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EP-3964578-B1 - METHYLATION-BASED MODIFIED TUMOR MARKER STAMP-EP8 AND APPLICATION THEREOF

EP3964578B1EP 3964578 B1EP3964578 B1EP 3964578B1EP-3964578-B1

Inventors

  • LI, ZHENYAN
  • DONG, SHIHUA

Dates

Publication Date
20260513
Application Date
20200429

Claims (14)

  1. A method for pan-cancer detection in vitro, comprising detecting the 5-methylation profile of a target sequence, wherein said target sequence is selected from: (i) a polynucleotide, its nucleotide sequence is shown in SEQ ID No. 1, (ii) a fragment of the polynucleotide of (i) consisting of or containing the bases at positions 204 to 223 or 1 to 478 of SEQ ID NO: 1; and/or (iii) a nucleic acid complementary to the polynucleotide or fragment of (i) - (ii).
  2. The method for pan-cancer detection in vitro according to claim 1, wherein said target sequence is a fragment of the polynucleotide of (i) consisting of bases at positions 204 to 223.
  3. The method for pan-cancer detection in vitro according to claim 1 or 2, wherein said pan-cancer comprises at least a cancer selected from the group consisting of: hematologic cancers such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleuroma; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems tumors such as skin cancer, melanoma, osteosarcoma, liposarcoma and thyroid cancer.
  4. The method for pan-cancer detection in vitro according to any one of claims 1 - 3, comprising detecting the 5-methylation profile of a sample in vitro, comprising: (i) providing the sample and extracting nucleic acid; (ii) detecting the 5-methylation profile on a CpG site(s) of the target sequence(s) according to claim 1 or 2, preferably wherein said target sequence is a fragment of the polynucleotide of (i) consisting of bases at positions 204 to 223.
  5. The method for pan-cancer detection in vitro according to any one of claims 1-4, wherein detecting the 5-methylation profile comprises pyrosequencing, bisulfite conversion sequencing, a method using a methylation chip, qPCR, digital PCR, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing technology, HPLC, MassArray or methylation specific PCR.
  6. The method for pan-cancer detection in vitro according to claim 4, wherein, step (ii) comprises: treating the product of (i) to convert unmodified cytosine into uracil; preferably, treating the nucleic acid of step (i) with bisulfite; and analyzing the 5-methylation profile of the target sequence in the nucleic acid.
  7. The method for pan-cancer detection in vitro according to claim 4 or 6, wherein, step (ii) comprises the use of primers shown in SEQ ID NO: 15 and 16; primers shown in SEQ ID NO: 5 and 6.
  8. The method for pan-cancer detection in vitro according to claim 7, wherein, step (ii) comprises the use of primers shown in SEQ ID NO: 15 and 16.
  9. A method of preparing a pan-cancer 5-methylation detection agent, comprising providing one or more polynucleotides comprising the target sequence(s) according to claims 1-2 and designing a detection agent for specifically detecting said target sequence(s), wherein the target sequence has at least the bases at positions 204 to 223 of SEQ ID NO: 1; wherein the detection agent comprises primers or probes.
  10. A combination of pan-cancer 5-methylation detection agents, wherein the combination of agents specifically detects the 5-methylation on CpG site(s) of said target sequence(s) according to any one of claims 1-2, wherein said combination of detection agents are: the primers shown in SEQ ID NO: 15 and 16; or the primers shown in SEQ ID NO: 5 and 6.
  11. The combination of pan-cancer 5-methylation detection agents according to claim 10 for use in pan-cancer detection, wherein said pan-cancer comprises at least a cancer selected from the group consisting of hematologic cancers such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleuroma; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems tumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.
  12. Use of one or more polynucleotides comprising the sequence(s) according to any one of claims 1-2, and a combination of pan-cancer 5-methylation detection agents according to claim 10, in the manufacture of a pan-cancer detection kit, preferably wherein said pan-cancer comprises at least a cancer selected from the group consisting of hematologic cancers such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleuroma; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems tumors such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.
  13. The use according to claim 12, wherein samples of the cancer comprise: tissue samples, paraffin embedded samples, blood samples, pleural effusion samples, and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, tissue and cell biopsy samples.
  14. A pan-cancer 5-methylation detection kit, comprising container(s) and the combination of pan-cancer 5-methylation detection agents, according to claim 10 in the container(s).

Description

Technical field The disclosure is in the field of markers for diagnosing diseases. More specifically, the disclosure relates to a methylation based tumor marker STAMP, Specific Tumor Aligned Methylation of Pan-cancer, and use thereof. BACKGROUND OF DISCLOSURE Tumors have been considered a genetic disease for decades. Several large-scale systematic sequencings for human have confirmed that the number of somatic mutations in cancer tissues is significantly less than expected. These results suggest that cancer is not a simple genetic disease. In order to diagnose tumor, many new tumor markers have been discovered and used for clinical diagnosis in recent years. Before 1980, tumor markers were mainly hormones, enzymes, proteins and other cell secretions, such as carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP) used as markers of gastric cancer, liver cancer and other tumors, carbohydrate antigen 125 (CA125) used as a marker of cervical cancer, and prostate specific antigen (PSA) used as a marker of prostate cancer. Although these tumor markers are still used in clinic, their sensitivity and accuracy have been difficult to meet the clinical needs. More and more evidences show that small changes in epigenetic regulation play an important role in tumors. Epigenetics is a subject that studies that the heritable change of gene function without a change of DNA sequence, which eventually leads to the change of phenotype. Epigenetics mainly includes DNA methylation, histone modification, changes of microRNA level and other biochemical processes. DNA methylation is one of the epigenetic mechanisms, refers to the process of transferring methyl from S-adenosylmethionine (methyl donor) to specific bases under the catalysis of DNA methyltransferase. However, DNA methylation in mammals mainly occurs at the C of 5'-CpG-3', which results in 5-methylcytosine (5mC). Fluid biopsy is a technique for the diagnosis and prediction of tumors using circulating tumor cells or circulating tumor DNA as detection targets. The technology has many shortcomings. First, the sensitivity and specificity are not good enough. The tumor itself is heterogeneous, including a variety of subtypes of cell populations. The proportion of tumor DNA in clinical samples, especially blood samples, is very low. The existing tumor markers are difficult to meet the sensitivity of clinical requirements, and it is easy to cause misdiagnosis. Second, one marker has good effect only for one or a few kinds of tumors. As the DNA sources in blood are very complex, the existing tumor markers cannot solve the complex problems of tumor source and metastasis. Because of these complexities, it is difficult for many DNA methylation tumor markers to have a unified standard in clinical application, which seriously affects the sensitivity and accuracy of the markers. In the previous study of the inventor, some tumor markers based on methylation modification were found. WO 2016/083360, WO 2016/207656, WO 2017/072292 and WO 2017/143296 disclose the use of NKX2-6/chromosome 8 sequences for the detection of lung, bladder, breast or prostate cancer, respectively. However, to provide more ways for tumor diagnosis, it is still necessary to find more new tumor markers. SUMMARY OF DISCLOSURE The object of the disclosure is to provide a method for detecting tumor based on abnormal hypermethylation of specific sites in tumor using DNA methylation modification as a tumor marker. The first aspect of the present disclosure provides the method according to claim 1. Disclosed, but not claimed, is an isolated first polynucleotide, including: (a) a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 1; (b) a fragment of the polynucleotide of (a), having at least one (such as 2-191, more specifically 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170, 180, 190) modified CpG site; and/or (c) a nucleic acid (such as the polynucleotide with a nucleotide sequence as shown in SEQ ID No: 3) complementary to the polynucleotide or fragment of (a) or (b). Disclosed, but not claimed, modifications include 5-hydroxymethylation(5hmC), 5-formylcytosine(5fC) or 5-carboxylcytosine(5-caC). In a preferable embodiment, the fragment of the polynucleotide contains bases at: positions 204 to 223 (including methylation sites 021 to 024). Disclosed, but not claimed, fragments include: positions 1 to 478 (including methylation sites 001 to 040) of SEQ ID NO: 1 or 2; positions 513 to 1040 (including methylation sites 041 to 077) of SEQ ID NO: 1 or 2; positions 1082 to 1602 (including methylation sites 078 to 114) of SEQ ID NO: 1 or 2; positions 1621 to 2117 (including methylation sites 115 to 153) of SEQ ID NO: 1 or 2; positions 2160 to 2700 (including methylation sites 154 to 191) of SEQ ID NO: 1 or 2. Disclosed, but not claimed, is an isolated polynucleotide, which is converted from the first polynucleotide, and as compared with the sequence of the first polynucleotide, its cytosine