CN113388685A - Methylation marker for diagnosing esophageal cancer - Google Patents
Methylation marker for diagnosing esophageal cancer Download PDFInfo
- Publication number
- CN113388685A CN113388685A CN202110905091.3A CN202110905091A CN113388685A CN 113388685 A CN113388685 A CN 113388685A CN 202110905091 A CN202110905091 A CN 202110905091A CN 113388685 A CN113388685 A CN 113388685A
- Authority
- CN
- China
- Prior art keywords
- methylation
- samples
- coefficient
- esophageal
- esophageal cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000011987 methylation Effects 0.000 title claims abstract description 74
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 74
- 208000000461 Esophageal Neoplasms Diseases 0.000 title claims abstract description 35
- 206010030155 Oesophageal carcinoma Diseases 0.000 title claims abstract description 35
- 201000004101 esophageal cancer Diseases 0.000 title claims abstract description 35
- 239000003550 marker Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 22
- 238000003745 diagnosis Methods 0.000 claims description 20
- 210000001519 tissue Anatomy 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 9
- 238000012164 methylation sequencing Methods 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 5
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 4
- 238000007855 methylation-specific PCR Methods 0.000 claims description 4
- 210000003296 saliva Anatomy 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 208000036764 Adenocarcinoma of the esophagus Diseases 0.000 claims description 2
- 206010003445 Ascites Diseases 0.000 claims description 2
- 206010030137 Oesophageal adenocarcinoma Diseases 0.000 claims description 2
- 208000002151 Pleural effusion Diseases 0.000 claims description 2
- 206010036790 Productive cough Diseases 0.000 claims description 2
- 238000011529 RT qPCR Methods 0.000 claims description 2
- 210000000941 bile Anatomy 0.000 claims description 2
- 238000001574 biopsy Methods 0.000 claims description 2
- 238000001369 bisulfite sequencing Methods 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 238000004364 calculation method Methods 0.000 claims description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000007847 digital PCR Methods 0.000 claims description 2
- 208000028653 esophageal adenocarcinoma Diseases 0.000 claims description 2
- 201000002726 esophagus leiomyosarcoma Diseases 0.000 claims description 2
- 201000005621 esophagus lymphoma Diseases 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 208000037819 metastatic cancer Diseases 0.000 claims description 2
- 208000011575 metastatic malignant neoplasm Diseases 0.000 claims description 2
- 238000012175 pyrosequencing Methods 0.000 claims description 2
- 238000007790 scraping Methods 0.000 claims description 2
- 208000024794 sputum Diseases 0.000 claims description 2
- 210000003802 sputum Anatomy 0.000 claims description 2
- 238000007671 third-generation sequencing Methods 0.000 claims description 2
- 210000002700 urine Anatomy 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 description 18
- 201000011510 cancer Diseases 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 11
- 230000007067 DNA methylation Effects 0.000 description 8
- 238000012549 training Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 108091029523 CpG island Proteins 0.000 description 5
- 208000036765 Squamous cell carcinoma of the esophagus Diseases 0.000 description 5
- 208000007276 esophageal squamous cell carcinoma Diseases 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000001973 epigenetic effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091029430 CpG site Proteins 0.000 description 3
- 208000019505 Deglutition disease Diseases 0.000 description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- 206010061876 Obstruction Diseases 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000007637 random forest analysis Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 101100495925 Schizosaccharomyces pombe (strain 972 / ATCC 24843) chr3 gene Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000621 bronchi Anatomy 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 230000035622 drinking Effects 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 230000009747 swallowing Effects 0.000 description 2
- 210000003437 trachea Anatomy 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 102100024387 AF4/FMR2 family member 3 Human genes 0.000 description 1
- 208000008035 Back Pain Diseases 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010008479 Chest Pain Diseases 0.000 description 1
- 102100027995 Collagenase 3 Human genes 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010013952 Dysphonia Diseases 0.000 description 1
- 206010016717 Fistula Diseases 0.000 description 1
- 108091005772 HDAC11 Proteins 0.000 description 1
- 102100039385 Histone deacetylase 11 Human genes 0.000 description 1
- 208000010473 Hoarseness Diseases 0.000 description 1
- 102100029426 Homeobox protein Hox-C10 Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000833166 Homo sapiens AF4/FMR2 family member 3 Proteins 0.000 description 1
- 101000855412 Homo sapiens Carbamoyl-phosphate synthase [ammonia], mitochondrial Proteins 0.000 description 1
- 101000577887 Homo sapiens Collagenase 3 Proteins 0.000 description 1
- 101000989027 Homo sapiens Homeobox protein Hox-C10 Proteins 0.000 description 1
- 101001022948 Homo sapiens LIM domain-binding protein 2 Proteins 0.000 description 1
- 101000983292 Homo sapiens N-fatty-acyl-amino acid synthase/hydrolase PM20D1 Proteins 0.000 description 1
- 101000624960 Homo sapiens Nesprin-3 Proteins 0.000 description 1
- 101001133605 Homo sapiens Parkin coregulated gene protein Proteins 0.000 description 1
- 101000861263 Homo sapiens Steroid 21-hydroxylase Proteins 0.000 description 1
- 101000626703 Homo sapiens YEATS domain-containing protein 2 Proteins 0.000 description 1
- 101000760251 Homo sapiens Zinc finger protein 578 Proteins 0.000 description 1
- 101000988419 Homo sapiens cAMP-specific 3',5'-cyclic phosphodiesterase 4D Proteins 0.000 description 1
- 208000016495 Horner Syndrome Diseases 0.000 description 1
- 241000521257 Hydrops Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 102100035113 LIM domain-binding protein 2 Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100026873 N-fatty-acyl-amino acid synthase/hydrolase PM20D1 Human genes 0.000 description 1
- 208000012266 Needlestick injury Diseases 0.000 description 1
- 102100023307 Nesprin-3 Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 102100034314 Parkin coregulated gene protein Human genes 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091006255 SLC8A3 Proteins 0.000 description 1
- 102100032576 Sodium/calcium exchanger 3 Human genes 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100024781 YEATS domain-containing protein 2 Human genes 0.000 description 1
- 102100024722 Zinc finger protein 578 Human genes 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000001680 brushing effect Effects 0.000 description 1
- 102100029170 cAMP-specific 3',5'-cyclic phosphodiesterase 4D Human genes 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000013502 data validation Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000003890 fistula Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 235000021059 hard food Nutrition 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 210000002416 recurrent laryngeal nerve Anatomy 0.000 description 1
- 101150054338 ref gene Proteins 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000000331 sympathetic ganglia Anatomy 0.000 description 1
- 230000037426 transcriptional repression Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the fields of biotechnology and biomedicine, in particular to a group of methylation markers for diagnosing esophageal cancer; the methylation marker combination comprises at least one of the following methylation sites: cg05446471, cg10085326, cg11926456, cg12126990, cg13480465, cg19310604, cg20473977, cg21041579, cg21553182, cg24276395, cg25724842, cg 26033932.
Description
Technical Field
The invention relates to the fields of biotechnology and biomedicine, in particular to a group of methylation markers for diagnosing esophageal cancer.
Background
DNA methylation is one of the most studied epigenetic regulatory systems in mammals, and the pattern of writing, reading and maintaining such covalent epigenetic markers has been fully elucidated. CpG island (CGI) is an extension of the DNA sequence rich in CpG dinucleotides, and is present in most mammalian promoter regions, and the regulatory relationship between methylation and gene expression was preliminarily analyzed through studies on DNA methylation at these positions. Methylation of CpG islands often leads to long-term stable gene silencing, however, DNA methylation patterns located outside the promoter region were poorly studied in a variety of dynamic biological processes before corresponding epigenetic research approaches were developed. Currently, studies relating to genome-wide methylation profiles have fully revealed dynamic alterations in DNA methylation in enhancers, genomes, and partially methylated regions, suggesting a regulatory role in ontogeny and disease processes. Although DNA methylation has been widely recognized as an inhibitory epigenetic marker, the regulatory mechanisms underlying its mediated transcriptional repression have not been fully elucidated. Some studies have shown that DNA methylation-mediated transcriptional regulation may be involved in its interaction with sequence-specific transcription factors.
Esophageal cancer is a common tumor of the digestive tract, and about 30 million people die of esophageal cancer every year worldwide. The morbidity and mortality varies greatly from country to country. China is one of the high-incidence areas of esophageal cancer in the world, and the average death rate of people is about 15 ten thousand every year. More men than women, the onset age is usually over 40 years. Early symptoms of esophageal cancer are often not evident, but there can be varying degrees of discomfort when swallowing coarse and hard food, including dysphagia, post-sternal burning, needle stick, or traction and friction-like pain. The food passes slowly and has a feeling of stagnation or foreign body sensation. Dysphagia and stasis are usually relieved and eliminated by swallowing water. The symptoms are mild, severe and slow in progression. The typical symptoms of esophageal cancer in the middle and late stages are progressive dysphagia, i.e., hard dry food, semifluid food, and water and saliva which cannot be swallowed. Mucus-like phlegm is usually spitted, and is the secretion of the saliva and esophagus of the lower pharynx. The patient gradually becomes emaciated, dehydrated and powerless. Persistent chest or back pain is indicated as late stage symptoms, with cancer having invaded the extra-esophageal tissues. When inflammatory edema caused by cancer and obstruction subsides temporarily or part of cancer and obstruction subsides, the obstruction symptoms can be relieved temporarily, and the condition is often mistakenly considered to be improved. Hoarseness can occur if cancer invades the recurrent laryngeal nerve; horner syndrome can develop if the cervical sympathetic ganglion is compressed; if the liquid invades the trachea and the bronchus, esophagus, trachea or bronchus fistula can be formed, severe choking cough can occur when water or food is swallowed, and respiratory system infection can occur. Finally, a cachexia state occurs. If there is metastasis of liver, brain and other organs, jaundice, hydrops in abdominal cavity, coma and other states may occur.
China starts the research of prevention and treatment of esophageal cancer at the end of the 50 s of the 20 th century, and prevention and treatment research points are established in rural areas in high incidence areas. The method adopts propaganda and education and applies an esophageal cytology diagnosis method to carry out general investigation on population in high-incidence areas so as to find out early detection and early treatment and improve the cure rate.
Disclosure of Invention
Diagnostic marker combinations
In one aspect, the present invention provides a methylation marker combination for diagnosing esophageal cancer, wherein the methylation marker combination comprises at least one of the following methylation sites: cg05446471, cg10085326, cg11926456, cg12126990, cg13480465, cg19310604, cg20473977, cg21041579, cg21553182, cg24276395, cg25724842, cg 26033932.
Preferably, the methylation marker combination is a combination of the following methylation sites: cg05446471, cg10085326, cg11926456, cg12126990, cg13480465, cg19310604, cg20473977, cg21041579, cg21553182, cg24276395, cg25724842 and cg 26033932; in particular, the degree of methylation of the above methylation sites in patients and healthy subjects was different (FDR value < 0.05).
Preferably, the difference comprises a degree of methylation in the patient that is at least 1-fold that in a healthy person, or a degree of methylation in the patient that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% that in a healthy person.
Preferably, the at least 1 fold includes 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, or 3.5 fold or more.
In one embodiment of the invention, esophageal cancer is diagnosed by evaluating the methylation degree of at least one marker in the methylation marker combination in a subject, and the diagnosis comprises determining whether the subject is ill, risk of ill, probability of ill, course of ill, or type of ill. The subject may be diagnosed with esophageal cancer and may exhibit one or more symptoms of esophageal cancer. Alternatively, the subject may be healthy. It should be understood that the term "healthy" as used herein is relative to the esophageal cancer status, and "healthy" is not defined as any absolute assessment or status.
The term "subject" as used herein refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Preferably, the subject described herein is a human.
Preferably, the esophageal cancer includes, but is not limited to, esophageal squamous carcinoma, esophageal adenocarcinoma, esophageal lymphoma, esophageal leiomyosarcoma, and esophageal metastatic cancer.
Preferably, the esophageal cancer is esophageal squamous carcinoma.
Diagnostic system
In another aspect, the invention provides an esophageal cancer diagnosis system, which includes a calculation module for calculating a prediction value of a diagnosis model according to the methylation degree of at least one methylation marker in the aforementioned methylation marker combination.
Preferably, the diagnostic model predicted value is calculated using a LASSO constructed model, and the coefficients at each of the positions are as follows: cg05446471(-4.843834), cg10085326(5.328574), cg11926456(7.431151), cg12126990(0.2826689), cg13480465(0.9789349), cg19310604(-3.254839), cg20473977(2.170933), cg21041579(0.167167), cg21553182(-0.2435546), cg24276395(1.641063), cg25724842(2.531114) and cg26033932 (1.138211).
Preferably, the model constructed using LASSO includes the results of detection of cancerous tissue and tissue adjacent to the cancerous tissue in at least 50 patients; more preferably, at least 90 instances.
Preferably, the detection is methylation sequencing.
More preferably, the detection is methylation sequencing using Illumina 450K methylation chip.
Preferably, the esophageal cancer diagnosis system further comprises at least one of any of the following modules:
1) a detection module for performing methylation detection;
2) a collection module that collects methylation detection results of a subject;
3) and the output module is used for outputting the predicted value of the diagnosis model.
Preferably, the collection module may be a collection module of the prior art, including but not limited to a methylation specific PCR module, a real-time methylation specific PCR module, a PCR module for methylated DNA specific binding proteins, a methylation chip module, a methylation sensitive restriction enzyme module.
Reagent kit
In another aspect, the present invention provides a kit for diagnosing whether a subject has esophageal cancer, the kit comprising reagents for detecting the methylation degree of at least one methylation marker in the aforementioned methylation marker combination;
preferably, the methylation detection reagent comprises a reagent used in any one or more of the following methylation detection methods, including: pyrosequencing, bisulfite conversion sequencing, methylation chip methods, qPCR methods, digital PCR methods, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection methods, simplified bisulfite sequencing techniques, HPLC methods, MassArray, methylation specific PCR (msp), or combinations thereof.
Preferably, the kit further comprises bisulfite or bisulfite, DNA purification reagent, DNA extraction reagent, PCR amplification reagent and instruction for use; the specification designates detection operation steps and result determination criteria.
Preferably, the diagnosis of esophageal cancer in the subject is based on a determination of the extent of methylation in a sample from the subject.
Preferably, the sample from the subject includes, but is not limited to, tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples, and alveolar lavage samples, ascites and lavage samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cytology smear samples, cervical scraping or brushing samples, tissue and cell biopsy samples.
Applications of
In another aspect, the invention provides the application of the methylation marker combination, the esophageal cancer diagnosis model and the kit in preparing products for diagnosing esophageal cancer.
Method
In another aspect, the invention provides a method of diagnosing whether a subject has esophageal cancer, the method comprising calculating a predictive value for a diagnostic model of the subject according to a formula. Whether the disease is affected, the risk of the disease, the probability of the disease, the course of the disease or the type of the disease can be judged according to the predicted value of the diagnosis model.
Implementation of the method and/or system of embodiments of the present invention may include performing or completing selected tasks manually, automatically, or a combination thereof.
Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the present invention, a number of selected tasks could be implemented by hardware, by software, or by firmware, or by a combination thereof using an operating system. For example, a chip or a circuit may be classified according to a hardware category for performing a selected task. As software, selected tasks according to embodiments of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system.
In one embodiment, one or more tasks according to exemplary embodiments of methods and/or systems as described herein may be performed by a data processor, such as a computing platform for executing a plurality of instructions. Optionally, the data processor includes a volatile memory for storing instructions and/or data, and/or a non-volatile memory, such as a magnetic hard disk and/or removable media, for storing instructions and/or data. Optionally, a network connection is also provided. Also optionally, a display and/or a user input device such as a keyboard or mouse is provided.
Drawings
FIG. 1 is a graph of the results of evaluating the diagnostic performance of the model using a training set, a test set, and a TCGA-ESCC data set; a is the result statistics of the training set, B is the result statistics of the test set, C is the result statistics of the TCGA-ESCC data set, D is the ROC curve for the training set diagnosis, E is the ROC curve for the test set diagnosis, and F is the ROC curve for the TCGA-ESCC data set diagnosis.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 data Collection and construction of diagnostic model
Study object
During the period from 2010 to 2014, we collected 91 patients with esophageal squamous carcinoma from the tumor hospital of the Chinese medical academy of sciences and the tumor hospital of Zhejiang province. The local, ethnic, sex, diagnosis age, drinking, smoking, tumor occurrence and clinical stage of all subjects were obtained from the medical records of each patient. The patients in this study group all had known informed consent, and the ethical review committees of the tumor hospital of the Chinese medical academy of sciences and the tumor hospital of Zhejiang province have approved the relevant studies.
We performed ESCC patient clinical staging interpretation on the seventh edition AJCC standard, defining the smoking and drinking status of patients according to the following criteria: persons who smoke <1 cigarette per day and have a duration of <1 year are considered non-smokers, and vice versa; the person who drinks more than or equal to 2 times per week and drinks more than or equal to 1 year is judged to be drunk, and the person who does not drink is judged to be drunk otherwise. We completed patient survival follow-up by: the last time when the study subject was followed by the patient was 2018, 11 months, the admission record of the study subject, the confirmation information provided by the family members and the confirmation information provided by the department related to the patient's household location.
We judge the pathological type of the patient by taking clinical pathological diagnosis report as a standard. None of the study patients had been treated with chemotherapeutic drugs or radiation prior to surgery. After esophageal cancer resection, we selected cancer tissue and paracancerous tissue (5 cm away from the tumor site margin) from each patient for subsequent study. We proceed the grouping and screening of samples according to the strict process, and the distribution of the basic clinical data of the subject selected into patients is shown in Table 1.
TABLE 1 distribution of clinical pathological data of esophageal squamous carcinoma patients
*An upper section: 20-25 cm; middle section: 25-30 cm; the following steps: 30-40 cm.
#Staging of tumor TNM was assessed according to esophageal cancer AJCC, seventh edition.
Tumor cell content identification
First, we obtained cancer and paracancerous tissues cryopreserved at-80 ℃ for patients enrolled; then, using a freezing embedding medium to process unfrozen tissues in time, and carrying out frozen section after the embedding medium is fixed; then, H & E staining was performed according to the laboratory routine procedure, and the stained sections were mounted with neutral gum; finally, we selected more than two pathologists to judge cancer cell content to satisfy the following two rules: (1) cancer cell content in cancer tissue is more than or equal to 70 percent, and (2) cancer cells are not contained in tissues beside the cancer.
Methylation sequencing and analysis
Extracting DNA, and confirming that the DNA can meet the quality requirement of subsequent DNA methylation detection by means of NanoDrop 2000 detection, Qubit detection, electrophoresis and the like. After sulfite transformation of the DNA sample, Methylation sequencing was performed using Illumina 450K Methylation chip (Illumina Human Methylation 450K beacon chip).
The Illumina 450K methylation chip contains 485,512 methylation sites in total, covers 99% of encoding genes, and also comprises other genome positions: (1) 96% CpG islands; (2) sites other than CpG islands; (3) non-CpG sites present in stem cells; (4) sites where there is a difference between normal tissue and various tumor tissues; (5) FANTOM 4 promoter; (6) dnase hypersensitive sites; (7) a miRNA promoter region. The detection accuracy of the 450K chip has been independently verified by two research institutes (Bibikova et al, 2011; Sandoval et al, 2011).
Data analysis and validation
The correlation between the methylation level of a single CpG and the expression level of the gene in which it is located was assessed by Spearman rank correlation analysis. For paired samples, we calculated differential methylation and differential expression levels for cancer and paracancer samples, respectively, and performed methylation-gene expression correlation analysis. Screening criteria: (1) differential methylation of CpG sites at and near cancer (FDR values <0.05,. DELTA.. beta. > 0.2); (2) differential expression of gene expression between carcinoma and paracarcinoma (FDR value <0.05, FC >2 or < 0.5); (3) CpG site methylation is related to the expression of the gene (FDR value is less than 0.05, and correlation coefficient | r | >0.3), and 1034 differential methylation sites which can regulate the expression of the gene are screened out finally.
In order to fully explore the diagnostic efficacy of the DNA methylation marker in esophageal squamous carcinoma, a strict statistical process is constructed to identify the diagnosis marker based on the methylation site of the regulatory gene expression:
(1) 91 patients with esophageal squamous carcinoma were randomly divided into a training set (n-60) and a testing set (n-31) according to a ratio of 2: 1; performing multiple random forest iteration analysis in the training set data based on the screened 1034 differential methylation sites, and removing 1/3 features behind the scores in each iteration process according to the importance scores of the single sites given by a random forest algorithm;
(2) based on 91 characteristics obtained by random forest screening, selecting a LASSO (last Absolute Shrinkage and Selection Operator, LASSO) regression method to construct an ideal diagnosis model on the basis of the existing characteristics;
(3) finally, a diagnosis model containing 12 methylation markers (specific information is shown in table 2) is constructed according to the training set data;
(4) the model was applied to the training set, test set and TCGA-ESCC data set (download website: http:// gdac. branched property. org/, 450K methylated chip data containing 95 esophageal squamous carcinoma and 14 paracarcinoma samples in total) to evaluate the diagnostic efficacy of the model, and the diagnostic results and ROC curve are shown in FIG. 1.
TABLE 2.12 detailed information of methylation markers
| Markers | Chr | Position | Ref gene | Relation to island | Group | Enhancer | DHS |
| cg05446471 | chr3 | 13522740 | HDAC11 | S_Shore | 5'UTR | ||
| cg10085326 | chr11 | 102826680 | MMP13 | TSS1500 | |||
| cg11926456 | chr2 | 211432972 | CPS1 | Body | TRUE | ||
| cg12126990 | chr2 | 100588044 | AFF3 | Body | TRUE | ||
| cg13480465 | chr4 | 16795757 | LDB2 | Body | TRUE | ||
| cg19310604 | chr12 | 54383389 | HOXC10 | S_Shelf | 3'UTR | ||
| cg20473977 | chr5 | 59193490 | PDE4D | S_Shelf | Body | ||
| cg21041579 | chr14 | 95907997 | SYNE3 | Body | TRUE | ||
| cg21553182 | chr19 | 52956821 | ZNF578 | Island | TSS200 | TRUE | |
| cg24276395 | chr3 | 183419891 | YEATS2 | S_Shelf | 5'UTR | ||
| cg25724842 | chr6 | 163574564 | PACRG | S_Shelf | Body | ||
| cg26033932 | chr14 | 70547465 | SLC8A3 | Body |
5) Confirmation of Individual methylation sites
The differential expression level of the 12 methylated sites and the diagnosed AUC value, specificity and sensitivity (shown in Table 3) are confirmed again in the detection results of 91 patients, and the 12 methylated sites can be used as diagnostic markers independently and have good diagnostic effect.
TABLE 3.12 differential expression levels of the methylated sites and AUC values, specificity and sensitivity of the diagnosis
Claims (10)
1. A methylation marker combination for diagnosing esophageal cancer, the methylation marker combination comprising at least one of the following methylation sites: cg05446471, cg10085326, cg11926456, cg12126990, cg13480465, cg19310604, cg20473977, cg21041579, cg21553182, cg24276395, cg25724842, cg 26033932;
preferably, the methylation marker combination is a combination of the following methylation sites: cg05446471, cg10085326, cg11926456, cg12126990, cg13480465, cg19310604, cg20473977, cg21041579, cg21553182, cg24276395, cg25724842 and cg 26033932.
2. The methylation marker combination of claim 1, wherein the methylation sites are differentially methylated in the patient and in a healthy subject.
3. The methylation marker combination of claim 2, wherein the difference comprises a degree of methylation in the patient that is at least 1-fold that in a healthy subject, or a degree of methylation in the patient that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% that in a healthy subject.
4. The methylation marker combination of claim 1, wherein the esophageal cancer includes, but is not limited to, esophageal squamous carcinoma, esophageal adenocarcinoma, esophageal lymphoma, esophageal leiomyosarcoma, and esophageal metastatic cancer;
preferably, the esophageal cancer is esophageal squamous carcinoma.
5. An esophageal cancer diagnostic system comprising a calculation module that calculates a diagnostic model predictive value for the degree of methylation of at least one methylation marker in the methylation marker combination of claim 1;
preferably, the diagnostic model predicted value is calculated using a LASSO constructed model, and the coefficients at each of the positions are as follows: coefficient-4.843834 for cg05446471, coefficient 5.328574 for cg10085326, coefficient 7.431151 for cg11926456, coefficient 0.2826689 for cg12126990, coefficient 0.9789349 for cg13480465, coefficient-3.254839 for cg19310604, coefficient 2.170933 for cg20473977, coefficient 0.167167 for cg21041579, coefficient-0.2435546 for cg 53182, coefficient 1.641063 for cg 24276395), coefficient 2.531114 for cg25724842, and coefficient 1.138211 for cg 26033932.
6. The esophageal cancer diagnostic system of claim 5, further comprising at least one of any of the following modules:
1) a detection module for performing methylation detection;
2) a collection module that collects methylation detection results of a subject;
3) and the output module is used for outputting the predicted value of the diagnosis model.
7. A kit for diagnosing whether a subject has esophageal cancer, the kit comprising a methylation detection reagent that detects the degree of methylation of at least one methylation marker in the methylation marker combination of claim 1.
8. The kit of claim 7, wherein the methylation detection reagent comprises a reagent used in any one or more of the following methods comprising: pyrosequencing, bisulfite conversion sequencing, methylation chip methods, qPCR methods, digital PCR methods, second generation sequencing, third generation sequencing, whole genome methylation sequencing, DNA enrichment detection methods, simplified bisulfite sequencing techniques, HPLC methods, MassArray, methylation specific PCR, or combinations thereof.
9. The kit of claim 7, wherein the diagnosis of esophageal cancer in the subject is based on a determination of the extent of methylation in a sample from the subject;
the sample from the subject includes, but is not limited to, tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples, and alveolar lavage samples, ascites and lavage samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cytology smear samples, cervical scraping or swabbing samples, tissue and cell biopsy samples.
10. Use of the methylation marker combination according to claim 1, the esophageal cancer diagnosis model according to claim 5 and the kit according to claim 7 for preparing products for diagnosing esophageal cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110905091.3A CN113388685B (en) | 2021-08-08 | 2021-08-08 | Methylation markers for diagnosing esophageal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110905091.3A CN113388685B (en) | 2021-08-08 | 2021-08-08 | Methylation markers for diagnosing esophageal cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113388685A true CN113388685A (en) | 2021-09-14 |
| CN113388685B CN113388685B (en) | 2023-10-31 |
Family
ID=77622500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110905091.3A Active CN113388685B (en) | 2021-08-08 | 2021-08-08 | Methylation markers for diagnosing esophageal cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113388685B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948561A (en) * | 2023-02-10 | 2023-04-11 | 武汉艾米森生命科技有限公司 | Reagent for esophageal squamous carcinoma diagnosis or auxiliary diagnosis, detection kit and application thereof |
| CN117165689A (en) * | 2023-10-26 | 2023-12-05 | 深圳市睿法生物科技有限公司 | Methylation marker for early screening of pan-solid tumor and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745700A (en) * | 2015-03-27 | 2015-07-01 | 南京医科大学 | Esophageal-cancer-related methylated biomarker and application thereof |
| CN109830264A (en) * | 2019-03-15 | 2019-05-31 | 杭州慕谷科技有限公司 | The method that tumor patient is classified based on methylation sites |
-
2021
- 2021-08-08 CN CN202110905091.3A patent/CN113388685B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745700A (en) * | 2015-03-27 | 2015-07-01 | 南京医科大学 | Esophageal-cancer-related methylated biomarker and application thereof |
| CN109830264A (en) * | 2019-03-15 | 2019-05-31 | 杭州慕谷科技有限公司 | The method that tumor patient is classified based on methylation sites |
Non-Patent Citations (3)
| Title |
|---|
| GETING WU等: "CPS1 expression and its prognostic significance in lung adenocarcinoma", 《ANN TRANSL MED.》 * |
| HO-CHANG KUO等: "Epigenetic hypomethylation and upregulation of matrix metalloproteinase 9 in Kawasaki disease", 《ONCOTARGET.》 * |
| ILLUMINA, INC.: "Infinium HD Methylation SNP List", 《PRODUCT INFORMATION SHEET, DATA SHEET, AND PRODUCT FILES FOR THE HUMANMETHYLATION450 BEADCHIP》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948561A (en) * | 2023-02-10 | 2023-04-11 | 武汉艾米森生命科技有限公司 | Reagent for esophageal squamous carcinoma diagnosis or auxiliary diagnosis, detection kit and application thereof |
| CN115948561B (en) * | 2023-02-10 | 2024-01-02 | 武汉艾米森生命科技有限公司 | Reagent and detection kit for esophageal squamous carcinoma diagnosis or auxiliary diagnosis and application thereof |
| CN117165689A (en) * | 2023-10-26 | 2023-12-05 | 深圳市睿法生物科技有限公司 | Methylation marker for early screening of pan-solid tumor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113388685B (en) | 2023-10-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lu et al. | Tongue coating microbiome data distinguish patients with pancreatic head cancer from healthy controls | |
| US20220365067A1 (en) | Analysis of cell-free dna in urine and other samples | |
| Shoda et al. | Molecular, endoscopic, histologic, and circulating biomarker-based diagnosis of eosinophilic gastritis: Multi-site study | |
| CN109897899B (en) | Marker for prognosis judgment of locally advanced esophageal squamous carcinoma and application thereof | |
| KR20210045953A (en) | Cell-free DNA for the evaluation and/or treatment of cancer | |
| CN109906276A (en) | For detecting the recognition methods of somatic mutation feature in early-stage cancer | |
| US20150211053A1 (en) | Biomarkers for diabetes and usages thereof | |
| Colaco et al. | Correlation of gene expression with bladder capacity in interstitial cystitis/bladder pain syndrome | |
| CN106156543B (en) | A kind of tumour ctDNA information statistical method | |
| CN104195145B (en) | Biomarker of liver cirrhosis, and application thereof | |
| Barnicle et al. | Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis | |
| CN113388685B (en) | Methylation markers for diagnosing esophageal cancer | |
| CN111500705B (en) | IgAN intestinal flora marker, igAN metabolite marker and application thereof | |
| CN112899368B (en) | Biomarker for early diagnosis of primary hepatocellular carcinoma, detection reagent and application thereof | |
| US20200172980A1 (en) | Prostatic cancer marker, pcdh9, and application thereof | |
| Affara et al. | MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms | |
| WO2017202185A1 (en) | Peripheral blood gene marker for screening benign and malignant small pulmonary nodules and use thereof | |
| Hu et al. | Assessment of spatial and temporal variation in the skin transcriptome of atopic dermatitis by use of 1.5 mm Minipunch biopsies | |
| Mougeot et al. | Haemophilus pittmaniae and Leptotrichia spp. constitute a multi-marker signature in a cohort of human papillomavirus-positive head and neck cancer patients | |
| WO2017156739A1 (en) | Isolated nucleic acid application thereof | |
| KR20170067137A (en) | METHOD FOR DISCOVERING miRNA BIOMARKER FOR CANCER DIAGNOSIS AND USE THEREOF | |
| CN111968702B (en) | Malignant tumor early screening system based on circulating tumor DNA | |
| CN112553351A (en) | Bile duct cancer non-invasive marker based on relative abundance of intestinal microorganisms, screening method and application | |
| US20150284779A1 (en) | Determination of a tendency to gain weight | |
| CN111778340A (en) | Biomarker for early cervical cancer diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |