New tumor detection marker tagme and its use
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI EPIPROBE BIOTECH CO LTD
- Filing Date
- 2022-09-22
- Publication Date
- 2026-07-03
Smart Images
Figure BDA0003859567940000111 
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene epigenetic modification, and more specifically, this invention relates to a novel tumor detection biomarker and its application. Background Technology
[0002] Endometrial cancer (EC) is one of the most common malignant tumors of the female reproductive tract, second only to cervical cancer in incidence. With improved living conditions, the incidence of EC is rising annually. It is estimated that by 2030, the global economic and medical burden due to EC will increase by 60%. Although most EC occurs in postmenopausal women, the incidence has recently increased significantly in women aged 40 or younger, ranging from 2-14%. For young women of reproductive age, if EC is diagnosed early, without myometrial invasion or extrauterine spread, there is still a chance to preserve the uterus and / or ovaries. Therefore, early diagnosis of EC is crucial, as it can reduce female mortality, secure treatment opportunities for younger patients, and preserve fertility or reproductive endocrine function.
[0003] However, currently, there is a lack of reliable, effective, and non-invasive early screening and auxiliary diagnostic methods in clinical practice. In the early stages of endometrial cancer, most patients do not have obvious related positive signs. Multiple studies have shown that 90% of patients with endometrial cancer will experience various types of vaginal bleeding, but among women who experience abnormal vaginal bleeding, only 5-10% will be diagnosed with endometrial cancer or precancerous lesions. Furthermore, there is currently a lack of reliable, effective, and non-invasive screening and auxiliary diagnostic methods in clinical practice. Existing traditional detection methods include: (1) Ultrasound examination. Transvaginal ultrasound (TVS) is often the initial examination for postmenopausal bleeding patients and is also the most commonly used non-invasive auxiliary examination method. However, due to its high false positive rate, it cannot reliably distinguish between benign and malignant endometrium. At the same time, EC can also occur in women whose endometrium does not thicken; (2) Imaging examination: Imaging examinations including magnetic resonance (pelvic MRI), CT, PET-CT, etc. can clarify the size and specific location of the lesion, but it is difficult to diagnose early EC or precancerous lesions. It is generally used for classification and grading; (3) Cytological examination: As a non-invasive detection method, it has high specificity and low sensitivity. The positive rate of vaginal exfoliated cytology examination is low, and cancer cells exfoliated from the uterine cavity are easy to dissolve and degenerate, and are not easy to identify after staining. (4) Serum tumor markers: There are no specific and sensitive diagnostic markers for endometrial cancer. Some patients may have abnormalities in CA125, CA19-9, CA153, or HE4, which are correlated with histological type, depth of myometrial invasion, and extrauterine invasion, but have poor correlation with benign or malignant endometrial tissue. Due to the lack of further minimally invasive triage methods, all patients with abnormal vaginal bleeding must undergo invasive examinations—curettage and / or hysteroscopic biopsy—to perform pathological examination of endometrial tissue for the diagnosis of endometrial cancer. Most endometrial cancers develop from endometrial hyperplasia to atypical hyperplasia (precancerous lesions), a progression that lasts for many years. During the long course of the disease, these patients with abnormal vaginal bleeding often undergo multiple invasive endometrial biopsies, leading to triple pressure on their physical, psychological, and economic well-being. Therefore, there is an urgent need to find new methods for the early detection of cancer. Summary of the Invention
[0004] The purpose of this invention is to provide a novel set of DNA methylation tumor markers, TAGMe-1 and TAGMe-2, and their combined application. These markers are hypomethylated in normal tissues and hypermethylated in endometrial cancer samples, and can be used for the detection of endometrial cancer. Sample types for detection include, but are not limited to, tissues, cervical exfoliated cells, uterine curettage, and vaginal secretions.
[0005] In a first aspect of the invention, the use of isolated nucleic acids or nucleic acids derived therefrom in the preparation of reagents or kits for tumor detection (including screening, diagnosis, detection or prognostic assessment) is provided; wherein the nucleic acid is: (1) a nucleic acid or combination of nucleic acids with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5; or (2) a nucleic acid or combination of nucleic acids that is sequence-complementary to the nucleic acid of (1); wherein the nucleic acid derived from the isolated nucleic acid is a nucleic acid 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.
[0006] In one or more embodiments, SEQ ID NO:1 or SEQ ID NO:5 further includes a sequence variant or homologous sequence thereof. Preferably, 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 or SEQ ID NO:5. Accordingly, it also includes polynucleotides 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).
[0007] In one or more embodiments, the nucleic acids sequence complementary to the nucleic acid of (1) are nucleic acids of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:6; the present invention also includes sequence variants or homologous sequences of SEQ ID NO:2 or SEQ ID NO:6 that have more than 80%, more than 85%, more than 90%, more than 92%, more than 95%, more than 96%, more than 98%, more than 99%, more than 99.5%, or more than 99.8% sequence identity with the sequences shown in SEQ ID NO:2 or SEQ ID NO:6. Accordingly, it also includes polynucleotides derived from the transformation of the sequence variants or homologous sequences (conversion of unmodified cytosine to T or U, while the cytosine C at the modified CpG site remains unchanged).
[0008] In one or more embodiments, the tumors include: endometrial cancer, cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck tumors, colon cancer, colorectal cancer, esophageal cancer, lung cancer (lung squamous cell carcinoma and lung adenocarcinoma), and bile duct cancer.
[0009] In one or more embodiments, the tumor detection targets a tumor (such as endometrial cancer) or a precancerous lesion (such as endometrial atypical hyperplasia).
[0010] In one or more embodiments, the samples targeted for tumor detection include (but are not limited to): blood samples, tissue samples (such as paraffin-embedded samples), cervical samples, uterine cavity samples, pleural effusion samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, and cell samples; preferably, the samples include (but are not limited to): cervical scraping or brushing samples, cervical swabs, uterine cavity tissue (scrapes), cervical exfoliated cells, uterine cavity scrapings, uterine cavity lavage fluid, and vaginal secretions.
[0011] In another aspect of the present invention, a method for detecting the methylation level of a sample to be tested is provided, comprising: extracting nucleic acid from the sample to be tested; and detecting the CpG site modification status of a target sequence or fragment thereof in the extracted nucleic acid, wherein the target sequence is a nucleic acid or combination of nucleic acids derived from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5, wherein the unmodified cytosine is converted to T or U corresponding to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5, while the cytosine C at the modified CpG site remains unchanged.
[0012] In one or more embodiments, methods for detecting CpG site modifications of target sequences in extracted nucleic acids include: pyrosequencing, bisulfite conversion sequencing, methylation array method, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, or combinations thereof.
[0013] In one or more embodiments, the method for detecting the CpG site modification status of the target sequence in the extracted nucleic acid includes: (i) processing the extracted nucleic acid to convert unmodified cytosine into uracil.
[0014] In one or more embodiments, the modification includes 5-methylation (5mC), 5-hydroxymethylation (5hmC), 5-aldehyde methylation (5-fC), or 5-carboxymethylation (5-caC).
[0015] In one or more embodiments, the nucleic acid described in step (i) is treated with bisulfite; and (ii) the modification of the target sequence described in the nucleic acid treated with (i) is analyzed.
[0016] In one or more embodiments, the nucleic acid is a combination of “SEQ ID NO:1 or its reverse complementary sequence” and “SEQ ID NO:5 or its reverse complementary sequence”.
[0017] In one or more embodiments, an abnormal methylation profile refers to the high methylation of C in the nucleic acid CpG.
[0018] In one or more embodiments, other methylation detection methods and future newly developed methylation detection methods may also be applied to this invention.
[0019] In one or more embodiments, the methylation profile method is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis of a disease.
[0020] In one or more embodiments, the method for detecting the methylation profile of the sample is an in vitro method.
[0021] 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: one or more of HpaII, AciI, Bsu15I, Hin1I, Hin6I, HpyCH4IV, NarI, etc.
[0022] In another aspect of the present invention, a method for preparing a reagent for the detection of tumors (including screening, diagnosis, detection, or prognostic assessment) is provided, the method comprising: providing a nucleic acid with a nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5, using the full length or fragment of the nucleic acid as a target sequence, and designing a detection reagent specifically for detecting CpG site modifications of the target sequence.
[0023] In one or more embodiments, the detection reagents include, but are not limited to, primers, probes, chips, or test strips.
[0024] In one or more embodiments, one or more sets of reagents may be prepared for the full length or a fragment of SEQ ID NO:1.
[0025] In one or more embodiments, one or more sets of reagents may be prepared for the full length or a fragment of SEQ ID NO:5.
[0026] In one or more embodiments, the detection reagent is integrated onto a chip.
[0027] In another aspect of the invention, a reagent or combination of reagents is provided that specifically detects the CpG site modification of a target sequence, wherein the target sequence is a nucleic acid or combination of nucleic acids derived from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5.
[0028] In one or more embodiments, the reagent or combination of reagents is targeted at a gene sequence containing the target sequence.
[0029] In one or more embodiments, the gene sequence includes a gene panel or a gene group.
[0030] In one or more embodiments, preferably, the reagent or combination of reagents is selected from one or more of the following groups: primers of the sequences shown in SEQ ID NO:9 and SEQ ID NO:10; primers of the sequences shown in SEQ ID NO:11 and SEQ ID NO:12; primers of the sequences shown in SEQ ID NO:13 and SEQ ID NO:14; or primers of the sequences shown in SEQ ID NO:15 and SEQ ID NO:16.
[0031] In another aspect of the invention, the use of the said reagent or combination of reagents is provided for preparing a kit for detecting (including screening, diagnosis, detection or prognostic assessment) tumors; preferably, the tumors include: endometrial cancer, cervical cancer, colorectal cancer, and lung cancer.
[0032] In another aspect of the invention, a kit is provided for tumor detection (including screening, diagnosis, detection, or prognostic assessment), the kit comprising the aforementioned reagents or combinations of reagents.
[0033] In one or more embodiments, the kit may also include, but is not limited to: DNA purification reagent, DNA extraction reagent, bisulfite, and PCR amplification reagent.
[0034] In one or more embodiments, the kit further includes: instructions for specifying the detection operation steps and result judgment criteria.
[0035] In another aspect of the invention, isolated nucleic acids or nucleic acids derived therefrom are provided, wherein the nucleic acids are: (1) nucleic acids or combinations of nucleic acids with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5; or (2) nucleic acids or combinations of nucleic acids that are sequence-complementary to the nucleic acid of (1); wherein the nucleic acid derived from the nucleic acid is a nucleic acid 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.
[0036] In one or more embodiments, the modified CpG site includes a CpG site that has undergone 5-aldehyde methylation, 5-hydroxymethylation, 5-methylation, or 5-carboxymethylation.
[0037] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description
[0038] Figure 1 Heatmap of candidate biomarkers for the diagnosis of endometrial cancer and precancerous lesions.
[0039] Figure 2 Performance graphs of AD, TAGMe-1, and TAGMe-2 as biomarkers in other cancer types in the TCGA database.
[0040] Figure 3 AD, Target NGS results in Example 3.
[0041] Figure 4 AD, TAGMe-1 analysis results in Example 4.
[0042] Figure 5 AD, Me-qPCR results of cervical cells in Example 5.
[0043] Figure 6 AD, Me-qPCR results of uterine cells in Example 6.
[0044] Figure 7 Sequence information of TAGMe-1 methylated tumor markers.
[0045] Figure 8 Sequence information of TAGMe-2 methylated tumor markers. Detailed Implementation
[0046] This invention discloses that DNA methylation variations play a crucial role in the development and progression of endometrial cancer, and can serve as biomarkers for early screening, auxiliary diagnosis, efficacy evaluation, and recurrence monitoring of endometrial cancer. Based on the analysis of a large number of clinical samples, this invention has isolated novel DNA methylation tumor biomarkers. These biomarkers are all in a hypomethylated state in normal tissues, but exhibit a significant difference in methylation status in tumor samples (especially endometrial cancer) and in patients with tumors, showing a statistically significant difference. The biomarkers show even better results when used in combination for detection. Therefore, the tumor biomarkers of this invention can serve as markers for the diagnosis, screening, classification, detection, and prognosis of clinical tumors, as well as as novel molecules for auxiliary clinical diagnosis or prognosis of tumors, or for the design of diagnostic reagents and kits.
[0047] In this invention, the term "sample" or "sample" includes substances obtained from any individual (preferably a human) or isolated tissues, cells, or bodily fluids (such as plasma) suitable for detecting DNA methylation status. For example, the sample may include, but is not limited to: blood samples, tissue samples (such as paraffin-embedded samples), cervical samples, uterine cavity samples, pleural effusion samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, and cell samples; preferably, the sample includes (but is not limited to): cervical scraping or brush samples, cervical swabs, uterine cavity tissue (scrapes), cervical exfoliated cells, uterine cavity scrapings, uterine cavity lavage fluid, and vaginal secretions.
[0048] In this invention, the term "highly methylated" refers to the presence of highly methylated, hydroxymethylated, aldehyde-methylated, or carboxymethylated CpG modifications 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 highly methylated state. For example, in real-time quantitative methylation-specific PCR, the determination of a highly methylated state can be based on statistically significant differences in the relative methylation status of control samples.
[0049] In this invention, the term "tumor" refers to a type of tumor whose genome exhibits a hypermethylated state as described in this invention, in the region of SEQ ID NO:1 or SEQ ID NO:5.
[0050] As used in this invention, "detection" includes screening, diagnosis, testing, or prognostic assessment.
[0051] As used in this invention, the term "endometrial cancer" broadly includes "endometrial cancer and its precancerous lesions, the latter also referred to as "endometrial atypical hyperplasia" or "early endometrial cancer"; however, it is understood that in specific cases, such as some embodiments, the term "endometrial cancer" is listed separately from "endometrial cancer precancerous lesions," in which case "endometrial cancer" refers to cancer that has passed the precancerous lesion stage, such as being in the advanced stage.
[0052] This invention proposes that certain significant epigenetic changes are important factors in the development of endometrial cancer. Compared with DNA mutations, specific regions of the genome exhibit a hypermethylated state, which occurs earlier and is more stable in the early diagnosis of tumors. Abnormal DNA methylation patterns can be used to predict the risk of atypical hyperplasia transforming into endometrial cancer. This DNA hypermethylation state persists throughout the entire process of normal cells transforming into tumor cells, and occurs in the very early stages of tumor development, preceding gene mutations. Therefore, tumor detection based on DNA hypermethylation can be applied to very early tumor screening, auxiliary diagnosis, efficacy evaluation, and recurrence monitoring, covering the entire process of tumor diagnosis and treatment.
[0053] Based on this, the present invention provides a set of methylation biomarkers, TAGMe-1 and TAGMe-2, as molecular markers for the detection of endometrial cancer. These biomarkers are hypomethylated in normal and adjacent endometrial tissues, and hypermethylated in endometrial cancer and precancerous lesions (dysplasia). They can be used to non-invasively assist in the diagnosis of early endometrial cancer and precancerous lesions through exfoliated cells from the cervix and uterine cavity, filling a gap in existing endometrial cancer screening technologies. Furthermore, with further development, a relatively simple, inexpensive, reliable, and high-throughput clinical test can be achieved.
[0054] In this invention, the methylation status of the nucleotide sequence or a portion thereof shown in SEQ ID NO:1 or SEQ ID NO:5 differs significantly between tumor and non-tumor tissues. When an abnormally high methylation status is detected in the gene sequence region, the subject is considered to have a tumor or belong to a high-risk group for tumors. This significant difference in methylation status shown in the gene sequence or a portion thereof shown in SEQ ID NO:1 or SEQ ID NO:5 is highly evident in endometrial cancer (including early stages); simultaneously, this significant difference in methylation status also exists in some other tumors, including: cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck tumors, colon cancer, colorectal cancer, esophageal cancer, lung cancer (lung squamous cell carcinoma and lung adenocarcinoma), and cholangiocarcinoma.
[0055] This invention also includes "conservative variant sequences" having high sequence identity with the nucleotide sequences shown in SEQ ID NO:1 (or its reverse complementary sequence) or SEQ ID NO:5 (or its reverse complementary sequence). "High sequence identity" is defined as, for example, higher than 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.
[0056] Based on the above, the present invention provides a nucleic acid derived from a specific region of the human genome, having the gene sequence shown in SEQ ID NO:1 or SEQ ID NO:5 or a portion thereof, and also including its antisense strand. Within tumor cells, 5-methylcytosine (5mC) is generated at multiple 5'-CpG-3' base C positions in this nucleic acid sequence.
[0057] Detection of one or more CpGs provided by this invention is possible; therefore, this invention also includes nucleic acid fragments of the nucleotide sequence, including at least one methylated CpG site. The at least one may include 2 to 53 of SEQ ID NO:1 or its reverse complementary sequence, more specifically 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, and 50. The at least one may include 2 to 52 of SEQ ID NO:5 or its reverse complementary sequence, more specifically 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, and 50. Those skilled in the art will understand that 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 obtainable according to the content provided by this invention.
[0058] With the information on the specific segments in the human genome provided by the present invention, those skilled in the art can easily obtain and apply the CpG sites. The 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.
[0059] This invention also includes gene panels or gene groups containing the nucleotide sequences or sequence fragments shown in SEQ ID NO:1 or SEQ ID NO:5, or their complementary sequences (SEQ ID NO:2 or SEQ ID NO:6). For the aforementioned gene panels or gene groups, characteristics of normal cells and tumor cells can also be obtained by detecting DNA methylation status.
[0060] It should be understood that a wide variety of techniques for analyzing methylation status can be applied in this invention, and this invention does not impose any particular limitation on such detection techniques. 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 tumors.
[0061] The nucleic acid or fragment thereof, or its complementary sequence, described in SEQ ID NO:1 or SEQ ID NO:5 of the present invention can be converted to uracil after bisulfite treatment, while the 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 with nucleotide sequences shown in SEQ ID NO:3 or SEQ ID NO:7, or nucleic acids or nucleic acid fragments with nucleotide sequences shown in SEQ ID NO:4 or SEQ ID NO:8. These nucleic acids can serve as more direct targets for designing detection reagents or detection kits.
[0062] The nucleic acids and / or complementary nucleic acids and / or one or more fragments of the nucleotide sequences shown in SEQ ID NO:1 or SEQ ID NO:5 of this invention can 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 such nucleic acid sets 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.
[0063] The nucleic acids of the present invention, derived from the nucleotide sequences shown in SEQ ID NO:1 or SEQ ID NO:5 and / or their complementary nucleic acids and / or one or more fragments thereof (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 such nucleic acid sets 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.
[0064] As a preferred embodiment of the present invention, nucleic acids and / or complementary nucleic acids and / or one or more fragments thereof from the nucleotide sequence shown in SEQ ID NO:1 are used in combination with nucleic acids and / or complementary nucleic acids and / or one or more fragments thereof from the nucleotide sequence shown in SEQ ID NO:5 for detection.
[0065] Based on the target genes and their epigenetic characteristics provided in this invention, techniques known in the art, as well as some techniques that are about to be developed, can all 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 that are still under development or will 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: 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, etc., which are conventional methods in this field. Although some preferred embodiments of this invention are provided, the overall scheme of this invention is not limited thereto.
[0066] 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.
[0067] 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).
[0068] The method of this invention can be used to: test subject samples to assess whether the subject has a tumor; or to identify high-risk groups for tumors. 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.
[0069] 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.
[0070] As a preferred embodiment of the present invention, a method for detecting the methylation status of nucleic acids in a sample in vitro is also provided, wherein the method is methylation-sensitive restriction endonuclease (MSRE) digestion. When a methylated base is present at its cleavage site, the methylation-sensitive restriction endonuclease cannot cleave DNA. 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 with a methylation-sensitive type II endonuclease and its isoenzymes (insensitive to methylation), and then analyzed by DNA blotting. The advantages of this method include: no need to know detailed information about the primary structure of the target DNA, and the ability to provide a direct evaluation of the methylation status of CpG islands, including obtaining some quantitative analytical information on the methylation of the gene being tested.
[0071] 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.
[0072] This invention provides a method for preparing a tumor 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 that can be accomplished by those skilled in the art.
[0073] Once the sequence of the nucleic acid is known, primer design 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 Tables 2 and 6. Besides primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] The methods and reagents of this invention exhibit very high accuracy when used to diagnose clinical tumors, as demonstrated in the detection of various clinical tumor samples in the embodiments of this invention. This invention can be applied to fields such as pre-tumor screening, efficacy assessment, auxiliary diagnosis, and prognostic monitoring, or, as mentioned above, situations where the purpose is not to obtain a direct disease diagnosis.
[0078] The biomarkers for tumors such as endometrial cancer provided by this invention exhibit very high specificity and sensitivity when used alone. Furthermore, the sensitivity and specificity are further enhanced when two biomarkers are used in combination. Therefore, the biomarkers of this invention have significant application value in fields such as tumor adjuvant diagnosis, efficacy assessment, and prognostic monitoring.
[0079] This invention provides biomarkers for tumors such as endometrial cancer, applicable to the detection of cervical exfoliated cells, etc. The samples are easy to obtain and are non-invasive. Compared to clinical procedures requiring surgical tissue sampling, this significantly reduces patient discomfort, improves compliance, and simplifies the procedure for clinicians. Clearly, this represents a very significant advancement.
[0080] The present invention will be further illustrated below with reference to 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. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.
[0081] Example 1: Screening of biomarkers for endometrial cancer detection
[0082] Forty-three frozen tissue samples were collected from five groups: normal endometrium, ordinary hyperplastic endometrium, atypical hyperplastic endometrium (precancerous lesion), endometrial cancer, and paired adjacent normal tissue. Degenerate expression bisulfite sequencing (RRBS) was used to analyze the methylation data at the whole genome level to screen for DNA methylation markers specific to endometrial cancer and precancerous lesions.
[0083] 1. Obtaining clinical samples
[0084] Frozen tissue samples were collected from five groups: normal endometrium, common hyperplastic endometrium, atypical hyperplastic endometrium (precancerous lesion), endometrial cancer, and paired adjacent cancerous tissue. Each sample was about the size of a soybean.
[0085] 2. DNA extraction
[0086] Genomic DNA was extracted from the sample using the Epiprobe Biotech K-21 Genomic DNA Extraction Kit (but the nucleic acid extraction technique of this invention is not limited to this method).
[0087] 3. RRBS sequencing library construction
[0088] The extracted gDNA sample was digested with MspI restriction enzyme, and then magnetic beads were used to sort the appropriately sized fragments. Subsequent steps included end-completion, addition of dA, and addition of adapters.
[0089] 4. Bisulfite treatment
[0090] The above library samples were modified with disulfite. In this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research (catalog number D5006) was used, and the operation was strictly carried out according to the instructions in the user manual (but this invention is not limited to this kit).
[0091] 5. PCR amplification and product purification
[0092] Each library sample was amplified using primers with different molecular tags. The amplification products were sorted and purified using magnetic beads, and the concentrations were measured before sequencing.
[0093] 6. RRBS sequencing
[0094] Genomic RRBS libraries from tissue samples were sequenced at 2×150bp using an Illumina Hiseq 2000. After sequencing, the data underwent quality control and alignment to analyze basic information such as the number of valid sequences, the number of CpG sites measured, and the number of coverage layers in each sample, as shown in Table 1. The total data volume of the RRBS libraries reached 198.31G. The average sequencing sequences obtained from the normal endometrial group (Group A, NE), the common hyperplasia group (Group B, EH), the atypical hyperplasia group (Group C, AH), the endometrial cancer group (Group D, EC), and the adjacent normal tissue (Group E, PC) were 16,686,194M, 16,847,536M, 14,942,502M, 14,729,170M, and 14,637,777M, respectively. The number of CpG sites with a coverage layer of 5 or more were 4,055,958M, 4,169,395M, 4,369,673M, 3,984,048M, and 4,271,290M, respectively. Statistical data and preliminary experimental analysis indicate that the overall quality of this RRBS sequencing was good, with good sequencing depth and CpG coverage layer numbers, uniform data distribution across groups, and high reliability of the results.
[0095] Table 1
[0096]
[0097]
[0098] 7. Marker Screening
[0099] By analyzing methylation sequencing data across different stages and combining it with experimental analysis, specific methylation markers present in atypical hyperplasia of the endometrium (precancerous lesions) and / or cancer stages were screened. For example... Figure 1 A heatmap of a small subset of candidate biomarkers for the diagnosis of endometrial cancer and precancerous lesions is shown. The inventors obtained two groups of candidate methylation biomarkers for the detection of endometrial cancer: Precancer-DMRs and Cancer-DMRs. These biomarkers correspond to DMRs that are significantly hypermethylated only in AH and EC or only in EC stages, while being hypomethylated in other stages, including adjacent tissues.
[0100] (1) TAGMe-1
[0101] The nucleotide sequence of the obtained TAGMe-1 methylated tumor marker is shown in SEQ ID NO:1. Figure 7 The sites marked with the background color are CG sites that may undergo methylation (group 53).
[0102] The inverse complementary sequence of SEQ ID NO:1 is as shown in SEQ ID NO:2. Figure 7 The sites marked with the background color are CG sites that may undergo methylation.
[0103] The sequence of SEQ ID NO:1 after sulfite conversion is shown in SEQ ID NO:3. Figure 7 The sites indicated by the background color are CG sites that may undergo methylation, and Y represents C or T.
[0104] The reverse complementary sequence of SEQ ID NO:3, after sulfite conversion, yields the sequence shown in SEQ ID NO:4. Figure 7 The sites indicated by the background color are CG sites that may undergo methylation, and Y represents C or T.
[0105] (2) TAGMe-2
[0106] The nucleotide sequence of the obtained TAGMe-2 methylated tumor marker is shown in SEQ ID NO:5. Figure 8 The sites marked with the background color are CG sites that may undergo methylation (52 groups).
[0107] The reverse complementary sequence of SEQ ID NO:5 is as shown in SEQ ID NO:6, with the background color indicating the CG sites that are potential sites for methylation.
[0108] The sequence of SEQ ID NO:1 after sulfite conversion is shown in SEQ ID NO:7. Figure 8 The sites indicated by the background color are CG sites that may undergo methylation, and Y represents C or T.
[0109] The reverse complementary sequence of SEQ ID NO:7, after sulfite conversion, is shown in SEQ ID NO:8. Figure 8 The sites indicated by the background color are CG sites that may undergo methylation, and Y represents C or T.
[0110] 8. Validate the clinical performance of candidate DNA methylation biomarkers using pyrosequencing.
[0111] To further verify whether the candidate biomarkers selected above have clinical application value, the biomarkers of this invention were compared with other Precancer-DMRs that were also analyzed and considered to have methylation differences and significance. Figure 1(Ranked high in the Precancer-DMRs heatmap), comparative experiments were conducted using pyrosequencing technology on additionally collected clinical tissue samples. Forty-five clinical tissue samples were collected, including tissues from 14 subjects with normal endometrium (NE), 11 subjects with hyperplasia (without dysplasia, EH), 5 subjects with dysplasia (AH), and 15 subjects with endometrial cancer (EC).
[0112] The primers for pyrosequencing amplification are shown in Table 2.
[0113] Table 2
[0114]
[0115]
[0116] Table 3 shows the performance indicators of candidate methylation markers in clinical tissue samples for diagnosing precancerous lesions of the endometrium and above.
[0117] Table 3
[0118]
[0119] Based on the above, the AUCs of the two DMR sites, TAGMe-1 and TAGMe-2, were 0.938 and 0.925, respectively, both higher than 0.9. Surprisingly, they can achieve very high AUCs for patients with early-stage endometrial cancer, demonstrating their excellent performance as methylation markers for early screening of endometrial cancer.
[0120] Example 2: Performance Verification of TAGMe-1 and TAGMe-2
[0121] The TCGA database, a recognized tumor database, includes data on various tumor types; for a specific tumor, it records a series of expression data, miRNA expression data, methylation data, mutation data, and copy number data. Therefore, the inventors used this database to verify the performance of TAGMe-1 and TAGMe-2.
[0122] Download of methylation data for multiple cancer types from the TCGA database: Methylation 450K microarray data for all tumor samples in the TCGA database were downloaded, including 11,087 tumor and control normal samples. The 35 tumor types involved and their sample sizes are shown in Table 4.
[0123] Table 4. Information on methylation datasets for 35 types of tumors
[0124]
[0125]
[0126] Calculation of methylation values for TAGMe-1 and TAGMe-2: First, the average methylation values of TAGMe-1 and TAGMe-2 in different types of tumor tissue samples and control normal tissue samples are calculated. Then, the difference between the average methylation values of TAGMe-1 and TAGMe-2 in the tumor samples and control samples of the same type is calculated.
[0127] Diagnostic performance analysis of TAGMe-1 and TAGMe-2 in other cancers: Differences in methylation values of TAGMe-1 and TAGMe-2 between tumor samples and control samples from different cancer types, as shown in the figure. Figure 2 As shown in A and 2C, two-sided Mann-Whitney t-test analysis revealed statistically significant differences in methylation levels between the tumor and control groups for TAGMe-1 in various tumor types, including endometrial cancer, cervical cancer, cholangiocarcinoma, colorectal cancer (colon cancer, rectal cancer), esophageal cancer, lung cancer (squamous cell carcinoma, adenocarcinoma), head and neck tumors, and pancreatic cancer. TAGMe-2, on the other hand, showed statistically significant differences in methylation levels between the tumor and control groups in colorectal cancer, endometrial cancer, and cervical cancer, respectively. The corresponding diagnostic AUC and performance are shown in [reference needed]. Figure 2 B, 2D and Table 5.
[0128] Table 5. Performance of Biomarkers in Other Cancer Types
[0129]
[0130] Therefore, TAGMe-1 and TAGMe-2 have diagnostic value in many types of tumors.
[0131] Example 3: Detection of DNA methylation in endometrial cancer-adjacent tissue samples: target-NGS method
[0132] In this embodiment, the target-NGS method was used to analyze DNA methylation in endometrial cancer-adjacent tissue samples. The steps are as follows:
[0133] 1. Obtaining clinical samples
[0134] Twenty normal endometrial tissues / adjacent tissues, six endometrial atypical hyperplasia samples, and twenty endometrial cancer tissue samples were obtained from clinical settings. The normal / adjacent samples served as the control group, while the atypical hyperplasia and cancer tissue samples served as the tumor detection experimental group.
[0135] 2. DNA extraction
[0136] DNA was extracted from the experimental group and the control group respectively. DNA extraction was performed using the Epiprobe Biotech K-21 genomic DNA extraction kit (but this invention is not limited to this method).
[0137] 3. Bisulfite treatment
[0138] The extracted DNA was modified with bisulfite. In this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research (catalog number D5006) was used, and the procedure was strictly followed according to the instruction manual (but this invention is not limited to this kit).
[0139] 4. PCR amplification and library preparation
[0140] After modification, the target region was amplified and enriched using PCR primers designed for bisulfite treatment. The primer sequences are shown in Table 6.
[0141] Table 6
[0142]
[0143] The product was then purified and sequencing adapters were ligated to construct the NGS sequencing library.
[0144] 5. NGS sequencing
[0145] The test was performed using the Illumia HiSeq X high-throughput sequencer, and the procedure was strictly followed according to the instruction manual (but this invention is not limited to this model of high-throughput sequencer).
[0146] 6. Calculation of TAGMe-1 and TAGMe-2 methylation values
[0147] The Target NGS sequencing data includes the methylation status of all individual CpG sites within the target region. The TAGMe-1 and TAGMe-2 methylation levels are calculated as follows: Methylation level of a single site = number of methylated reads at that site / total number of reads. The methylation level of the target region = the average methylation level of all sites.
[0148] 7. Analysis of results from a single biomarker
[0149] Organization Target NGS results as follows Figure 3 The differences in TAGMe-1 and TAGMe-2 methylation levels between the adjacent normal / normal control group and the tumor experimental group were compared, and ROC analysis was performed. The TAGMe-1 results are shown in the figure. Figure 3A. In clinical samples of endometrial cancer, the methylation values of TAGMe-1 in atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. The ROC analysis results of TAGMe-1 are as follows: Figure 3 B. The AUC of TAGMe-1 in distinguishing between the endometrial cancer group and the control group was 0.995, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.983, both showing good discriminative performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-1 in distinguishing between the endometrial cancer group and the control group were 95% and 100%, respectively; the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 83.33% and 90%, respectively.
[0150] TAGMe-2 results are as follows Figure 3 C, In clinical samples of endometrial cancer, the methylation values in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. The ROC analysis results of TAGMe-2 are as follows: Figure 3 D. The AUC of TAGMe-2 in distinguishing between the endometrial cancer group and the control group was 0.990, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.967, both showing good discriminatory performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-2 in distinguishing between the endometrial cancer group and the control group were 95% and 100%, respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 83.33% and 100%, respectively.
[0151] 8. Analysis of the combined application of markers
[0152] The combined detection of TAGMe-1 and TAGMe-2 is shown in Table 7.
[0153] Table 7. Performance of Combined Analysis of TAGMe-1 and TAGMe-2
[0154]
[0155] The results showed that the combination of the two had a synergistic effect, further increasing the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions.
[0156] Example 4: Detection of DNA methylation in endometrial cancer-adjacent tissue samples: pyrosequencing method
[0157] In this embodiment, pyrosequencing was used to detect DNA methylation in endometrial cancer-adjacent tissue samples for biomarker analysis. The steps are as follows:
[0158] 1. Obtaining clinical samples: 25 normal cervical exfoliated cell samples, 5 cervical exfoliated cell samples from patients with atypical endometrial hyperplasia, and 15 cervical exfoliated cell samples from patients with endometrial cancer were obtained from clinical practice. The normal cervical exfoliated cell samples served as the control group, while the cervical exfoliated cell samples from patients with atypical hyperplasia and endometrial cancer served as the tumor detection experimental group.
[0159] 2. DNA extraction: Extract DNA from clinical samples; in this experiment, the Epiprobe Genomic DNA Extraction Kit (EpiprobeBiotech, K-21) was used for DNA extraction (but this invention is not limited to this method).
[0160] 3. Bisulfite treatment
[0161] The extracted DNA was modified with bisulfite. In this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research, catalog number D5006, was used, and the procedure was strictly followed according to the instruction manual (but this invention is not limited to this method).
[0162] 4. Primer design
[0163] Based on the TAGMe-1 and TAGMe-2 sequences (SEQ ID NO:1 and SEQ ID NO:5), amplification primers and pyrosequencing primers were designed respectively. The methylation value of the corresponding CpG site on the target sequence was detected as a representative of its methylation value. The PCR primer amplification sequence, pyrosequencing primer sequence, pyrosequencing detection sequence and detection site are the same as in Example 1.
[0164] 5. PCR amplification and agarose gel electrophoresis
[0165] The sample treated with bisulfite was used as a template for PCR amplification. The specificity of the PCR amplification was identified by agarose gel electrophoresis after amplification.
[0166] 6. Pyrosequencing
[0167] The test was performed using a Qiagen PyroMark Q96ID pyrosequencing instrument, strictly following the instructions.
[0168] 7. Calculation of methylation value
[0169] Pyrosequencing can independently detect the methylation status of individual CpG sites within a target region, and calculate the average methylation value of all CpG sites as the methylation values of TAGMe-1 and TAGMe-2 in the sample.
[0170] 8. Results Analysis
[0171] The differences in TAGMe-1 and TAGMe-2 methylation levels between the adjacent normal / normal control group and the tumor experimental group were compared, and ROC analysis was performed.
[0172] The TAGMe-1 analysis results are as follows: Figure 4 A. In clinical samples of endometrial cancer, the methylation values of TAGMe-1 in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. ROC analysis results are as follows: Figure 4 B. The AUC of TAGMe-1 in distinguishing between the endometrial cancer group and the control group was 0.955, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.952, both showing good discriminative performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-1 in distinguishing between the endometrial cancer group and the control group were 100% and 84%, respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 100% and 84%, respectively.
[0173] The TAGMe-2 analysis results are as follows: Figure 4 C. In clinical samples of endometrial cancer, the methylation values of TAGMe-2 in atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. The ROC analysis results of TAGMe-2 are as follows: Figure 4 D. The AUC of TAGMe-2 in distinguishing between the endometrial cancer group and the control group was 0.943, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.928, both showing good discriminatory performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-2 in distinguishing between the endometrial cancer group and the control group were 86.67% and 88%, respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 100% and 80%, respectively.
[0174] 9. Combined analysis of TAGMe-1 and TAGMe-2
[0175] The combined detection of TAGMe-1 and TAGMe-2 was further analyzed to assess the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions. The combined performance is shown in Table 8.
[0176] Table 8. Performance of the combined analysis of TAGMe-1 and TAGMe-2
[0177]
[0178]
[0179] Adjacent samples cannot be simply considered normal samples. During clinical sampling, adjacent samples are prone to contamination with cancerous tissue or exhibit epigenetic variations due to the location of the cancerous lesion, which can lead to a decrease in specificity. Therefore, the specificity of some samples in the table may fluctuate compared to the aforementioned examples. Nevertheless, the combined detection of the two markers significantly improves the specificity, overall concordance rate, and positive predictive value in detecting endometrial atypical hyperplasia.
[0180] Example 5: Detection of DNA methylation in endometrial cancer-cervical exfoliated cell samples
[0181] In this embodiment, Me-qPCR was used to detect DNA methylation in endometrial cancer-cervical exfoliated cell samples for biomarker analysis. The steps are as follows:
[0182] 1. Obtaining clinical samples: 38 normal cervical exfoliated cell samples, 8 cervical exfoliated cell samples from patients with atypical endometrial hyperplasia, and 33 cervical exfoliated cell samples from patients with endometrial cancer were obtained from clinical practice. The normal cervical exfoliated cell samples served as the control group, while the cervical exfoliated cell samples from patients with atypical hyperplasia and endometrial cancer served as the tumor detection experimental group.
[0183] 2. DNA extraction: Extract DNA from clinical samples; in this experiment, the Epiprobe Genomic DNA Extraction Kit (EpiprobeBiotech, K-21) was used for DNA extraction (but this invention is not limited to this method).
[0184] 3. Enzyme digestion reaction: DNA is digested using methylation-sensitive restriction endonucleases. Unmethylated restriction sites will be cleaved. In this experiment, HpaII enzyme (NEB, R0171) was used for the enzyme digestion reaction (but this invention is not limited to this method).
[0185] 4. Primer design: Based on the TAGMe-1 and TAGMe-2 sequences, corresponding amplification primers were designed to amplify the methylation-sensitive restriction endonuclease digestion products. The abundance of the digested DNA fragments was detected and used as representatives of the methylation values of TAGMe-1 and TAGMe-2, respectively. The GAPDH gene was used as an internal control.
[0186] 5. qPCR amplification: The enzyme-digested sample was used as a template for qPCR amplification. In this experiment, the ABI 7500 qPCR instrument from Thermo Scientific was used for detection (but this invention is not limited to this method).
[0187] 6. Methylation value calculation: The DNA methylation level of each sample is assessed using the following formula: ΔCt_ 本发明标记物 =Ct_ 本发明标记物 -Ct_ GAPDHThe smaller the ΔCt, the higher the methylation level.
[0188] 7. Results Analysis: The differences in TAGMe-1 and TAGMe-2 methylation levels between the control group and the tumor experimental group were compared, and ROC analysis was performed.
[0189] Cervical cell Me-qPCR results are as follows Figure 5 The TAGMe-1 results are as follows: Figure 5 A. In clinical samples of endometrial cancer, the methylation values of TAGMe-1 in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. The TAGMe-1 ROC analysis results are as follows: Figure 5 B. The AUC of TAGMe-1 in distinguishing between the endometrial cancer group and the control group was 0.916, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.870, both showing good discriminative performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-1 in distinguishing between the endometrial cancer group and the control group were 84.85% (68.1%-94.89%) and 89.47% (75.2%-97.06%), respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 87.5% (47.35%-99.68%) and 76.32% (59.76%-88.56%), respectively. The results for TAGMe-2 are as follows... Figure 5 C. In clinical samples of endometrial cancer, the methylation values of TAGMe-2 in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. The TAGMe-2 ROC analysis results are as follows: Figure 5 D. The AUC of TAGMe-2 in distinguishing between the endometrial cancer group and the control group was 0.921, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.845, both showing good discriminatory performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-2 in distinguishing between the endometrial cancer group and the control group were 87.88% and 86.84%, respectively, and the sensitivity and specificity in distinguishing between atypical hyperplasia and the control group were 100% and 57.89%, respectively.
[0190] 8. Combined analysis of TAGMe-1 and TAGMe-2: TAGMe-1 and TAGMe-2 were combined for detection to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions. The performance of the combined model is shown in Table 9.
[0191] Table 9. Performance of Combined Analysis of TAGMe-1 and TAGMe-2
[0192]
[0193] Based on the above, the combined detection of the two markers can significantly improve the overall concordance rate in detecting atypical endometrial hyperplasia, as well as the sensitivity and concordance rate in detecting endometrial cancer.
[0194] Example 6: Detection of DNA methylation in endometrial cancer exfoliated cell samples (Me-qPCR method)
[0195] Nineteen normal cervical exfoliated cell samples, six samples from patients with atypical endometrial hyperplasia, and 21 samples from patients with endometrial cancer were obtained from clinical settings. The normal cervical exfoliated cell samples served as the control group, while the samples from patients with atypical hyperplasia and endometrial cancer served as the tumor detection experimental group. Following the Me-qPCR detection procedure in Example 5, the differences in TAGMe-1 and TAGMe-2 methylation levels between the control group and the tumor experimental group were compared, and ROC analysis was performed.
[0196] Me-qPCR results of uterine cavity cells are as follows Figure 6 The TAGMe-1 results are as follows: Figure 6 A. In clinical samples of endometrial cancer, the methylation values of TAGMe-1 in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. ROC analysis results are as follows: Figure 6 B. The AUC of TAGMe-1 in distinguishing between the endometrial cancer group and the control group was 0.988, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.921, both showing good discriminative performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-1 in distinguishing between the endometrial cancer group and the control group were 90.48% and 100%, respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 83.33% and 94.74%, respectively.
[0197] TAGMe-2 results are as follows Figure 6 C, In clinical samples of endometrial cancer, the methylation values of TAGMe-2 in both atypical hyperplasia and cancerous tissue samples were significantly higher than those in the control group. ROC analysis results are as follows: Figure 6 D. The AUC of TAGMe-2 in distinguishing between the endometrial cancer group and the control group was 0.985, and the AUC in distinguishing between the atypical hyperplasia group and the control group was 0.973, both showing good discriminatory performance. Taking the maximum Youden coefficient as the threshold, the sensitivity and specificity of TAGMe-2 in distinguishing between the endometrial cancer group and the control group were 95.24% and 94.74%, respectively, and the sensitivity and specificity in distinguishing between the atypical hyperplasia group and the control group were 100% and 84.21%, respectively.
[0198] The combined detection of TAGMe-1 and TAGMe-2 was used to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions. The performance of the combined model is shown in Table 10.
[0199] Table 10. Performance of the combined analysis of TAGMe-1 and TAGMe-2
[0200]
[0201] Based on the above, the combined detection of the two markers can significantly improve the overall concordance rate in detecting atypical endometrial hyperplasia.
[0202] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively 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 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. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.
Claims
1. The use of a detection reagent in the preparation of a reagent for identifying tumors, comprising: (a) Provide an isolated nucleic acid, which is a nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1, or a combination of nucleic acids of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5; (b) Using the nucleic acid in (a) as the target sequence, design a detection reagent that specifically detects the CpG site modification status of the target sequence; The tumor is endometrial cancer.
2. Use according to claim 1, characterized in that, Detection is performed on tumors or their precancerous lesions.
3. Use according to claim 1, characterized in that, The samples to be tested include: cervical smears or brushes, cervical swabs, uterine tissue, cervical exfoliated cells, uterine curettage, uterine irrigation fluid, and vaginal secretions.
4. A method of detecting methylation level of a test sample, characterized by, The method described is not a disease diagnosis method, and includes: Extracting nucleic acid from the sample to be tested; and The modification status of the CpG site of the target sequence or its fragment in the extracted nucleic acid is detected. The target sequence is selected from: (1) the nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1, or (2) the combination of the nucleic acid of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5; its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.
5. The method of claim 4, wherein, Methods for detecting CpG site modifications in target sequences of extracted nucleic acids include: pyrosequencing, bisulfite conversion sequencing, methylation array, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, or combinations thereof.
6. The method as described in claim 4, characterized in that, A method for detecting CpG site modifications of target sequences in extracted nucleic acids includes: (i) processing the extracted nucleic acids to convert unmodified cytosine into uracil; and (ii) analyzing the modification status of the target sequences in the nucleic acids processed in (i).
7. The method of claim 6, wherein, The nucleic acid described in step (i) is treated with bisulfite.
8. A method for preparing a reagent for the detection of a tumor, characterized in that, The method includes: Provide nucleic acid combinations of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5, and design detection reagents to specifically detect CpG site modifications of the target sequence using the nucleic acids or nucleic acid combinations as target sequences; the detection reagents include: primers, probes, chips or test strips; The tumor in question is endometrial cancer.
9. Use of a reagent or combination of reagents for specifically detecting CpG site modifications of a target sequence in the preparation of a kit for identifying tumors, wherein the tumor is endometrial cancer, and the target sequence is a nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1, or a combination of nucleic acids of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5; wherein the reagent or combination of reagents is directed to a gene sequence containing the target sequence.
10. Use according to claim 9, characterized in that, The gene sequence mentioned includes gene panels or gene groups.
11. Use according to claim 9, characterized in that, The reagents or combinations of reagents mentioned are selected from: Primers of the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10; or Primers for the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10; and primers for the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; or Primers for the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14; or Primers for the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, and primers for the sequences shown in SEQ ID NO: 15 and SEQ ID NO:
16.
12. A kit for performing tumor detection, characterized by, The tumor is endometrial cancer, and the kit includes: a reagent or combination of reagents for specifically detecting the CpG site modification of a target sequence, wherein the target sequence is a nucleic acid combination of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO:
5.
13. The kit for performing tumor detection according to claim 12, wherein The reagents or combinations of reagents mentioned are selected from: Primers for the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10; and primers for the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; or Primers for the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, and primers for the sequences shown in SEQ ID NO: 15 and SEQ ID NO:
16.
14. The kit for performing tumor detection according to claim 12, wherein, The kit also includes: bisulfite, DNA purification reagent, DNA extraction reagent, and PCR amplification reagent.