CN110106244A - A kind of noninvasive molecule parting kit of breast cancer and method - Google Patents
A kind of noninvasive molecule parting kit of breast cancer and method Download PDFInfo
- Publication number
- CN110106244A CN110106244A CN201910493271.8A CN201910493271A CN110106244A CN 110106244 A CN110106244 A CN 110106244A CN 201910493271 A CN201910493271 A CN 201910493271A CN 110106244 A CN110106244 A CN 110106244A
- Authority
- CN
- China
- Prior art keywords
- breast cancer
- type
- noninvasive
- dna
- nucleosome
- 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.)
- Pending
Links
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
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/30—Unsupervised data analysis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Evolutionary Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Zoology (AREA)
- Data Mining & Analysis (AREA)
- Analytical Chemistry (AREA)
- Theoretical Computer Science (AREA)
- Wood Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Bioethics (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Artificial Intelligence (AREA)
- Microbiology (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Databases & Information Systems (AREA)
- Epidemiology (AREA)
- Evolutionary Computation (AREA)
- Public Health (AREA)
- Software Systems (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of noninvasive molecule parting kit of breast cancer provided by the invention and method, it is desirable to provide a kind of to use simple, the high kit of Detection accuracy；And sample is easy to get, sampling flowsheet is simple, clinical experience is preferable and can procedure operation, the method for being very suitable to the noninvasive molecule parting of breast cancer of the non-disease diagnostic purpose of clinical application；Its technical solution includes that plasma DNA extracts reagent, library construction reagent, sequencing reagent, sequence testing chip, this method is based on to plasma DNA high-flux sequence data, pass through bioinformatic analysis, the nucleosome footprint different information in full-length genome range Natural promoter area in plasma DNA is obtained, and then lumen A type, lumen Type B, HER2 concentration type and the other noninvasive molecule parting of four type of basal cell type are realized to patient with breast cancer according to the difference of footprint information；Belong to field of biotechnology.
Description
Technical field
The present invention provides a kind of noninvasive molecule parting products of breast cancer, specifically, being a kind of based on plasma DNA
The noninvasive molecule parting product of the breast cancer of high throughput sequencing technologies, the present invention also relate to the construction method of the molecule product, belong to
In field of biotechnology.
Background technique
Breast cancer is the most common invasive cancer of women.It is reported within 2015 according to the World Health Organization, the whole world is annual new
The breast cancer case of increasing about 1,150,000, death about 410,000, and disease incidence increases year by year.Breast cancer is also Chinese Human-Female
The most common cancer of property, annual Chinese Breast Cancer newly send out quantity and The dead quantity and account for global 12.2% and 9.6% respectively.
The molecule parting of breast cancer and the treatment of breast cancer and prognosis are closely related.Breast cancer molecular classifying method master at present
It will be using defined breast cancer classification standard in breast cancer international conference in 2013.Immunohistochemical method is used, according to
Estrogen receptor (estrogen receptor, ER) in cancerous tissue, progesterone receptor (progester-one receptor,
PR) and the testing result of HER-2, breast cancer is divided into 4 kinds of molecular isoforms: lumen A type (luminalA), lumen Type B
(luminal B), HER2 concentration type (HER2-enriched) and basal cell type (basal-like).Molecule parting result is wide
The general biological behaviour for the different subtypes breast cancer such as the determination of clinical treatment, curative effect and Index for diagnosis, therapeutic strategy
There are notable differences with prognosis.Cast is that expression of hormonal receptors is positive, prognosis bona；HER-2 type be hormone receptor without expression but
HER-2 expression is positive, though prognosis it is poor compared with conduit type can by specific target treatment be improved significantly.Basaloid
Type, i.e., usually said triple negative breast cancer, therapeutic effect and prognosis are poor.
Current molecular typing methods are all based on invasive diagnostic method, that is, pass through Needle-sucking cytology checks, cut work
Inspection etc. directly has the detection methods of wound to tissue, it is therefore an objective to obtain tumour cell or tumor tissues, metastatic lymph node.This
The invasive diagnostic method of kind increases the risk of the pain and tumour spread of patient.Additionally due to tumor tissues are with larger heterogeneous
Property, the Analysis of Biopsies locally punctured is likely to cause result bias, for biopsy pathological diagnosis be also required to have it is good
Pathology theoretical basis and practical experience.The patient of secondary biopsy is needed it needs to be determined that tumour progression position, for having performed the operation
Patient's stage after cutting can not obtain available tissue specimen and come judging prognosis and transfer case.
When cell death or quickly division, free DNA can be discharged and enter peripheral blood, be distributed mainly on blood plasma and serum
In.In normal person, dissociative DNA is mainly derived from hematopoietic cell.Since meronecrosis or cell turnover speed become faster, sense
When dye, ischemic, tumour, autoimmune disease, obesity and pregnancy, internal dissociative DNA content be will increase.It is sent out in tumour
During exhibition, tumour cell also can released dna into peripheral blood, i.e., it is free to obtain DNA of tumor cell (cell free tumor
DNA,ctDNA).The discovery of cfDNA and ctDNA and the development of ARMS-PCR and high throughput sequencing technologies bring for Non-invaive examination
New opportunity.CtDNA can replace tumor tissues to be widely used in the early screening of tumour, auxiliary diagnosis, targeted drug selection
With prognosis curative effect monitoring.
So tumor patient blood plasma dissociative DNA is mainly derived from the hematopoietic cell and tumour cell of tachymetabolism.That is blood plasma
After dissociative DNA is cell death, chromatin forms DNA fragmentation after endonuclease bamhi, and half-life period is very short, and half an hour will be by
It removes.The DNA sequence dna wherein fixed by nucleosome degradation speed due to nucleosome is protected is slower, and exposed DNA sequence dna is quickly
It is degraded, so dissociative DNA length is generally the integral multiple of nucleosome regular length.It is different tumor tissues, different types of
The nucleosome footprint of tumour cell, plasma DNA has differences.It can be by high throughput sequencing technologies to blood plasma
CfDNA carries out genome sequencing, obtains nucleosome location information, carries out molecule parting to patient by location information.
Summary of the invention
In view of the above deficiencies, simple, the high reagent of Detection accuracy is used the first purpose of the invention is to provide a kind of
Box.
A second object of the present invention is to provide a kind of samples to be easy to get, sampling flowsheet is simple, clinical experience is preferable and can flow
Journeyization operation, the method for being very suitable to the noninvasive molecule parting of breast cancer of the non-disease diagnostic purpose of clinical application.
For this purpose, first technical solution provided by the invention is such that
A kind of kit of the noninvasive molecule parting of breast cancer, including plasma DNA extract reagent, library construction reagent,
Sequencing reagent, sequence testing chip.
Second technical solution provided by the invention is such that
A kind of method of the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose, based on to plasma DNA high throughput
Sequencing data obtains the nucleosome foot in full-length genome range Natural promoter area in plasma DNA by bioinformatic analysis
Mark different information, so according to the difference of footprint information to patient with breast cancer realize lumen A type, lumen Type B, HER2 concentration type and
The other noninvasive molecule parting of four type of basal cell type.
Further, the method for the noninvasive molecule parting of breast cancer of above-mentioned non-disease diagnostic purpose, the nucleosome
Footprint difference be the region each gene transcription start site TSSs within the scope of plasma DNA full-length genome nucleosome distribution with
The difference in remaining region of gene falls to cause difference to generate with the region of nucleosome dissociation by fast degradation.
Further, the method for the noninvasive molecule parting of breast cancer of above-mentioned non-disease diagnostic purpose, specifically includes following
Step:
(1) high-flux sequence of plasma DNA
Reagent is extracted using plasma DNA described in claim 1, extracts the dissociative DNA of blood plasma, end is carried out and repairs
Multiple, connector connection, PCR amplification form sequencing library, carry out high-flux sequence, each sample at least detects the data of 6M reads
Amount；
(2) nucleosome engram analysis
By the RefSeq database of UCSC, the promoter region of current human protein coding gene, database note are obtained
Release the genomic locations in the region transcription initiation site TSSs upstream and downstream 1Kb of each gene of information counting statistics, and by the region
It is defined as original promoter region；The initial data being sequenced using claim 1 carries out sequencing data using Bowtie software
It compares, removes the repetitive sequence in comparison data, count the reads in the original promoter region of each protein coding gene
Number, and data are standardized using RPKM method, original promoter area coverage can prompt nucleosome trace, covering
It is less to spend lower nucleosome combination number, otherwise nucleosome number is more；
(3) nucleosome footprint variance analysis
According to nucleosome footprint difference, lumen A is filtered out using the Kruskal-Wallis nonparametric one-factor analysis of variance
There are the gene promoter region of coverage difference in type, lumen Type B, HER2 concentration type and basal cell type group, pass through sum of ranks
It examines 4 groups of coverage numerical value of the same gene to be compared two-by-two, and P value is corrected using holm method, filter out FDR
It is worth the gene promoter region less than 0.1；
(4) clustering
It is standardized using gene promoter coverage data of the Cluster software to differential expression, it is poly- using grade
Class method carries out clustering to the data after standardization according to the correlation between sample, and with R language pheatmap packet to data
It is visualized；According to the trace difference of the area TSSs nucleosome, sample type cluster is lumen A type, lumen Type B, HER2
4 class of concentration type and three female.
Further, the method for the noninvasive molecule parting of breast cancer of above-mentioned non-disease diagnostic purpose, described in step (1)
High-flux sequence is carried out according to conventional plasma DNA genome high-flux sequence method, and final obtain is greater than 6M
The data volume of reads.
Further, the method for the noninvasive molecule parting of breast cancer of above-mentioned non-disease diagnostic purpose, described in step (1)
The detection platform of high-flux sequence is ion torrent proton microarray dataset.
Further, the method for the noninvasive molecule parting of breast cancer of above-mentioned non-disease diagnostic purpose, the blood plasma
The high-flux sequence of dissociative DNA method particularly includes:
Step 1) peripheral blood blood plasma preparation
Blood plasma separation is carried out using two step centrifugal process
Step 2) plasma DNA extraction
Plasma DNA is extracted using plasma DNA extracts kit, after the completion of extraction, is carried out using Qubit 3.0
Concentration mensuration；
Step 3) library construction and quantitative
End reparation is carried out to DNA using Ion Plus Fragment Library Kit kit, is made after magnetic beads for purifying
Upper sequence measuring joints are added at the both ends DNA with DNA ligase, connection product is expanded after carrying out magnetic beads for purifying, after magnetic beads for purifying
Library construction is completed, concentration mensuration is carried out；Each library is diluted to 100pM according to concentration, carries out QPCR using ABI7500
After quantitative, every 10 library equivalent is mixed, its final concentration of 100pM is made；
Step 4) high-flux sequence
Use Ion PITMHi-QTMOT2200Kit carries out emulsion-based PCR to mixing library, is combined into library with sequencing ISP
ISP template is simultaneously expanded, and Dynabeads is then usedTMMyOneTMStreptavidin C1 carries out the enrichment of ISP template, uses
Ion PITMHi-QTMSequencing Kit initializes sequenator, and ISP template is mixed with mass controlled template, sequencing primer
After annealing, on loading to PI chip, in Ion Torrent ProtonTMSequenator selects the plan (journey pre-set
Sequence), carry out sequencing reaction；
Further, it is above-mentioned based on plasma DNA high throughput sequencing technologies to the parting of the noninvasive molecule of breast cancer
Method, two step centrifugal process carry out blood plasma separation and specifically include:
(1) at 4 DEG C, 1600g centrifugation 10min separates blood plasma with leucocyte, blood platelet, red blood cell, and blood plasma is turned
It moves on in 2mL centrifuge tube；
(2) 4 DEG C, 16000g is centrifuged 10min and removes residual cells, and supernatant is transferred in 2mL centrifuge tube to get to periphery
Blood blood plasma.
Further, it is above-mentioned based on plasma DNA high throughput sequencing technologies to the parting of the noninvasive molecule of breast cancer
Method, the program flow number are 300, and each sample data volume is greater than 6M.
Compared with prior art, technical solution provided by the invention has the advantages that
1, technical solution provided by the invention passes through to plasma DNA high-flux sequence, bioinformatic analysis and machine
Device study, is realized to patient with breast cancer's lumen A type (luminalA), lumen Type B (luminal B), HER2 concentration type (HER2-
Enriched) and the noninvasive accurate parting of basal cell type (basal-like), select patient with breast cancer clinical convenient for medical staff
The mode for the treatment of.
2, technical solution provided by the invention overcomes existing breast cancer molecular parting product using the office of invasive method
It is sex-limited, especially need invasive puncture to sample the material of breast cancer molecular parting, clinical experience is bad and has
The disadvantages of can not solving Tumor Heterogeneity, puncturing contraindication, tumor spread is caused to shift risk；Technical method provided by the invention
Raw material be 3-5ml peripheral blood, sample is easy to get, sampling flowsheet is simple, clinical experience is preferable and can procedure operation, it is non-
Often suitable clinical application.
3, detection sample of the present invention is peripheral blood blood plasma, and fresh periphery is preferably acquired with EDTA anticoagulant tube
The plasma specimen that whole blood sample obtains after being centrifuged also can be the plasma specimens of -70 DEG C or less preservations in 2 years.
4, technical solution provided by the invention passes through the biological information credit to plasma DNA high-flux sequence data
Analysis obtains the nucleosome footprint information in full-length genome range Natural promoter area in plasma DNA, and then according to footprint information
Difference realizes lumen A type, lumen Type B, HER2 concentration type and the other noninvasive molecule of four type of basal cell type to patient with breast cancer
Parting；It is Peripheral Blood In Patients With Breast Cancer that it, which detects material, belongs to Non-invasive detection scope, can be used for mammary cancer chemotherapy, endocrine is controlled
The selection gist for the treatment of, targeted therapy or operation plan.
5, technical solution provided by the invention passes through every within the scope of full-length genome in analysis plasma of breast cancer patients dissociative DNA
The nucleosome distributional difference information in a gene transcription start site region (TSSs) and remaining region of gene is to patient with breast cancer
Noninvasive molecule parting is carried out, foundation can be provided for the suitable therapeutic scheme of selection.
Detailed description of the invention
Fig. 1 is the TSS area coverage of HBEGF gene and MMP11 gene；
Fig. 2 is the cluster of patient with breast cancer's plasma DNA full-length genome nucleosome footprint difference before being treated based on 47
As a result.
Specific embodiment
With specific embodiment, the invention will be further described below, but the invention is not limited to these embodiments.
Reagent provided by the invention is this field conventional reagent, can pass through commercially available acquisition.
Embodiment 1
The noninvasive molecule parting product of breast cancer provided by the invention includes that plasma DNA extracts reagent (blood plasma
Dissociative DNA extracts the DNA extraction kit that reagent is MagaBio company), (Life technologies is public for library construction reagent
The Ion Plus Fragment Library Kit kit of department), sequencing reagent, sequence testing chip.Wherein library construction Kit
It is repaired including end, connector connection, PCR amplification reagent.
It should be noted that above-mentioned plasma DNA extract reagent and library construction reagent and sequencing reagent can also be with
Using other producers, such as:
Ion torrentproton microarray dataset: the sequencing reagent Ion PITMHi-QTMOT2200Kit article No. used:
A26434 and Ion PITMHi-QTMSequencing 200Kit article No.: A26433, sequence testing chip Ion PITMChip Kit
V3 article No.: A26771.
Illumina Miseq microarray dataset: the sequencing reagent MiSeq Reagent Kit v2 (300-cycles) used
Article No.: MS-102-2002.
Sample illustrate: collect clinical immunization groupization confirmation Peripheral Blood In Patients With Breast Cancer 47, wherein 12, lumen A type,
Lumen Type B 26, HER2 concentration type 3 and three female 6.It is specific as shown in table 1.
The noninvasive molecule parting of embodiment breast cancer
Step 1: blood plasma separation: carrying out (1) 4 DEG C of blood plasma separation using two step centrifugal process, 1600g is centrifuged 10min for blood plasma
It is separated with leucocyte, blood platelet, red blood cell, blood plasma is transferred in 2mL centrifuge tube；(2) 4 DEG C, 16000g is centrifuged 10min
Residual cells are removed, supernatant are transferred in 2mL centrifuge tube to get peripheral blood blood plasma is arrived.
Step 2: plasma DNA extracts: extracting blood plasma using plasma DNA extracts kit (MagaBio company)
Dissociative DNA, stringent by specification is operated, and after the completion of extraction, is carried out using Qubit 3.0 (Life Technologies)
Concentration mensuration.
Step 3: library construction and quantitative: using Ion Plus Fragment Library Kit (Life
Technologies company) kit carries out end reparation to DNA, and (this method is that routine magnetic bead in this field is pure after magnetic beads for purifying
Change method) using DNA ligase, in the upper sequence measuring joints of the both ends DNA addition, (the sequence measuring joints linker reagents are Ion
XpressTMBarcode Adapters 1-96Kit), connection product is expanded after carrying out magnetic beads for purifying, after magnetic beads for purifying i.e.
Library construction is completed, carries out concentration mensuration using Qubit 3.0.Each library is diluted to 100pM according to concentration, is used
After ABI7500 (Thermofisher) carries out QPCR quantitatively, every 10 library equivalent is mixed, its final concentration of 100pM is made.
Step 4: high-flux sequence: using Ion PITMHi-QTMOT2200Kit (Life Technologies) to mixing
Library carries out emulsion-based PCR, so that library is combined into ISP template with sequencing ISP and is expanded, is then used
DynabeadsTMMyOneTMStreptavidin C1 (Invitrogen) carries out the enrichment of ISP template.Use Ion PITMHi-
QTMSequencing Kit (Life Technologies) initializes sequenator, by ISP template and mass controlled template, sequencing primer
After carrying out mixing annealing, on loading to PI chip, pre-set in the selection of Ion Torrent ProtonTM sequenator
Program flow number is 300, and each sample data volume is greater than 6M), carry out sequencing reaction.Sequencing result is as shown in table 1.
1 47 breast cancer plasma specimen information of table and sequencing reads number averagely read long message
Step 5: bioinformatic analysis
(1) nucleosome engram analysis
By the RefSeq database of UCSC, obtain the promoter region of current human protein coding gene, we according to
The genomic locations in the region transcription initiation site (TSSs) upstream and downstream 1Kb of each gene of database annotation information counting statistics,
And the region is defined as original promoter region.After we obtain the initial data of sequencing, using Bowtie software to sequencing
Data are compared, and remove the repetitive sequence in comparison data, count the original promoter region of each protein coding gene
Reads number, and data are standardized using RPKM method.
With heparin-binding epidermal growth factor (the heparin-binding epidermal of original coverage difference
Growth factor-like growth factor, HB-EGF) for gene, lumen Type B patient is in original promoter
Coverage is lower than the coverage of its lumen A type patient, as shown in Figure 1a.HB-EGF gene is epidermal growth factor (epidermal
Growth factor, EGF) family member.HB-EGF gene can directly activate EGFR and ErbB4 or indirect activation
The heterodimeric receptor body of ErbB2 and ErbB3 composition.
By taking MMP-11 (matrix metalloproteinase-11, MMP-11) gene as an example, equally
It is lower than it in the coverage of lumen A type patient, as shown in Figure 1 b in the coverage of original promoter in lumen Type B patient.
MMP11 gene over-expresses in kinds of tumors, takes part in the infiltration and transfer process of tumour cell, generation and hair with tumour
It opens up closely related.
(2) nucleosome trace variance analysis
The nucleosome absent region site JiTSS is cance high-expression gene compared with the region that adjacent domain is substantially reduced, it is on the contrary then
For low expression gene.First by the Kruskal-Wallis nonparametric one-factor analysis of variance filter out lumen A type, lumen Type B,
There are the gene promoter regions of coverage difference in HER2 concentration type and basal cell type group, pass through the same base of rank sum test
4 groups of coverage numerical value of cause are compared two-by-two, and are corrected using holm method to P value, filter out FDR value less than 0.1
Gene promoter region.
(3) clustering
It is standardized using gene promoter coverage data of the Cluster software to differential expression, it is poly- using grade
Class method carries out clustering to the data after standardization according to the correlation between sample.And with R language pheatmap packet to data
It is visualized.
The high-throughput detection of plasma DNA based on 47 breast cancer carries out the coverage variance analysis in the region TSSs,
295, the region difference TSSs of lumen A type and lumen Type B, three female and lumen A type are found by comparing two-by-two refering to table 2
104, the region difference TSSs, 116, the region difference TSSs of three female and lumen Type B, HER2 concentration type and lumen A type
2, the region difference TSSs, 21, the region difference TSSs of HER2 concentration type and lumen Type B, the difference of HER2 concentration type and three female
Different TSSs areal is 0, this may be less related with the Sample size of HER2 concentration type.By using difference expression gene into
Row Unsupervised clustering, as a result, it has been found that sample can be polymerized to 4 kinds of classifications, lumen A type, lumen Type B, HER2 based on 474 genes
Concentration type and three female can be clustered respectively as 1 branch.The mode significant difference of coverage differential gene between each branch.
2. difference coverage of table analyzes the region TSSs and corresponding list of genes
Lumen A type, that is, Immunohistochemical detection ER is positive or PR is positive, and HER-2 is negative, Ki67 low expression.This type is
The most common molecular isoform of breast cancer, disease incidence 44.5%-69.0%.Lumen A type type is sensitive to endocrine therapy, efficient
Up to 40%, and ER level and the sensibility of endocrine therapy are positively correlated.Because HER-2 level be feminine gender, be not suitable for into
Row molecular targeted therapy.
Lumen Type B, that is, Immunohistochemical detection ER is positive or PR is positive, and HER2 is also positive, is more common in advanced age breast cancer
Patient.This hypotype also belongs to the tumour of endocrine therapy sensitivity, but due to the HER2 positive, to the curative effect of tamoxifen compared with lumen A
Type is poor, preferable to the effect of arimedex.
HER2 concentration type, that is, Immunohistochemical detection ER, PR is negative, and HER2 is positive, and Ki-67 is mostly high expression.Primary
In patient with breast cancer, with the presence of 20%-30% HER2 overexpression, and that researches show that prognosis is poor.Majority is late case, is easy
There is axillary lymphatic metastasis.Controlling for Trastuzumab combined chemotherapy is widely used to HER2 targeted drug rdativery sensitive in this type at present
Treatment scheme.
Basal cell type, that is, immunohistochemistry detection ER, PR, HER-2 is feminine gender, but is not equal to triple negative breast cancer, is had
There is 5%-45%ER for the positive in studies have shown that basal cell type patient with breast cancer, 14%HER2 is positive, and triple negative breast cancer
In only 80%-90% belong to basal cell type breast cancer.Its age of onset is low compared with other hypotypes, accounts in breast cancer
18.9%-28.8%.Basal cell type is considered as 1 worst hypotype of Prognosis in Breast Cancer all the time, DFS phase and
Overall survival is short compared with other hypotypes, is easy to appear the DISTANT METASTASES INs such as lung, brain.It is insensitive to current breast cancer treatment regimen,
Chemotherapy is unique systemic therapy approach.General clinical prognosis is poor.And in terms of molecular targeted therapy, it is possible to search out new
Therapy target.
The molecule parting of breast cancer is the main foundation of follow-up clinical therapeutic scheme selection.Breast cancer molecular parting is wide at present
General use standard is defined breast cancer classification standard in breast cancer international conference in 2013, is based on immunohistochemistry molecule parting knot
Fruit.But the material that immunohistochemistry uses is puncturing tissue, be it is invasive, clinical experience is bad.In addition, according to different immune groups
Chemoattractant molecule genotyping result takes the larger scale clinical application discovery of corresponding treatment scheme, still has the therapeutic effect of some patientss not
Obviously, or instead side effect is strengthened, therefore clinically needs finer molecule parting.The present invention passes through plasma free
The noninvasive molecule parting to breast cancer may be implemented in the bioinformatic analysis of DNA high-flux sequence data.And this method is
To being scanned within the scope of full-length genome, more information are obtained than traditional immunohistochemistry, are expected to further by the later period
Analysis obtains finer molecule parting, can get the therapeutic scheme of more individuation.In short, to have started breast cancer noninvasive by the present invention
The new method of molecule parting facilitates the accurate treatment of breast cancer.
Claims (9)
1. a kind of kit of the noninvasive molecule parting of breast cancer, which is characterized in that extract reagent, library including plasma DNA
Construct reagent, sequencing reagent, sequence testing chip.
2. a kind of method of the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose, which is characterized in that this method is based on to blood
Dissociative DNA high-flux sequence data are starched, by bioinformatic analysis, obtains in plasma DNA and is opened within the scope of full-length genome
The nucleosome footprint different information in mover area, and then lumen A type, lumen are realized to patient with breast cancer according to the difference of footprint information
Type B, HER2 concentration type and the other noninvasive molecule parting of four type of basal cell type.
3. the method for the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose according to claim 2, which is characterized in that
The nucleosome footprint difference is the region each gene transcription start site TSSs within the scope of plasma DNA full-length genome
The difference of nucleosome distribution and remaining region of gene is fallen difference is caused to generate with the region of nucleosome dissociation by fast degradation
's.
4. the method for the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose according to claim 2, which is characterized in that
Specifically includes the following steps:
(1) high-flux sequence of plasma DNA
Reagent is extracted using plasma DNA described in claim 1, extracts the dissociative DNA of blood plasma, end reparation is carried out, connects
Head connection, PCR amplification form sequencing library, carry out high-flux sequence, and each sample at least detects the data volume of 6M reads；
(2) nucleosome engram analysis
By the RefSeq database of UCSC, the promoter region of current human protein coding gene, database annotation letter are obtained
The genomic locations in the region transcription initiation site TSSs upstream and downstream 1Kb of each gene of counting statistics are ceased, and the region is defined
For original promoter region；The initial data being sequenced using claim 1, compares sequencing data using Bowtie software
It is right, the repetitive sequence in comparison data is removed, the reads number in the original promoter region of each protein coding gene is counted,
And data are standardized using RPKM method, original promoter area coverage can prompt nucleosome trace, coverage
Lower nucleosome combination number is less, otherwise nucleosome number is more；
(3) nucleosome footprint variance analysis
According to nucleosome footprint difference, lumen A type, pipe are filtered out using the Kruskal-Wallis nonparametric one-factor analysis of variance
It is same by rank sum test there are the gene promoter region of coverage difference in chamber Type B, HER2 concentration type and basal cell type group
4 groups of coverage numerical value of one gene are compared two-by-two, and are corrected using holm method to P value, are filtered out FDR value and are less than
0.1 gene promoter region；
(4) clustering
It is standardized using gene promoter coverage data of the Cluster software to differential expression, using hierarchical agglomerate method
Clustering is carried out to the data after standardization according to the correlation between sample, and data are carried out with R language pheatmap packet
It visualizes；According to the trace difference of the area TSSs nucleosome, sample type cluster is lumen A type, lumen Type B, HER2 enrichment
4 class of type and three female.
5. the method for the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose according to claim 4, which is characterized in that
High-flux sequence described in step (1) is carried out according to conventional plasma DNA genome high-flux sequence method, is finally obtained
Obtain the data volume greater than 6M reads.
6. the method for the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose according to claim 4, which is characterized in that
The detection platform of high-flux sequence described in step (1) is ion torrent proton microarray dataset.
7. the method for the noninvasive molecule parting of breast cancer of non-disease diagnostic purpose according to claim 4, which is characterized in that
The high-flux sequence of the plasma DNA method particularly includes:
Step 1) peripheral blood blood plasma preparation
Blood plasma separation is carried out using two step centrifugal process
Step 2) plasma DNA extraction
Plasma DNA is extracted using plasma DNA extracts kit, after the completion of extraction, carries out concentration using Qubit 3.0
Measurement；
Step 3) library construction and quantitative
End reparation is carried out to DNA using on Plus Fragment Library Kit kit, uses DNA after magnetic beads for purifying
Ligase adds upper sequence measuring joints at the both ends DNA, and connection product is expanded after carrying out magnetic beads for purifying, completes after magnetic beads for purifying
Library construction carries out concentration mensuration；Each library is diluted to 100pM according to concentration, it is quantitative to carry out QPCR using ABI7500
Afterwards, every 10 library equivalent is mixed, and makes its final concentration of 100pM；
Step 4) high-flux sequence
Use Ion PITMHi-QTMOT2200Kit carries out emulsion-based PCR to mixing library, and library and sequencing ISP is made to be combined into ISP mould
Plate is simultaneously expanded, and Dynabeads is then usedTM MyOneTMStreptavidin C1 carries out the enrichment of ISP template, uses Ion
PITM Hi-QTMSequencing Kit initializes sequenator, and ISP template mix moving back with mass controlled template, sequencing primer
After fire, on loading to PI chip, in Ion Torrent ProtonTMSequenator selects the plan pre-set, is surveyed
Sequence reaction.
8. it is according to claim 7 based on plasma DNA high throughput sequencing technologies to the parting of the noninvasive molecule of breast cancer
Method, which is characterized in that two step centrifugal process carry out blood plasma separation and specifically include:
(1) at 4 DEG C, 1600g centrifugation 10min separates blood plasma with leucocyte, blood platelet, red blood cell, and blood plasma is transferred to
In 2mL centrifuge tube；
(2) 4 DEG C, 16000g is centrifuged 10min and removes residual cells, supernatant is transferred in 2mL centrifuge tube to get peripheral blood blood is arrived
Slurry.
9. it is according to claim 7 based on plasma DNA high throughput sequencing technologies to the parting of the noninvasive molecule of breast cancer
Method, which is characterized in that the program flow number is 300, and each sample data volume is greater than 6M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910493271.8A CN110106244A (en) | 2019-06-06 | 2019-06-06 | A kind of noninvasive molecule parting kit of breast cancer and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910493271.8A CN110106244A (en) | 2019-06-06 | 2019-06-06 | A kind of noninvasive molecule parting kit of breast cancer and method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110106244A true CN110106244A (en) | 2019-08-09 |
Family
ID=67494260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910493271.8A Pending CN110106244A (en) | 2019-06-06 | 2019-06-06 | A kind of noninvasive molecule parting kit of breast cancer and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110106244A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110570951A (en) * | 2019-09-06 | 2019-12-13 | 广州市雄基生物信息技术有限公司 | Method for constructing classification model of new auxiliary chemotherapy curative effect of breast cancer |
| CN111378754A (en) * | 2020-04-23 | 2020-07-07 | 嘉兴市第一医院 | TCGA (TCGA-based genetic algorithm) database-based breast cancer methylation biomarker and screening method thereof |
| CN115295074A (en) * | 2022-10-08 | 2022-11-04 | 南京世和基因生物技术股份有限公司 | Application of gene marker in malignant pulmonary nodule screening, construction method of screening model and detection device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293910A (en) * | 2014-07-23 | 2015-01-21 | 周彤 | Gene group evaluating breast cancer molecule typing and detection kit thereof |
| CN109680049A (en) * | 2018-12-03 | 2019-04-26 | 东南大学 | A kind of method and its application based on the dissociative DNA in blood high-flux sequence analysis affiliated individual physiological state of cfDNA |
-
2019
- 2019-06-06 CN CN201910493271.8A patent/CN110106244A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293910A (en) * | 2014-07-23 | 2015-01-21 | 周彤 | Gene group evaluating breast cancer molecule typing and detection kit thereof |
| CN109680049A (en) * | 2018-12-03 | 2019-04-26 | 东南大学 | A kind of method and its application based on the dissociative DNA in blood high-flux sequence analysis affiliated individual physiological state of cfDNA |
Non-Patent Citations (1)
| Title |
|---|
| ROBERTA MALTONI等: "Cell-free DNA detected by "liquid biopsy" as a potential prognostic biomarker in early breast cancer", 《ONCOTARGET》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110570951A (en) * | 2019-09-06 | 2019-12-13 | 广州市雄基生物信息技术有限公司 | Method for constructing classification model of new auxiliary chemotherapy curative effect of breast cancer |
| CN111378754A (en) * | 2020-04-23 | 2020-07-07 | 嘉兴市第一医院 | TCGA (TCGA-based genetic algorithm) database-based breast cancer methylation biomarker and screening method thereof |
| CN115295074A (en) * | 2022-10-08 | 2022-11-04 | 南京世和基因生物技术股份有限公司 | Application of gene marker in malignant pulmonary nodule screening, construction method of screening model and detection device |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101027412B (en) | Urine markers for detection of bladder cancer | |
| ES2691404T3 (en) | Non-invasive cancer diagnosis | |
| CN110106244A (en) | A kind of noninvasive molecule parting kit of breast cancer and method | |
| CN107663533A (en) | A kind of lung cancer EGFR L858R and 19Del ddPCR detection methods and application | |
| Leprieur et al. | Sequential ctDNA whole-exome sequencing in advanced lung adenocarcinoma with initial durable tumor response on immune checkpoint inhibitor and late progression | |
| CN111833963A (en) | cfDNA classification method, device and application | |
| CN108027360A (en) | quantitative Her2 protein for optimal treatment of cancer | |
| CN109280702A (en) | Determine the method and system of individual chromosome textural anomaly | |
| CN114164276B (en) | Kit, device and method for lung cancer diagnosis | |
| CN106841593B (en) | Application of the LACC1 and FHL2 genes in preparing scoliosis detection product | |
| CN108949979A (en) | A method of judging that Lung neoplasm is good pernicious by blood sample | |
| Sanderson et al. | Improving the diagnosis of endometrial hyperplasia using computerized analysis and immunohistochemical biomarkers | |
| CN107287345B (en) | Detection kit for accurate diagnosis and treatment of acute myeloid leukemia and TSEN34 clinical application | |
| Boris et al. | Immunophenotypic portrait of leukemia‐associated‐phenotype markers in B acute lymphoblastic leukemia | |
| CN117701694A (en) | Glioblastoma prognosis prediction scoring model based on extracellular matrix expression and application | |
| CN117568481A (en) | Group of plasma exosome tsRNAs markers related to liver cancer and application thereof | |
| Chen et al. | cDNA microarray analysis and immunohistochemistry reveal a distinct molecular phenotype in serous endometrial cancer compared to endometrioid endometrial cancer | |
| CN106244675B (en) | Kit for adult AML risk stratification and clinical prognosis evaluation and application of CPNE3 | |
| CN111690746A (en) | Platelet RNA marker related to lung cancer and application thereof | |
| CN115482935B (en) | Lung adenocarcinoma patient prognosis model for predicting small cell transformation and establishment method thereof | |
| CN110570951A (en) | Method for constructing classification model of new auxiliary chemotherapy curative effect of breast cancer | |
| Sherman et al. | Morphological and molecular classification of human cancer | |
| CN105753960B (en) | A kind of tumor markers and its application | |
| CN104962612B (en) | G.41256139delT, BRCA1 gene frameshift mutation and its is preparing the application in Computer-aided Diagnosis of Breast Cancer kit | |
| CN106498085A (en) | A kind of kit for predicting that patients with esophageal squamous cell carcinoma of can performing the operation in early days recurs and shifts risk |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190809 |