WO2020020072A1 - 基于甲基化修饰的肿瘤标记物stamp-ep2 - Google Patents

基于甲基化修饰的肿瘤标记物stamp-ep2 Download PDF

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WO2020020072A1
WO2020020072A1 PCT/CN2019/096797 CN2019096797W WO2020020072A1 WO 2020020072 A1 WO2020020072 A1 WO 2020020072A1 CN 2019096797 W CN2019096797 W CN 2019096797W WO 2020020072 A1 WO2020020072 A1 WO 2020020072A1
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cancer
samples
tumors
methylation
polynucleotide
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French (fr)
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李振艳
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上海奕谱生物科技有限公司
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Priority to EP19839876.0A priority patent/EP3828273A4/en
Priority to US17/250,458 priority patent/US20210292850A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

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  • the present invention belongs to the field of disease diagnostic markers, and more specifically, the present invention relates to methylated modified tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer).
  • STAMP Specific Tumor Aligned Methylation of Pan-cancer
  • the occurrence and development of tumors is a complex, multi-layered, multi-factor, and dynamic process, which includes the intricate interaction of multiple factors such as the external environment, genetic variation, and epigenetic variation.
  • External environmental factors include carcinogens such as physicochemical biology and unhealthy living habits
  • genetic mutations include gene mutations, copy number changes, chromosomal misalignment, etc.
  • Epigenetic mutations mainly include DNA methylation, histone modification, non-coding RNA and other factors.
  • environmental factors, genetic factors and epigenetic factors complement each other and work together to cause a series of inactivation of tumor suppressor genes and activation of proto-oncogenes, which in turn leads to tumors.
  • tumor problems still face many challenges for humans. Although new surgical methods and targeted treatments, immunotherapy, etc. in recent years Some gratifying progress has been made, but there are still many misunderstandings about people's understanding of tumors. Major issues such as tumor metastasis, recurrence, heterogeneity, and drug resistance need to be urgently addressed.
  • Tumors occur in most tissues of the human body, and different types of tumors are divided into numerous subtypes. Especially in recent years, due to the development of tumor molecular biology, tumor classification has become more Refinement. For different types, different stages, or different molecular subtypes of tumors, the treatment options are also very different.
  • tumor markers were mainly cell secretions such as hormones, enzymes, and proteins.
  • CCA125 carcinoembryonic antigen
  • AFP alpha-fetoprotein
  • CA125 can be used as a marker for cervical cancer
  • PSA prostate specific antigen
  • Liquid biopsy technology uses circulating tumor cells in the blood or circulating tumor DNA as detection targets to diagnose and predict tumors.
  • This technology is still in its infancy and has many shortcomings.
  • Clinical samples, especially blood samples, tumors The proportion of DNA is very small, the existing tumor markers are difficult to meet the sensitivity required by clinical requirements, and it is easy to cause misdiagnosis in the clinic.
  • one marker has a good effect on only one or a few tumors, and the blood
  • the source of DNA is very complicated, so existing tumor markers cannot cope with the problems of complex tumor origin, metastasis and so on.
  • An object of the present invention is to provide a method for detecting a tumor by using DNA methylation modification as a tumor marker and utilizing a phenomenon of abnormal hypermethylation of specific sites in the tumor.
  • an isolated polynucleotide including: (a) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; (b) a nucleotide sequence shown in SEQ ID NO: 2 (C) a fragment of the polynucleotide of (a) to (b) above, and at least one modified CpG site (such as 2 to 45, more specifically, 3, 5, 10, etc.) 15, 20, 25, 30, 40); (d) a nucleic acid complementary to the polynucleotide or fragment (a) to (c) above (such as the nucleotides shown in SEQ ID NO: 5 or SEQ ID NO: 6) Acid sequence of the polynucleotide).
  • the modification includes a 5-methylation modification, a 5-hydroxymethylation modification, a 5-aldehyde methylation modification, or a 5-carboxymethylation modification.
  • an isolated polynucleotide is provided, which is transformed from the polynucleotide.
  • the sequence corresponding to the aforementioned first aspect has a cytosine C at a modified CpG site that is unchanged. Modified cytosines are converted to T or U.
  • the polynucleotide includes: (e) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7; (f) SEQ ID NO: 4 or SEQ ID: NO: 8 polynucleotide of the nucleotide sequence; (g) the fragment of the polynucleotide of (e) to (f) above, and there is at least one modified CpG site (such as 2 to 45) , More specifically 3, 5, 10, 15, 20, 25, 30, 40).
  • the use of the polynucleotide according to the first aspect or the second aspect is provided for preparing a tumor detection reagent or kit.
  • the tumor includes (but is not limited to): hematological tumors such as leukemia, lymphoma, multiple myeloma; 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, pleural tumor; Nervous system tumors such as glioma, neuroblastoma, meningiomas; Head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; Gynecological and reproductive systems Tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, Thyroid cancer.
  • hematological tumors such as leukemia, lympho
  • the tumor samples include: 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 Fluid samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or swab samples, tissue and cell biopsy samples.
  • a method for preparing a tumor detection reagent comprising: providing the polynucleotide according to the first aspect or the second aspect, and a full length or a fragment of the polynucleotide As a target sequence, a detection reagent that specifically detects the modification of the CpG site of the target sequence is designed; wherein at least one of the target sequences (such as 2 to 45, more specifically, 3, 5, 10, 15) , 20, 25, 30, 40) modified CpG sites; preferably, the detection reagent includes (but is not limited to): primers, probes.
  • a reagent or a combined reagent which specifically detects the modification of the CpG site of a target sequence, wherein the target sequence is the polynucleotide according to any one of the first aspect or the second aspect.
  • the reagent or the combined reagent is directed to a gene sequence (designed based on the gene sequence) including the target sequence, and the gene sequence includes a gene panel or a gene group.
  • the detection reagent includes: a primer and a probe.
  • the primers are: the primers shown in SEQ ID NOs: 5 and 6.
  • the use of the reagent or the combined reagent according to the fifth aspect of the present invention is provided for preparing a kit for detecting a tumor; preferably, the tumor includes (but is not limited to): 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, pleural tumors; blood system tumors such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive systems Tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; neurological tumors such as glioma, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, Laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer,
  • Digestive system tumors such
  • a detection kit which includes: a container, and the aforementioned reagent or reagent combination located in the container; preferably, each reagent is located in a separate container.
  • the kit further includes: bisulfite or bisulfite, a DNA purification reagent, a DNA extraction reagent, a PCR amplification reagent, and / or an instruction manual (indicating the detection operation steps and Results judgment criteria).
  • a method for detecting a methylation profile of a sample in vitro comprising: (i) providing a sample and extracting a nucleic acid; (ii) detecting a CpG site of a target sequence in the nucleic acid of (i)
  • the target sequence is the polynucleotide according to the first aspect or the polynucleotide according to the second aspect, which is transformed from the polynucleotide.
  • the analysis method includes: pyrosequencing method, bisulfite conversion sequencing method, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, and third-generation sequencing method.
  • Method whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation-specific PCR, or a combination thereof, and part of the sequence shown in SEQ ID NO.1
  • In vitro detection method and in vivo trace detection method of combined gene group of all or all methylation sites can also be applied in the present invention.
  • step (ii) includes: (1) processing the product of (i) to convert unmodified cytosine to uracil; preferably, the modification includes 5-methyl Modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification, or 5-carboxymethylation modification; preferably, the process described in step (i) is treated with bisulfite or bisulfite.
  • Nucleic acid (2) Analyze the modification of the CpG site of the target sequence described in (1) the treated nucleic acid.
  • the abnormality of the methylation pattern means that C in the polynucleotide CpG is highly methylated.
  • the method of methylation profile is not for the purpose of directly obtaining the diagnosis result of the disease, or it is not a diagnostic method.
  • a tumor diagnostic kit comprising a primer pair designed using the sequence shown in the first aspect or the second aspect of the present invention and a gene panel or gene group containing the sequence. Detection of basal state obtains the characteristics of normal cells and tumor cells.
  • FIG. 1 Twenty pairs of paracancerous-lung cancer samples were obtained clinically. The paracancerous samples were used as the lung cancer control group, and the lung cancer samples were used as the lung cancer experimental group. The STAMP-EP2 methylation value (left) and the detection specificity of the control group and the experimental group were compared Sensitivity (right).
  • Figure 3 Clinically obtained 10 samples of normal stomach (or gastritis) as the control group, 10 cases of gastric cancer surgical margin samples as the experimental group 1, and 20 samples of gastric cancer as the experimental group 2, comparing the STAMP-EP2 methyl groups in the three groups of samples ⁇ ⁇ The value.
  • FIG. 6 Twenty pairs of paracancerous-breast cancer samples were obtained clinically. The paracancerous samples were used as the breast cancer control group, and the breast cancer samples were used as the breast cancer experimental group. The STAMP-EP2 methylation values of the control group and the experimental group were compared (left) and Detection specificity and sensitivity (right panel).
  • FIG. 7 Clinically obtained 18 pairs of para-cancerous-pancreatic cancer samples, the para-cancerous samples were used as the pancreatic cancer control group, and the pancreatic cancer samples were used as the pancreatic cancer experimental group.
  • the STAMP-EP2 methylation values of the control group and the experimental group were compared (left) Detection specificity and sensitivity (right panel).
  • Figure 8 Clinically obtained 11 pairs of paracancerous-head and neck cancer samples, including 5 cases of laryngeal cancer, 2 cases of tonsil cancer, 2 cases of epiglottic cancer, 1 case of root cancer of the tongue, and 1 case of hypopharyngeal cancer. Tissues were used as the experimental group, and the STAMP-EP2 methylation value was compared between the head and neck cancer control group and the experimental group.
  • Figure 9 Clinically obtained bile samples from 10 non-cancer patients, bile samples from 10 patients with gallbladder cancer, non-cancer samples as the gallbladder cancer control group, gallbladder cancer samples as the gallbladder cancer experimental group, and the control group and the experimental group STAMP-EP2 methyl Values (left) and detection specificity and sensitivity (right).
  • Figure 10 Clinically obtained 10 non-leukemia bone marrow smear samples as a control group and 20 leukemia bone marrow smear samples as an experimental group. Compare the STAMP-EP2 methylation value (left) and detection specificity of the leukemia control group with the experimental group. And sensitivity (right).
  • FIG. 11 Clinically obtained 8 cases of kidney cancer adjacent control samples were used as the control group, 14 cases of renal cancer samples were taken as the experimental group, and the STAMP-EP2 methylation value (left) and the detection specificity were compared between the renal cancer control group and the experimental group. Sensitivity (right).
  • FIG. 12 Five cases of bladder cancer adjacent samples and non-cancerous urine samples were clinically obtained as the control group, and 7 cases of bladder cancer tissue samples and bladder cancer urine samples were obtained as the experimental group.
  • the bladder cancer control group and the experimental group STAMP-EP2 Methylation value.
  • Plasma samples from 20 normal humans were used as a control group, and plasma samples from patients with different tumor types included 10 liver cancer plasma samples, 10 pancreatic cancer plasma samples, 10 lung cancer plasma samples, 10 colorectal cancer plasma samples, and 10 cases.
  • Breast cancer plasma samples were compared for STAMP-EP2 methylation values in each group.
  • the present inventor is committed to the study of tumor markers, and after extensive research and screening, provides a universal DNA methylation tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer).
  • STAMP is low in normal tissues. Methylation status.
  • STAMP is hypermethylated in tumor tissues. It can be used for clinical tumor detection and as a basis for designing tumor diagnostic reagents.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • sample includes a substance suitable for detection of DNA methylation status obtained from any individual or isolated tissue, cell, or body fluid (such as plasma).
  • high (degree) methylation refers to the presence of a high methylation, methylolation, aldehyde methylation, or carboxymethylation modification of CpG in a gene sequence.
  • MSP methylation-specific PCR
  • a PCR reaction with methylation-specific primers can obtain a positive PCR result, and the DNA (gene) region of the test can be considered to be in a hypermethylated state.
  • the determination of hypermethylation status can be analyzed for statistical differences based on the relative value of the methylation status of its control sample.
  • the tumors include but are not limited to: hematological tumors such as leukemia, lymphoma, multiple myeloma; 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, pleural tumor; neurological tumors such as glioma, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, throat cancer, nasopharyngeal cancer; gynecological and reproductive system tumors such as breast Cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.
  • hematological tumors such as leukemia, lymphoma, multiple myel
  • the inventors have conducted extensive and intensive research and determined the target STAMP-EP2.
  • the methylation status of the STAMP-EP2 gene sequence region is significantly different between tumor tissues and non-tumor tissues, as long as abnormal promoter methylation status (high methylation) of one of the aforementioned genes is detected, It can be determined that the subject is a high-risk person.
  • the significant differences between tumor tissues and non-tumor tissues presented by STAMP-EP2 exist broadly in various types of tumors, including solid tumors and non-solid tumors.
  • the present invention provides an isolated polynucleotide, which is derived from the human genome and has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 (the reverse of SEQ ID NO: 1).
  • SEQ ID NO: 1 the reverse of SEQ ID NO: 2
  • SEQ ID NO: 6 the reverse complementary sequence of SEQ ID NO: 2
  • multiple 5 ' -CpG-3 'at the base C position produces 5-methylcytosine (5mC).
  • the present invention also includes a fragment of a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6, and at least one of them (such as 2 ⁇ 45, more specifically 3, 5, 10, 15, 20, 25, 30, 40) methylated CpG sites.
  • the above polynucleotides or fragments can also be used for designing detection reagents or detection kits.
  • the polynucleotide fragment is, for example, a fragment including bases 247 to 305 in SEQ ID NO: 1 (including CpG 17 to 27 in SEQ ID NO: 1). Site); a fragment containing bases 249 to 307 in SEQ ID NO: 2 (containing CpG sites 18 to 28 in SEQ ID NO: 2). Antisense strands of the aforementioned fragments are also available.
  • these fragments are examples of preferred embodiments of the present invention. According to the information provided by the present invention, other fragments may also be selected.
  • a gene panel or a gene group comprising a nucleotide sequence or a sequence fragment shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID ID NO: 5, or SEQ ID NO: 6 is also included in the present invention.
  • the characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation status.
  • polynucleotides can be used as a key region for people to analyze methylation status in the genome, and their methylation status can be analyzed by various techniques known in the art. Any technique that can be used to analyze the methylation state can be applied in the present invention.
  • cytosine that has not been methylated is converted into uracil, and cytosine that has been methylated remains unchanged.
  • the present invention also provides a polynucleotide obtained after the above polynucleotide is treated with bisulfite or bisulfite, including: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 8.
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 7 SEQ ID NO: 7
  • SEQ ID NO: 8 The polynucleotide of the nucleotide sequence shown in SEQ ID NO: 8.
  • the present invention also includes a fragment of the polynucleotide obtained by subjecting the polynucleotide or the antisense strand thereof to bisulfite or bisulfite treatment, and at least one methylated CpG site is present therein.
  • a detection reagent designed based on the polynucleotide sequence is also provided for detecting the methylation profile of the polynucleotide in a sample in vitro. Detection methods and reagents for determining the sequence of the genome and the methylation state known in the art can be used in the present invention.
  • the present invention provides a method for preparing a tumor detection reagent, including: providing the polynucleotide, using the full length or a fragment of the polynucleotide as a target sequence, and designing a detection reagent that specifically detects the target sequence. ; Wherein, at least one methylated CpG site exists in the target sequence.
  • the detection reagents of the present invention include, but are not limited to, primers, probes, and the like.
  • the reagent is, for example, a primer pair.
  • designing the primer is known to those skilled in the art.
  • the two primers are on both sides of the specific sequence of the target gene to be amplified (including CpG Within the sequence, it is complementary to the gene region where CpG is originally methylated, and complementary to TpG where it is the gene region originally demethylated). It should be understood that, according to the new discovery of the present invention, a person skilled in the art can design a variety of primers or probes or other types of detection reagents for CpG sites or combinations thereof at different positions on the target sequence. It is included in the technical solution of this invention.
  • the reagent may also be a reagent combination (primer combination), including more than one set of primers, so that the multiple polynucleotides described above can be amplified separately.
  • the invention also provides a kit for detecting methylation profiles of polynucleotides in a sample in vitro.
  • the kit includes a container and the above-mentioned primer pair located in the container.
  • the kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification and the like.
  • kit may further include an instruction manual, in which detection operation steps and result determination standards are marked, so as to facilitate application by those skilled in the art.
  • Determining the methylation profile of a polynucleotide can be performed by existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or others are still under development and will Developed technology to proceed.
  • MSP methylation-specific PCR
  • Methylight real-time quantitative methylation-specific PCR
  • Quantitative methylation-specific PCR can also be used to detect methylation levels. This method is based on a continuous optical monitoring of fluorescent PCR, which is more sensitive than the MSP method. Its high throughput avoids analysis of its results by electrophoresis.
  • a method for detecting a methylation profile of a polynucleotide in a sample in vitro is also provided.
  • the method is based on the principle that bisulfite or bisulfite can convert unmethylated cytosine to uracil, which is converted to thymine during subsequent PCR amplification, and methylation
  • the cytosine remains unchanged; therefore, after treating the polynucleotide with bisulfite or bisulfite, the methylated site produces a polynucleotide polymorphism (SNP) similar to a C / T.
  • SNP polynucleotide polymorphism
  • the method of the present invention comprises: (a) providing a sample and extracting genomic DNA; (b) processing the genomic DNA described in step (a) with bisulfite or bisulfite, so that the genomic DNA is not Methylated cytosine is converted to uracil; (c) Analyze whether there is an abnormal methylation pattern in the genomic DNA treated in step (b).
  • the method of the present invention can be used for: (i) testing a sample of a subject to analyze whether the subject has a tumor; and (ii) distinguishing a high-risk group of tumors.
  • the method described may also be a situation where the purpose is not to obtain a direct diagnosis of the disease.
  • DNA methylation is detected by PCR amplification and pyrosequencing.
  • PCR amplification and pyrosequencing Those skilled in the art should understand that this method is not limited to practical applications, and other DNA methylation detection methods are also possible.
  • the primers used are not limited to those provided in the examples.
  • the invention can be applied to the fields of tumor auxiliary diagnosis, curative effect determination, prognosis monitoring and the like, and has high clinical application value.
  • SEQ ID NO: 5 The reverse complement of the nucleotide sequence shown in the above SEQ ID NO: 1 is as follows SEQ ID NO: 5:
  • SEQ ID ID NO: 6 The reverse complement of the nucleotide sequence shown in the above SEQ ID ID NO: 2 is as follows SEQ ID ID NO: 6:
  • SEQ ID NO: 5 The sequence of the above SEQ ID NO: 5 after bisulfite treatment (where Y represents C or U) is as follows SEQ ID NO: 4 (where Y represents C or U):
  • SEQ ID NO: 6 The sequence of the above SEQ ID NO: 6 after bisulfite treatment (where Y represents C or U) is as follows SEQ ID NO: 8 (where Y represents C or U):
  • DNA extraction extract the DNA of the experimental group and the control group separately; this experiment uses the phenol-chloroform extraction method, but is not limited to this method;
  • Primer design According to the characteristics of the STAMP-EP2 sequences SEQ ID NO: 1 and SEQ ID NO: 2, design PCR amplification primers and pyrosequencing primers. Because SEQ ID NO: 1 and SEQ ID NO: 2 are relatively large Similarity, the primers designed in this experiment can detect the 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 CpG sites in SEQ: ID NO: 1 at the same time. CpG sites at 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 are methylated.
  • pyrosequencing was used to detect the 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 CpG sites in SEQ ID NO: 1 and 18, 19, and 20 in SEQ ID NO: 2 , 21, 22, 23, 24, 25, 26, 27, 28 methylation value of CpG sites, as a representative of the STAMP-EP2 methylation value, PCR primer amplification sequence, pyrosequencing primer sequence, pyro
  • the detection sequences and detection sites of Pyrosequencing are shown in Table 1.
  • PCR amplification and agarose gel electrophoresis The samples treated with bisulfite are used as PCR products for PCR amplification. The amplified products are identified by agarose gel electrophoresis for specificity of PCR amplification. The size of the amplified fragment should be 143bp;
  • STAMP-EP2 methylation value calculation Pyrosequencing can detect the target region SEQ ID ID NO: 1, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 CpG sites With the methylation of CpG sites at 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 in SEQ ID NO: 2, calculate the average value as the STAMP-EP2 in this sample.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Obtaining clinical samples Obtain bile samples from 10 non-cancer patients and bile samples from 10 patients with gallbladder cancer. Non-cancer samples were used as gallbladder cancer control group, and gallbladder cancer samples were used as gallbladder cancer experimental group.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • the inventors collected 20 normal human plasma as a control group, and the plasma samples of patients with different tumor types include 10 liver cancer plasma samples, 10 pancreas Cancer plasma samples, 10 lung cancer plasma samples, 10 colorectal cancer plasma samples, 10 breast cancer plasma samples;
  • Step 2.3.4.5.6.7 is the same as that in Example 2.
  • Design amplification primers according to the sequences of SEQ ID NOs: 1 and 2. For different sequence regions, design primers for amplification using conventional methods.
  • FIG. 14 shows that the average methylation value of SEQ ID ID: 1 region in normal liver cell lines is 3.0%, and the average methylation value in liver cancer cell line HepG2 is 78.3%.
  • the methylation level of liver cancer cell lines Significantly higher than normal liver cell lines.
  • the average methylation value of SEQ ID NO: 2 region in normal liver cell lines is 1.8%, and the average methylation value in liver cancer cell line HepG2 is 78.7%.
  • the methylation level of liver cancer cell lines is significantly higher than normal liver cell lines. .

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Abstract

提供了一种甲基化肿瘤标志物STAMP-EP2及其在制备肿瘤诊断试剂中的应用。该肿瘤标志物STAMP-EP2，在所有肿瘤类型中均高甲基化，对应的正常组织中均低甲基化，具有高敏感性与特异性。检测该STAMP-EP2的引物可用于制备肿瘤诊断试剂盒。

Description

基于甲基化修饰的肿瘤标记物STAMP-EP2 技术领域
本发明属于疾病诊断标记物领域，更具体地，本发明涉及基于甲基化修饰的肿瘤标记物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)。
背景技术
肿瘤的发生发展是一个复杂的、多层面多因素的、动态的过程，包含了外界环境、遗传学变异以及表观遗传学变异等多种因素错综复杂的相互作用。外界环境因素包括物理化学生物等致癌因素以及不健康的生活习惯等，遗传学变异包括基因突变、拷贝数变化、染色体错位等，表观遗传学变异主要包括DNA甲基化、组蛋白修饰、非编码RNA等因素。在肿瘤的发生发展过程中，环境因素、遗传学因素以及表观遗传学因素相辅相成，共同作用，导致一系列抑癌基因的失活以及原癌基因的激活，进而导致肿瘤。在肿瘤的发展过程中，虽然三种因素皆贯穿其中，互相作用。但是在肿瘤的发生及早期，三种因素的作用重点是有先后顺序的，目前来说，肿瘤问题对于人类来讲还面临诸多挑战，尽管近年来新的手术方法以及靶向治疗、免疫治疗等方面取得了一些喜人的进展，但是人们对肿瘤的认识还存在许多误区，肿瘤的转移、复发、异质性、耐药性等等重大问题都急需解决。
人体的肿瘤有很多种，人体的大部分组织都有肿瘤的发生，而不同类型的肿瘤又分为众多的亚型，尤其是近年来获益于肿瘤分子生物学领域的发展，肿瘤的分类更加细化。对于不同类型、不同分期或不同分子亚型的肿瘤，其治疗方案也截然不同。
随着人们对肿瘤认识的加深以及科学技术的进步，许多新型的肿瘤标记物被发现并用于临床诊断。1980年之前，肿瘤标记物主要是一些激素、酶类、蛋白质等细胞分泌物，例如癌胚抗原(CEA)、甲胎抗原(AFP)等可以作为肝癌胃癌等多种肿瘤的标记物，糖类抗原125(CA125)可以作为宫颈癌的标记物，前列腺特异性抗原(PSA)可作为前列腺癌标记物，目前这一类肿瘤标记物虽然临床仍在用，但其敏感性与准确性已难以满足临床需求。
液体活检技术是以血中循环肿瘤细胞或循环肿瘤DNA为检测靶标对肿瘤进行诊断和预测的技术。该技术目前还处于起步阶段，存在诸多不足：首先，敏感性与特异性不够高，肿瘤本身有很大的异质性，包含多种亚型的细胞群，而临床样本尤其是血液样本，肿瘤DNA所占比例非常小，现有的肿瘤标记物难以满足临床要求的敏感性，在临床容易造成误诊；其次，一种标记物只针对一种或少数几种肿瘤有较好效果，而血液中的DNA来源非常复杂，因此现有的肿瘤标记物无法应对复杂的肿瘤来源、转移等问题。由于这些复杂情况的存在，使得很多DNA甲基化肿瘤标记物在应用于临床时难以有统一的使用标准，严重影响标记物的敏感度以及准确性。人体 的肿瘤有很多种，不同的肿瘤既有特性又有共性，如果能找到一种不同肿瘤共同的标记物，对于肿瘤的筛查、诊断、治疗以及疗效判定等等方面意义重大。
因此，肿瘤诊断领域亟待开发具有普适性的，易于判断且准确性高的新型肿瘤标志物。
发明内容
本发明的目的在于提供以DNA甲基化修饰作为肿瘤标记物，利用肿瘤中特异性位点异常高甲基化现象来检测肿瘤的方法。
在本发明的第一方面，提供分离的多核苷酸，包括：(a)SEQ ID NO:1所示核苷酸序列的多核苷酸；(b)SEQ ID NO:2所示核苷酸序列的多核苷酸；(c)上述(a)～(b)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～45个，更具体如3，5，10，15，20，25，30，40个)；(d)与上述(a)～(c)的多核苷酸或片段互补的核酸(如SEQ ID NO:5或SEQ ID NO:6所示核苷酸序列的多核苷酸)。
在一个优选例中，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰。
在本发明的第二方面，提供分离的多核苷酸，其由所述的多核苷酸转变而来，对应于前述第一方面的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T或U。
在一个优选例中，其由对应于前述第一方面的多核苷酸经过亚硫酸氢盐或重亚硫酸氢盐处理转变而来。在另一优选例中，所述的多核苷酸包括：(e)SEQ ID NO:3或SEQ ID NO:7所示核苷酸序列的多核苷酸；(f)SEQ ID NO:4或SEQ ID NO:8所示核苷酸序列的多核苷酸；(g)上述(e)～(f)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～45个，更具体如3，5，10，15，20，25，30，40个)。
在本发明的第三方面，提供前述第一方面或第二方面所述的多核苷酸的用途，用于制备肿瘤的检测试剂或试剂盒。
在一个优选例中，所述肿瘤包括(但不限于)：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在另一优选例中，所述肿瘤的样本包括：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组 织及细胞活检样本。
在本发明的第四方面，提供一种制备肿瘤检测试剂的方法，所述方法包括：提供前述第一方面或第二方面所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个(如2～45个，更具体如3，5，10，15，20，25，30，40个)修饰的CpG位点；较佳地，所述的检测试剂包括(但不限于)：引物，探针。
在本发明的第五方面，提供试剂或组合的试剂，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面或第二方面任一所述的多核苷酸的全长或片段，其中存在至少1个(如2～45个，更具体如3，5，10，15，20，25，30，40个)修饰的CpG位点。
在一个优选例中，所述的试剂或组合的试剂针对包含所述靶序列的基因序列(基于所述基因序列而设计)，所述的基因序列包括基因Panel或基因群组。
在另一优选例中，所述的检测试剂包括：引物，探针。
在另一优选例中，所述的引物为：SEQ ID NO:5和6所示的引物。
在本发明的第六方面，提供本发明的第五方面所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括(但不限于)：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在本发明的第七方面，提供一种检测试剂盒，其包括：容器，以及位于容器中的前面所述的试剂或试剂组合；较佳地，每一种试剂位于一个独立的容器中。
在另一优选例中，所述的试剂盒中还包括：亚硫酸氢盐或重亚硫酸氢盐，DNA纯化试剂，DNA提取试剂，PCR扩增试剂和/或使用说明书(标明检测操作步骤和结果判定标准)。
在本发明的第八方面，提供一种体外检测样品的甲基化谱式的方法，包括：(i)提供样品，提取核酸；(ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面所述的多核苷酸或由其转变而来的前述第二方面所述的多核苷酸。
在一个优选例中，步骤(3)中，分析的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR、或它们的组合以及SEQ ID NO.1所示序列中部分或全 部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。并且，其它其他甲基化检测方法及未来新开发的甲基化检测方法也可被应用于本发明中。
在另一优选例中，步骤(ii)包括：(1)对(i)的产物进行处理，使其中未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的核酸；(2)分析经(1)处理的核酸中所述的靶序列的CpG位点的修饰情况。
在另一优选例中，所述的甲基化谱式异常是指该多核苷酸CpG中的C发生高度甲基化。
在另一优选例中，所述的甲基化谱式的方法不以直接获得疾病的诊断结果为目的，或不是诊断性的方法。
在本发明的第九方面，提供一种肿瘤诊断试剂盒，包括利用本发明的第一方面或第二方面所示序列设计的引物对以及包含该序列的基因Panel或基因群组，通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、临床获取20对癌旁-肺癌样本，癌旁样本作为肺癌对照组，肺癌样本作为肺癌实验组，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图2、临床获取10例结直肠癌癌旁样本作为对照组，30例结直肠癌样本作为实验组，比较结直肠癌对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图3、临床获取10例正常胃(或胃炎)样本作为对照组，10例胃癌手术切沿样本作为实验组1，获取20例胃癌样本作为实验组2，比较三组样本中STAMP-EP2甲基化值。
图4、临床获取10例结直肠癌癌旁样本作为对照组，30例结直肠癌样本作为实验组，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图5、临床获取10例正常胃(或胃炎)样本作为对照组，10例胃癌手术切沿样本作为实验组1，20例胃癌样本作为实验组2，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图6、临床获取20对癌旁-乳腺癌样本，癌旁样本作为乳腺癌对照组，乳腺癌样本作为乳腺癌实验组，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异 性和敏感性(右图)。
图7、临床获取18对癌旁-胰腺癌样本，癌旁样本作为胰腺癌对照组，胰腺癌样本作为胰腺癌实验组，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图8、临床获取11对癌旁-头颈部癌样本，包括5例喉癌、2例扁桃体癌、2例会厌癌、1例舌根癌、1例下咽癌，癌旁样本作为对照，癌组织作为实验组，比较头颈部癌对照组与实验组STAMP-EP2甲基化值。
图9、临床获取10例非癌患者胆汁样本，10例胆囊癌患者胆汁样本，非癌样本作为胆囊癌对照组，胆囊癌样本作为胆囊癌实验组，比较对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图10、临床获取10例非白血病骨髓涂片样本作为对照组，20例白血病骨髓涂片样本作为实验组，比较白血病对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图11、临床获取8例肾癌癌旁对照样本作为对照组，14例肾癌样本作为实验组，比较肾癌对照组与实验组STAMP-EP2甲基化值(左图)及检测特异性和敏感性(右图)。
图12、临床获取5例膀胱癌癌旁样本与非癌尿液样本作为对照组，获取7例膀胱癌组织样本与膀胱癌尿液样本作为实验组，比较膀胱癌对照组与实验组STAMP-EP2甲基化值。
图13、采集20例正常人血浆作为对照组，以及不同肿瘤类型患者血浆样本包括10例肝癌血浆样本、10例胰腺癌血浆样本、10例肺癌血浆样本、10例结直肠癌血浆样本、10例乳腺癌血浆样本，比较各组的STAMP-EP2甲基化值。
图14、CpG位点在肿瘤细胞系与非肿瘤细胞系的甲基化差异。其中深色方格相应位点呈现“甲基化”，而浅色方格表示相应位点呈现“非甲基化”。
具体实施方式
本发明人致力于肿瘤标志物的研究，经过广泛的研究筛选，提供一种通用型的DNA甲基化肿瘤标志物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)，在正常的组织中STAMP处于低甲基化状态，在肿瘤组织中STAMP呈高甲基化状态，可用于临床肿瘤的检测，以及用于作为设计肿瘤诊断试剂的基础。
术语
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离 纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“样本”或“样品”包括从任何个体或分离的组织、细胞或体液(如血浆)中获得的、适合于DNA甲基化状态检测的物质。
如本文所用，“高(度)甲基化”是指在一个基因序列中CpG存在高度甲基化、羟甲基化、醛甲基化或羧甲基化修饰。例如，以甲基化特异PCR(MSP)分析手段而言，以甲基化特异性引物所进行的PCR反应可获得阳性的PCR结果即可认为该受试的DNA(基因)区处于高甲基化状态。例如，以实时定量甲基化特异性PCR而言，高甲基化状态的判定可根据其对照样品的甲基化状态的相对值分析统计学差异。
如本文所用，所述的肿瘤包括但不限于：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
基因标志物
为了寻找对于诊断肿瘤有用的靶标，本发明人经过了广泛而深入的研究，确定了STAMP-EP2这一靶标。STAMP-EP2基因序列区域的甲基化状态在肿瘤组织和非肿瘤组织之间存在显著的差异，只要检测到其中一个上述基因的启动子区域发生异常的甲基化状态(高度甲基化)，即可判定该受检者为肿瘤高危人员。并且，STAMP-EP2呈现的这种在在肿瘤组织和非肿瘤组织之间存的显著差异广谱地存在于各种种类的肿瘤中，包括实体瘤以及非实体瘤。
因此，本发明提供了分离的多核苷酸，所述的多核苷酸来自于人基因组，具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5(为SEQ ID NO:1的反向互补序列)或SEQ ID NO:6(为SEQ ID NO:2的反向互补序列)所示的核苷酸序列，在肿瘤患者的肿瘤细胞内，该多核苷酸序列中，多处5’-CpG-3’的碱基C位置上，生成5-甲基胞嘧啶(5mC)。本发明也包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5或SEQ ID NO:6所示核苷酸序列的多核苷酸的片段，且其中存在至少1个(如2～45个，更具体如3，5，10，15，20，25，30，40个)甲基化CpG位点。上述的多核苷酸或片段也可以应用于设计检测试剂或检测试剂盒。
在本发明的一些具体实施例中，所述的多核苷酸的片段例如是：包含SEQ ID NO: 1中第247～305位碱基的片段(包含SEQ ID NO：1中17～27号CpG位点)；包含SEQ ID NO:2中第249～307位碱基的片段(含有SEQ ID NO：2中18～28号CpG位点)。上述片段的反义链也是可用的。并且，这些片段是本发明的较佳实施方式的举例，根据本发明提供的信息，也可以选择其它的片段。
此外，包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5或SEQ ID NO:6所示核苷酸序列或序列片段的基因Panel或基因群组也被包含在本发明中。针对所述的基因Panel或基因群组，也可以通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
上述的多核苷酸可以作为基因组中人们分析甲基化状态的关键区域，通过各种本领域已知的技术来分析它们的甲基化状态。任何可用于分析甲基化状态的技术均可被应用于本发明中。
上述的多核苷酸在经过亚硫酸氢盐或重亚硫酸氢盐处理后，其中未发生甲基化的胞嘧啶转化为尿嘧啶，而发生甲基化的胞嘧啶保持不变。
因此，本发明还提供了上述多核苷酸经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸，包括：SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:7或SEQ ID NO:8所示核苷酸序列的多核苷酸。这些多核苷酸也可以应用于设计检测试剂或检测试剂盒。
本发明也包含上述多核苷酸或其反义链经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸的片段，且其中存在至少1个甲基化CpG位点。
检测试剂及试剂盒
基于本发明的新发现，还提供了基于所述的多核苷酸序列设计的检测试剂，用于体外检测样品中多核苷酸的甲基化谱式。本领域已知的确定基因组的序列及甲基化状态的检测方法和试剂均可被应用于本发明中。
因此，本发明提供了一种制备肿瘤检测试剂的方法，包括：提供所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的检测试剂；其中，所述的靶序列中存在至少1个甲基化CpG位点。
本发明所述的检测试剂包括但不限于：引物，探针，等等。
所述的试剂例如是引物对，在得知了多核苷酸的序列后，设计引物是本领域技术人员已知的，两个引物在将被扩增的目标基因特定序列的两侧(包含CpG序列在内，与其中CpG互补为针对原为甲基化的基因区，而与其中TpG互补为针对原为去甲基化的基因区)。应理解，根据本发明的新发现，针对所述靶序列上的不同位置的CpG 位点或其组合，本领域技术人员可以设计出多种引物或探针或其它类型的检测试剂，这些均应被包含在本发明的技术方案中。
所述的试剂也可以是试剂组合(引物组合)，包括多于一组的引物，从而可分别扩增上述的多条多核苷酸。
本发明还提供了体外检测样品中多核苷酸的甲基化谱式的试剂盒，该试剂盒包括：容器，以及位于容器中的上述引物对。
此外，所述的试剂盒中还可包括用于提取DNA、DNA纯化、PCR扩增等所需的各种试剂。
此外，所述的试剂盒中还可包括使用说明书，其中标明检测操作步骤和结果判定标准，以便于本领域技术人员应用。
检测方法
测定多核苷酸的甲基化谱式可通过已有的技术(如甲基化特异性PCR(MSP)或实时定量甲基化特异性PCR，Methylight)来进行，或其它仍在发展中和将被开发出来的技术来进行。
检测甲基化水平时也可使用定量甲基化特异性PCR(QMSP)的方法。这种方法是基于一种荧光PCR的持续性的光学监控，其较MSP方法更为敏感。其通量高并避免了用电泳方法对其结果进行分析。
其他可用的技术还有：焦磷酸测序法、重亚硫酸盐转化测序法、qPCR法、二代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术或HPLC法以及组合基因群组检测法等该领域常规方法。应理解，在本发明的新揭示的基础上，本领域公知的这些技术以及即将发展的一些技术，均可被应用于本发明中。
作为本发明的优选方式，还提供了一种体外检测样品中多核苷酸的甲基化谱式的方法。所述的方法基于的原理是：亚硫酸氢盐或重亚硫酸氢盐可以将未甲基化的胞嘧啶转化为尿嘧啶，在后续的PCR扩增过程中转变为胸腺嘧啶，而甲基化的胞嘧啶保持不变；因而，经过亚硫酸氢盐或重亚硫酸氢盐处理多核苷酸后，甲基化的位点产生类似于一个C/T的多核苷酸多态性(SNP)。基于上述原理来鉴定检测样品中多核苷酸的甲基化谱式，可以有效区分出甲基化与非甲基化的胞嘧啶。
本发明所述的方法包括：包括：(a)提供样品，提取基因组DNA；(b)利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(a)所述的基因组DNA，从而基因组DNA中未甲基化的胞嘧啶转化为尿嘧啶；(c)分析经步骤(b)处理的基因组DNA中是否存在甲基化谱式异常。
本发明的方法可用于：(i)对受试者样品进行检测，分析受试者是否患有肿瘤；(ii) 区分肿瘤高危人群。所述的方法也可以是不以获得直接的疾病诊断结果为目的的情形。
在本发明的优选实施例中，通过PCR扩增及焦磷酸测序法检测DNA甲基化，本领域人员应理解，实际应用中并不限于该方法，其它DNA甲基化检测方法亦可。在进行PCR扩增中，所应用的引物也不限于是实施例中所提供的。
由于基因组DNA经过重亚硫酸盐处理后，非甲基化的胞嘧啶转变为尿嘧啶，在后续的PCR过程中又转换为胸腺嘧啶，会降低基因组的序列复杂度，使得PCR扩增出特异目标片段的难度增大。因此优选地可采用巢式PCR扩增，设计外围与内围两对引物，进行两轮PCR扩增反应，以第一轮的扩增产物作为第二轮扩增的模板，可以有效提高扩增的效率与特异性。然而应理解，本发明中可用的检测方法并不限于此。
经过针对临床样本的研究验证，本发明的方法用于诊断临床肿瘤时，准确性非常高。本发明可应用于肿瘤辅助诊断、疗效判定、预后监测等等领域，具有很高的临床应用价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、针对STAMP-EP2检测的核酸序列
提供STAMP-EP2肿瘤标记物的序列，如下SEQ ID NO:1(chr5:140797163～140797701(hg19/Human))所示，其中下划线标示碱基为甲基化CpG位点，下划线下面的数字表示该位点的编号。
Figure PCTCN2019096797-appb-000001
上述序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:3：
Figure PCTCN2019096797-appb-000002
提供STAMP-EP2肿瘤标记物的序列，如下SEQ ID NO:2(chr5:140787504～ 140788044(hg19/Human))所示，其中下划线标示碱基为甲基化CpG位点，下划线下面的数字表该该位点的编号；
Figure PCTCN2019096797-appb-000003
上述序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:4：
Figure PCTCN2019096797-appb-000004
上述SEQ ID NO:1所示核苷酸序列的反向互补序列如下SEQ ID NO:5：
Figure PCTCN2019096797-appb-000005
上述SEQ ID NO:2所示核苷酸序列的反向互补序列如下SEQ ID NO:6：
Figure PCTCN2019096797-appb-000006
上述SEQ ID NO:5序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:4(其中Y代表C或U)：
Figure PCTCN2019096797-appb-000007
上述SEQ ID NO:6序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:8(其中Y代表C或U)：
Figure PCTCN2019096797-appb-000008
实施例2、STAMP-EP2：肺癌-临床病例样本验证-焦磷酸测序法
1.获取临床样本：从临床获取20对癌旁-肺癌样本，癌旁样本作为肺癌对照组，肺癌样本20例作为肺癌实验组；
2.DNA提取：分别提取实验组和对照组DNA；本实验用酚氯仿抽提法，但不限于该方法；
3.重亚硫酸盐处理：以重亚硫酸盐处理提取的DNA样本，严格按照步骤操作；本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，但不限于该试剂盒；
4.引物设计：根据STAMP-EP2序列SEQ ID NO：1与SEQ ID NO：2特点，设计PCR扩增引物与焦磷酸测序引物，由于SEQ ID NO：1与SEQ ID NO：2有比较大的相似性，本实验设计引物可同时检测SEQ ID NO：1中17、18、19、20、21、22、23、24、25、26、27号CpG位点与SEQ ID NO：2中18、19、20、21、22、23、24、25、26、27、28号CpG位点甲基化。后续通过焦磷酸测序法检测SEQ ID NO：1中17、18、19、20、21、22、23、24、25、26、27号CpG位点与SEQ ID NO：2中18、19、20、21、22、23、24、25、26、27、28号CpG位点的甲基化值，作为STAMP-EP2甲基化值的代表，PCR引物扩增序列、焦磷酸测序引物序列、焦磷酸测序上机检测序列以及检测位点如表1所示；
表1
Figure PCTCN2019096797-appb-000009
5.PCR扩增及琼脂糖凝胶电泳：以重亚硫酸盐处理后的样本作为PCR的产物，进行PCR扩增，扩增后的产物通过琼脂糖凝胶电泳鉴定PCR扩增的特异性，扩增片段大小应为143bp；
6.焦磷酸测序：通过QIAGEN公司的Pyro Mark Q96ID焦磷酸测序仪进行检测，严格按照说明书步骤进行操作；
7.STAMP-EP2甲基化值计算：焦磷酸测序可以检测出目标区域SEQ ID NO：1中17、18、19、20、21、22、23、24、25、26、27号CpG位点与SEQ ID NO：2中18、19、20、21、22、23、24、25、26、27、28号CpG位点的甲基化情况，计算平均值作为STAMP-EP2在该样本中的甲基化值；
8.结果分析：比较肺癌对照组与实验组STAMP-EP2甲基化值，如图1，结果显 示，在肺癌临床样本中，STAMP-EP2在肺癌实验组中甲基化值显著增高，P<0.0001，检测敏感性95％，特异性100％。
实施例2、STAMP-EP2：结直肠癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取10例结直肠癌癌旁样本作为对照组，获取30例结直肠癌样本作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较结直肠癌对照组与实验组STAMP-EP2甲基化值，如图2，结果显示在结直肠癌临床样本中，STAMP-EP2在结直肠癌实验组中甲基化值显著增高，P<0.0001,检测敏感性93.33％，特异性100％。
实施例3、STAMP-EP2：胃癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取10例正常胃(或胃炎)样本作为对照组，获取10例胃癌手术切沿样本作为实验组1，获取20例胃癌样本作为实验组2；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较正常胃(或胃炎)对照组与手术切沿实验组1以及胃癌实验组2三组样本中STAMP-EP2甲基化值，如图3，结果显示在胃癌临床样本中，STAMP-EP2在胃癌实验组2中甲基化值显著增高，P<0.0001。同时，手术切沿组实验组1的甲基化情况介于正常胃(或胃炎)对照组与胃癌实验组2之间，说明STAMP-EP2甲基化在手术切沿位置已开始有变化，STAMP-EP2作为胃癌标记物，一方面可用于胃癌检测，另一方面可以用于胃癌手术切沿判定。
实施例4、STAMP-EP2：宫颈癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取12例宫颈癌癌旁样本作为对照组，获取16例宫颈癌样本作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较宫颈癌对照组与实验组STAMP-EP2甲基化值，如图4，结果显示在宫颈癌临床样本中，STAMP-EP2在宫颈癌实验组中甲基化值显著增高，P<0.0001，检测敏感性93.33％，特异性100％。
实施例5、STAMP-EP2：肝癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取22对癌旁-肝癌样本，癌旁样本作为肝癌对照组，肝癌样本作为肝癌实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较肝癌对照组与实验组STAMP-EP2甲基化值，如图5，结果显示在肝癌临床样本中，STAMP-EP2在肝癌实验组中甲基化值显著增高，P<0.0001，检测敏感性100％，特异性100％。
实施例6、STAMP-EP2：乳腺癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取20对癌旁-乳腺癌样本，癌旁样本作为乳腺癌对照组，乳腺癌样本作为乳腺癌实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较乳腺癌对照组与实验组STAMP-EP2甲基化值，如图6，结果显示在乳腺癌临床样本中，STAMP-EP2在乳腺癌实验组中甲基化值显著增高，P<0.0001，检测敏感性100％，特异性100％。
实施例7、STAMP-EP2：胰腺癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取18对癌旁-胰腺癌样本，癌旁样本作为胰腺癌对照组，胰腺癌样本作为胰腺癌实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较胰腺癌对照组与实验组STAMP-EP2甲基化值，如图7，结果显示在胰腺癌临床样本中，STAMP-EP2在胰腺癌实验组中甲基化值显著增高，P<0.0001，检测敏感性88.9％，特异性100％。
实施例8、STAMP-EP2：头颈部癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取11对癌旁-头颈部癌样本，包括5例喉癌、2例扁桃体癌、2例会厌癌、1例舌根癌、1例下咽癌，癌旁样本作为对照，癌组织作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较头颈部癌对照组与实验组STAMP-EP2甲基化值，如图8，结果显示在头颈部癌临床样本中，STAMP-EP2在头颈部癌实验组中甲基化值显著增高，P<0.0001，检测敏感性100％，特异性100％。
实施例9、STAMP-EP2：胆囊癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取10例非癌患者胆汁样本，10例胆囊癌患者胆汁样本，非癌样本作为胆囊癌对照组，胆囊癌样本作为胆囊癌实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较胆囊癌对照组与实验组STAMP-EP2甲基化值，如图9，结果 显示在胆囊癌临床样本中，STAMP-EP2在胆囊癌实验组中甲基化值显著增高，P<0.0001，检测敏感性90％，特异性100％。
实施例10、STAMP-EP2：白血病-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取10例非白血病骨髓涂片样本作为对照组，获取20例白血病骨髓涂片样本作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较白血病对照组与实验组STAMP-EP2甲基化值，如图10，结果显示在白血病临床样本中，STAMP-EP2在白血病实验组中甲基化值显著增高，P<0.0001，检测敏感性100％，特异性100％。
实施例11、STAMP-EP2：肾癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取8例肾癌癌旁对照样本作为对照组，获取14例肾癌样本作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较肾癌对照组与实验组STAMP-EP2甲基化值，如图11，结果显示在肾癌临床样本中，STAMP-EP2在肾癌实验组中甲基化值显著增高，P<0.0001，检测敏感性92.86％，特异性100％。
实施例12、STAMP-EP2：膀胱癌-临床样本验证-焦磷酸测序法
1.获取临床样本：从临床获取5例膀胱癌癌旁样本与非癌尿液样本作为对照组，获取7例膀胱癌组织样本与膀胱癌尿液样本作为实验组；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：比较膀胱癌对照组与实验组STAMP-EP2甲基化值，如图12，结果显示在膀胱癌临床样本中，STAMP-EP2在膀胱癌实验组中甲基化值显著增高。
实施例13、STAMP-EP2，血浆样本-临床样本验证
1.为了证明SATMP-C肿瘤标记物同样可通过液体活检的方式进行检测，本发明人采集20例正常人血浆作为对照组，以及不同肿瘤类型患者血浆样本包括10例肝癌血浆样本、10例胰腺癌血浆样本、10例肺癌血浆样本、10例结直肠癌血浆样本、10例乳腺癌血浆样本；
2.步骤2.3.4.5.6.7同实施例2中步骤；
8.结果分析：如图13所示，与正常人血浆对照相比，在肝癌、胰腺癌、肺癌、结直肠癌以及乳腺癌组中，STAMP-EP2甲基化值均显著增高。
实施例14、STAMP-EP2：CpG位点在肿瘤细胞系与非肿瘤细胞系的甲基化差异
1.提取肺癌细胞系HepG2与正常肺细胞系基因组DNA；
2.分别用重亚硫酸盐处理提取的HepG2与正常肺细胞系基因组DNA，作为后续PCR扩增的模板；
3.根据SEQ ID NO:1和2的序列设计扩增引物，针对不同序列区域，常规方法设计引物进行扩增。
4.PCR扩增之后，2％琼脂糖凝胶电泳检测PCR片段特异性，切胶回收目的片段，连接插入T载体，转化感受态大肠杆菌，涂菌板，第二天挑克隆测序，每个片段挑取10个克隆进行Sanger测序；
结果如图14，显示SEQ ID N O:1区域在肝正常细胞系中平均甲基化值为3.0％，在肝癌细胞系HepG2中平均甲基化值为78.3％，肝癌细胞系甲基化水平显著高于肝正常细胞系。SEQ ID NO:2区域在肝正常细胞系中平均甲基化值为1.8％，在肝癌细胞系HepG2中平均甲基化值为78.7％，肝癌细胞系甲基化水平显著高于肝正常细胞系。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 分离的多核苷酸，其特征在于，包括：
    (a)SEQ ID NO:1所示核苷酸序列的多核苷酸；
    (b)SEQ ID NO:2所示核苷酸序列的多核苷酸；
    (c)上述(a)～(b)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点；
    (d)与上述(a)～(c)的多核苷酸或片段互补的核酸。
  2. 如权利要求1所述的分离的多核苷酸，其特征在于，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰。
  3. 分离的多核苷酸，其特征在于，其由权利要求1或2所述的多核苷酸转变而来，对应于权利要求1的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T或U。
  4. 如权利要求3所述的多核苷酸，其特征在于，包括：
    (e)SEQ ID NO:3或SEQ ID NO:7所示核苷酸序列的多核苷酸；
    (f)SEQ ID NO:4或SEQ ID NO:8所示核苷酸序列的多核苷酸；
    (g)上述(e)～(f)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点。
  5. 权利要求1～4任一所述的多核苷酸的用途，其特征在于，用于制备肿瘤的检测试剂或试剂盒。
  6. 如权利要求5所述的用途，其特征在于，所述肿瘤包括：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  7. 如权利要求5或6所述的应用，其特征在于，所述肿瘤的样本包括：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
  8. 一种制备肿瘤检测试剂的方法，其特征在于，所述方法包括：提供权利要求1～4任一所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个修饰的CpG位点。
  9. 试剂或组合的试剂，其特征在于，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是权利要求1～4任一所述的多核苷酸的全长或片段，其中存在至少1 个修饰的CpG位点。
  10. 如权利要求9所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试剂针对包含所述靶序列的基因序列，所述的基因序列包括基因Panel或基因群组。
  11. 如权利要求9所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试剂包括：引物或探针；较佳地，所述的引物为：SEQ ID NO:5和6所示的引物。
  12. 权利要求9～11任一所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  13. 一种检测试剂盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求9～11任一所述的试剂或试剂组合。
  14. 一种体外检测样品甲基化谱式的方法，其特征在于，包括：
    (i)提供样品，提取核酸；
    (ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是权利要求1或2所述的多核苷酸或由其转变而来的权利要求3或4所述的多核苷酸。。
  15. 如权利要求14所述的方法，其特征在于，步骤(3)中，分析的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、MassArray、甲基化特异PCR、或它们的组合以及SEQ ID NO.1所示序列中部分或全部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。
  16. 如权利要求14所述的方法，其特征在于，步骤(ii)包括：
    (1)对(i)的产物进行处理，使其中未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的核酸；
    (2)分析经(1)处理的核酸中所述的靶序列的修饰情况。
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