CN105986024A - Genes for prognosis of triple negative breast cancer and application thereof - Google Patents
Genes for prognosis of triple negative breast cancer and application thereof Download PDFInfo
- Publication number
- CN105986024A CN105986024A CN201510091976.9A CN201510091976A CN105986024A CN 105986024 A CN105986024 A CN 105986024A CN 201510091976 A CN201510091976 A CN 201510091976A CN 105986024 A CN105986024 A CN 105986024A
- Authority
- CN
- China
- Prior art keywords
- gene
- seq
- breast cancer
- negative breast
- prognosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses genes for prognosis of triple negative breast cancer and the application thereof. The genes include gene GSTM1, gene BAG1, gene CD68, gene HDGFRP3, gene MAOB and gene TMEFF2. The genes can be applied to a multi-parallel testing liquid-phase gene chip for prognosis of triple negative breast cancer, a kit for prognosis of triple negative breast cancer, and a gene evaluation system for prognosis of triple negative breast cancer. By the adoption of the genes, accurate prognosis and classification of the relapse conditions of early-stage triple negative breast cancer patients in China five years after surgery can be achieved, and clinical medication can be effectively and timely guided.
Description
Technical field
The present invention relates to a kind of gene and application thereof, particularly relate to one group for the gene of three negative breast cancer prognosis in early days and
Application.
Background technology
Breast carcinoma is the whole world most commonly seen malignant tumor of women and the cause of death.Along with China's expanding economy and life style
Changing, the sickness rate of the breast carcinoma in domestic particularly flourishing city presents rapid increase trend (speed with annual 5% rises),
Year, neopathy number was close to 180,000.In megalopolis such as Shanghai, breast carcinoma has leapt to first of female malignant sickness rate.By
In raising and the raising for the treatment of level of current common people's examination consciousness, the mortality rate of breast carcinoma declines therewith, breast carcinoma of early stage
5 annual survival rates are up to 90%.
Three negative breast cancer (triple-negative breast cancer, TNBC) refer to estrogen receptor ER, progesterone receptor
PR and human epidermal growth factor acceptor (HER2) are a kind of Special Types of Breast Cancer of feminine gender, account for all breast carcinoma
15-20%.Within 2008, can perform the operation according to Tumor Hispital Attached to Fudan Univ the statistical result of breast cancer case, TNBC accounts for be owned
The 18% of breast carcinoma.Showing according to the existing research for TNBC, the breast carcinoma differentiation of hormone receptor-negative is poor, histology
Classification is higher, and easily recurrence and overall survival are relatively low, and Endocrine Drug therapy is insensitive is the Major Clinical of TNBC breast carcinoma
Performance.It is strong that TNBC not only has aggressive, the clinical characters of poor prognosis, and cannot accept endocrine because lacking hormone receptor
Treatment, lacks HER-2 gene loci and cannot accept Biological target therapy, the most at present cannot be again to three under the conditions of SABC
Negative breast cancer is segmented, and simultaneously for three negative breast cancer, there is no unified standard treatments at present, becomes breast carcinoma and control
The difficult point treated, recurring in early days, shifting in early days and ratio easily occur easily occur in a portion three Breast Cancer Patients with Negative Axillaries in early days
The transfer and relapse of the important organs such as more serious lung, liver.According to the common recognition of current breast carcinoma, the treatment master of TNBC breast carcinoma
Will be based on chemotherapy.
Current up-to-date research thinks that three negative breast cancer are probably the miscellaneous of a big class breast carcinoma, has height heterogeneity, its
The poor prognosis opposite sex is very big, and traditional Clinico Pathologic index there is no method and distinguishes the prognosis of three negative breast cancer.High-throughout entirely transcribe
Group gene chip again because the costliness of its price, quantity of information huge, it is impossible to apply in the clinic of reality.Abroad still there is no pin
Chip express spectra to three negative breast cancer.Simultaneously because in retrospective study at present, for fresh the freezing of chip express spectra research
Deposit specimen the rarest, be difficult to study for having had the case completely following up a case by regular visits to information.
It addition, the Oncotype DX that the U.S. has listed at present is directed to the breast carcinoma of estrogen receptor positive, according to difference
Scoring criterion can be that the breast carcinoma judging prognosis of estrogen receptor positive is with further direction of medication usage.But Oncotype is DX
Using the technology of more complicated RT-PCR, need to extract RNA, the process of reverse transcription is more complicated, can reduce its effect
Rate, so to three negative patients inapplicable.And Mammaprint has 70 genes that patient can be divided into high-risk and low danger wind
Danger group, the molecule parting important for breast carcinoma does not limit.Owing to it exists the gene of too much unknown function, analyzing effect
Rate and practice have the biggest restriction, are widely popularized utilization and limit bigger.And the sample that Mammaprint needs is facing
It is more difficult to obtain on bed, needs fresh frozen specimen, further limit clinical application.
Therefore, it is badly in need of the method that exploitation is a kind of more practical, easy and is quickly used for detecting the gene of three negative breast cancer prognosis.
Summary of the invention
The technical problem to be solved in the present invention is to provide one group of gene for three negative breast cancer prognosis and application thereof.Pin of the present invention
To be the difficult point in breast cancer treatment that is three negative breast cancer, in particular in clinical pathology prompting three negative breast in early days
Cancer, by the multiple parallel quantitative measurement technology for custom chip, can carry out multiple parallel quantitative study to mRNA, and
The specimen used can be the paraffin section that Pathology Deparment is widely present, and therefore, the present invention has more Clinical practicability, efficient, clever
Operation quick, easy, the advantage such as accurately.
For solving above-mentioned technical problem, a first aspect of the present invention, it is provided that one group (includes three the moon in early days for three negative breast cancer
Property breast carcinoma) gene of prognosis, comprising: gene GSTM1, BAG1, CD68, HDGFRP3, MAOB and TMEFF2.
Recurrence in early stage three Breast Cancer Patients with Negative Axillary of Chinese population after surgery 5 years and situation about not recurring can be entered by the combination of this gene
Row prognosis and classification, in order to intervene in time, effective guiding clinical treatment.
Therefore, for the genome of the present invention, it is possible to provide a series of application, as included: following for three negative breast cancer
The multiple parallel detection liquid phase gene chip of prognosis, for the test kit of three negative breast cancer prognosis and pre-for three negative breast cancer
After the evaluation system etc. of gene.In addition, it is necessary to explanation, heretofore described early stage three negative breast cancer refers in early days
Three negative breast cancer (T1-2N0-1)。
A second aspect of the present invention, it is provided that one is multiple for three negative breast cancer (including three negative breast cancer in early days) prognosis
Parallel testing (such as multiple parallel detection by quantitative) liquid phase gene chip (this chip is to detect for said gene group, with
Just detection gene GSTM1, BAG1, CD68, HDGFRP3, MAOB and TMEFF2), including probe；Wherein,
Described probe includes one of following three groups of nucleotide sequences:
(1) sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(2) complementary strand of every sequence in sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(3) there is the sequence of at least 70% homology with every sequence in the sequence shown in SEQ ID NO.1～SEQ ID NO.12
Row.
Described chip also includes: the probe of sequence shown in SEQ ID NO.13～SEQ ID NO.16.
A third aspect of the present invention, it is provided that a kind of reagent for three negative breast cancer (including three negative breast cancer in early days) prognosis
Box, including the probe of one of following three groups of nucleotide sequences:
(1) sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(2) complementary strand of every sequence in sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(3) there is the sequence of at least 70% homology with every sequence in the sequence shown in SEQ ID NO.1～SEQ ID NO.12
Row.
Described test kit also includes: the probe of sequence shown in SEQ ID NO.13～SEQ ID NO.16.
It addition, this test kit may also include for detect gene GSTM1, BAG1, CD68, HDGFRP3, MAOB and
Other corresponding reagent of TMEFF2, such as Proteinase K, Lysis Mixture etc..
A fourth aspect of the present invention, it is provided that a kind of gene for three negative breast cancer (including three negative breast cancer in early days) prognosis
Evaluation system, including recurrence risk figures module and evaluation module；
Described recurrence risk figures module, is used for obtaining recurrence risk figures, and wherein, this recurrence risk figures is to be surveyed by said gene chip
The respective mRNA value of gene GSTM1, BAG1, CD68, HDGFRP3, MAOB and TMEFF2 of the sample obtained
The value being multiplied with its effect estimated value in Cox returns and obtain；
Described evaluation module, for the recurrence risk figures that will obtain in recurrence risk figures module, passes through Receiver operating curve
(ROC) optimal marginal value (cutoff), can be divided into good prognosis and poor prognosis two groups by sample.
Preferably, the computing formula of described recurrence risk figures can be as follows:
FFPE_SCORE=-0.1636*GSTM1+0.5272*BAG1+1.6144*CD68-4.4979* HDGFRP3-2.1337*MAO
B-3.6536*TMEFF2
Wherein, the GSTM1 in formula represents the mRNA value of the GSTM1 gene utilizing said gene chip detection to obtain；
BAG1 represents the mRNA value of the BAG1 gene utilizing said gene chip detection to obtain；
CD68 represents the mRNA value of the CD68 gene utilizing said gene chip detection to obtain；
HDGFRP3 represents the mRNA value of the HDGFRP3 gene utilizing said gene chip detection to obtain；
MAOB represents the mRNA value of the MAOB gene utilizing above-mentioned said gene chip detection to obtain；
TMEFF2 represents the mRNA value of the TMEFF2 gene utilizing said gene chip detection to obtain.
For said gene group, it is to obtain through following research:
By the Tumor Hispital Attached to Fudan Univ breast carcinoma specimen of 2006 to 2008 is started with, at full genome express spectra water
Flat and public database screens, and the molecular marker gene that phase Preliminary screening obtains in the past is pre-with the breast carcinoma that previous work is integrated
Rear related gene group, the function of these molecular marker genes relate to growth and propagation, extracellular matrix formation, chemokine receptors,
Multiple important biomolecule processes such as apoptosis, signal transduction and DNA reparation.The paraffin specimen of three negative breast cancer is carried out
Detection, filters out prognosis differential expression genes group.Filter out following base according to the specimen information of three ' negative ' specimens prognosis quality simultaneously
Because having prognostic value in three negative breast carcinoma of early stage, clinical meaning is as follows:
Gene GSTM1: be positioned on No. 1 chromosome, encodes mankind's glutathione S-transferase GSTM1_HUMAN egg
In vain.The Cytoplasm of glutathione S-transferase is by two different supergene families codings with the form that cell membrane combines.Participate in
Intracellular organic matter transhipment, the synthesis of hormone, the protection of cellular oxidation stress damage, and in cancer chemotherapeutic protection and drug resistance
Middle performance critical function.It addition, its polymorphism is also relevant to disease susceptibility.Document is reported, GSTM1 is in lobular carcinoma in situ
Express more.
Gene BAG1: be positioned on Chromosome 9, can encode BAG family molecule companion's actuator 1, and BAG1 is one
Planting the multi-functional associated proteins being identified, it has and BCL-2 forms complex, and promotes the ability of anti-apoptotic.
But BAG1 is also not belonging to BCL-2 family.BAG1 can combine with multiple hormone receptor, thus adjusts their function.
The positive expression amount mammary gland benign tumor to be exceeded of BAG-1 and normal galactophore tissue in mammary gland tissue.Can be as mammary gland tissue
The early diagnosis index of canceration.
Gene C D68: being positioned on No. 17 chromosome, (leukocyte differentiation antigen 68) is the low-density of a kind of Glycoprotein binding
Lipoprotein.The phagocytosis activity of tissue macrophages plays a role, intracellular lysosome metabolism and extracellular cell with
Between cell, the interaction of cell and pathogen.The specific agglutination element of conjunctive tissue and organ or selection, make macrophage
Subgroup is gone back to the nest specific site.
Gene HDGFRP3: be positioned on No. 15 chromosome, is the gene of coding HDGR3_HUMAN albumen, is hepatocarcinoma
Somatomedin associated protein 3, occurs in tumor tissues lymphocytic infiltration raising, there is expression in Various Tissues.This
Gene can strengthen DNA synthesis, can play a role in cell proliferation.
Gene M AOB: monoamine oxidase B gene, the protein of this gene code belongs to flavin amino oxidase family.It is one
Plant enzyme, be positioned at mitochondrial outer membrane.Biological and the different amine of the oxidative deamination of its catalysis and neural activity and vasoactive amines are maincenter god
Play an important role in the metabolism of system and peripheral tissues.
Gene TMEFF2 (transmembrance protein with EGF-like and two follistatin-like domains): it is compiled
The code protein of a kind of tomoregulin-2, is the gene that a class is relevant to neurodevelopment, was cloned first in 1999.
Epidermal growth factor (EGF) sample functional domain and EGF/Neuregulin family somatomedin in TMEFF2 have higher homology
Property.TMEFF2 express by androgenic regulation and control, and suppressed by c-Myc albumen, be likely to be of the function of antioncogene.
In the present invention, the multiple parallel quantitative measurement technology of use, is the nucleic acid hybridization of a kind of sandwich structure, the method
BDNA molecule is used to amplify the target RNA signal of capture.It can analyze various sample (tissue, cell, blood,
FFPE sample, RNA or DNA etc. of purification), only need to use specific cleavage liquid lysed sample, to sample size lower limit without wanting
Ask, be particularly suitable for needing trace sample is done the research experiment that polygenes is quantitative.Its fusion transcript and DNA copy number variation
Extracting and purifying without nucleic acid, it is not necessary to PCR, is only amplified by the hybridization and signal with probe, can carry out gene quantification, keep away
Exempt from the interference of a lot of anthropic factor, can quickly obtain experimental result accurately.It is applicable to the paraffin section sample preserved for many years.
The present invention, for the gene of three negative breast cancer prognosis, is hybridized by molecule, and the means that amplification is amplified carry out multiple parallel inspection
Survey, the expression of gene in retrospective three negative breast cancer tissue samples is changed situation and detects, thus the morning to Chinese population
The situation that phase three Breast Cancer Patients with Negative Axillary recurs in 5 years after surgery and do not recurs carries out accurate prognosis and classification, the most in time
Instruct clinical application.
Accompanying drawing explanation
The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings:
Fig. 1 is the cluster analysis figure of training set；
Fig. 2 is the cluster analysis figure of checking collection；
Fig. 3 is area under curve (the AUC)-Receiver operating curve (ROC) of training set, AUC=0.818 (95%
Credibility interval be 0.659-0.977)；
Fig. 4 is area under curve (the AUC)-Receiver operating curve (ROC) of training set, AUC=0.974 (95%
Credibility interval be 0.615-0.974)；
Fig. 5 is the Kaplan-Meier survival curve of training set, wherein, P value=0.003；
Fig. 6 is the Kaplan-Meier survival curve of checking collection, wherein, P value=0.020.
Detailed description of the invention
The present embodiment, with Chinese Breast Cancer crowd as object of study, is organized as sample with breast carcinoma pathology, with multiple parallel detection liquid
Phase gene chip instrument, carries out following experiment.
Wherein, reagent used in tests below if not otherwise specified, is then commercialization reagent.
It addition, all of breast cancer tissue specimen is all from the performed the operation breast that Tumor Hispital Attached to Fudan Univ Breast Surgery is accepted for medical treatment
The specimen of adenocarcinoma patients, pathological staging is T1-2N0-1Breast carcinoma of early stage, be defined as IBC specimen, its SABC
ER and PR is patient negative, that HER21+ or 2+Fish is detected as feminine gender.In conjunction with early stage breast cancer prognosis-related gene group
Work, use 10 folding cross validations (10-fold cross validation) to select and assessment models.With the mesh of cross validation
Be to obtain reliable and stable model.10 folding cross validations (10-fold cross validation), are divided into ten parts by data set,
Do training 1 part by wherein 9 parts in turn to test, filter out the gene expression profile that can determine whether Prognosis in Breast Cancer.Median follow-up time 71
Month.Each example all takes the paraffin-embedded tissue of primary tissue, confirms that cancerous cell is more than 50% through histopathology.
Specific experiment is as follows:
(1) sample collection and clinical information collection
Collecting tissue specimen retrospective in Tumor Hispital Attached to Fudan Univ Pathology Deparment, gather clinical information, clinical information includes:
Therapeutic scheme selection, complete clinical medical history, Classification, histological grade, with or without vascular infiltration, lymph node situation,
Hormone receptor situation, SABC index, treatment situation, Follow-up Data etc., and set up data base.Have collected from 2006 to
Modern three clear and definite negative breast cancer tissue specimens, did not accepted the specimen of any treatment before being operation, new adjuvant chemotherapy
Case is excluded, all of case postoperative pathology detection be lymphatic metastasis less than equal to 3 transfer performed the operation mammary gland
Cancer (T1-2N0-1).The case selected is local recurrence or the specimen of original place transfer clearly occur.The patient of local recurrence has cell
Clarify a diagnosis.Multiple only being determined by iconography of patient of metastasis, the pathology that need of single-shot are made a definite diagnosis.
Pick out altogether the specimen of the paraffin section of 60 examples.This 60 example specimen is randomly divided into two groups, wherein, and 30 example specimen conducts
Training set (good prognosis 19 example, poor prognosis 11 example in training set), as checking collection, (good prognosis 19 is concentrated in checking to 30 example specimen
Example, poor prognosis 11 example).
(2) for the specimen of above-mentioned 60 example paraffin sections, SABC albumen ER, PR, HER2 and ki67 are re-started,
Standard is as follows:
ER and PR antibody uses commercial antibody, Dako (clone ER 1D5 1:35 and PR 636 1:50).ER and PR
Negative judgment criteria is breast carcinoma nucleus positive < 1%.Two independent Pathologis pass judgment on the feminine gender of ER and PR jointly.
For the judgement that HER2 is positive, using monoclonal antibody (Dako, clone PN2A 1:400), the HER-2 positive needs
If tumor cell film is positive, positive ratio is more than 10%.HER2 is also carried out the semiquantitative judge of 0 to 3+, needs
According to film staining power.0 point (0) is considered not dye；1 point (1+) is considered as that staining power is weak, and cell membrane dyes
Imperfect；2 points of (2+) cell membrane staining powers are weak to medium, and at least colored proportion is more than 10%；3 points (3+) dyeing is strong
Degree uniformity, the ratio more than 30%).Under study for action, HER2 SABC 3+ is considered as that HER2 has amplification, 2+
Result need the result checked according to HER2/neu gene amplification FISH to judge further, HER2 is negative, and we are defined as exempting from
Epidemic disease group HER21+, and HER22+ and FISH is negative.
It is the patient that negative, HER21+ or HER22+ and Fish are detected as feminine gender by SABC ER and PR, confirms as
Three Breast Cancer Patients with Negative Axillaries.
(3)Multiple parallel detection by quantitativeThe preparation of technology specimen, can the condition described in laboratory manual routinely be carried out, specifically
Can be as follows.
1) preparation of specimens paraffin embedding slices tissue homogenate
1. measure the area of tissue on slide, take the clean scraper of 5 slides by FFPE sample (the most above-mentioned stone on slide
Wax section sample) scrape in the centrifuge tube of 1.5mL.Homogenate and the E.C. 3.4.21.64 of respective volume is added according to table 1 below
(10mg/mL).Wherein, homogenate is from commercial kit (Quantigene multiplex assay kit).
Table 1 homogenate and the consumption of E.C. 3.4.21.64
| Tissue area (mm2) | Homogenate (μ L) | E.C. 3.4.21.64 volume (μ L) |
| 25-100 | 300 | 3 |
| 100-225 | 600 | 6 |
| >225 | 900 | 9 |
The most momently after whirlpool concussion sample, sample being placed in 65 ± 1 DEG C of water-baths 6 hours, period whirlpool every passing hour shakes
1min。
3. maximum velocity centrifugation sample 5min under room temperature, precipitate cell debris, then, homogenate is transferred in new centrifuge tube,
Avoiding paraffin and fragment residual, fragment is removed in repetitive operation if necessary.
Use tissue homogenate the most immediately or be stored in-80 DEG C of refrigerators.
2) sampleMultiple parallel detection by quantitative(liquid phase gene chip) experimental procedure
1. required reagent is taken out from refrigerator, be placed in room temperature and melt balance 10min.Tissue homogenate, if freezing, first
By it after-80 DEG C of refrigerators take out and room temperature is melted, it is placed in 37 ± 1 DEG C 30 minutes.After hatching, vibration mixing sample.Place
In room temperature.
2. Lysis Mixture is placed in 37 ± 1 DEG C of water-baths preheat 30 minutes, and softly mixes.
3. according to sample number used, in conjunction with preparation working solution listed in table 2 below.
The component of table 2 working solution
Wherein, the Lysis Mixture in said components, Blocking Reagent, Proteinase K and Capture Beads from
Business-like test kit (Quantigene multiplex assay kit).
Probe involved in 2.0Probe Set (probe shown in SEQ ID NO.1-16) be for gene GSTM1, BAG1,
The capture probe of CD68, HDGFRP3, MAOB and TMEFF2 and hybridization probe and reference gene (ACTB and PPIA)
Capture probe and hybridization probe (table 3).Wherein, the concentration of every kind of probe is 1 μ g/mL.
Table 3 probe
4. hybridizing on plate in 96 holes, every hole adds 60 μ L working solutions and the 40 corresponding sample of μ L (above-mentioned specimens paraffin embedding slices groups
Knit the homogenate striking off specimen), after overlay film, 54 ± 1 DEG C of 600rpm vibration lucifuges hybridize 18-22 hour.
5., after hybridization, by the liquid in hybridization plate, proceed in the corresponding aperture of corresponding magnetic separation plate.
6. magnetic separation plate is placed on hand-held magnetic frame.Each reacting hole adds lavation buffer solution (from Quantigene multiplex
Assay kit) 100 μ L washing 20s, in triplicate.
7. add pre-amplifier working solution (from Quantigene multiplex assay kit) 100 μ L, after overlay film, 50 ± 1 DEG C of 600rpm
Vibration lucifuge temperature is bathed 1 hour.
8. repeat step 6.
9. add amplifier working solution (from Quantigene multiplex assay kit) 100 μ L, after overlay film, 50 ± 1 DEG C of 600rpm
Vibration lucifuge temperature is bathed 1 hour.
10. repeat step 6.
11. add Label Probe working solution (from Quantigene multiplex assay kit) 100 μ L, after overlay film, 50 ± 1 DEG C
600rpm vibration lucifuge temperature is bathed 1 hour.
12. repeat step 6.
13. add SAPE working solution (from Quantigene multiplex assay kit) 100 μ L, after overlay film, room temperature 600rpm
Vibration lucifuge temperature is bathed 0.5 hour.
Magnetic separation plate is placed on hand-held magnetic frame by 14., and each reacting hole adds SAPE lavation buffer solution (from Quantigene
Multiplex assay kit) 100 μ L washing 20s, in triplicate.
15. add 130 μ L SAPE lavation buffer solutions (from Quantigene multiplex assay kit) in each reacting hole,
After 800rpm vibrates 1 minute, it is placed in Bioplex 100 liquid chip and analyzes system, read fluorescent value data.
Wherein, in said chip detection, hybridization instrument: Labnet wortemp 56；Hybridization cleans instrument: Bioplex
ProII wash station；Fluorescence data reads instrument: Luminex 100.
The paraffin specimen that multiple parallel detection by quantitative is used is previously stored Pathology Deparment, and the paraffin specimen selected is all through Pathology Deparment
Doctor's the most re-reading HE stained of the high age and service seniority, it is ensured that selected paraffin specimen is in all specimen, shared by malignant tumor
Ratio is the highest, at least meets the requirement of 50% simultaneously.Ungratified specimen is rejected.
(4)Multiple parallel detection by quantitative(liquid phase gene chip) technology detects
Analyze system by Bioplex 100 liquid chip and read the fluorescent value of the related gene that above-mentioned detection obtains, and use internal reference base
Analyze because the geometrical mean of (ACTB and PPIA) carries out homogenization to each testing gene, to obtain each base of each sample
The expression of cause.
(5) data process:
Being sampled being randomly divided into two groups by existing specimen case employing random sampling method respectively to two kinds of tissue samples, one group is
Training set (training set 30 example), one group as independent checking collection (checking collection 30 examples).
By combining the work of early stage breast cancer prognosis-related gene group, use 10 folding cross validation (10-fold cross
Validation) select and assessment models.Utilize Cox proportional hazards regression models, build risk of recurrence computation model, pass through
Training set builds the gene profile of three negative patients, is then verified by independent checking collection.All statistical analysis processes are passed through
SPSS software (IBM SPSS 19) and Weka3.6.10 complete, with bilateral P < 0.05 for statistically significant dividing value.
Multiple parallel quantitative measurement technology is analyzed system by Bioplex 100 liquid chip and is read the fluorescent value of related gene, and uses
The geometrical mean of reference gene carries out homogenization and analyzes, to obtain the expression of each gene of each sample each testing gene.
Owing to same training set being set up model, all of single factor test survival analysis and multifactor survival analysis all use sampling with repetition
(Bootstrapping) internal verification is carried out 1000 times.Individual authentication collection carries out external certificate.Kaplan-Meier survival analysis enters
Linear trend test between row packet factor level, with bilateral P < 0.05 for statistically significant dividing value.
(6) data result
1, through early stage screening and optimization, there are 6 mRNA (GSTM1, BAG1, CD68, HDGFRP3, MAOB
And TMEFF2) include model construction in.Utilize Cox Proportional hazards regression equation, establish model based on above molecule, mould
Type result is as follows:
FFPE_SCORE (recurrence risk figures)
=-0.1636*GSTM1+0.5272*BAG1+1.6144*CD68-4.4979*HDGFRP3-2.13 37*MAOB-3.6536*
TMEFF2
Wherein, the GSTM1 in formula represents the mRNA of the GSTM1 gene utilizing above-mentioned multiple parallel detection by quantitative to obtain
Value；
BAG1 represents the mRNA value of the BAG1 gene utilizing above-mentioned multiple parallel detection by quantitative to obtain；
CD68 represents the mRNA value of the CD68 gene utilizing above-mentioned multiple parallel detection by quantitative to obtain；
HDGFRP3 represents the mRNA value of the HDGFRP3 gene utilizing above-mentioned multiple parallel detection by quantitative to obtain；
MAOB represents the mRNA value of the MAOB gene utilizing above-mentioned multiple parallel detection by quantitative to obtain；
TMEFF2 represents the mRNA value of the TMEFF2 gene utilizing above-mentioned multiple parallel detection by quantitative to obtain.
It is to say, readings and its with the mRNA obtained in above-mentioned multiple parallel detection by quantitative are in Cox Proportional hazards returns
Effect estimated value be multiplied, obtained scoring is exactly the recurrence risk figures of three Breast Cancer Patients with Negative Axillaries.
2, Dendrogram is passed through, it can be seen that in training set specimen, sample and the sample without Preventive of Preventive are each
From being polymerized to a class, illustrate that these 6 genes can be poly-well three negative breast cancer postoperative recurrences with do not recur sample area separately
Being sample number above class dendrogram, right row are the titles of targeted 6 differential genes of probe of the present invention, are to express further below
Collection of illustrative plates, black represents that gene upregulation (relative to the average of this gene), white represent and lowers, according to the model of training set, uses
Checking collection is verified, the accuracy of classification reached for 80% (as shown in Figure 1-2).
3, by the readings of mRNA obtained in detection multiple parallel in training set and its effect in Cox Proportional hazards returns
Really estimated value is multiplied the recurrence risk figures (FFPE_SCORE) of three obtained negative breast cancer, according to model score, calculates
The recurrence risk figures (FFPE_SCORE) of every patient is concentrated in training set and checking, and the area under curve (AUC) of training set is
0.818, the credibility interval of 95% is (0.659-0.977), and obtains optimal cutoff value according to ROC, is divided into height
Danger group and low danger group.By Kaplan-Meier curve, it can be seen that either training set still verifies collection, high-risk group and low danger
Group all can well distinguish different prognosis, and for having significant difference without disease developing time, (training set P=0.003 is tested
Card collection P=0.020) (as seen in figures 3-6).
4, we recurrence risk figures and other clinical significant prognostic indicators (menstruation state, axillary gland positive number,
Tumor size, Nuclear grading, with or without vascular cancer embolus, operative treatment mode) put into and carry out multinomial logistic regression, it can be seen that nothing
Opinion is to concentrate in training set or checking, and recurrence risk figures is independently of the prognostic indicator of other Prognostic Factors, and its P value is respectively less than
0.05, there is significant difference.Wherein, the result in training set is as shown in table 4, and the result that checking is concentrated is as shown in table 5.
It addition, in table 4-5, the estimated value of B=regression coefficient, the standard error of SE=estimated value, the Wald inspection system of Wald=estimated value
Variable, df=degree of freedom, Sig=significance level i.e. P value, the estimated value of the effect of Exp (B)=each factor or dummy variable.
From above-mentioned experiment, the present invention can be to the recurrence of the early stage three negative breast cancer prognosis of Chinese population and situation about not recurring
Carry out accurate prognosis and classification, for instructing clinical application to lay the foundation in time.
Variable in table 4 equation
Variable in table 5 equation
Claims (8)
1. one group of gene for three negative breast cancer prognosis, it is characterised in that including: gene GSTM1, BAG1, CD68,
HDGFRP3, MAOB and TMEFF2.
2. the multiple parallel detection liquid phase gene chip for three negative breast cancer prognosis, it is characterised in that: include probe；
Wherein, described probe includes one of following three groups of nucleotide sequences:
(1) sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(2) complementary strand of every sequence in sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(3) there is the sequence of at least 70% homology with every sequence in the sequence shown in SEQ ID NO.1～SEQ ID NO.12
Row.
3. chip as claimed in claim 2, it is characterised in that: described chip also includes: SEQ ID NO.13～SEQ ID
The probe of sequence shown in NO.16；
Described three negative breast cancer include: three negative breast cancer in early days.
4. the test kit for three negative breast cancer prognosis, it is characterised in that include one of following three groups of nucleotide sequences
Probe:
(1) sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(2) complementary strand of every sequence in sequence shown in SEQ ID NO.1～SEQ ID NO.12；
(3) there is the sequence of at least 70% homology with every sequence in the sequence shown in SEQ ID NO.1～SEQ ID NO.12
Row.
5. test kit as claimed in claim 4, it is characterised in that: described test kit also includes: SEQ ID NO.13～SEQ
The probe of sequence shown in ID NO.16；
Described three negative breast cancer include: three negative breast cancer in early days.
6. test kit as claimed in claim 4, it is characterised in that: described test kit also includes: E.C. 3.4.21.64.
7. the evaluation system for the gene of three negative breast cancer prognosis, it is characterised in that including: recurrence risk figures module
And evaluation module；
Described recurrence risk figures module, is used for obtaining recurrence risk figures, and wherein, this recurrence risk figures is by described in claim 2
Gene GSTM1, BAG1, CD68, HDGFRP3, MAOB and TMEFF2 of sample of recording of gene chip each
The value being multiplied with its effect estimated value in Cox returns from mRNA value and obtain；
Described evaluation module, for the recurrence risk figures that will obtain in recurrence risk figures module, passes through Receiver operating curve
Optimal marginal value, sample can be divided into good prognosis and poor prognosis two groups.
Evaluate system the most as claimed in claim 7, it is characterised in that: described three negative breast cancer include: three negative breast in early days
Adenocarcinoma；
The computing formula of described recurrence risk figures is as follows:
FFPE_SCORE=-0.1636*GSTM1+0.5272*BAG1+1.6144*CD68-4.4979* HDGFRP3-2.1337
*MAOB-3.6536*TMEFF2
Wherein, the GSTM1 in formula represents the GSTM1 gene that utilizes the genechip detection described in claim 2 to obtain
MRNA value；
BAG1 represents the mRNA value of the BAG1 gene utilizing the genechip detection described in claim 2 to obtain；
CD68 represents the mRNA value of the CD68 gene utilizing the genechip detection described in claim 2 to obtain；
HDGFRP3 represents the mRNA value of the HDGFRP3 gene utilizing the genechip detection described in claim 2 to obtain；
MAOB represents the mRNA value of the MAOB gene utilizing the genechip detection described in the claims 2 to obtain；
TMEFF2 represents the mRNA value of the TMEFF2 gene utilizing the genechip detection described in claim 2 to obtain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091976.9A CN105986024B (en) | 2015-02-27 | 2015-02-27 | One group of gene and its application for triple negative breast cancer prognosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091976.9A CN105986024B (en) | 2015-02-27 | 2015-02-27 | One group of gene and its application for triple negative breast cancer prognosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105986024A true CN105986024A (en) | 2016-10-05 |
| CN105986024B CN105986024B (en) | 2019-10-25 |
Family
ID=57038779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510091976.9A Active CN105986024B (en) | 2015-02-27 | 2015-02-27 | One group of gene and its application for triple negative breast cancer prognosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105986024B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220445A (en) * | 2018-03-12 | 2018-06-29 | 中国科学院上海高等研究院 | A kind of evaluation triple negative breast cancer methods of risk assessment |
| CN109880905A (en) * | 2019-03-22 | 2019-06-14 | 复旦大学附属肿瘤医院 | One group of gene and its application for triple negative breast cancer immunohistochemistry parting |
| CN112802600A (en) * | 2021-02-07 | 2021-05-14 | 山东第一医科大学附属省立医院(山东省立医院) | Whole-course supervision system and method for soft tissue tumor informatization pathology auxiliary diagnosis |
| CN113061655A (en) * | 2021-03-25 | 2021-07-02 | 中国科学院合肥物质科学研究院 | Gene labels for predicting breast cancer neoadjuvant chemotherapy sensitivity and application thereof |
| CN116434880A (en) * | 2023-03-06 | 2023-07-14 | 哈尔滨理工大学 | High-entropy alloy hardness prediction method based on fuzzy self-consistent clustering integration |
| RU2811257C1 (en) * | 2023-07-14 | 2024-01-11 | Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") | Method of predicting risk of developing triple-negative breast cancer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195825A (en) * | 2007-12-10 | 2008-06-11 | 上海华冠生物芯片有限公司 | Gene for prognosis of breast cancer and uses thereof |
| CN102443627A (en) * | 2004-08-10 | 2012-05-09 | 加的夫生物学有限公司 | Methods and kit for the prognosis of breast cancer |
| WO2013134774A1 (en) * | 2012-03-09 | 2013-09-12 | Cornell University | Modulation of breast cancer growth by modulation of xbp1 activity |
-
2015
- 2015-02-27 CN CN201510091976.9A patent/CN105986024B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443627A (en) * | 2004-08-10 | 2012-05-09 | 加的夫生物学有限公司 | Methods and kit for the prognosis of breast cancer |
| CN101195825A (en) * | 2007-12-10 | 2008-06-11 | 上海华冠生物芯片有限公司 | Gene for prognosis of breast cancer and uses thereof |
| WO2013134774A1 (en) * | 2012-03-09 | 2013-09-12 | Cornell University | Modulation of breast cancer growth by modulation of xbp1 activity |
Non-Patent Citations (1)
| Title |
|---|
| 崔壮等: "GSTT1基因、GSTM1基因多态性与乳腺癌", 《天津医药》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220445A (en) * | 2018-03-12 | 2018-06-29 | 中国科学院上海高等研究院 | A kind of evaluation triple negative breast cancer methods of risk assessment |
| CN109880905A (en) * | 2019-03-22 | 2019-06-14 | 复旦大学附属肿瘤医院 | One group of gene and its application for triple negative breast cancer immunohistochemistry parting |
| CN109880905B (en) * | 2019-03-22 | 2022-08-30 | 复旦大学附属肿瘤医院 | Genes for immunohistochemical typing of triple negative breast cancer and application thereof |
| CN112802600A (en) * | 2021-02-07 | 2021-05-14 | 山东第一医科大学附属省立医院(山东省立医院) | Whole-course supervision system and method for soft tissue tumor informatization pathology auxiliary diagnosis |
| CN113061655A (en) * | 2021-03-25 | 2021-07-02 | 中国科学院合肥物质科学研究院 | Gene labels for predicting breast cancer neoadjuvant chemotherapy sensitivity and application thereof |
| CN113061655B (en) * | 2021-03-25 | 2022-04-19 | 中国科学院合肥物质科学研究院 | Gene labels for predicting breast cancer neoadjuvant chemotherapy sensitivity and application thereof |
| CN116434880A (en) * | 2023-03-06 | 2023-07-14 | 哈尔滨理工大学 | High-entropy alloy hardness prediction method based on fuzzy self-consistent clustering integration |
| CN116434880B (en) * | 2023-03-06 | 2023-09-08 | 哈尔滨理工大学 | High-entropy alloy hardness prediction method based on fuzzy self-consistent clustering integration |
| RU2811257C1 (en) * | 2023-07-14 | 2024-01-11 | Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") | Method of predicting risk of developing triple-negative breast cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105986024B (en) | 2019-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1781814B3 (en) | Methods and kit for the prognosis of breast cancer | |
| ES2905579T3 (en) | Expression profiling algorithm and test for prognosis of breast cancer relapse | |
| CN104620109B (en) | Carcinoma of urinary bladder detection composition, kit and related methods | |
| CN105087568B (en) | One group of gene and its application for tumor cells parting | |
| CN105319364B (en) | For predicting that the Combining diagnosis of microhepatia cancer recurrence is marked | |
| CN101410532B (en) | For detecting the urine gene expression ratios of cancer | |
| CN105986024B (en) | One group of gene and its application for triple negative breast cancer prognosis | |
| CN104263815B (en) | A group of genes used for prognosis of hormone receptor-positive breast cancer and applications thereof | |
| Bollmann et al. | Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer? | |
| Yin et al. | Twist expression in circulating hepatocellular carcinoma cells predicts metastasis and prognoses | |
| CN105986034A (en) | Application of group of gastric cancer genes | |
| CN109825586A (en) | DNA methylation qPCR kit and application method for lung cancer detection | |
| CN109504780A (en) | DNA methylation qPCR kit and application method for lung cancer detection | |
| CN106771248A (en) | High-level serous ovarian cancer diagnosis and/or the mark of Index for diagnosis | |
| CN109679957A (en) | IncRNALNC_004208 and its detection reagent are preparing the application in glioma prognostic agent | |
| CN109161593A (en) | The application of circular rna and microRNA in colorectal cancer sieving and diagnosis | |
| CN113355418B (en) | Gene for typing osteosarcoma and evaluating prognosis of osteosarcoma and application thereof | |
| Stackhouse et al. | Measurement of glut-1 expression using tissue microarrays to determine a race specific prognostic marker for breast cancer | |
| CN113156120B (en) | Application of B7H4 in preparation of endometrial cancer molecular typing reagent and system | |
| CN113046438B (en) | Endometrial cancer prognosis evaluation system incorporating molecular typing and immune scoring | |
| CN104975082B (en) | One group of gene and its application for assessing lung cancer for prognosis | |
| CN103687963A (en) | A method of determining the prognosis of hepatocellular carcinomas using a multigene signature associated with metastasis | |
| CN110244058B (en) | Application of ENPP1 in preparation of high-grade serous ovarian cancer diagnosis and prognosis kit | |
| CN107299129A (en) | Circle nucleic acid as breast cancer biomarker application | |
| CN113249479B (en) | Pancreatic cancer related lncRNA marker, probe and application of detection kit in pancreatic cancer diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |