CN107034295A - For early diagnosis of cancer and the DNA methylation index of Hazard degree assessment and its application - Google Patents
For early diagnosis of cancer and the DNA methylation index of Hazard degree assessment and its application Download PDFInfo
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Abstract
It is used in subject cancer diagnosis the present invention relates to a kind of and/or suffers from the method for cancer Hazard degree assessment, the described method comprises the following steps：A) methylation state of DNA in the sample from subject and in the sample from healthy control group at least one DNA methylation site is determined；B) and by the methylation state of DNA in the sample of subject at the DNA methylation site it is compared with the methylation state of DNA in the sample of healthy control group at the corresponding DNA methylation site, if above-mentioned there is the significant difference of methylation state in both, show the subject with cancer or in the danger for suffering from cancer.Present invention also offers the kit for the above method.
Description
Technical field
It is used in subject cancer diagnosis the present invention relates to a kind of and/or suffers from the method and its reagent of cancer Hazard degree assessment
Box.
Background technology
Oophoroma is gynecological tumor (1) most fatal in global range ((1) is reference numbers, as follows).Height is caused
Dead rate is mainly comprising two reasons：Lack early detection means and chemotherapy resistance (2).Although initial chemotherapy is to human ovarian cancer patients
With effect, but about 25% patient will produce chemotherapy resistance (3) within six months.Tumor tissues are goed deep into
Research finds the change that there is various gene and epigenetic in ovary carcinogenesis and progress.It was found that effective early detection
Mark, especially in blood, to improvement suffer from oophoroma prognosis be in the urgent need to.
It is well known that oncolysis released dna fragment (cfDNA, dissociative DNA) and miRNA and cell factor etc.
Other molecules (4) into the circulatory system.Tumour correlation cfDNAs mutation is based on known in late tumor in the circulatory system
Mutation.The cfDNA that the tumour of original site is discharged into blood be it is limited, so abrupt climatic change come detect early-stage cancer be tool
It is challenging.In addition, the driving event of early-stage cancer is substantially probably epigenetic, possibly it can not be carried out based on gene mutation
Detection.
It is a systematic disease that the success of nearest checkpoint immunotherapy, which makes us re-recognize cancer, can be by
Immune system is directly affected (5).Nearest evidence shows the immunocyte of specific type in human epithelial ovarian carcinoma cells proliferation and transfer
Play important role (6-8).So, immunocyte perhaps can be used as pry tumor pathogenesis and the window of physiological equilibrium
Mouthful.The event related to immune system perhaps can be as the preferable mark of tumour, especially in early stage.It is envisioned that
, the gene or epigenetic of tumour cell, which change, will excite immunosurveillance, and immunocyte will carry out apparent something lost
Biography reprograms to resist the appearance of tumour cell.
Recently, peripheral blood DNA methylation spectrum have been found that with several cancers such as breast cancer, lung cancer, stomach cancer, oophoroma with
Pancreas has relevance (9-15).Previous being associated based on the full apparent gene group that blood DNA methylates to ovarian cancer risk
Research, is (12,16-18) implemented on a low-density platform comprising 27,000CpGs.So, we set about present
Study and go to explore potential value of the leucocyte DNA methylation in epithelial origin oophoroma using high density platform.
The content of the invention
The present inventor is implemented one in Chinese epithelial origin human ovarian cancer patients and ground based on full apparent gene group association
The two benches case-control study studied carefully.In the exploratory stage, full-length genome methylates 450,000 CpG site of experiment screening, and
And verified again in Qualify Phase for the methylated CpG site that there were significant differences.
Therefore, the present invention provides the following：
1. a kind of be used in subject cancer diagnosis and/or suffer from the method for cancer Hazard degree assessment, methods described includes following
Step：
A) determine in the sample from subject and in the sample from healthy control group at least one be selected from by
Methylation state of DNA at the DNA methylation site of group consisting of：
Cg11792281, cg08450091, cg14559409, cg13888226, cg17383024, cg09182724,
Cg22639787, cg07786355, cg04983687, cg21933078, cg26097381, cg07930620, cg05526438,
Cg22119466, cg01381374, cg02488385, cg06415087, cg00832928, cg19138325, cg09770068,
Cg14645856, cg10408178, cg16409562, cg05091585, cg09712234, cg25607383, cg21581312,
Cg20430870, cg06749803, cg21166544, cg19716090, cg12946518, cg20391833, cg17597901,
Cg06023279, cg24816464, cg15837838, cg15613100, cg07455406, cg09249800, cg24908166,
Cg22972055, cg03002688, cg26548134, cg15591386, cg10001590, cg26772894, cg12397924,
Cg06784563, cg00414077, cg26366833, cg16898066, cg00066854, cg20956594, cg15125566,
Cg05721476, cg00207226, cg23327334, cg03181118, cg25784280, cg15050398, cg17833106,
Cg01535567, cg20807852, cg20595268, cg07588614, cg24059119, cg18275732, cg12717729,
Cg08365438, cg12104982, cg01286319, cg04515608, cg22534374, cg14380045, cg16962115,
Cg22441770, cg12049550, cg02756683, cg01950479, cg26313233, cg13223682, cg14023999,
Cg23045991, cg12643917, cg16541275, cg22686939, cg21990144, cg11937033, cg02659854,
Cg17863551, cg27376514, cg20513976, cg27109284, cg11453546, and cg22211672；With
B) by the methylation state of DNA in the sample of subject at the DNA methylation site with coming from health
Methylation state of DNA in the sample of control group at the corresponding DNA methylation site is compared, if both above-mentioned
There is the significant difference of methylation state, then show the subject with cancer or in the danger for suffering from cancer.
2. the method described in 1 according to more than, is consisted of wherein the DNA methylation site is selected from including at least one
Group DNA methylation site：Cg09249800, cg12717729, cg12049550, cg22441770, cg17833106,
Cg09182724, cg22534374, cg16962115, cg08450091, cg03002688, cg00207226, cg23327334,
Cg25607383, cg22972055, cg06415087, cg11937033, cg14380045, cg13888226, cg08365438,
Cg20430870, cg14559409, cg02756683, cg00066854, cg04515608, cg21933078, cg19138325,
Cg20956594, cg15125566, cg16409562, cg22211672, cg14023999, cg01535567, cg26772894,
Cg27376514, cg16541275cg06784563, cg01286319, cg12104982, cg24059119, cg17863551,
Cg00414077, cg22639787, cg02488385, cg11453546, cg19716090, and cg01950479；
It is more preferably selected from the group consisted of：
Cg00207226, cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787；
Or selected from the group consisted of：Cg06784563, cg16541275, cg00207226, and cg00066854；Or be
Cg00207226 or cg22441770.
3. the method described in 1 or 2 according to more than, wherein the subject is mammal, preferably it is people, it is more preferably sub-
People from continent, more preferably Chinese, most preferably the Hans；And/or the cancer is early stage, mid-term or advanced cancer, for example in fact
Body knurl, is preferably selected from breast cancer, lung cancer, stomach cancer, oophoroma and cancer of pancreas etc., more preferably oophoroma, for example early ovarian cancer or
Advanced ovarian cancer, or selected from serous ovarian cancer, endometrioid ovarian cancer and mucous ovarian cancer.
4. the method according to any one of above 1-3, wherein the sample of the subject and the healthy control group
For the base of the genomic DNA of genomic DNA, preferably peripheral blood, urine, saliva or hair, most preferably peripheral blood (leucocyte)
Because of a group DNA.
5. the method according to any one of above 1-4, wherein the methylation state includes hyper-methylation (methyl
Change) and hypomethylation (demethylation).
6. for cancer diagnosis in subject and/or suffering from the kit of cancer Hazard degree assessment, the kit includes：
(a) it is used to determine to select at least one in the sample from subject and in the sample from healthy control group
The reagent or reaction system of methylation state of DNA at the DNA methylation site of free group consisting of：
Cg11792281, cg08450091, cg14559409, cg13888226, cg17383024, cg09182724,
Cg22639787, cg07786355, cg04983687, cg21933078, cg26097381, cg07930620, cg05526438,
Cg22119466, cg01381374, cg02488385, cg06415087, cg00832928, cg19138325, cg09770068,
Cg14645856, cg10408178, cg16409562, cg05091585, cg09712234, cg25607383, cg21581312,
Cg20430870, cg06749803, cg21166544, cg19716090, cg12946518, cg20391833, cg17597901,
Cg06023279, cg24816464, cg15837838, cg15613100, cg07455406, cg09249800, cg24908166,
Cg22972055, cg03002688, cg26548134, cg15591386, cg10001590, cg26772894, cg12397924,
Cg06784563, cg00414077, cg26366833, cg16898066, cg00066854, cg20956594, cg15125566,
Cg05721476, cg00207226, cg23327334, cg03181118, cg25784280, cg15050398, cg17833106,
Cg01535567, cg20807852, cg20595268, cg07588614, cg24059119, cg18275732, cg12717729,
Cg08365438, cg12104982, cg01286319, cg04515608, cg22534374, cg14380045, cg16962115,
Cg22441770, cg12049550, cg02756683, cg01950479, cg26313233, cg13223682, cg14023999,
Cg23045991, cg12643917, cg16541275, cg22686939, cg21990144, cg11937033, cg02659854,
Cg17863551, cg27376514, cg20513976, cg27109284, cg11453546, and cg22211672；Optionally
(b) specification, this description describes by the sample of subject at the DNA methylation site
Methylation state of DNA and the DNA methylation shape in the sample of healthy control group at the corresponding DNA methylation site
State is compared, if both above-mentioned significant difference that there is methylation state, shows that the subject suffers from cancer or place
In suffer from cancer it is dangerous in.
7. the kit described in 6 according to more than, is selected from by with the following group wherein the DNA methylation site includes at least one
Into group DNA methylation site：
Cg09249800, cg12717729, cg12049550, cg22441770, cg17833106, cg09182724,
Cg22534374, cg16962115, cg08450091, cg03002688, cg00207226, cg23327334, cg25607383,
Cg22972055, cg06415087, cg11937033, cg14380045, cg13888226, cg08365438, cg20430870,
Cg14559409, cg02756683, cg00066854, cg04515608, cg21933078, cg19138325, cg20956594,
Cg15125566, cg16409562, cg22211672, cg14023999, cg01535567, cg26772894, cg27376514,
Cg16541275cg06784563, cg01286319, cg12104982, cg24059119, cg17863551, cg00414077,
Cg22639787, cg02488385, cg11453546, cg19716090, and cg01950479；
It is more preferably selected from the group consisted of：
Cg00207226, cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787；
Or selected from the group consisted of：Cg06784563, cg16541275, cg00207226, and cg00066854；Or be
Cg00207226 or cg22441770.
8. the kit described in 6 or 7 according to more than, wherein the subject is mammal, preferably it is people, more preferably
Asian, more preferably Chinese, most preferably the Hans；And/or the cancer is early stage, mid-term or advanced cancer, for example
Solid tumor, is preferably selected from breast cancer, lung cancer, stomach cancer, oophoroma and cancer of pancreas etc., more preferably oophoroma, such as early ovarian cancer
Or advanced ovarian cancer, or selected from serous ovarian cancer, endometrioid ovarian cancer and mucous ovarian cancer.
9. the kit according to any one of above 6-8, wherein the sample of the subject and the healthy control group
Product are genomic DNA, are preferred from the genomic DNA of peripheral blood, urine, saliva or hair, (white most preferably from peripheral blood
Cell) genomic DNA.
10. the kit according to any one of above 6-9, wherein the reagent or reaction system are to be used for following
A kind of method determines methylation state of DNA (hyper-methylation or hypomethylation)：
1) methylation status of PTEN promoter；
2) methylation-specific digestion with restriction enzyme；
3) bisulfites DNA sequencing；
4) methylation sensitive mononucleotide primer extends；
5) restriction enzyme boundary mark genome scanning；
6) otherness methylation hybridization；
7) the methylation profiles analysis based on chip；With
8) base specific cutting/mass spectral analysis.
Brief description of the drawings
Fig. 1 methylate biomarker explore and checking research flow chart；
The full apparent gene group association research of Fig. 2 epithelial origin oophoromas.Manhattan figure illustrates full apparent gene group and closed
Join the analysis result (B) of all cases and check analysis result (A) of Journal of Sex Research, early-stage cases and control, late case and right
According to result (C), red long horizontal line represents P<1.0x 10-4；
Fig. 3 Qualify Phases have the methylation sites of significant difference.Hotspot graph illustrates DNA methylation difference analysis
As a result, including all cases and check analysis result (total), early-stage cases and control (early stage), late case and control are (late
Phase), serous ovarian cancer and control (serosity), endometrioid ovarian cancer and control (endometrial-like), mucus ovary
Cancer and control (mucus).The sequence of otherness methylation sites is the false discovery rate according to all cases and control (total)；
Receiver operating curve's analysis of 6 significant difference methylation sites of Fig. 4.(A) all cases and control
The Receiver operating curve of (total), early-stage cases and control (early stage), late case and control (late period).(B) serosity
Oophoroma and control (serosity), endometrioid ovarian cancer and control (endometrial-like), mucous ovarian cancer and control are (viscous
Fluidity) Receiver operating curve；
The path analysis of Fig. 5 gene ontologies (GO) and otherness methylation sites.(A) Venn diagram illustrates differential expression
The lap of methylation sites.(B) (biological processes) is annotated to the obvious related GO of 46 differential expression methylation sites.It is vertical
Axle represents GO classification, and transverse axis represents the enrichment times annotated with conspicuousness GO.(C) base that path includes is annotated in GO
Cause；
The correlation that Fig. 6 .DNA methylate with clotting factor/platelet parameter.With ovarian cancer risk/existence in hotspot graph
Degree of correlation based on-log10 (false discovery rate) represent.Risk and existence for oophoroma, each focus
Color code have specified that white/light red as priming the pump, peony is used as highest risk.For clotting factor and
Blood platelet, color code scope is from blueness to white, then to red, and red represents positive correlation, blue to represent negatively correlated.From white
Positive correlation enhancing is represented to red, represents that negative correlation strengthens from white to navy blue.PLCR:Blood platelet maxicell ratio,
PDW:MPW, MPV:Mean platelet volume, DD:DDi, PLT:Platelet count, PCT:Blood platelet ratio
Product, Fbg:Plasma fibrinogen, PT:Prothrombin time, AT:Antithrombin III；
The Receiver Operating Characteristics of 40 significant difference methylation sites between all cases and control in Fig. 7 embodiments
Curve；
The Receiver Operating Characteristics of 24 significant difference methylation sites between early-stage cases and control in Fig. 8 embodiments
Curve；
In Fig. 9 embodiments 39 late case and control between significant difference methylation sites Receiver Operating Characteristics
Curve；
The subject of 32 significant difference methylation sites between serous ovarian cancer case and control in Figure 10 embodiments
Performance curve；
In Figure 11 embodiments 34 significant difference methylation sites between endometrioid ovarian carninomatosis example and control by
Examination person's performance curve；
The subject of 11 significant difference methylation sites between mucous ovarian cancer case and control in Figure 12 embodiments
Performance curve；With
The correlation that Figure 13 .DNA methylate with clotting factor/platelet parameter.With ovarian cancer risk/existence in hotspot graph
Degree of correlation based on-log10 (false discovery rate) represent.Risk and existence for oophoroma, each focus
Color code have specified that white/light red as priming the pump, peony is used as highest risk.For clotting factor and
Blood platelet, color code scope is from blueness to white, then to red, and red represents positive correlation, blue to represent negatively correlated.From white
Positive correlation enhancing is represented to red, represents that negative correlation strengthens from white to navy blue.
Embodiment
Solid tumor is just gradually being considered as a kind of systematic disease, can be metabolized in protein and blood by DNA, RNA
The change of product is recognized.Although many researchs are it has been reported that gene mutation event in the circulatory system, few to grind
Study carefully and be absorbed in epigenetic DNA methylation mark in the circulatory system.In order to recognize that oophoroma peripheral blood DNA methylates mark
Thing, this research implements the full apparent gene group association study in two stages.In the first stage, we have detected on 24
485 in the peripheral blood cells of the normal healthy controls of skin source property oophoroma case and 24 age-matcheds, more than 000 DNA methylation
Site simultaneously filters out 96 CpG sites with the significance difference opposite sex.In second stage, we use Illumina ' s Custom
VeraCode methylation detecting methods are verified in 206 epithelial origin oophoromas and 205 controls, and are determined 46
Individual CpG sites.We set up forecast model using 46 CpG sites, and final Receiver operating curve's display uses wherein 6
The predictablity rate for the model that individual CpG sites are set up is 77.3% (95% credibility interval：72.9%-81.8%).We lead to
Cross and path analysis is carried out to the related gene in 46 CpG sites, it was found that the enrichment of immune system related gene, including LYST
(cg16962115, FDR=1.24E-04), CADM1 (cg21933078, FDR=1.22E-02), NFATC1 (cg06784563,
FDR=1.46E-02).In addition, peripheral blood DNA methylation level and platelet parameter/clotting factor level have correlation.This
Item research discloses one group of epigenetic liquid biopsy mark and the overall Immunity of patient and platelet parameter/blood coagulation system
Etc. closely related, it is possible to for detect all epithelial origin oophoromas by stages and hypotype.
Therefore, in one aspect, the invention provides one kind is for cancer diagnosis in subject and/or suffers from cancer risk assessment
The method of valency, the described method comprises the following steps：A) determine in the sample from subject and from healthy control group
In sample at least one (1 to 96, for example, 1,2,3,4,5,6,7,8,9,10,11,
12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27
It is individual, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42
It is individual, 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57
It is individual, 58,59,60,61,62,63,64,65,66,67,68,69,70,71,72
It is individual, 73,74,75,76,77,78,79,80,81,82,83,84,85,86,87
It is individual, 88,89,90,91,92,93,94,95,96) selected from the DNA methylation of group consisted of
Methylation state of DNA at site：Cg11792281, cg08450091, cg14559409, cg13888226, cg17383024,
Cg09182724, cg22639787, cg07786355, cg04983687, cg21933078, cg26097381, cg07930620,
Cg05526438, cg22119466, cg01381374, cg02488385, cg06415087, cg00832928, cg19138325,
Cg09770068, cg14645856, cg10408178, cg16409562, cg05091585, cg09712234, cg25607383,
Cg21581312, cg20430870, cg06749803, cg21166544, cg19716090, cg12946518, cg20391833,
Cg17597901, cg06023279, cg24816464, cg15837838, cg15613100, cg07455406, cg09249800,
Cg24908166, cg22972055, cg03002688, cg26548134, cg15591386, cg10001590, cg26772894,
Cg12397924, cg06784563, cg00414077, cg26366833, cg16898066, cg00066854, cg20956594,
Cg15125566, cg05721476, cg00207226, cg23327334, cg03181118, cg25784280, cg15050398,
Cg17833106, cg01535567, cg20807852, cg20595268, cg07588614, cg24059119, cg18275732,
Cg12717729, cg08365438, cg12104982, cg01286319, cg04515608, cg22534374, cg14380045,
Cg16962115, cg22441770, cg12049550, cg02756683, cg01950479, cg26313233, cg13223682,
Cg14023999, cg23045991, cg12643917, cg16541275, cg22686939, cg21990144, cg11937033,
Cg02659854, cg17863551, cg27376514, cg20513976, cg27109284, cg11453546, cg22211672；
And b) by the methylation state of DNA in the sample of subject at one or more DNA methylation sites with coming from
Methylation state of DNA in the sample of healthy control group at corresponding one or more DNA methylation sites is compared
Compared with, if it is above-mentioned both there is the significant difference of methylation state, show the subject with cancer or in suffering from cancer
In danger.
In a preferred embodiment of the invention, the DNA methylation site includes at least one (1 to 46, example
Such as, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17
It is individual, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32
It is individual, 33,34,35,36,37,38,39,40,41,42,43,44,45,46) be selected from
The DNA methylation site of the group consisted of：Cg09249800, cg12717729, cg12049550, cg22441770,
Cg17833106, cg09182724, cg22534374, cg16962115, cg08450091, cg03002688, cg00207226,
Cg23327334, cg25607383, cg22972055, cg06415087, cg11937033, cg14380045, cg13888226,
Cg08365438, cg20430870, cg14559409, cg02756683, cg00066854, cg04515608, cg21933078,
Cg19138325, cg20956594, cg15125566, cg16409562, cg22211672, cg14023999, cg01535567,
Cg26772894, cg27376514, cg16541275cg06784563, cg01286319, cg12104982, cg24059119,
Cg17863551, cg00414077, cg22639787, cg02488385, cg11453546, cg19716090, and
Cg01950479, the methylation level in this 46 CpG sites is compareed in all types of oophoromas relative to health volunteer
All there is significant otherness.
In a preferred embodiment of the invention, the DNA methylation site is further preferably selected from what is consisted of
Group：Cg00207226, cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787.This 6
CpG sites be used to build oophoroma forecast model in the present invention, successfully predict ovarian cancer risk, and in oophoroma
Different chromatographic analysis with histological subtypes by stages when diagnosis effect it is preferable.Therefore, in the method for the invention, preferably detect
1-6 (such as 1,2,3,4,5 or 6) are selected from following DNA methylation site：Cg00207226,
Cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787.The detection can also be detected simultaneously
One or more other any DNA methylation sites listed above.
In a preferred embodiment of the invention, the DNA methylation site is selected from the group consisted of：
Cg06784563, cg16541275, cg00207226, and cg00066854.The present inventor has found first, this 4 CpG sites
The change of DNA methylation level and 5 platelet parameter (PLT：Platelet count, PCT:Mean platelet volume, MPV：Average blood is small
Plate bulk, PDW：MPW, PLCR：Large platelet ratio) it is related.It may be speculated that the DNA first in these CpG sites
Change of the base state relative to normal healthy controls is determined for the platelet change of subject, and/or for diagnosing/
It is determined that being detected the disease (such as inflammatory disease) related to blood platelet of subject.Therefore, in the method for the invention, preferably
Detect that 1-4 (such as 1,2,3 or 4) are selected from following DNA methylation site：Cg06784563, cg16541275,
Cg00207226, and cg00066854.The detection can also detect one or more other any DNA first listed above simultaneously
Base site.Particularly preferred above DNA methylation site is cg00207226, because the methylation state of DNA in the site is relative
Diagnosis of ovarian cancer is can be not only used in the change of normal healthy controls and/or the Hazard degree assessment that has ovarian cancer, and can be used in examining
It is disconnected/to determine to be detected the disease or illness related to blood platelet of subject.
In a preferred embodiment of the invention, the DNA methylation site is cg22441770.The present inventor is first
It is secondary to find, change and the clotting factor parameter PT (prothrombin time) and AT III of the DNA methylation level in the CpG sites
(Antithrombin III) has correlation.Therefore, change of the methylation state of DNA in the site relative to normal healthy controls not only can be with
For diagnosis of ovarian cancer and/or the Hazard degree assessment that has ovarian cancer, and it can be used in determining clotting factor (particularly with parameter PT
With AT III) state change, it is and/or related to clotting factor (particularly and parameter PT and AT III) for diagnosing/determining
Disease or illness, such as blood coagulation disorders.
In the present invention, subject can be any people or non-human mammal.The example of non-human mammal includes spirit
Long class, livestock animals (such as horse, ox, sheep, pig, donkey), laboratory test animal (such as mouse, rat, rabbit, cavy), pet are (such as
Dog, cat) and capture wild animal (such as deer, fox).Preferably, mammal is people.There is no special limit for the population of people
System, it is preferred that being Asian, more preferably Chinese, most preferably the Hans.
In the present invention, term " cancer " not only includes the malignant tumour originating from epithelial tissue, and including originating from
The malignant tumour of mesenchymal tissue, in addition to other malignant tumours such as nephroblastoma, malignant teratoma etc..For the class of cancer
Type, is not particularly limited, but is preferably in the present invention solid tumor, including but not limited to breast cancer, lung cancer, stomach cancer, ovary
Cancer, cancer of pancreas, liver cancer, lymthoma, hemangioma etc..
In the present invention, term " diagnosis " refers to judge whether subject suffers from certain disease or its symptom, sign etc..
In the present invention, for determining side of the methylation state of DNA relative to the change of normal healthy controls at DNA methylation site of the present invention
Method, reagent or reaction system can combine use individually or with other one or more any Method for cancer diagnostics/marks
In the diagnosis of cancer.In the latter case, in certain form of cancer diagnosis, method of the invention can be a kind of diagnosis
The householder method of cancer.
In the present invention, term " suffering from cancer Hazard degree assessment " refers to predict possibility or risk of the subject with cancer
High low degree.
In the present invention, term " sample " refers to any biomaterial or genomic DNA containing genomic DNA in itself,
Including but not limited to cell material, biofluid (such as blood), excrement, tissue biopsy specimen, surgery operating sample or introducing are dynamic
In object and the fluid (for example, the solution regained from enema wash) then removed or the genome extracted therefrom
DNA.The biological sample tested according to the inventive method can directly be tested or may need some form of place before test
Reason.For example, biopsy or surgical operation sample may need homogenizing before test, or it may need section with in-situ test
Qualitative expression's level of each gene.Or, cell sample may need permeabilization before test.In addition, with regard to biological sample not
It is that it may need addition reagent (such as buffer) so that sample for liquid form (if necessary to test such form)
Circulation.Preferably, the sample is genomic DNA, is preferred from the genomic DNA of peripheral blood, urine, saliva or hair, most
Preferably come from the genomic DNA of peripheral blood (leucocyte).The sample of subject and control group is typically same type of sample.
In the present invention, term " healthy control group " refers to the health volunteer for not suffering from cancer.At one of the present invention
In embodiment, the health volunteer was without being previously clinically diagnosed with cancer and with detected subject in year
Match in terms of in age frequency.The determination and selection of healthy control group are conventional for the technical staff of medical domain
's.
In the present invention, term " DNA methylation " has implication known in the art.In the catalysis of transmethylase
Under, the cytimidine of DNA two nucleotides of CG is optionally added methyl, forms 5-methylcytosine, this is common in gene
5 '-CG-3 ' sequences.Most vertebrate genomic DNA has a small amount of methylated cytosine, is concentrated mainly on gene 5 '
The noncoding region at end, and cluster presence.Methylating for DNA can cause the inactivation of gene, and DNA methylation causes some districts domain dna
Conformation change, so as to have impact on protein and DNA interaction, causes gene to inactivate.In addition, sequence specific methylation knot
Hop protein (MBD/MeCP) can be combined with the methylated CpG of promoter region, prevent transcription factor from being acted on promoter, so as to prevent
Gene transcription process.The 70% to 80% of all CpG methylates.CpG can concentrate cluster, be referred to as on " CpG islands ".
In the present invention, term " DNA methylation site " refers to the site of CpG on genomic DNA.Specifically, in this hair
In bright, the cytosine residues in DNA methylation site are represented with cgxxxxxxxx, the site of its chip that methylated for Illunima
Number, it is clear to those skilled in the art.For DNA methylation site of the present invention, Figure of description 3
In also list chromosome number and related gene information where these sites.Due to DNA double-strand complementary structure, so this hair
Bright DNA methylation site also includes corresponding on complementary dna chain with the above-mentioned cytosine residues represented with cgxxxxxxxx
The methylation sites of corresponding cytimidine at (n+1)th.Therefore, as the DNA for referring to the cytosine residues that cgxxxxxxxx is represented
During methylation sites (n), while being additionally included in the methylation sites of corresponding cytimidine at (n+1)th on complementary dna chain.
In the present invention, term " methylation state of DNA " is interpreted as referring to a specific nucleotide or many in region of DNA domain
The presence that is methylated in individual nucleotides, it is not present and/or measures.In one embodiment of the invention, the DNA methylation position
Methylation state at point includes but is not limited to hyper-methylation and hypomethylation.In the present invention, " hyper-methylation " and " methyl
Change " it is used interchangeably, it is intended that the CpG at some nucleotides is present and high methylation；And " hypomethylation " and " demethyl
Change " it is used interchangeably, it is intended that CpG is not present at some nucleotides and minuent methylates.As described above, determine one with
During the methylation state of DNA of the cytosine residues in the DNA methylation site (n) that cgxxxxxxxx is represented, while also including true
It is scheduled on the methylation state of the methylation sites of corresponding cytimidine at (n+1)th on complementary dna chain.
Method for assessing methylation state of DNA is well known by persons skilled in the art, is included but is not limited to lower section
Method：1) methylation status of PTEN promoter；2) methylation-specific digestion with restriction enzyme；3) bisulfites DNA sequencing；4) first
Base sensitiveness mononucleotide primer extends；5) restriction enzyme boundary mark genome scanning；6) otherness methylation hybridization；7) it is based on core
The methylation profiles analysis of piece (example includes, BeadArray platform technologys (Illumina, USA))；Cut with 8) base specific
Cut/mass spectral analysis (Sequenom, USA).Reagent or reaction system for these methods are that those of ordinary skill in the art are ripe
Know, and provided generally in the form of commercially available kit.Therefore, the present invention is also provided for assessing DNA methylation shape
Application of the reagent or reaction system of state in the method for the present invention or the kit of the preparation present invention.
In the present invention, term " significant difference that there is methylation state " refers to and control group (such as healthy control group)
The corresponding DNA methylation site of sample at methylation state of DNA (hyper-methylation/hypomethylation) compare, from subject
Sample in methylation state of DNA (hyper-methylation/hypomethylation) at the DNA methylation site exist statistically
Significant difference, such as P<0.05, preferably P<0.01.In a particularly preferred embodiment of the present invention, P<1.0x
10-4。
Embodiment
The present invention is described in detail hereinafter with reference to embodiment, the embodiment is only intended to illustrate the present invention, without
It is intended to limit the scope of the present invention.The scope of the present invention is specifically limited by appended claims.
Material and method
Study population
Oophoroma case is that the age that histodiagnosis is made a definite diagnosis is passed through by tumour hospital of 2007-2013 Medical University Of Tianjin
25-85 Sui original epithelial origin oophoroma women (all Chinese han populations).Case make a definite diagnosis have within first 6 months treatment of cancer history or
Person's blood transfusion history forecloses.The blood sample of case group is collected in operating room, and blood routine and blood clotting test obtain blood count, blood coagulation
Carried out in factor values, and these all before surgery one week.Control group is that tumor free subject is (complete in the community resident of Tianjin
Portion is Chinese han population), it be not diagnosed as cancer previously and with case group in age frequency (5 year interval) on
Match somebody with somebody.Each subject completes survey (including demographic information, health status, life style and eating habit) and provided
The blood sample of research.This research has passed through tumour hospital of Medical University Of Tianjin and Cancer Institute Institutional Review Board
Approval, and the informed consent form that acquisition is studied with clinical sample.
In the exploratory stage, 24 ovarian epithelial carcinoma case groups (12 FIGO I-II and 12 FIGO III- by stages by stages
) and the control group of age distribution matching is picked out progress full-length genome methylation analysis by random IV.In Qualify Phase, 206
Example ovarian epithelial carcinoma case group and the control group of 205 age distribution matchings are used for verifying that I-stage is select CpG
Point.
DNA is extracted and bisulfite conversion
The sample genomic dna of collection be by kit QiazenDNA Blood Mini Kit (Qiazen,
Valencia, CA, USA) extracted from leucocyte, -80 DEG C are stored in before bisulf iotate-treated.According to reagent sulfurous acid
Hydrogen salt conversion reagent box EZ-96DNA Methylation-Gold Kit's (D5008) (Zymo Research, Orange, CA)
Handbook handles genomic DNA, wherein unmethylated Cytosines are into uracil, and the cytimidine methylated then keeps former
Shape.The DNA of bisulfite conversion is stored in -20 DEG C, is used in post-conversion in one week.
Full-length genome methylates express spectra
In the exploratory stage, the full-length genome express spectra that methylates is to use Illumina Infinium
The processing of HumanMethylation450 (San Diego, CA) chip.We use Illumina BeadArrayTMReader is obtained
To chip image, data are read with Illumina ' s GenomeStudio softwares.All samples all have passed through quality control survey
Examination, including Dyeing control, extension control, object removal control, hybridization control, bisulfites conversion and control, specificity control,
Feminine gender control, non-polymorphism combines control, and each hole carries out background mark with negative control pearl.The DNA methyl in each CpG sites
Change value (β value) scope is, from 0 (not methylating) to 1 (methylating), to represent the methylated alleles signal for subtracting background intensity
To the relative ratio of total fluorescence signal.
Verify the CpG sites of screening
In Qualify Phase, Illunima ' s Custom VeraCode methylation assays screen for confirming 96
CpG sites.Illunima ' s BeadXpress Reader and Illumina ' s GenomeStudio are each used for scanning core
Piece and reading data.Implemented in analysis including 9 internal controls (specific alleles extension control, PCR uniformity controls
System, room extension control, sexual control, first hybridization control, it is secondary hybridization control, feminine gender control, pollution detection control) matter
Control test.
Statistical analysis：
To compare the Demographics of case group and control group, classified variable and continuous variable are blocked respectively
Examine and independent samples t test side.In the initial exploratory stage, compared using independent samples t test between case group and control group
The difference of the median β value in each CpG sites, and (I-II, III-IV) carries out chromatographic analysis by stages according to ovarian epithelial carcinoma.
Then we select 96 methylated CpG sites that most there were significant differences from full-length genome data and are verified that condition is as follows：
(a)P<1.0×10-4；(b) compared with control group, the variation tendency between I-II phases and III-IV phase ovarian epithelial carcinoma cases
Unanimously；(c) Illumina Custom VeraCode methylation tests can be used to determine Genotyping.In Qualify Phase, make
With independent samples t test come the difference for the value that methylated between evaluation studies group.Pearson correlation analyses are carried out, to assess DNA first
Baseization and the correlation of blood count/clotting factor.In order to correct the error repeatedly tested, using Benjamini and
Hochberg bearing calibrations calculate false discovery rate (FDR), FDR<It is meaningful when 0.05.
In addition, in order to assess the classification performance that Qualify Phase has the methylated CpG site of otherness, we utilize
Logistic regression models establish Receiver operating curve (ROC) forecast model.Using progressively and manually selecting
Method choice DNA methylation mark.
In addition, the common point in order to determine gene in biological pathways, we are carried out using DAVID gene ontologies instrument
Path enrichment analysis, the analysis is used for the gene of the related differential methylation CpG location proximates of ovarian cancer risk.All analyses make
Carried out with the version of SAS softwares 9.3 (SAS Institute, Cary, NC, USA) and R software 3.1.2 versions.
As a result
In order to determine the otherness of oophoroma peripheral white blood cells DNA methylation, we implement two stage case-right
According to full apparent gene group association research (EWAS, Epigenome wide association study), Fig. 1 illustrates research
The flow chart of design.In the exploratory stage most started, the scanning of full-length genome is in 24 ovarian epithelial carcinomas and 24 ages
Carried out in the normal healthy controls of matching using Illumina ' s InfiniumHumanmethylation 450K chips.In case
And control group, age, BMI, smoking history, the otherness of cancer family history and menopause history is not statistically significant.All diseases
Example is all serous ovarian cancer, and 12 early stages, 12 late periods (table 1), Manhattan figure illustrates full apparent gene group analysis result
(Fig. 2).The methylation level for having 242 CpG sites between 24 ovarian epithelial carcinomas and 24 control groups is shown substantially
Significant difference (P<1.0×10-4).When to neoplasm staging progress chromatographic analysis, compared with 24 control groups, 39 CpG
Site has significant difference in the epithelial ovarian of 12 early stages (I＆II phases), and 375 CpG sites are 12 late periods
(III＆IV) there is significant difference in.We have selected the most obvious CpG sites of 96 differences and are tested in the second phase
Demonstrate,prove (being shown in Table 2).
In an independent sample comprising 206 ovarian epithelial carcinomas and 205 controls, we apply Illunima ' s
Custom VeraCode methylation detecting methods analyze the 96 CpG methylation sites selected.The year of case and control group
Age, BMI, smoking history, cancer family history, menopause history is all consistent.We have found that 40 CpG sites and ovarian epithelial carcinoma wind
Danger is substantially related, wherein 16 hyper-methylations, 24 hypomethylations (see Fig. 3, table 3).Chromatographic analysis, hair are carried out to neoplasm staging
Existing 88 early-stage cases (I＆II phases) have compared 24 CpG sites (9 hyper-methylations and 15 hypomethylations) first with control group
Base level has significant otherness, and 115 late cases (III＆IV phases) compare 39 CpG sites (21 of discovery with control
Individual hyper-methylation and 18 hypomethylations) methylation level have significance difference the opposite sex.This research is also to case and the first of control group
Base level has carried out the chromatographic analysis of histological classification.Compared with 205 control groups, we are in 85 serous ovarian cancers
32 CpG sites are found, is found in 59 ovarian epithelial carcinomas in 34 CpG sites, 24 mucous ovarian cancers and finds 11
The methylation level in individual CpG sites has significant otherness (Fig. 3, table 4).On the whole, we determined that 46 CpG sites
Methylation level, have significant otherness in all types of compare all.
The representative performance curve (ROC) of the subject in single CpG sites is shown in Fig. 7-12.Use subject's work
Make under indicatrix area be 0.773 (0.729-0.818) forecast model filter out 6 CpG sites (cg00207226,
Cg01535567, cg19716090, cg20956594, cg22534374, cg22639787), show that this 6 CpG sites are compared
There is higher predictive ability on ovarian cancer risk according to group (77.3%).This 6 CpG sites it is different by stages and histological subtypes
Chromatographic analysis when diagnosis effect it is preferable：Infantile tumour TG-AUC is 0.754 (0.693-0.816), late tumor curve
Lower area is 0.803 (0.754-0.853), and serous ovarian cancer TG-AUC is 0.773 (0.713-0.833), intrauterine
Film sample oophoroma TG-AUC is 0.799 (0.735-0.864), and mucous ovarian cancer TG-AUC is 0.725 (0.609-
0.841) (see Fig. 4).
In order to determine that this 46 methylated CpG sites assume the general character of 56 genes of regulation, we use DAVID gene sheets
Body method has carried out path enrichment analysis.Enrichment analysis multiple highest is the cytotoxicity of leucocyte regulation, on this path
Including two genes：LYST (cg16962115, FDR=1.24E-04) and CADM1 (cg21933078, FDR=1.22E-02).
Most enrichment analysis result all contains the related path of immune system；And we have found that gene NFATC1
(cg06784563, FDR=1.46E-02) is also relevant with immune system (see Fig. 5) in Reactom pathway databases.In addition,
We have evaluated the correlation (being shown in Table 5) between the difference of methylation sites and blood cell number/clotting factor.In DNA methyl
Change between blood cell count, other are all without significant correlation (Figure 13) in addition to platelet count.We have found that several CpG
Site and platelet parameter and clotting factor have correlation (Fig. 6).5 platelet parameter (PLT：Platelet count, PCT:Blood
Platelet specific volume, MPV：Mean platelet volume, PDW：MPW, PLCR：Large platelet ratio) and 4 blood coagulations because
Son (PT：Prothrombin time, D-dimer：DDi, Fbg：Fibrinogen, AT III：Antithrombin III) index
Incorporate correlation analysis.We have found that four CpG sites (cg06784563, cg16541275, cg00207226,
) and all platelet parameters have correlation cg00066854.For clotting factor, neither one site and this four blood coagulations
The factor has correlation.The DNA methylation level and PT and AT III in cg22441770 sites have correlation, and have 11
CpG sites and DDi have correlation.
Discuss
In this two stage full apparent gene group association research of case-control, we determined that 46 with Han nationality female
The property significantly correlated new CpG methylation sites of ovarian cancer risk.We are also constructed one relatively high using 6 CpG sites
The risk forecast model of accuracy, be particularly suitable for use in early ovarian cancer.There is the methylation sites gene of significant difference to 46
Path analysis is carried out, it is a part for immune system related pathways to find them.The related otherness of ovarian cancer risk methylates
CpG sites and blood count are unrelated, but have been found that with platelet parameter/clotting factor there is correlation.As far as we know, this
It is to disclose peripheral white blood cells DNA methylation and the report first of the correlation of platelet parameter/clotting factor.
The peripheral blood DNA mark that methylates all has been reported that in a few class cancers, including breast cancer, head and neck cancer, oophoroma, stomach
Cancer, colorectal cancer and cancer of pancreas etc., but its biological significance is unclear (19).It is reported that, DNA methylation and life style, day
Normal diet is related to environmental exposure (20).Recently, more and more researchs show that DNA methylation has correlation with smoking and BMI
(21,22).Previous research conjecture, the change that peripheral blood DNA methylates is possibly due to the conversion of blood cell composition.To disclose
This mechanism, Koestler et al. compare normal human peripheral blood's WBC sub-population (including B cell, granulocyte, monocyte,
NK cells, CD4+T cells, CD8+T cells and Pan-T cells) DNA methylation, it is determined that 50 CpG sites are different white thin
The methylation level of born of the same parents' subgroup has otherness (11).However, this 50 CpG sites are in our study without statistics
Meaning is learned, this is also consistent with the report before in breast cancer (9).In our study, the methyl related to oophoroma
Changing site and WBC sub-population does not have relevance.What is interesting is, it has been found that peripheral blood DNA methylates and platelet parameter and solidifying
Blood factor has certain contact.Blood platelet in blood is it is reported that participating in blood coagulation, immune response, and inflammation and tumour progression/
The effect (8,23) of key is played in transfer.In oophoroma, it has been reported that number of platelets is pre- with epithelial origin oophoroma
Afterwards related (24) and blood platelet can directly facilitate human epithelial ovarian carcinoma cells proliferation (6).When ovary carcinogenesis Epithelial and stromal is converted,
The main change of DNA methylation can be induced (25) by TGF β 1, and the TGF β 1 of secretion of platelet can promote the upper of tumour cell
Skin mesenchymal transformation (26).However, the mechanism of DNA methylation and blood platelet relation is unclear, it is necessary to further in peripheral blood
Research.
In this research, we determined that where otherness CpG sites related to ovarian cancer risk or neighbouring base
Because being that take part in the related path of immune system, include the cytotoxicity of leucocyte mediation, cell killing, immunological effect process.
Wherein there are three genes, NFATC1 (cg06784563), LYST (cg16962115), and CADM1 (cg21933078) immune
In system related pathways.Cg06784563 is the nuclear transcription factor in NFATC1 5 ' end promoter regulation area ciphering activation T cells
Son.NFAT albumen, which has had been reported that, to be proved to play important function (27) in immune system, and NFAT transcription factors including
There is extremely important effect in many paths including tumour.In oophoroma, NFACT1 in tumor tissues relative to pairing just
Often tissue is significant is overexpressed, and promotes tumor cell in vitro by activating ERK1/2/p38/MAPK signal paths up-regulation c-myc
Breed (29).Cg21933078 is that at No. 11 chromosome CADM1 upstream 15kb, CADM1 is a cell adhesion molecule and recognized
To be a tumor suppressor gene (30,31).CADM1 low expressions are probably because of promoter zone methylation and therefore promoted swollen
Tumor cell proliferation/invasion and attack (30,31).In oophoroma, CADM1, which is lowered, can be used as the bad mark of prognosis (32).CADM1 leads to
Crossing makes tumour cell is more sensitive to immune surveillance mechanisms to play the New function for suppressing metastases, and it is tumour that CADM1, which loses,
A crucial step (33) for immunoediting.Cg16962115 is located at 1q42.3 and encodes lysosome transhipment regulatory protein LYST upstreams
At 35kb.LYST is mutated (34) related to Xie Diyake-east syndrome.There are not LYST and oophoroma or other cancer phases at present
The report of pass.Require further study and explore this gene function related to oophoroma.
The previous full apparent gene group association study of oophoroma has determined that several peripheral blood otherness methylated CpG sites
(12,16,17).However, neither one reaches selection standard (P when we verify<1.0×10-4).The exploration rank studied in the past
Section is methylated chip based on Illumina ' s 27K, and the coverage of the 450K chips used in us is than big in the past
It is many.We pick out checking 96 CpG sites only one of which in studying are included in 27K chips.
In general, we have determined that the DNA methylation mark of one group of haematogenous and epithelial origin oophoroma wind
Danger is related.Therefore, peripheral blood DNA methylate express spectra can turn into ovarian cancer risk assess and early detection new tool.Greatly
Type prospective cohort study and deep biological mechanism research need to verify these new biomarkers, and are accurate
Medical science provides oophoroma apparent gene group therapeutic strategy.
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The full apparent gene group detection of table 1. and the research object basic condition and Clinical symptoms of checking
Claims (10)
1. a kind of be used in subject cancer diagnosis and/or suffer from the method for cancer Hazard degree assessment, it the described method comprises the following steps：
A) determine to be selected from by following at least one in the sample from subject and in the sample from healthy control group
Methylation state of DNA at the DNA methylation site of the group of composition：Cg11792281, cg08450091, cg14559409,
Cg13888226, cg17383024, cg09182724, cg22639787, cg07786355, cg04983687, cg21933078,
Cg26097381, cg07930620, cg05526438, cg22119466, cg01381374, cg02488385, cg06415087,
Cg00832928, cg19138325, cg09770068, cg14645856, cg10408178, cg16409562, cg05091585,
Cg09712234, cg25607383, cg21581312, cg20430870, cg06749803, cg21166544, cg19716090,
Cg12946518, cg20391833, cg17597901, cg06023279, cg24816464, cg15837838, cg15613100,
Cg07455406, cg09249800, cg24908166, cg22972055, cg03002688, cg26548134, cg15591386,
Cg10001590, cg26772894, cg12397924, cg06784563, cg00414077, cg26366833, cg16898066,
Cg00066854, cg20956594, cg15125566, cg05721476, cg00207226, cg23327334, cg03181118,
Cg25784280, cg15050398, cg17833106, cg01535567, cg20807852, cg20595268, cg07588614,
Cg24059119, cg18275732, cg12717729, cg08365438, cg12104982, cg01286319, cg04515608,
Cg22534374, cg14380045, cg16962115, cg22441770, cg12049550, cg02756683, cg01950479,
Cg26313233, cg13223682, cg14023999, cg23045991, cg12643917, cg16541275, cg22686939,
Cg21990144, cg11937033, cg02659854, cg17863551, cg27376514, cg20513976, cg27109284,
Cg11453546, and cg22211672；With
B) by the methylation state of DNA in the sample of subject at the DNA methylation site with coming from normal healthy controls
Methylation state of DNA in the sample of group at the corresponding DNA methylation site is compared, if both above-mentioned presence
The significant difference of methylation state, then show the subject with cancer or in the danger for suffering from cancer.
2. according to the method described in claim 1, wherein the DNA methylation site include at least one be selected from consist of
Group DNA methylation site：Cg09249800, cg12717729, cg12049550, cg22441770, cg17833106,
Cg09182724, cg22534374, cg16962115, cg08450091, cg03002688, cg00207226, cg23327334,
Cg25607383, cg22972055, cg06415087, cg11937033, cg14380045, cg13888226, cg08365438,
Cg20430870, cg14559409, cg02756683, cg00066854, cg04515608, cg21933078, cg19138325,
Cg20956594, cg15125566, cg16409562, cg22211672, cg14023999, cg01535567, cg26772894,
Cg27376514, cg16541275cg06784563, cg01286319, cg12104982, cg24059119, cg17863551,
Cg00414077, cg22639787, cg02488385, cg11453546, cg19716090, and cg01950479；
It is more preferably selected from the group consisted of：
Cg00207226, cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787；Or choosing
Free group consisting of：Cg06784563, cg16541275, cg00207226, and cg00066854；Or be cg00207226
Or cg22441770.
3. method according to claim 1 or 2, wherein the subject is mammal, preferably it is people, it is more preferably sub-
People from continent, more preferably Chinese, most preferably the Hans；And/or the cancer is early stage, mid-term or advanced cancer, for example in fact
Body knurl, is preferably selected from breast cancer, lung cancer, stomach cancer, oophoroma and cancer of pancreas etc., more preferably oophoroma, for example early ovarian cancer or
Advanced ovarian cancer, or selected from serous ovarian cancer, endometrioid ovarian cancer and mucous ovarian cancer.
4. the method according to any one of claim 1-3, wherein the sample of the subject and the healthy control group
For genomic DNA, the genomic DNA of peripheral blood, urine, saliva or hair is preferred from, it is (white thin most preferably from peripheral blood
Born of the same parents) genomic DNA.
5. the method according to any one of claim 1-4, wherein the methylation state includes hyper-methylation and low first
Base.
6. for cancer diagnosis in subject and/or suffering from the kit of cancer Hazard degree assessment, the kit includes：
(a) be used for determine in the sample from subject and in the sample from healthy control group at least one be selected from by
The reagent or reaction system of methylation state of DNA at the DNA methylation site of group consisting of：
Cg11792281, cg08450091, cg14559409, cg13888226, cg17383024, cg09182724,
Cg22639787, cg07786355, cg04983687, cg21933078, cg26097381, cg07930620, cg05526438,
Cg22119466, cg01381374, cg02488385, cg06415087, cg00832928, cg19138325, cg09770068,
Cg14645856, cg10408178, cg16409562, cg05091585, cg09712234, cg25607383, cg21581312,
Cg20430870, cg06749803, cg21166544, cg19716090, cg12946518, cg20391833, cg17597901,
Cg06023279, cg24816464, cg15837838, cg15613100, cg07455406, cg09249800, cg24908166,
Cg22972055, cg03002688, cg26548134, cg15591386, cg10001590, cg26772894, cg12397924,
Cg06784563, cg00414077, cg26366833, cg16898066, cg00066854, cg20956594, cg15125566,
Cg05721476, cg00207226, cg23327334, cg03181118, cg25784280, cg15050398, cg17833106,
Cg01535567, cg20807852, cg20595268, cg07588614, cg24059119, cg18275732, cg12717729,
Cg08365438, cg12104982, cg01286319, cg04515608, cg22534374, cg14380045, cg16962115,
Cg22441770, cg12049550, cg02756683, cg01950479, cg26313233, cg13223682, cg14023999,
Cg23045991, cg12643917, cg16541275, cg22686939, cg21990144, cg11937033, cg02659854,
Cg17863551, cg27376514, cg20513976, cg27109284, cg11453546, and cg22211672；Optionally
(b) specification, this description describes by the DNA first in the sample of subject at the DNA methylation site
Base state is entered with the methylation state of DNA in the sample of healthy control group at the corresponding DNA methylation site
Row compares, if both above-mentioned significant difference that there is methylation state, shows the subject with cancer or in trouble
In the danger of cancer.
7. kit according to claim 6, is selected from by with the following group wherein the DNA methylation site includes at least one
Into group DNA methylation site：
Cg09249800, cg12717729, cg12049550, cg22441770, cg17833106, cg09182724,
Cg22534374, cg16962115, cg08450091, cg03002688, cg00207226, cg23327334, cg25607383,
Cg22972055, cg06415087, cg11937033, cg14380045, cg13888226, cg08365438, cg20430870,
Cg14559409, cg02756683, cg00066854, cg04515608, cg21933078, cg19138325, cg20956594,
Cg15125566, cg16409562, cg22211672, cg14023999, cg01535567, cg26772894, cg27376514,
Cg16541275cg06784563, cg01286319, cg12104982, cg24059119, cg17863551, cg00414077,
Cg22639787, cg02488385, cg11453546, cg19716090, and cg01950479；
It is more preferably selected from the group consisted of：
Cg00207226, cg01535567, cg19716090, cg20956594, cg22534374, and cg22639787；Or choosing
Free group consisting of：Cg06784563, cg16541275, cg00207226, and cg00066854；Or be cg00207226
Or cg22441770.
8. the kit according to claim 6 or 7, wherein the subject is mammal, preferably it is people, more preferably
Asian, more preferably Chinese, most preferably the Hans；And/or the cancer is early stage, mid-term or advanced cancer, for example
Solid tumor, is preferably selected from breast cancer, lung cancer, stomach cancer, oophoroma and cancer of pancreas etc., more preferably oophoroma, such as early ovarian cancer
Or advanced ovarian cancer, or selected from serous ovarian cancer, endometrioid ovarian cancer and mucous ovarian cancer.
9. the kit according to any one of claim 6-8, wherein the sample of the subject and the healthy control group
Product are genomic DNA, are preferred from the genomic DNA of peripheral blood, urine, saliva or hair, (white most preferably from peripheral blood
Cell) genomic DNA.
10. the kit according to any one of claim 6-9, wherein the reagent or reaction system are to be used for following
A kind of method determines methylation state of DNA (hyper-methylation or hypomethylation)：
1) methylation status of PTEN promoter；
2) methylation-specific digestion with restriction enzyme；
3) bisulfites DNA sequencing；
4) methylation sensitive mononucleotide primer extends；
5) restriction enzyme boundary mark genome scanning；
6) otherness methylation hybridization；
7) the methylation profiles analysis based on chip；With
8) base specific cutting/mass spectral analysis.
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| CN114736968A (en) * | 2022-06-13 | 2022-07-12 | 南京世和医疗器械有限公司 | Application of plasma free DNA methylation marker in lung cancer early screening and lung cancer early screening device |
| WO2024027796A1 (en) * | 2022-08-04 | 2024-02-08 | 江苏鹍远生物科技股份有限公司 | Use of marker in diagnosing breast cancer or predicting breast cancer risks |
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