DNA methylation markers tagme-14, 15, and 16 and use thereof in tumor analysis

Abstract

Provided are DNA methylation markers TAGMe-14, 15, and 16 and use thereof in tumor analysis. Whether the markers are in abnormally high methylation states in tumor tissue is analyzed to separate the population at high risk of developing cancer.

Description

DNA methylation markers TAGMe-14,15,16 and their applications in tumor analysis Technical Field

[0001] This invention belongs to the fields of epigenetics and oncology, specifically relating to DNA methylation markers TAGMe-14,15,16 and their use in tumor analysis. Background Technology

[0002] A tumor is a new growth formed when a cell in a local tissue loses its normal regulation of growth at the gene level under the influence of various carcinogenic factors, leading to its clonal abnormal proliferation. With the increasing aging population, the number of cancer patients worldwide is constantly rising, and cancer has become a global public health challenge. Its etiology is extremely complex, and prevention of most cancers from the perspective of their causes is difficult. Furthermore, the development of cancer is a gradual process, and most patients are already in the middle or late stages when they seek medical attention, missing the opportunity for fundamental treatment. Early detection, diagnosis, and treatment are key to improving cure rates and treatment prognosis. Therefore, early cancer screening and timely intervention can effectively block cancer progression and reduce morbidity and mortality.

[0003] Epigenetics is a branch of genetics that studies heritable changes in gene expression without altering the nucleotide sequence of the gene, ultimately leading to phenotypic changes. Epigenetics includes DNA methylation, histone modification, genomic imprinting, chromosomal remodeling, and non-coding RNA regulation. It primarily influences gene function and characteristics by regulating gene transcription or translation, thereby affecting tumorigenesis and development.

[0004] DNA methylation is an important epigenetic modification, referring to the chemical modification process in which an active methyl group is transferred to a specific base in the DNA strand under the catalysis of DNA methyltransferase (DNMT), using S-adenosylmethionine as a methyl donor. In mammals, DNA methylation mainly occurs at the 5′ end of the cytosine island of cytosine-phosphate-guanine (CpG), generating 5′-methylcytosine (m5C). As an important epigenetic phenomenon, DNA methylation participates in various important biological processes, playing a crucial role in regulating gene expression, maintaining genome stability, regulating DNA spatial conformation, and influencing the higher-order structure of chromatin. Numerous studies have shown that early tumorigenesis is accompanied by an increase in the methylation level of tumor suppressor genes or a decrease in the methylation level of proto-oncogenes. Alterations in methylation patterns are considered the first detectable tumor-related indicator, and these patterns further change with increasing tumor malignancy.

[0005] Aberrant DNA methylation is closely related to the occurrence, development, and carcinogenesis of tumors, mainly due to the following reasons: 1. Cytosine in methylated CpG island dinucleotides undergoes deamination to thymine at a higher frequency, causing gene mutations; 2. Tumor suppressor genes and DNA repair genes are silenced due to hypermethylation; 3. Oncogenes are activated due to decreased methylation levels; 4. Decreased overall genomic methylation levels lead to the activation of transposons and repetitive sequences, resulting in decreased chromosome stability. Therefore, DNA methylation can serve as a biomarker and prognostic indicator for early diagnosis of tumors, and is of great significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis, and treatment monitoring. Although early tumor screening based on DNA methylation molecular markers has gradually gained attention, very few programs are actually applied clinically, so tumor screening is considered a specialized test. Furthermore, most existing tumor markers are only for specific tumor types, and there are almost no markers that can be used for multi-cancer screening. Therefore, finding molecular targets that can be used for diagnosis, prognosis, and prediction of tumor development and progression is of great significance for early tumor screening, clinical intervention, and guiding patient treatment. Summary of the Invention

[0006] The purpose of this invention is to provide an epigenetic modification-related tumor marker that utilizes the abnormal hypermethylation at specific sites of the marker in the tumor to detect the tumor.

[0007] In a first aspect of the present invention, a method for preparing a reagent for detecting tumors is provided, comprising:

[0008] (a) Providing a methylation modification marker or a nucleic acid derived therefrom, said methylation modification marker comprising:

[0009] (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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

[0010] (2) Methylation modification markers or fragments that are sequence complementary to the methylation modification markers or fragments in (1);

[0011] The nucleic acid derived therefrom is a methylation 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;

[0012] (b) Using the methylation modification markers in (a) as targets, design a detection reagent that specifically detects the CpG site modification status of the target.

[0013] In one or more embodiments, SEQ ID NO: 1, 2 or 3 may also include sequence variants or homologous sequences thereof.

[0014] In one or more embodiments, 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 with the sequence shown in SEQ ID NO: 1, 2, or 3. Accordingly, methylation 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.

[0015] In one or more embodiments, the apparent modification includes 5-methylation (5mC), 5-hydroxymethylation (5hmC), 5-aldehyde methylation (5-fC), or 5-carboxymethylation (5-caC).

[0016] In one or more embodiments, the at least one modified CpG site is any CpG site selected from numbers 1 to 43 in the sequence shown in SEQ ID NO: 1, or a combination thereof (e.g., 2 to 43, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40); any CpG site selected from numbers 1 to 57 in the sequence shown in SEQ ID NO: 2, or a combination thereof (e.g., 2 to 57, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55); any CpG site selected from numbers 1 to 63 in the sequence shown in SEQ ID NO: 3, or a combination thereof (e.g., 2 to 63, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60); preferably, it is SEQ ID NO: 1. SEQ ID NO: 1 shows any CpG site selected from 8 to 21 or 8 to 22 or any combination thereof; SEQ ID NO: 2 shows any CpG site selected from 43 to 56 or 43 to 57 or any combination thereof; SEQ ID NO: 3 shows any CpG site selected from 16 to 28 or 14 to 28 or any combination thereof.

[0017] In one or more embodiments, the methylation modification marker fragment is selected from the following sequences: the sequence shown at positions 247-382 or 217-412 in SEQ ID NO: 1, the sequence shown at positions 635-734 or 606-762 in SEQ ID NO: 2, and the sequence shown at positions 179-303 or 149-333 in SEQ ID NO: 3.

[0018] In one or more embodiments, the nucleic acid derived from the methylation modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6.

[0019] In one or more embodiments, the detection includes diagnosis, identification, screening, or prognostic assessment.

[0020] In one or more embodiments, one or more sets of reagents may be prepared for the target sequence.

[0021] In one or more embodiments, the reagents for detecting tumors include, but are not limited to, primers, probes, chips, or test strips.

[0022] In one or more embodiments, the detection reagent is integrated onto a chip.

[0023] In another aspect of the invention, the use of methylation modification markers or nucleic acids derived therefrom in the preparation of reagents or kits for detecting tumors is provided; wherein the methylation modification markers include: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) methylation modification markers that are sequence-complementary to the methylation modification markers or fragments of (1); wherein the derived nucleic acid is a methylation 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.

[0024] In one or more embodiments, the tumors include (but are not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, hematological system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors, or skeletal system tumors; preferably, the tumors 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.

[0025] In one or more embodiments, the samples targeted for tumor detection include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0026] In one or more embodiments, the nucleic acid derived from the methylation modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6.

[0027] In one or more embodiments, the tumor detection is targeted at tumors (including early, intermediate, or late-stage tumors) or their precancerous lesions.

[0028] In one or more embodiments, 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-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) a methylation modification marker that is sequence-complementary to the methylation modification marker or fragment of (1).

[0030] In one or more embodiments, methods for analyzing CpG site modifications of target sequences or fragments in a 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] In one or more embodiments, the method for analyzing the CpG site modification status of the target sequence in the extracted methylation modification marker includes: (i) processing the extracted methylation modification marker to convert unmodified cytosine into uracil; preferably, the modification includes 5-methylation, 5-hydroxymethylation, 5-aldehyde methylation, or 5-carboxymethylation; more preferably, treating the methylation modification marker in step (i) with Bisulfite; and (ii) analyzing the modification status of the target sequence in the nucleic acid treated in (i).

[0032] In one or more embodiments, an abnormal methylation level refers to the high methylation of C in the methylation modification marker CpG.

[0033] In one or more embodiments, 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., and also including combinations of one or more of them.

[0034] In one or more embodiments, 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] In one or more embodiments, 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 tumors is provided, which specifically detects the CpG site modification status of a target sequence, wherein the target sequence is a methylation modification marker or a nucleic acid derived therefrom, the methylation modification marker comprising: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) a methylation modification marker that is sequence-complementary to the methylation modification marker or fragment of (1); wherein the derived nucleic acid is a methylation 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 the gene Pane1 or a 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 247–382 or 217–412 of SEQ ID NO: 1, positions 635–734 or 606–762 of SEQ ID NO: 2, and positions 179–303 or 149–333 of SEQ ID NO: 3.

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

[0038] In one or more embodiments, the reagent is: primers of the sequences SEQ ID NO: 7 and SEQ ID NO: 8; primers of the sequences SEQ ID NO: 9 and SEQ ID NO: 10; primers of the sequences SEQ ID NO: 11 and SEQ ID NO: 12;

[0039] In one or more embodiments, 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 reagent is provided for preparing a kit for detecting tumors.

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

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

[0043] In one or more embodiments, the kit may further include: instructions for specifying the detection procedures and result determination criteria.

[0044] In another aspect of the invention, isolated methylation modification markers or nucleic acids derived therefrom are provided, the methylation modification markers comprising: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) methylation modification markers that are sequence-complementary to the methylation modification markers or fragments of (1); wherein the derived nucleic acid is a methylation 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.

[0045] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0046] Figure 1. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in lung cancer with controls (left); results of sensitivity and specificity analysis (right).

[0047] Figure 2. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in liver cancer with controls (left); results of sensitivity and specificity analysis (right).

[0048] Figure 3. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in prostate cancer with controls (left); results of sensitivity and specificity analysis (right).

[0049] Figure 4. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in cervical cancer with controls (left); results of sensitivity and specificity analysis (right).

[0050] Figure 5. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in endometrial cancer with controls (left); results of sensitivity and specificity analysis (right).

[0051] Figure 6. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in urothelial carcinoma with controls (left); results of sensitivity and specificity analysis (right).

[0052] Figure 7. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in biliary tract tumors with controls (left); results of sensitivity and specificity analysis (right).

[0053] Figure 8. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in gastric cancer with controls (left); results of sensitivity and specificity analysis (right).

[0054] Figure 9. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in breast cancer with controls (left); results of sensitivity and specificity analysis (right).

[0055] Figure 10. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in esophageal cancer with controls (left); results of sensitivity and specificity analysis (right).

[0056] Figure 11. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in gliomas with controls (left panel); results of sensitivity and specificity analysis (right panel).

[0057] Figure 12. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in colorectal cancer with controls (left); results of sensitivity and specificity analysis (right).

[0058] Figure 13. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in leukemia with controls (left); results of sensitivity and specificity analysis (right).

[0059] Figure 14. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in pancreatic cancer with controls (left); results of sensitivity and specificity analysis (right).

[0060] Figure 15. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in thyroid cancer with controls (left); results of sensitivity and specificity analysis (right).

[0061] Figure 16. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in melanoma with controls (left); results of sensitivity and specificity analysis (right).

[0062] Figure 17. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in nasopharyngeal carcinoma with controls (left); results of sensitivity and specificity analysis (right).

[0063] Figure 18. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in oral cancer with controls (left); results of sensitivity and specificity analysis (right).

[0064] Figure 19. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in laryngeal cancer with controls (left); results of sensitivity and specificity analysis (right).

[0065] Figure 20. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in osteosarcoma with controls (left); results of sensitivity and specificity analysis (right).

[0066] Figure 21. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in lymphoma with controls (left); results of sensitivity and specificity analysis (right).

[0067] Figure 22. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in renal cell carcinoma with controls (left); results of sensitivity and specificity analysis (right).

[0068] Figure 23. Comparison of methylation values ​​of TAGMe-14, 15, and 16 in ovarian cancer with controls (left); results of sensitivity and specificity analysis (right).

[0069] Figure 24. Sequencing analysis of methylation levels of methylation sites (CpG) 001-007 and 022-042 in SEQ ID NO: 1 in cancer cells after bisulfite treatment, with normal cells (Para) as a control.

[0070] Figure 25. Sequencing analysis of methylation levels of methylation sites 001-042 in SEQ ID NO: 2 in cancer cells after bisulfite treatment, with normal cells as a control.

[0071] Figure 26. Sequencing analysis of methylation levels of methylation sites 001-015 and 029-063 in SEQ ID NO: 3 in cancer cells after bisulfite treatment, with normal cells as controls. Detailed Implementation

[0072] The inventors have focused on screening methylation modification biomarkers and have provided novel biomarkers: TAGMe-14, TAGMe-15, and TAGMe-16. By analyzing whether these biomarkers exhibit abnormally high methylation in tumor tissue, it is possible to assess whether a 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 and non-solid tumors. Detection reagents can be designed using these biomarkers as targets.

[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] 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.

[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 82%, 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] The embodiments of the present invention provide a series of sequence fragments containing CpG sites. These fragments 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. With the information on the specific fragments in the human genome provided by the present invention, those skilled in the art are already capable of obtaining and applying the CpG sites.

[0078] After this 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 this invention. Detection of one or more CpGs provided in this invention is possible; therefore, this invention also includes fragments of nucleic acids containing the nucleotide sequence, and includes at least one methylated CpG site.

[0079] Based on the disclosures of this invention, a wide variety of techniques can be used to analyze methylation status. 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. As one approach, this invention also includes the gene Pane1 or gene group containing the aforementioned epigenetic modification marker sequence or sequence fragment or its complementary sequence. For the aforementioned gene Pane1 or gene group, DNA methylation status detection can also be used to obtain characteristics of normal cells and cancer cells.

[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, preferably 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 targeted analytical reagents obtained from the sequences of the present invention can also be integrated into one or more units, such as one or more kits. 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 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.

[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 some preferred embodiments of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing. However, in practical applications, this method is not limited to the present invention; other DNA methylation detection methods known in the art or currently being improved may also be used. In the PCR amplification, the primers used are not limited to those provided in the examples; primers that differ in sequence from those provided in the examples 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 detection reagents of the present invention can be formed into reagent combinations, such as primer combinations. For example, the combinations may include more than one set of primers, thereby enabling the amplification of the aforementioned multiple nucleic acids separately.

[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 when used for 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 direct disease diagnosis results.

[0096] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and examples. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0097] Example 1: Determination of methylation detection targets

[0098] 1. Obtain the DNA sequences (human) of TAGMe-14, 15 and 16, SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0099] TAGMe-14 sequence (SEQ ID NO: 1):

[0100] 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 43 (methylated CpG sites 1 to 43). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold area corresponds to the detection target region (positions 247 to 382; including "CG" 8 to 21) in some embodiments.

[0101] TAGMe-15 sequence (SEQ ID NO: 2):

[0102] 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 57 (methylated CpG sites 1 to 57). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold area corresponds to the detection target region (positions 635 to 734; including "CG" 43 to 56) in some embodiments.

[0103] TAGMe-16 sequence (SEQ ID NO: 3):

[0104] 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 63 (methylated CpG sites 1 to 63). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold area corresponds to the detection target region (positions 179 to 303; including positions 16-28 "CG") in some embodiments.

[0105] The above are the gene sequences of TAGMe 14, TAGMe-15, and TAGMe-16, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0106] 2. The sequence after bisulfite treatment is as follows (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6) (where Y represents C or U);

[0107] TAGMe-14 sequence (SEQ ID NO: 4):

[0108] 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 43 (transformed methylated CpG sites 1 to 43). 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.

[0109] TAGMe-15 sequence (SEQ ID NO: 5):

[0110] 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 57 (transformed methylated CpG sites 1 to 57). 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.

[0111] TAGMe-16 sequence (SEQ ID NO: 6):

[0112] 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 63 (transformed methylated CpG sites 1 to 63). 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.

[0113] 3. Design primers upstream and downstream of the detection region based on the above sequence and the identified detection region.

[0114] Example 2 Primer Design and Synthesis

[0115] 2.1 Design first-round PCR primers with a length of 25-35 bp and appropriate CG content, and make the amplification length 100-300 bp;

[0116] 2.2 Add a barcode (Sample-ID) to the end of the first round of primers;

[0117] 2.3 Add a tag sequence to the end of the first round of primers for library construction;

[0118] 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 (Table 1).

[0119] Table 1

[0120] In the table, TAGMe-14 F1 and TAGMe-14R1 amplify the sequence segment from position 247 to 382 (containing CpGs 8 to 21) of SEQ ID NO: 4, and the sequence segment containing the primer complementary region is the sequence segment from position 217 to 412 (containing CpGs 8 to 22).

[0121] In the table, TAGMe-15 F1 and TAGMe-15R1 amplify the sequence segment from position 635 to 734 (containing CpGs 43 to 56) of SEQ ID NO: 5, and the sequence segment containing the primer complementary region is the sequence segment from position 606 to 762 (containing CpGs 43 to 57).

[0122] In the table, TAGMe-16 F1 and TAGMe-16R1 amplify the sequence segment from positions 179 to 303 (containing CpGs 16 to 28) of SEQ ID NO: 6, and the sequence segment containing the primer complementary region is the sequence segment from positions 149 to 333 (containing CpGs 14 to 28).

[0123] Example 3 Primer Validation

[0124] A primer pair was synthesized for two rounds of PCR reactions using positive and negative references:

[0125] The PCR system and reaction conditions for the first round of PCR are shown in Table 2.

[0126] Table 2

[0127] The second round of PCR, the PCR system and reaction conditions are shown in Table 3.

[0128] Table 3

[0129] Example 4: Differential NGS sequencing of TAGMe-14 / 15 / 16 CpG site methylation between tumor tissues and non-tumor cells performed the following steps:

[0130] 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.

[0131] DNA extraction: DNA was extracted from the experimental group and the control group respectively; this experiment used the adsorption column method for extraction, but is not limited to this method;

[0132] 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 of ZYMO Research, catalog number D5006, was used, but the kit is not limited to this one.

[0133] The NGS library was constructed using two rounds of PCR amplification using primers with different barcodes (Sample-ID) (first-round PCR primers) and universal sequencing primers for the Illumina system (second-round PCR primers).

[0134] 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.

[0135] Sequencing results analysis: Extracting sequencing information from samples based on primer barcode sequences;

[0136] TAGMe-14 / 15 / 16 methylation value calculation: NGS sequencing can independently detect the methylation status of individual CpG sites in the target region, and calculate the median value of methylation of all CpG sites as the TAGMe-14 / 15 / 16 methylation value in the sample;

[0137] Results analysis: The methylation values ​​of TAGMe-14 / 15 / 16 in non-tumor tissues and tumor tissues were compared, and the cutoff value was determined by ROC curve.

[0138] Example 5 TAGMe-14 / 15 / 16: Clinical Sample Validation for Lung Cancer - NGS Sequencing

[0139] For the biomarkers TAGMe-14, 15, and 16, 20, 20, and 20 lung cancer adjacent normal samples were obtained clinically as the control group (normal), and 20, 20, and 20 lung cancer samples were obtained as the experimental group (cancer). Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of lung cancer clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0140] The results are shown in Figure 1. In clinical lung cancer samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (****P<0.0001, ****P<0.0001, ****P<0.0001).

[0141] Example 6: TAGMe-14 / 15 / 16: Validation of Clinical Samples from Liver Cancer - NGS Sequencing

[0142] For the biomarkers TAGMe-14, 15, and 16, 20, 20, and 15 adjacent normal samples of liver cancer were obtained clinically as control groups, and 20, 20, and 15 liver cancer samples were obtained as experimental groups. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of clinical liver cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0143] The results are shown in Figure 2, which shows that in clinical samples of liver cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, ****P<0.0001, **P<0.01).

[0144] Example 7 TAGMe-14 / 15 / 16: Clinical Sample Validation for Prostate Cancer - NGS Sequencing

[0145] For the biomarkers TAGMe-14, 15, and 16, 15, 15, and 10 adjacent normal prostate cancer samples were obtained clinically as the control group, and 15, 13, and 10 prostate cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical prostate cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0146] The results are shown in Figure 3, which indicates that in clinical samples of prostate cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, ****P<0.0001, *P<0.05).

[0147] Example 8 TAGMe-14 / 15 / 16: Cervical Cancer Clinical Sample Validation - NGS Sequencing

[0148] For the biomarkers TAGMe-14, 15, and 16, 20, 19, and 19 cervical adenocarcinoma adjacent normal samples were obtained clinically as the control group, and 20, 18, and 20 cervical cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of cervical cancer clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0149] The results are shown in Figure 4, which shows that in cervical cancer clinical samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (****P<0.0001, ****P<0.0001, **P<0.01).

[0150] Example 9 TAGMe-14 / 15 / 16: Clinical Sample Validation of Endometrial Cancer - NGS Sequencing

[0151] For the biomarkers TAGMe-14, 15, and 16, 15, 11, and 9 cases of adjacent normal endometrial cancer samples were obtained clinically as control groups, and 18, 18, and 9 cases of endometrial cancer samples were obtained as experimental groups. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of clinical endometrial cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0152] The results are shown in Figure 5. In clinical samples of endometrial cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (***P<0.001, ****P<0.0001, ****P<0.0001).

[0153] Example 10 TAGMe-14 / 15 / 16: Clinical Sample Validation of Urothelial Carcinoma - NGS Sequencing

[0154] For the biomarkers TAGMe-14, 15, and 16, 15, 20, and 20 samples of adjacent tissue from urothelial carcinoma were obtained clinically as the control group, and 15, 20, and 19 samples of urothelial carcinoma were obtained 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 urothelial carcinoma samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0155] The results are shown in Figure 6, which indicates that in clinical samples of urothelial carcinoma, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (**P<0.01, ****P<0.0001, *****p<0.0001).

[0156] Example 11 TAGMe-14 / 15 / 16: Clinical Sample Validation of Biliary Tract Tumors - NGS Sequencing

[0157] For the biomarkers TAGMe-14, 15, and 16, 15, 20, and 18 adjacent normal samples of biliary tract tumors were obtained clinically as control groups, and 15, 20, and 20 biliary tract tumor samples were obtained as experimental groups. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of clinical biliary tract tumor samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0158] The results are shown in Figure 7, which indicates that in clinical samples of biliary tract tumors, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissues were significantly higher than those in adjacent normal tissues (**P<0.01, ****P<0.0001, ****P<0.0001).

[0159] Example 12 TAGMe-14 / 15 / 16: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0160] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 cases of adjacent normal gastric cancer samples were obtained clinically as the control group, and 8, 8, and 8 cases of gastric cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of gastric cancer clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0161] The results are shown in Figure 8, which indicates that in clinical samples of gastric cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ***P<0.001, ***P<0.001).

[0162] Example 13 TAGMe-14 / 15 / 16: Clinical Sample Validation for Breast Cancer - NGS Sequencing

[0163] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 breast cancer adjacent normal tissue samples were obtained clinically as the control group, and 8, 8, and 8 breast cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of breast cancer clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0164] The results, as shown in Figure 9, indicate that in clinical breast cancer samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, **P<0.01, *P<0.05).

[0165] Example 14 TAGMe-14 / 15 / 16: Validation of Clinical Samples from Esophageal Cancer - NGS Sequencing

[0166] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 samples of adjacent esophageal cancer were obtained clinically as the control group, and 8, 8, and 8 samples of esophageal cancer were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical esophageal cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0167] The results are shown in Figure 10, which indicate that in esophageal cancer clinical samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, ***P<0.001, ***P<0.001).

[0168] Example 15 TAGMe-14 / 15 / 16: Clinical Sample Validation of Gliomas - NGS Sequencing

[0169] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 samples of glioma adjacent to normal tissue were obtained clinically as the control group, and 8, 8, and 8 samples of glioma were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical glioma samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0170] The results, as shown in Figure 11, indicate that in clinical samples of glioma, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ***P<0.001, ***P<0.001).

[0171] Example 16 TAGMe-14 / 15 / 16: Clinical Sample Validation for Colorectal Cancer - NGS Sequencing

[0172] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 colorectal cancer adjacent normal samples were obtained clinically as the control group, and 8, 8, and 8 colorectal cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct NGS libraries of colorectal cancer clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0173] The results, as shown in Figure 12, indicate that in clinical samples of colorectal cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (*P<0.05, ***P<0.001, ***P<0.001).

[0174] Example 17 TAGMe-14 / 15 / 16: Validation of Leukemia Clinical Samples - NGS Sequencing

[0175] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 non-leukemia bone marrow smear samples were obtained clinically as the control group, and 8, 8, and 8 leukemia bone marrow smear samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of leukemia clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0176] The results, as shown in Figure 13, indicate that in clinical leukemia samples, the methylation values ​​of TAGMe-14 / 15 / 16 in leukemia bone marrow smears were significantly higher than those in non-leukemia bone marrow smears (***P<0.001, ***P<0.001, ***P<0.001).

[0177] Example 18 TAGMe-14 / 15 / 16: Clinical Sample Validation of Pancreatic Cancer - NGS Sequencing

[0178] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 pancreatic cancer adjacent tissue samples were obtained clinically as the control group, and 8, 8, and 8 pancreatic cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical pancreatic cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0179] The results, as shown in Figure 14, indicate that in clinical pancreatic cancer samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, ***P<0.001, ***P<0.001).

[0180] Example 19 TAGMe-14 / 15 / 16: Clinical Sample Validation for Thyroid Cancer - NGS Sequencing

[0181] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 samples of adjacent tissue from thyroid cancer were obtained clinically as the control group, and 8, 8, and 8 samples of thyroid cancer were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical thyroid tumor samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0182] The results, as shown in Figure 15, indicate that in clinical samples of thyroid cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (***P<0.001, ***P<0.001, ***P<0.001).

[0183] Example 20 TAGMe-14 / 15 / 16: Clinical Sample Validation of Melanoma - NGS Sequencing

[0184] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 normal skin tissue samples were obtained clinically as the control group, and 8, 8, and 8 skin melanoma samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical melanoma samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0185] The results, as shown in Figure 16, indicate that in clinical melanoma samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (**P<0.01, ***P<0.001, ***P<0.001).

[0186] Example 21 TAGMe-14 / 15 / 16: Validation of Nasopharyngeal Carcinoma Clinical Samples - NGS Sequencing

[0187] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 cases of nasopharyngeal carcinoma adjacent normal tissue samples were obtained clinically as the control group, and 8, 8, and 8 cases of nasopharyngeal carcinoma samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of nasopharyngeal carcinoma clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0188] The results are shown in Figure 17. In clinical samples of nasopharyngeal carcinoma, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, **P<0.01, ***P<0.001).

[0189] Example 22 TAGMe-14 / 15 / 16: Clinical Sample Validation for Oral Cancer - NGS Sequencing

[0190] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 samples of adjacent normal oral cancer were obtained clinically as the control group, and 8, 8, and 8 samples of oral cancer were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical oral cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0191] The results are shown in Figure 18, which show that in clinical samples of oral cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent tissue (*P<0.05, *P<0.05, ***P<0.001).

[0192] Example 23 TAGMe-14 / 15 / 16: Clinical Sample Validation for Laryngeal Cancer - NGS Sequencing

[0193] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 samples of laryngeal cancer adjacent to the normal tissue were obtained clinically as the control group, and 8, 8, and 8 samples of laryngeal cancer were obtained 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-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0194] The results, as shown in Figure 19, indicate that in clinical samples of laryngeal cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ***P<0.001, ***P<0.001).

[0195] Example 24 TAGMe-14 / 15 / 16: Clinical Sample Validation of Osteosarcoma - NGS Sequencing

[0196] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 osteosarcoma adjacent normal tissue samples were obtained clinically as the control group, and 8, 8, and 8 osteosarcoma samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of osteosarcoma clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing procedure.

[0197] The results, as shown in Figure 20, indicate that in osteosarcoma clinical samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, ***P<0.001, ***P<0.001).

[0198] Example 25 TAGMe-14 / 15 / 16: Clinical Sample Validation for Lymphoma - NGS Sequencing

[0199] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 normal lymphocyte samples were obtained clinically as the control group, and 8, 8, and 8 lymphoma samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of lymphoma clinical samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0200] The results, as shown in Figure 21, indicate that in clinical lymphoma samples, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, **P<0.01, ***P<0.001).

[0201] Example 26 TAGMe-14 / 15 / 16: Clinical Sample Validation of Renal Cell Carcinoma - NGS Sequencing

[0202] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 cases of renal cell carcinoma adjacent normal tissue samples were obtained clinically as the control group, and 8, 8, and 8 cases of renal cell carcinoma samples were obtained 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-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0203] The results, as shown in Figure 22, indicate that in clinical samples of renal cell carcinoma, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (*P<0.05, **P<0.01, ***P<0.001).

[0204] Example 27 TAGMe-14 / 15 / 16: Clinical Sample Validation for Ovarian Cancer - NGS Sequencing

[0205] For the biomarkers TAGMe-14, 15, and 16, 8, 8, and 8 adjacent normal ovarian cancer samples were obtained clinically as the control group, and 8, 8, and 8 ovarian cancer samples were obtained as the experimental group. Following the method described in Example 2 above, two rounds of PCR reactions were performed to construct an NGS library of clinical ovarian cancer samples. The methylation levels of TAGMe-14 / 15 / 16 were analyzed according to the NGS sequencing steps.

[0206] The results, as shown in Figure 23, indicate that in clinical samples of ovarian cancer, the methylation values ​​of TAGMe-14 / 15 / 16 in cancerous tissue were significantly higher than those in adjacent normal tissue (***P<0.001, **P<0.01, ***P<0.001).

[0207] Example 28: Detection Performance Analysis

[0208] For the biomarker TAGMe-14, using clinical samples of tumor tissues and control tissues obtained in the aforementioned examples, the feasibility of combining CpG sites 8 to 21 (CpG sites in the region 247–382 of SEQ ID NO: 1) and each site as a single CpG site for tumor detection was analyzed. The methylation modification status of single CpG sites was determined using NGS sequencing. The results are shown in Table 4.

[0209] Table 4

[0210] For the biomarker TAGMe-15, using clinical samples of tumor tissues and control tissues obtained in the aforementioned examples, the feasibility of combining CpG sites 43 to 56 (CpG sites in segments 635 to 734 of SEQ ID NO: 2) and using each as a single CpG site for tumor detection was analyzed. The methylation modification status of single CpG sites was determined using NGS sequencing. The results are shown in Table 5.

[0211] Table 5

[0212] For the biomarker TAGMe-16, using clinical samples of tumor tissues and control tissues obtained in the aforementioned examples, the feasibility of combining CpG sites 16 to 28 (CpG sites in segments 179 to 303 of SEQ ID NO: 3) and using each as a single CpG site for tumor detection was analyzed. The methylation modification status of single CpG sites was determined using NGS sequencing. The results are shown in Table 6.

[0213] Table 6

[0214] According to Tables 4 to 6, the single CpG site in the detected region has high sensitivity and / or specificity. The single CpG site can also be used as a target for methylation modification analysis and can be used as a clinical auxiliary diagnostic analysis.

[0215] Example 29: Differential methylation of TAGMe-14 / 15 / 16 CpG sites in tumor and non-tumor cells – sequencing after bisulfite treatment.

[0216] Analysis was performed using bisulfite-treated sequencing PCR (BSP-Bisulfite Sequencing PCR), following these steps:

[0217] 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.

[0218] 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.

[0219] 3. Design amplification primers based on the sequence of SEQ ID NO: 2, as shown in Table 7, and perform amplification.

[0220] 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.

[0221] Table 7. BSP Primers

[0222] Figure 24 shows the BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 001-007 in the region of SEQ ID NO: 1 of TAGMe-14. The results show that the methylation level of TAGMe-14 in cancer cells is significantly higher than that in normal cells.

[0223] Figure 24 shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 022-043 in the region of SEQ ID NO: 1 of TAGMe-14. The results show that the methylation level of TAGMe-14 in cancer cells is significantly higher than that in normal cells.

[0224] Figure 25 shows the BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 001-042 in the region of TAGMe-15 in SEQ ID NO: 2. The results show that the methylation level of TAGMe-15 in cancer cells is significantly higher than that in normal cells.

[0225] Figure 26 shows the BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 001-015 in the region of SEQ ID NO: 3 of TAGMe-16. The results show that the methylation level of TAGMe-16 in cancer cells is significantly higher than that in normal cells.

[0226] Figure 26 shows the BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 029-063 in the region of SEQ ID NO: 3 of TAGMe-16. The results show that the methylation level of TAGMe-16 in cancer cells is significantly higher than that in normal cells.

[0227] 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 tumors, comprising: (a) Providing a methylation modification marker or a nucleic acid derived therefrom, said methylation modification marker comprising: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) Methylation modification markers or fragments that are sequence complementary to the methylation modification markers or fragments in (1); The nucleic acid derived therefrom is a methylation 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 methylation modification markers in (a) as targets, design a detection reagent that specifically detects the CpG site modification status of the target.

2. The method as described in claim 1, characterized in that, The at least one modified CpG site is any CpG site selected from numbers 1 to 43 in the sequence shown in SEQ ID NO: 1, any CpG site selected from numbers 1 to 57 in the sequence shown in SEQ ID NO: 2, or any CpG site selected from numbers 1 to 63 in the sequence shown in SEQ ID NO: 3; preferably, it is any CpG site selected from numbers 8 to 21 or 8 to 22 in the sequence shown in SEQ ID NO: 1, any CpG site selected from numbers 43 to 56 or 43 to 57 in the sequence shown in SEQ ID NO: 2, or any CpG site selected from numbers 16 to 28 or 14 to 28 in the sequence shown in SEQ ID NO: 3, or any CpG site selected from numbers 16 to 28 or 14 to 28 in the sequence shown in SEQ ID NO:

3.

3. The method as described in claim 1, characterized in that, The fragments of the methylation modification markers are selected from the sequences shown at positions 247-382 or 217-412 in SEQ ID NO: 1, positions 635-734 or 606-762 in SEQ ID NO: 2, and positions 179-303 or 149-333 in SEQ ID NO:

3.

4. The method as described in claim 1, characterized in that, The nucleic acid derived from the methylation modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5 or 6.

5. The application of methylation modification markers or nucleic acids derived from them in the preparation of reagents or kits for tumor detection; among which, The methylation modification markers include: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) A methylation modification marker that is sequence-complementary to the methylation modification marker or fragment of (1); wherein the transformed nucleic acid is a methylation modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C at its modified CpG site remains unchanged.

6. The application as described in claim 5, characterized in that, The tumors include: respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, hematological system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors, or skeletal system tumors; preferably, the tumors 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; or, the nucleic acid derived from the methylation modification marker is a nucleic acid with the nucleotide sequence shown in SEQ ID NO: 4, 5, or 6.

7. The application as described in claim 5, characterized in that, The samples used for tumor 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-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) a methylation modification marker or fragment that is sequence-complementary to the methylation 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 9, characterized in that, The method for analyzing the CpG site modification status of the target sequence in the extracted methylation modification markers includes: (i) processing the extracted methylation 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 methylation 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).

11. A reagent for detecting tumors, specifically detecting CpG site modification of a target sequence, wherein the target sequence is a methylation modification marker or a nucleic acid derived therefrom, the methylation modification marker comprising: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) a methylation modification marker or fragment that is sequence-complementary to the methylation modification marker or fragment of (1); wherein the derived nucleic acid is a methylation 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 for 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 247-382 or 217-412 of SEQ ID NO: 1, the sequence shown in positions 635-734 or 606-762 of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4. Primers for the sequence fragments shown in positions 179–303 or 149–333 of NO:

3.

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

12.

13. The reagent as described in claim 12, characterized in that, The reagents are: 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 reagents further include primers of SEQ ID NO: 13 and SEQ ID NO: 14 sequences.

14. Use of the reagent according to any one of claims 11-13 for preparing a kit for detecting tumors; preferably, the tumor comprises: Tumors 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 tumors 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.

15. An isolated methylation modification marker or a nucleic acid derived therefrom, said methylation modification marker comprising: (1) TAGMe-14 of the sequence shown in SEQ ID NO: 1, TAGMe-15 of the sequence shown in SEQ ID NO: 2, TAGMe-16 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) A methylation modification marker that is sequence-complementary to the methylation modification marker or fragment of (1); wherein the transformed nucleic acid is a methylation modification marker corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C at its modified CpG site remains unchanged.

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