion of unmodified cytosine to T or U, while the cytosine C at the modified CpG site remains unchanged) are also included.

[0016] In one embodiment, the modification includes 5-methylation (5mC), 5-hydroxymethylation (5hmC), 5-aldehyde methylation (5-fC), or 5-carboxymethylation (5-caC).

[0017] In one embodiment, the nucleic acid derived from the epigenetic modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6.

[0018] As one implementation, the at least one modified CpG site is any CpG site or combination thereof selected from numbers 1 to 24 in the sequence shown in SEQ ID NO: 1 (e.g., 2 to 24, more specifically 3, 5, 10, 11, 12, 15, 20, 22), any CpG site or combination thereof selected from numbers 1 to 41 in the sequence shown in SEQ ID NO: 2 (e.g., 2 to 41, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 38), and any CpG site or combination thereof selected from numbers 1 to 44 in the sequence shown in SEQ ID NO: 3 (e.g., 2 to 44, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 42); preferably, it is any CpG site or combination thereof selected from numbers 10 to 17 in the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 2 ... SEQ ID NO:2 shows any CpG site selected from 6 to 22 or 7 to 22 or any combination thereof, and SEQ ID NO:3 shows any CpG site selected from 1 to 17 or 2 to 16 or any combination thereof.

[0019] In one embodiment, the apparent modification marker fragment is selected from the following sequences: the sequence shown in positions 453-549 or 422-578 of SEQ ID NO: 1, the sequence shown in positions 141-256 or 111-284 of SEQ ID NO: 2, and the sequence shown in positions 45-175 or 16-205 of SEQ ID NO: 3.

[0020] As one implementation, the reagents for detecting cancer include, but are not limited to, primers, probes, chips, or test strips.

[0021] As one implementation method, one or more sets of reagents can be prepared for the target sequence.

[0022] In one implementation, the detection reagent is integrated onto a chip.

[0023] In one implementation, the detection includes diagnosis, identification, screening, or prognostic assessment.

[0024] In another aspect of the invention, the use of epigenetic modification markers or nucleic acids derived therefrom in the preparation of reagents or kits for detecting cancer is provided; wherein the epigenetic modification markers include: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or fragments containing at least one modified CpG site of SEQ ID NO: 1, 2 or 3; or (2) epigenetic modification markers that are sequence-complementary to the epigenetic modification markers or fragments of (1); wherein the derived nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.

[0025] In one implementation, the cancers include (but are not limited to): respiratory system cancers, digestive system cancers, urinary system cancers, gynecological and reproductive system cancers, hematological system cancers, nervous system cancers, head and neck cancers, skin system cancers, endocrine system cancers, or skeletal system cancers; preferably, the cancers include: lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, gastric cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer.

[0026] As one implementation method, the samples targeted for cancer detection include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0027] In one implementation, the cancer detection targets cancer (including early, intermediate, or late-stage cancer) or its precancerous lesions.

[0028] In one implementation, the samples include (but are not limited to): paraffin-embedded samples, pleural effusion samples and bronchoalveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, tissue and cell biopsy samples, etc.

[0029] In another aspect of the present invention, a method for analyzing the methylation level of a sample to be tested is provided, comprising: (i) acquiring the sample to be tested; and (ii) analyzing the CpG site modification of a target sequence or fragment thereof in the sample to be tested, wherein the target sequence comprises: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site of SEQ ID NO: 1, 2 or 3; or (2) an epigenetic modification marker or fragment thereof that is sequence-complementary to the epigenetic modification marker or fragment thereof of (1).

[0030] As one implementation method, the methods for analyzing the CpG site modification of the target sequence or its fragments in the sample to be tested include: pyrosequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, methylation microarray, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, or combinations thereof.

[0031] As one implementation, the method for analyzing the CpG site modification status of the target sequence in the extracted epigenetic modification markers includes: (i) processing the extracted epigenetic modification markers to convert unmodified cytosine into uracil; preferably, the modification includes 5-methylation, 5-hydroxymethylation, 5-aldehyde methylation, or 5-carboxymethylation; more preferably, treating the epigenetic modification markers described in step (i) with Bisulfite; and (ii) analyzing the modification status of the target sequence in the nucleic acid treated in (i).

[0032] As one implementation, abnormal methylation level refers to the high methylation of C in the epigenetic modification marker CpG.

[0033] As one implementation, the methylation-sensitive restriction endonuclease is a restriction endonuclease that is sensitive to methylated bases at its recognition site; including but not limited to: HhaI, BmgBI, HaeII, RruI, TaiI, Bsu15I, Hin6I, HpyCH4IV, NarI, etc., as well as combinations of one or more of them.

[0034] As one implementation method, the method for analyzing methylation levels is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis of a disease.

[0035] As one implementation method, the method for detecting the methylation level of the sample is an in vitro method.

[0036] In another aspect of the present invention, a reagent for detecting cancer is provided, which specifically detects the CpG site modification status of a target sequence, wherein the target sequence is an epigenetic modification marker or a nucleic acid derived therefrom, the epigenetic modification marker comprising: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site of SEQ ID NO: 1, 2 or 3; or (2) an epigenetic modification marker or fragment that is sequence-complementary to the epigenetic modification marker or fragment of (1); wherein the derived nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged; preferably, the reagent targets a gene sequence containing the target sequence, preferably, the gene sequence includes a gene panel or gene group; preferably, the reagent comprises: amplifying SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 ... Primers for the sequences shown in positions 453-549 or 422-578 of SEQ ID NO: 1, positions 141-256 or 111-284 of SEQ ID NO: 2, and positions 45-175 or 16-205 of SEQ ID NO: 3.

[0037] As one embodiment, the reagent is: a primer containing the sequence of positions 29-59 in SEQ ID NO: 7 and the sequence of positions 28-56 in SEQ ID NO: 8; a primer containing the sequence of positions 29-58 in SEQ ID NO: 9 and the sequence of positions 28-55 in SEQ ID NO: 10; and a primer containing the sequence of positions 29-57 in SEQ ID NO: 11 and the sequence of positions 28-57 in SEQ ID NO: 12.

[0038] As one embodiment, the reagent is: primers of SEQ ID NO: 7 and SEQ ID NO: 8 sequences; primers of SEQ ID NO: 9 and SEQ ID NO: 10 sequences; primers of SEQ ID NO: 11 and SEQ ID NO: 12 sequences;

[0039] As one embodiment, the reagent further includes primers of the sequences SEQ ID NO: 13 and SEQ ID NO: 14.

[0040] In another aspect of the invention, the use of the said reagent is provided for preparing a kit for detecting cancer.

[0041] In another aspect of the invention, a kit for detecting cancer is provided, comprising the aforementioned reagent.

[0042] As one implementation, the kit may also include, but is not limited to: DNA purification reagents, DNA extraction reagents, Bisulfite, and PCR amplification reagents.

[0043] As one implementation method, the kit also includes an instruction manual that specifies the detection procedure and result determination criteria.

[0044] In another aspect of the invention, isolated epigenetic modification markers or nucleic acids derived therefrom are provided, the epigenetic modification markers comprising: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or fragments containing at least one modified CpG site of SEQ ID NO: 1, 2 or 3; or (2) epigenetic modification markers that are sequence-complementary to the epigenetic modification markers or fragments of (1); wherein the derived nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged. Attached Figure Description

[0045] Figure 1. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in lung cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0046] Figure 2. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in liver cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0047] Figure 3. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in prostate cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0048] Figure 4. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in cervical cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0049] Figure 5. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in endometrial cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0050] Figure 6. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in urothelial carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0051] Figure 7. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in biliary tract tumors and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0052] Figure 8. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in gastric cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0053] Figure 9. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in breast cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0054] Figure 10. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in esophageal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0055] Figure 11. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in glioma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0056] Figure 12. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in colorectal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0057] Figure 13. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in leukemia and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0058] Figure 14. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in pancreatic cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0059] Figure 15. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in thyroid cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0060] Figure 16. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in melanoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0061] Figure 17. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in nasopharyngeal carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0062] Figure 18. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in oral cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0063] Figure 19. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in laryngeal cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0064] Figure 20. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in osteosarcoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0065] Figure 21. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in lymphoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0066] Figure 22. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in renal cell carcinoma and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0067] Figure 23. Comparison of methylation values ​​of TAGMe-8, 9, and 10 in ovarian cancer and control tissues (left panel), and results of sensitivity and specificity analysis (right panel).

[0068] Figure 24. Sequencing analysis of methylation levels of methylation sites (CpG) 001-009 and 018-024 in SEQ ID NO: 1 in cancer cells after bisulfite treatment, with normal cells (Para) as a control.

[0069] Figure 25. Sequencing analysis of methylation levels of methylation sites 001-006 and 023-041 in SEQ ID NO: 2 in cancer cells after bisulfite treatment, with normal cells as controls.

[0070] Figure 26. Sequencing analysis of methylation levels of methylation sites 017-044 in SEQ ID NO: 3 in cancer cells after bisulfite treatment, with normal cells as a control. Detailed Implementation

[0071] Based on extensive analysis and clinical trials, this invention provides novel epigenetic modification biomarkers, named TAGMe-8, TAGMe-9, or TAGMe-10. Within cancer cells, 5-methylcytosine (5mC) or other similar epigenetic modifications are generated at multiple 5'-CpG-3' base C positions in the nucleic acid sequence. By analyzing the presence of abnormally high methylation in the TAGMe-8, TAGMe-9, or TAGMe-10 gene sequence regions, it is possible to determine whether the subject belongs to a high-risk group for cancer. Furthermore, the significant differences exhibited by these epigenetic modification biomarkers between cancerous and non-cancer tissues are broadly present in various types of pan-cancer, including both solid tumors and non-solid tumors.

[0072] As used herein, "sample" or "sample" includes substances obtained from an individual (such as a person) or isolated tissues, cells, or bodily fluids (such as plasma or serum) that are suitable for DNA extraction and can be used for methylation detection. For example, the sample shown may include, but is not limited to: tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples and bronchoalveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, and tissue and cell biopsy samples.

[0073] As used herein, the term "high methylation" refers to the presence of high methylation, hydroxymethylation, aldehyde methylation, or carboxymethylation of CpG in a gene sequence. For example, in methylation-specific PCR (MSP) analysis, a positive PCR result obtained using methylation-specific primers indicates that the tested DNA (gene) region is in a high methylation state. For example, in real-time quantitative methylation-specific PCR, the determination of high methylation can be based on statistically significant differences in the relative methylation levels of control samples.

[0074] In this invention, the term "cancer" refers to a broad range of cancers (Pan-cancer), whose genomes contain epigenetic modification marker segments exhibiting a hypermethylated state as described in this invention. These cancers can be solid tumors or non-solid tumors, and may include (but are not limited to): respiratory system cancers, digestive system cancers, urinary system cancers, gynecological and reproductive system cancers, hematological system cancers, nervous system cancers, head and neck cancers, skin system cancers, endocrine system cancers, or skeletal system cancers, etc.

[0075] With the information on the specific segments in the human genome provided by the present invention, those skilled in the art are able to obtain and apply the CpG sites. Embodiments of the present invention provide a series of sequence fragments containing CpG sites, which may serve as examples of preferred embodiments. However, it should be understood that variations can be made based on the information provided by the present invention, such as selecting longer sequences that contain the sequences of the present invention, or selecting sequences that overlap regionally with the sequences of the present invention.

[0076] This invention also includes "conservative variant sequences" of the epigenetic modification marker (or its reverse complementary sequence) sequence that are conserved or have high sequence identity with the sequence of the epigenetic modification marker (or its reverse complementary sequence). "High sequence identity" is, for example, higher than 85%, 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc. It should be understood that differences may exist at individual sequence sites between different biological individuals (e.g., some meaningless SNPs may exist), but this does not affect the detection based on the overall scheme of this invention.

[0077] It is understood that after the present invention provides CpG numbering based on a single DNA strand, the corresponding numbering of each CpG site on the positive strand in the complementary DNA strand is readily available according to the content provided in the present invention. Detection of one or more CpGs provided in the present invention is possible; therefore, the present invention also includes fragments of nucleic acids of the nucleotide sequence, including at least one methylated CpG site.

[0078] This invention also includes gene panels or gene groups containing epigenetic modification marker sequences or sequence fragments or their complementary sequences. For the aforementioned gene panels or gene groups, DNA methylation status detection can also be used to characterize normal cells and cancer cells.

[0079] In this invention, a wide variety of techniques can be used to analyze methylation status. It should be understood that the nucleic acids provided by this invention can serve as key regions in the genome for analyzing methylation status, and their methylation status can be analyzed using various techniques known in the art, thereby analyzing the occurrence or development of cancer.

[0080] The epigenetic modification markers of the present invention, or fragments thereof, or their complementary sequences, can be converted to uracil after bisulfite treatment, while methylated cytosine remains unchanged. Therefore, the present invention also provides nucleic acids obtained by treating the above-mentioned nucleic acids (including their complementary strands (antisense strands)) with bisulfite, comprising: nucleic acids or nucleic acid fragments of the nucleotide sequences shown in SEQ ID NO: 4, 5, or 6. These nucleic acids can serve as more direct targets for designing detection reagents or detection kits.

[0081] The epigenetic modification markers and / or their complementary nucleic acids and / or one or more fragments thereof of the present invention can be integrated into one or more wholes, such as one or more nucleic acid sets, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid set to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more wholes, such as one or more kits.

[0082] The epigenetic modification markers and / or their complementary nucleic acids and / or nucleic acids derived from one or more fragments of the present invention (e.g., via bisulfite conversion) can also be integrated into one or more whole units, such as one or more nucleic acid sets, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid set to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more whole units, such as one or more kits, or one or more chips.

[0083] Based on the target genes and their epigenetic characteristics provided in this invention, techniques known in the art, as well as some techniques under development, can be applied to this invention to detect methylation levels. The determination of nucleic acid methylation profiles can be performed using existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other techniques still under development or to be developed. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation levels; it is based on continuous optical monitoring of fluorescent PCR and is more sensitive than the MSP method. It has high throughput and avoids the need for electrophoresis analysis. In addition, other available techniques include conventional methods in the field such as qPCR (Me-qPCR), next-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite conversion sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, and combinatorial gene group detection. Although some preferred embodiments are provided in the embodiments of this invention, the overall scheme of this invention is not limited thereto.

[0084] As a preferred embodiment of the present invention, a method for in vitro detection of the methylation profile of nucleic acids in a sample is also provided. The method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted into thymine during subsequent PCR amplification, while methylated cytosine remains unchanged. Therefore, after nucleic acid treatment with bisulfite, the methylated sites produce a nucleic acid polymorphism (SNP) similar to a C / T ratio. Identifying the methylation profile of nucleic acids in a sample based on this principle can effectively distinguish between methylated and unmethylated cytosine.

[0085] The method described in this invention includes: first, providing a sample and extracting genomic DNA; second, treating the genomic DNA obtained in step (a) with bisulfite, thereby converting unmethylated cytosine in the genomic DNA into uracil; and third, analyzing whether there are abnormal methylation patterns in the genomic DNA treated in step (b).

[0086] The method of this invention can be used to: test subject samples to assess whether the subject has cancer; or to identify high-risk groups for cancer. The method can be used in situations where the goal is not to obtain a direct disease diagnosis, such as situations where the goal is not to determine the final outcome of the disease, population geographic analysis studies, scientific research, population censuses, etc.

[0087] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing. However, this method is not limited to practical applications; other DNA methylation detection methods known in the art or currently being improved may also be used. The primers used in the PCR amplification are not limited to those provided in the embodiments; primers that differ in sequence from those provided in the embodiments of the present invention, but still target the nucleic acid or corresponding CpG site indicated by the present invention, may also be obtained.

[0088] This invention also provides a method for detecting the methylation status of nucleic acids in an in vitro sample, the method being methylation-sensitive restriction endonuclease digestion (MSRE). The methylation-sensitive restriction endonuclease cannot cleave DNA when it contains a methylated base at its cleavage site. The MSRE method is based on the fundamental principle that methylation-sensitive type II restriction endonucleases cannot cleave sequences containing one or more methylated cleavage sites. Fragments containing one or more methylated CpG sequences are cleaved using a methylation-sensitive type II endonuclease and its isoenzymes (insensitive to methylation), followed by DNA blotting analysis. The advantages of this method include: no need for detailed information on the primary structure of the target DNA, and the ability to provide a direct assessment of the methylation status of CpG islands, including obtaining some quantitative analytical information on the methylation of the gene being tested.

[0089] In relation to the marker nucleic acid provided by this invention, other methods and reagents known to those skilled in the art for determining the sequence of a genome, its variations, and methylation status may also be included in this invention.

[0090] This invention provides a method for preparing a cancer detection reagent, comprising: providing the aforementioned nucleic acid; using the full length or a fragment of the nucleic acid as a target sequence; and designing a detection reagent specifically for detecting the target sequence; wherein the target sequence includes at least one methylation CpG site. The detection reagent may include, but is not limited to, chips, primers, probes, etc.; after obtaining the aforementioned marker, the selection of the detection reagent is a matter for those skilled in the art.

[0091] Once the sequence of a nucleic acid is known, designing primers is known to those skilled in the art. Two primers are positioned flanking a specific sequence of the target gene to be amplified (including the CpG sequence, where the primers are complementary to the CpG to target methylated gene regions, and complementary to the TpG to target demethylated gene regions). In a preferred embodiment of the invention, the reagent is a primer, preferably one listed in the examples. Besides primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0092] The reagents may also be combinations of reagents, such as primer combinations. For example, the combination may include more than one set of primers, thereby enabling the amplification of the multiple nucleic acids mentioned above.

[0093] The present invention also provides a kit for in vitro detection of methylation profiles of nucleic acids in samples, the kit comprising: a container, and the aforementioned primer pair located in the container.

[0094] The kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. Furthermore, the kit may include an instruction manual specifying the detection procedures and result interpretation criteria to facilitate application by those skilled in the art.

[0095] The methods and reagents of this invention exhibit very high accuracy in diagnosing clinical cancer, as demonstrated in the detection of various clinical cancer samples in the embodiments of this invention. This invention can be applied to fields such as precancerous screening, efficacy assessment, auxiliary diagnosis, and prognostic monitoring, or, as mentioned above, situations where the aim is not to obtain a direct disease diagnosis result.

[0096] The present invention is illustrated below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0097] Example 1: Determination of methylation detection targets

[0098] 1.1 TAGMe-8, 9, 10

[0099] Obtain the human sequences of TAGMe-8, 9, and 10 genes, as follows:

[0100] TAGMe-8 sequence (SEQ ID NO: 1):

[0101] In the above sequence (positive chain), each "CG" marked by a solid underline represents a methylated CpG site, numbered sequentially from 5' to 3' as "CG" 1 to 24 (methylated CpG sites 1 to 24). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold areas correspond to the detection target regions (positions 453 to 549; including "CG" 10 to 17) in some embodiments.

[0102] TAGMe-9 sequence (SEQ ID NO: 2):

[0103] In the above sequence (positive chain), each "CG" marked by a solid underline represents a methylated CpG site, numbered sequentially from 5' to 3' as "CG" 1 to 41 (methylated CpG sites 1 to 41). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold areas correspond to the detection target regions (positions 141 to 256, including "CG" 7 to 22) in some embodiments.

[0104] TAGMe-10 sequence (SEQ ID NO: 3):

[0105] In the above sequence (positive chain), each "CG" marked by a solid underline represents a methylated CpG site, numbered sequentially from 5' to 3' as "CG" 1 to 44 (methylated CpG sites 1 to 44). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold areas correspond to the detection target regions (positions 45 to 175, including "CG" 2 to 16) in some embodiments.

[0106] 1.2 Bisulfite transformation

[0107] The above sequence was transformed with Bisulfite to obtain the corresponding DNA sequence, where Y represents C or U(T);

[0108] TAGMe-8 sequence (SEQ ID NO: 4):

[0109] In the above sequence (positive chain), each "YG" marked by a solid underline represents a transformed methylated CpG site, numbered sequentially from 5' to 3' as "YG" 1 to 24 (transformed methylated CpG sites 1 to 24). Dashed underlines correspond to the upstream and downstream primer design regions in some embodiments; italicized bold areas correspond to the detection target regions in some embodiments.

[0110] TAGMe-9 sequence (SEQ ID NO: 5):

[0111] In the above sequence (positive chain), each "YG" marked by a solid underline represents a transformed methylated CpG site, numbered sequentially from 5' to 3' as "YG" 1 to 41 (the 1st to 41st transformed methylated CpG sites). The dashed underline corresponds to the upstream and downstream primer design regions in some of the embodiments; the italicized bold areas correspond to the detection target regions in some of the embodiments.

[0112] TAGMe-10 sequence (SEQ ID NO: 6):

[0113] In the above sequence (positive chain), each "YG" marked by a solid underline represents a transformed methylated CpG site, numbered sequentially from 5' to 3' as "YG" 1 to 44 (transformed methylated CpG sites 1 to 44). Dashed underlines correspond to the upstream and downstream primer design regions in some embodiments; italicized bold areas correspond to the detection target regions in some embodiments.

[0114] 1.3 Determine the detection area and design primers upstream and downstream of the detection area.

[0115] Example 2: Design and Synthesis of Detection Reagents

[0116] 2.1 Design first-round PCR primers with a length of 25–35 bp and appropriate CG content, and ensure the amplification length is 100–300 bp:

[0117] 2.2 Add a barcode (Sample-ID) to the end of the first round of primers;

[0118] 2.3 Add a tag sequence to the end of the first round of primers for library construction;

[0119] 2.4 Primers for the first round of PCR reaction and primers for the second round of PCR reaction with Illumina adapters and indexes were synthesized. The primer sequences are shown in Table 1.

[0120] Table 1

[0121] In the table, TAGMe-8 F1 and TAGMe-8 R1 amplify the sequence segment from position 453 to 549 (containing CpGs 10 to 17) of SEQ ID NO: 4, and the sequence segment containing the primer complementary region is the sequence segment from position 422 to 578 (containing CpGs 10 to 17).

[0122] In the table, TAGMe-9 F1 and TAGMe-9 R1 amplify the sequence segment from positions 141 to 256 (containing CpGs 7 to 22) of SEQ ID NO: 5, and the sequence segment containing the primer complementary region is the sequence segment from positions 111 to 284 (containing CpGs 6 to 22).

[0123] In the table, TAGMe-10 F1 and TAGMe-10 R1 amplify the sequence segment from position 45 to 175 (containing CpGs 2 to 16) of SEQ ID NO: 6, and the sequence segment containing the primer complementary region is the sequence segment from position 16 to 205 (containing CpGs 1 to 17).

[0124] In the TAGMe-8 F1 sequence above, positions 29-59 (underlined) are complementary bases of the target detection sequence (upstream and downstream), and positions 1-28 are two tag sequences; in the TAGMe-8 R1 sequence, positions 28-56 are complementary bases of the target detection sequence (upstream and downstream), and positions 1-27 are two tag sequences.

[0125] In the TAGMe-9 F1 sequence above, positions 29-58 (underlined) are complementary bases of the target detection sequence (upstream and downstream), and positions 1-28 are two tag sequences; in the TAGMe-9 R1 sequence, positions 28-55 are complementary bases of the target detection sequence (upstream and downstream), and positions 1-27 are two tag sequences.

[0126] In the TAGMe-10 F1 sequence above, positions 29-57 (underlined) are complementary bases of the target detection sequence (upstream and downstream), and positions 1-28 are two tag sequences; in the TAGMe-10 R1 sequence, positions 28-57 are complementary bases of the target detection sequence (upstream and downstream), and positions 1-27 are two tag sequences.

[0127] Example 3: Validation of the detection reagent

[0128] A primer pair was synthesized for two rounds of PCR reactions using positive and negative references:

[0129] The first round of PCR system and reaction conditions are shown in Table 2.

[0130] Table 2

[0131] The second round of PCR system and reaction conditions are shown in Table 3.

[0132] Table 3

[0133] Example 4: Differential methylation of TAGMe-8, 9, 10 CpG sites in tumor and non-tumor tissues using NGS sequencing.

[0134] 1. Obtaining clinical samples: Obtain adjacent / non-cancerous to cancerous tissue samples from clinical settings. The adjacent / non-cancerous samples serve as the control group, while the cancerous tissue samples serve as the tumor detection experimental group.

[0135] 2. DNA extraction: DNA was extracted from the experimental group and the control group respectively; adsorption column extraction was used in this experiment.

[0136] 3. Bisulfite treatment: The extracted DNA samples were treated with bisulfite, and the procedure was strictly followed. In this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research, catalog number D5006, was used.

[0137] 4. Using primers with different barcodes (Sample-ID) (first-round PCR primers) and universal sequencing primers for the Illumina system (second-round PCR primers), perform two rounds of PCR amplification using conventional methods to construct the NGS library.

[0138] 5. After PCR amplification, 2% agarose gel electrophoresis was used to detect the PCR fragment specificity. 5 μL of PCR product was taken from each sample, mixed, and the target fragment library was purified and recovered for NGS sequencing.

[0139] 6. Sequencing results analysis: Extract sequencing information from the sample based on the primer barcode sequence;

[0140] 7. Calculation of TAGMe-8, 9, 10 methylation values: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region, and calculate the median value of methylation at all CpG sites as the TAGMe-8, 9, 10 methylation values ​​in the sample.

[0141] 8. Results analysis: The methylation values ​​of TAGMe-8, 9, and 10 in non-tumor tissues and tumor tissues were compared, and the cutoff value was determined by ROC curve.

[0142] Example 5: TAGMe-8, 9, 10: Clinical Sample Validation for Lung Cancer - NGS Sequencing

[0143] For TAGMe-8, 9, and 10, 40, 40, and 40 samples were obtained clinically, respectively. 20, 20, and 20 lung cancer adjacent normal samples were used as the control group, and 20, 20, and 20 lung cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of lung cancer clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing steps.

[0144] The results are shown in Figure 1. In clinical lung cancer samples, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (****P<0.0001, ****P<0.0001, **P<0.01).

[0145] Example 6 TAGMe-8, 9, 10: Validation of Clinical Samples from Liver Cancer - NGS Sequencing

[0146] For TAGMe-8, 9, and 10, 29, 39, and 30 samples were obtained clinically, respectively. 14, 19, and 15 samples adjacent to liver cancer were used as the control group, and 15, 20, and 15 samples of liver cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical liver cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0147] The results are shown in Figure 2. In clinical samples of liver cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, **P<0.01, ****P<0.0001).

[0148] Example 7 TAGMe-8, 9, 10: Clinical Sample Validation for Prostate Cancer - NGS Sequencing

[0149] For TAGMe-8, 9, and 10, 19, 20, and 40 samples were obtained clinically, respectively. 10, 10, and 20 samples of adjacent normal prostate cancer samples were used as the control group, and 9, 10, and 20 samples of prostate cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical prostate cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0150] The results are shown in Figure 3, which indicates that in clinical samples of prostate cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ****P<0.0001, *P<0.05).

[0151] Example 8 TAGMe-8, 9, 10: Validation of Cervical Cancer Clinical Samples - NGS Sequencing

[0152] For TAGMe-8, 9, and 10, 39, 38, and 40 samples were obtained clinically, respectively. 19, 20, and 20 samples of adjacent cervical cancer were used as the control group, and 20, 18, and 20 samples of cervical cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of cervical cancer clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0153] The results are shown in Figure 4. In clinical samples of cervical cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, *P<0.05, ****P<0.0001).

[0154] Example 9 TAGMe-8, 9, 10: Clinical Sample Validation of Endometrial Cancer - NGS Sequencing

[0155] For TAGMe-8, 9, and 10, 39, 36, and 37 clinical samples were obtained, respectively. 19, 16, and 17 samples of adjacent normal endometrial cancer were used as the control group, and 20, 20, and 20 samples of endometrial cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of clinical endometrial cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0156] The results are shown in Figure 5. In clinical samples of endometrial cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, **P<0.01, **P<0.01).

[0157] Example 10 TAGMe-8, 9, 10: Clinical Sample Validation of Urothelial Carcinoma - NGS Sequencing

[0158] For TAGMe-8, 9, and 10, 30, 40, and 40 samples were obtained clinically, respectively. 15, 20, and 20 samples of adjacent tissue from urothelial carcinoma were used as the control group, and 15, 20, and 20 samples of urothelial carcinoma were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of clinical urothelial carcinoma samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0159] The results are shown in Figure 6. In clinical samples of urothelial carcinoma, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (***P<0.001, *P<0.05, *P<0.05).

[0160] Example 11 TAGMe-8, 9, 10: Clinical Sample Validation of Biliary Tract Tumors - NGS Sequencing

[0161] For TAGMe-8, 9, and 10, 26, 40, and 16 samples were obtained clinically, respectively. 13, 20, and 8 samples of adjacent normal biliary tract tumors were used as the control group, and 13, 20, and 8 samples of biliary tract tumors were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical biliary tract tumor samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0162] The results are shown in Figure 7, which indicates that in clinical samples of biliary tract tumors, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissues were significantly higher than those in adjacent normal tissues (**P<0.01, ****P<0.0001, *P<0.05).

[0163] Example 12 TAGMe-8, 9, 10: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0164] For TAGMe-8, 9, and 10, 16, 12, and 12 samples were obtained clinically, respectively. 8, 6, and 6 samples of adjacent normal gastric cancer samples were used as the control group, and 8, 6, and 6 samples of gastric cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical gastric cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0165] The results are shown in Figure 8, which shows that in clinical samples of gastric cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, **P<0.01, **P<0.01).

[0166] Example 13 TAGMe-8, 9, 10: Clinical Sample Validation for Breast Cancer - NGS Sequencing

[0167] For TAGMe-8, 9, and 10, 12, 10, and 16 samples were obtained clinically, respectively. 6, 5, and 8 breast cancer adjacent normal tissue samples were used as the control group, and 6, 5, and 8 breast cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of breast cancer clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0168] The results are shown in Figure 9. In clinical samples of breast cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, *P<0.05, **P<0.01).

[0169] Example 14 TAGMe-8, 9, 10: Validation of esophageal cancer clinical samples - NGS sequencing

[0170] For TAGMe-8, 9, and 10, 12, 12, and 12 samples were obtained clinically, respectively. 6, 6, and 6 samples of esophageal cancer adjacent to the lesion were used as the control group, and 6, 6, and 6 samples of esophageal cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of esophageal cancer clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0171] The results are shown in Figure 10. They indicate that in esophageal cancer clinical samples, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, *P<0.05, *P<0.05).

[0172] Example 15 TAGMe-8, 9, 10: Validation of clinical samples from gliomas using NGS sequencing

[0173] For TAGMe-8, 9, and 10, 16, 16, and 13 clinical samples were obtained, respectively. 8, 8, and 5 glioma adjacent normal tissue samples were used as the control group, and 8, 8, and 8 glioma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical glioma samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0174] The results are shown in Figure 11. In clinical samples of glioma, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (*P<0.05, *P<0.05, *P<0.05).

[0175] Example 16 TAGMe-8, 9, 10: Validation of Clinical Samples from Colorectal Cancer - NGS Sequencing

[0176] For TAGMe-8, 9, and 10, 15, 18, and 20 samples were obtained clinically, respectively. 8, 8, and 10 samples of adjacent normal colorectal cancer were used as the control group, and 7, 10, and 10 samples of colorectal cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of clinical colorectal cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0177] The results, as shown in Figure 12, indicate that in clinical samples of colorectal cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (*P<0.05, *P<0.05, ***P<0.001).

[0178] Example 17 TAGMe-8, 9, 10: Validation of Leukemia Clinical Samples - NGS Sequencing

[0179] For TAGMe-8, 9, and 10, 15, 14, and 12 samples were obtained clinically, respectively. 8, 7, and 6 non-leukemia bone marrow smear samples were used as the control group, and 7, 7, and 6 leukemia bone marrow smear samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of leukemia clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0180] The results are shown in Figure 13. The results show that in clinical samples of leukemia, the methylation values ​​of TAGMe-9 and 10 in bone marrow smears of leukemia patients were significantly higher than those in bone marrow smears of non-leukemia patients (*P<0.05, *P<0.05, *P<0.05).

[0181] Example 18 TAGMe-8, 9, 10: Clinical Sample Validation of Pancreatic Cancer - NGS Sequencing

[0182] For TAGMe-8, 9, and 10, 14, 20, and 20 samples were obtained clinically, respectively. 6, 10, and 10 pancreatic cancer adjacent tissue samples were used as the control group, and 8, 10, and 10 pancreatic cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of liposarcoma clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0183] The results are shown in Figure 14. In clinical samples of pancreatic cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, ****P<0.0001, ****P<0.0001).

[0184] Example 19 TAGMe-8, 9, 10: Clinical Sample Validation for Thyroid Cancer - NGS Sequencing

[0185] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 samples of adjacent normal tissue from thyroid cancer were used as the control group, and 8, 10, and 10 samples of thyroid cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of clinical thyroid tumor samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0186] The results, as shown in Figure 15, indicate that in clinical samples of thyroid cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, ****P<0.0001, ***P<0.001).

[0187] Example 20 TAGMe-8, 9, 10: Clinical Sample Validation of Melanoma - NGS Sequencing

[0188] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 normal skin tissue samples were used as the control group, and 8, 10, and 10 skin melanoma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of melanoma clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0189] The results, as shown in Figure 16, indicate that in clinical melanoma samples, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ****P<0.0001, ***P<0.001).

[0190] Example 21 TAGMe-8, 9, 10: Validation of Nasopharyngeal Carcinoma Clinical Samples - NGS Sequencing

[0191] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 samples of nasopharyngeal carcinoma adjacent to normal tissue were used as the control group, and 8, 10, and 10 samples of nasopharyngeal carcinoma were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of nasopharyngeal carcinoma clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0192] The results, as shown in Figure 17, indicate that in clinical samples of nasopharyngeal carcinoma, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ***P<0.001, **P<0.01).

[0193] Example 22 TAGMe-8, 9, 10: Clinical Sample Validation for Oral Cancer - NGS Sequencing

[0194] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 samples of adjacent normal oral cancer samples were used as the control group, and 8, 10, and 10 samples of oral cancer samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of clinical oral cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0195] The results are shown in Figure 18. They indicate that in clinical samples of oral cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (*P<0.05, ****P<0.0001, ***P<0.001).

[0196] Example 23 TAGMe-8, 9, 10: Clinical Sample Validation for Laryngeal Cancer - NGS Sequencing

[0197] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 samples of laryngeal cancer adjacent to the normal tissue were used as the control group, and 8, 10, and 10 samples of laryngeal cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of clinical laryngeal cancer samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0198] The results, as shown in Figure 19, indicate that in clinical samples of laryngeal cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (***P<0.001, ****P<0.0001, ****P<0.0001).

[0199] Example 24 TAGMe-8, 9, 10: Validation of osteosarcoma clinical samples - NGS sequencing

[0200] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 osteosarcoma adjacent normal tissue samples were used as the control group, and 8, 10, and 10 osteosarcoma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of osteosarcoma clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0201] The results are shown in Figure 20. In the clinical samples of osteosarcoma, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, ****P<0.0001, ****P<0.0001).

[0202] Example 25 TAGMe-8, 9, 10: Validation of Lymphoma Clinical Samples - NGS Sequencing

[0203] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 normal lymphocyte samples were used as the control group, and 8, 10, and 10 lymphoma samples were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct NGS libraries of lymphoma clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0204] The results are shown in Figure 21, which show that in clinical lymphoma samples, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, ****P<0.0001, ****P<0.0001).

[0205] Example 26 TAGMe-8, 9, 10: Clinical Sample Validation of Renal Cell Carcinoma - NGS Sequencing

[0206] For TAGMe-8, 9, and 10, 32, 40, and 20 clinical samples were obtained, respectively. 16, 20, and 10 renal cell carcinoma adjacent normal tissue samples were used as the control group, and 16, 20, and 10 renal cell carcinoma samples were used as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of clinical renal cell carcinoma samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0207] The results, as shown in Figure 22, indicate that in clinical samples of renal cell carcinoma, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (****P<0.0001, ****P<0.0001, ***P<0.001).

[0208] Example 27 TAGMe-8, 9, 10: Validation of Ovarian Cancer Clinical Samples - NGS Sequencing

[0209] For TAGMe-8, 9, and 10, 16, 20, and 20 samples were obtained clinically, respectively. 8, 10, and 10 samples of adjacent ovarian cancer were used as the control group, and 8, 10, and 10 samples of ovarian cancer were used as the experimental group. Two rounds of PCR reactions were performed according to the method described in Example 2 above to construct an NGS library of ovarian cancer clinical samples. The methylation levels of TAGMe-8, 9, and 10 were analyzed according to the NGS sequencing procedure.

[0210] The results, as shown in Figure 23, indicate that in clinical samples of ovarian cancer, the methylation values ​​of TAGMe-8, 9, and 10 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ****P<0.0001, **P<0.01).

[0211] Example 28: Detection performance analysis of a single CpG site

[0212] For TAGMe-8, using clinical samples of various cancer tissues and control tissues obtained in the aforementioned examples, the feasibility of using CpG sites 10 to 17 (CpG sites in the segment 453 to 549 of SEQ ID NO: 1) as single CpG sites for cancer detection was analyzed. The methylation modification status of single CpG sites was determined by NGS sequencing.

[0213] The results are shown in Table 4.

[0214] Table 4

[0215] For TAGMe-9, using clinical samples of various cancer tissues and control tissues obtained in the aforementioned examples, the feasibility of using CpG sites 7 to 22 (CpG sites within the segment 141 to 256 of SEQ ID NO: 2) as single CpG sites for cancer detection was analyzed. The methylation modification status of single CpG sites was determined by NGS sequencing.

[0216] The results are shown in Table 5.

[0217] Table 5

[0218] For TAGMe-10, using clinical samples of various cancer tissues and control tissues obtained in the aforementioned examples, the feasibility of using CpG sites 2 to 16 (CpG sites within the 45th to 175th region of SEQ ID NO: 3) as single CpG sites for cancer detection was analyzed. The methylation modification status of single CpG sites was determined by NGS sequencing.

[0219] The results are shown in Table 6.

[0220] Table 6

[0221] The results in Tables 4 to 6 indicate that single CpG sites in the detected regions have high sensitivity and / or specificity, and single CpG sites can also be used as targets for methylation modification analysis, which can be used as clinical auxiliary diagnostic analysis.

[0222] Example 29: Differential methylation of TAGMe-8 / 9 / 10 CpG sites in tumor cells and non-tumor cells - sequencing after bisulfite treatment (BSP-Bisulfite Sequencing PCR)

[0223] The sequencing steps after bisulfite treatment are as follows:

[0224] 1. Genomic DNA was extracted from hematologic malignancy cell lines (myeloid leukemia cell line K562), colorectal cancer cell line (HCT116), pancreatic cancer cell line (SW1990), human renal clear cell adenocarcinoma cell line (786-O), gastric cancer cell line (BGC-823), breast cancer cell line (BT-549), and cervical cancer cell line (HeLa) and their corresponding normal cell genomic DNA was extracted.

[0225] 2. Genomic DNA extracted from cancer cell lines and normal cell lines was treated with bisulfite, respectively, to serve as templates for subsequent PCR amplification;

[0226] 3. Design amplification primers based on the sequence of the corresponding CpG segment in SEQ ID NO: 4, 5 or 6, as shown in Table 7, and perform amplification.

[0227] 4. After PCR amplification, the PCR fragment specificity was detected by 2% agarose gel electrophoresis. The target fragment was excised and recovered, ligated into the insertion T vector, transformed into competent E. coli, plated, and clones were selected for sequencing the next day. More than 10 clones of each fragment were selected for Sanger sequencing.

[0228] Table 7. BSP Primers

[0229] Figure 24 (top) shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 001-009 in the region of SEQ ID NO: 1. The results show that the TAGMe-8 methylation level of cancer cells is significantly higher than that of normal cells.

[0230] Figure 24 (bottom) shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 018-024 in the region of SEQ ID NO: 1. The results show that the TAGMe-8 methylation level of cancer cells is significantly higher than that of normal cells.

[0231] Figure 25 (top) shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 001-006 in the region of SEQ ID NO: 2. The results show that the methylation level of TAGMe-9 in cancer cells is significantly higher than that in normal cells.

[0232] Figure 25 (bottom) shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 023-041 in the region of SEQ ID NO: 2. The results show that the methylation level of TAGMe-9 in cancer cells is significantly higher than that in normal cells.

[0233] Figure 26 shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 017-044 in the region of SEQ ID NO: 3. The results show that the methylation level of TAGMe-10 in cancer cells is significantly higher than that in normal cells.

[0234] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a reagent for detecting cancer, comprising: (a) Providing an epigenetic modification marker or a nucleic acid derived therefrom, said epigenetic modification marker comprising: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site of SEQ ID NO: 1, 2, or 3; or (2) The epigenetic modification markers or fragments that are sequentially complementary to those in (1); The nucleic acid derived therefrom is an epigenetic modification marker corresponding to (1) or (2), in which the unmodified cytosine is converted to T or U, while the cytosine C at the modified CpG site remains unchanged. (b) Using the epigenetic modification markers in (a) as targets, design a detection reagent that specifically detects the CpG site modification status of the targets.

2. The method as described in claim 1, characterized in that, The nucleic acid derived from the epigenetic modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6; The at least one modified CpG site is any CpG site selected from numbers 1 to 24 in the sequence shown in SEQ ID NO: 1, any CpG site selected from numbers 1 to 41 in the sequence shown in SEQ ID NO: 2, or any CpG site selected from numbers 1 to 44 in the sequence shown in SEQ ID NO: 3, or a combination thereof.

3. The method as described in claim 2, characterized in that, The at least one modified CpG site is any CpG site selected from numbers 10 to 17 in the sequence shown in SEQ ID NO: 1, any CpG site selected from numbers 6 to 22 or 7 to 22 in the sequence shown in SEQ ID NO: 2, or any CpG site selected from numbers 1 to 17 or 2 to 16 in the sequence shown in SEQ ID NO: 3, or a combination thereof.

4. The method as described in claim 3, characterized in that, The fragments of the apparent modification markers are selected from the following sequences: the sequence shown in positions 453-549 or 422-578 of SEQ ID NO: 1, the sequence shown in positions 141-256 or 111-284 of SEQ ID NO: 2, and the sequence shown in positions 45-175 or 16-205 of SEQ ID NO:

3.

5. The application of epigenetic modification markers or nucleic acids derived from them in the preparation of reagents or kits for cancer detection; among which, The epigenetic modification markers include: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or fragments containing at least one modified CpG site of SEQ ID NO: 1, 2 or 3; or (2) epigenetic modification markers that are sequence-complementary to the epigenetic modification markers or fragments of (1); wherein the transformed nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.

6. The application as described in claim 5, characterized in that, The cancers mentioned include: respiratory system cancers, digestive system cancers, urinary system cancers, gynecological and reproductive system cancers, hematological system cancers, nervous system cancers, head and neck cancers, skin system cancers, endocrine system cancers, or skeletal system cancers; preferably, the cancers include: lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, stomach cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer.

7. The application as described in claim 5, characterized in that, The samples used for cancer detection include: tissue samples, body fluid samples, and blood samples.

8. A method for analyzing the methylation level of a sample to be tested, comprising: (i) Obtain the sample to be tested; and (ii) Analyze the CpG site modification status of the target sequence or its fragment in the sample to be tested, wherein the target sequence includes: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site in SEQ ID NO: 1, 2 or 3; or (2) an epigenetic modification marker or fragment that is sequence-complementary to the epigenetic modification marker or fragment of (1).

9. The method as described in claim 8, characterized in that, Methods for analyzing the CpG site modification status of target sequences or fragments in test samples include: pyrosequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, methylation microarray, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, or combinations thereof.

10. The method as described in claim 8, characterized in that, The method for analyzing the CpG site modification status of the target sequence in the extracted epigenetic modification markers includes: (i) processing the extracted epigenetic modification markers to convert unmodified cytosine into uracil; preferably, the modification includes 5-methylation, 5-hydroxymethylation, 5-aldehyde methylation or 5-carboxymethylation.

11. The method as described in claim 8, characterized in that, The epigenetic modification markers described in step (i) were treated with Bisulfite; and (ii) the modification of the target sequences described in the nucleic acids treated in (i) were analyzed.

12. A reagent for detecting cancer, specifically detecting CpG site modification of a target sequence, wherein the target sequence is an epigenetic modification marker or a nucleic acid derived therefrom, the epigenetic modification marker comprising: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site of SEQ ID NO: 1, 2, or 3; or (2) an epigenetic modification marker or fragment that is sequence-complementary to the epigenetic modification marker or fragment of (1); wherein the transformed nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged; preferably, the reagent is directed to a gene sequence containing the target sequence, preferably, the gene sequence includes a gene panel or gene group; preferably, the reagent includes: amplifying the sequence shown in positions 453-549 or 422-578 of SEQ ID NO: 1, the sequence shown in positions 141-256 or 111-284 of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 453-549, or SEQ ID NO: 422-578, or SEQ ID NO: 2, or SEQ ID NO: 453-549, or SEQ ID NO: 422-578, or SEQ ID NO: 422-578, or SEQ ID NO: 422-578, or SEQ ID NO: 422-256 ... Primers for the sequence fragments shown in NO:3, positions 45–175 or 16–205.

13. The reagent as described in claim 12, characterized in that, The reagents are: primers containing the sequence of positions 29-59 in SEQ ID NO: 7 and the sequence of positions 28-56 in SEQ ID NO: 8; primers containing the sequence of positions 29-58 in SEQ ID NO: 9 and the sequence of positions 28-55 in SEQ ID NO: 10; and primers containing the sequence of positions 29-57 in SEQ ID NO: 11 and the sequence of positions 28-57 in SEQ ID NO:

12. Preferably, the reagent is: primers of SEQ ID NO: 7 and SEQ ID NO: 8 sequences; primers of SEQ ID NO: 9 and SEQ ID NO: 10 sequences; primers of SEQ ID NO: 11 and SEQ ID NO: 12 sequences; Preferably, the reagent further includes primers of the sequences SEQ ID NO: 13 and SEQ ID NO:

14.

14. Use of the reagent of claim 12 or 13 for preparing a kit for detecting cancer; preferably, the cancer includes: Cancers of the respiratory system, digestive system, urinary system, gynecological and reproductive system, hematologic system, nervous system, head and neck, skin system, endocrine system, or skeletal system; preferably, the cancers include: lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, stomach cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer.

15. An isolated epigenetic modification marker or a nucleic acid derived therefrom, said epigenetic modification marker comprising: (1) TAGMe-8 of the sequence shown in SEQ ID NO: 1, TAGMe-9 of the sequence shown in SEQ ID NO: 2, TAGMe-10 of the sequence shown in SEQ ID NO: 3, or a fragment containing at least one modified CpG site in SEQ ID NO: 1, 2 or 3; or (2) an epigenetic modification marker or fragment that is sequence-complementary to the epigenetic modification marker or fragment of (1); wherein the transformed nucleic acid is an epigenetic modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.

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