CN110004229A - Application of the polygenes as EGFR monoclonal antibody class Drug-resistant marker - Google Patents
Application of the polygenes as EGFR monoclonal antibody class Drug-resistant marker Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/156—Polymorphic or mutational markers
Abstract
Application the invention discloses PTEN or/and BRAF or/and PIK3CA gene as EGFR monoclonal antibody class Drug-resistant marker.The present invention passes through high-throughput, highly sensitive detection, it was found that this three kinds cause the appearance in the gene genetic mutational site of secondary resistance to do sth. in advance about 13 weeks than clinical imageology discovery secondary resistance situation using PTEN, BRAF and PIK3CA gene as targeting EGFR monoclonal antibodies Drug-resistant marker.Tracking and analysis PTEN, BRAF and PIK3CA genetic mutation, it can predict whether detected person generates EGFR monoclonal antibody class Drug-resistant, it also can be that patient strives for that longer clinical treatment optimizes window phase while saving unnecessary medical expense for patient, for improving advanced colorectal cancer patient clinical therapeutic effect, improving its survival region, the unnecessary medical treatment cost of reduction with important value.
Description
Technical field
The invention belongs to pharmaceutical technology field, it is related to a kind of polygenes as EGFR monoclonal antibody class Drug-resistant mark
The application of object, and in particular to a kind of PTEN or/and BRAF or/and PIK3CA gene are resistance to as EGFR monoclonal antibody class drug
The application of medicine marker.
Background technique
Targeting epidermal growth factor receptor (EGFR) monoclonal antibodies drug Cetuximab (Cetutuximab) and pa
Buddhist nun's monoclonal antibody (Panitumumab) can specifically bind and block activation of the upstream the EGFR ligand to intracellular signaling pathway, resistance
Hinder cell Proliferation, promote apoptosis, reduce invasion and transfer ability that new vessels formed, inhibited tumour cell, so as to improve evening
The survival region of phase colorectal cancer patients.Even researches show that classic chemotherapy is invalid or failure patient still has 35% to 55%
Patient still is able to benefit from EGFR targeted therapy, thus such drug individually or with classic chemotherapy scheme use in conjunction
As the proposed standard scheme in international treatment guidelines.However, such clinical drug is widely applied, there are two big difficulties: (1)
EGFR targeted drug is more expensive, monthly spends in tens of thousands of members；(2) and not all colorectal cancer patients can target medicine from EGFR
Benefit in object treatment, primary drug resistance and secondary resistance are the main reason for limiting EGFR targeted drug Clinical efficacy.Cause
This, finds prediction scheme that is effective, being reliably capable of Accurate Prediction EGFR targeted therapy curative effect, for clinically avoid due to
The western appropriate secondary drug resistance of former times drug and waste payment for medical care and early to patient using more efficiently therapeutic scheme, and then improve evening
The treatment and prognosis of phase colorectal cancer patients have great importance.
New gene mutation site occur in the research discovery tumor tissues of tumor cell gene group is to cause clinically
Secondary drug resistant major reason, however colorectal cancer patients with terminal is often difficult to obtain tumor tissues, even if obtaining biopsy,
Its clinical representativeness is also limited by materials position.At the same time, clinically also lack effective serum markers pair at present
The targeted therapy effect of EGFR is predicted.In recent years, the peripheral blood cycling tumor DNA from tumour cell (ctDNA,
Circulating tumor DNA) it is concerned due to carrying tumour-specific abrupt information.It is now recognized that tumor patient
Circulation dissociative DNA be mainly derived from the tumour cell release of molecular marker for increased proliferation, the necrosis of tumor micrometastasis stove or circulating tumor cell,
Apoptosis and cracking are the very high ctDNA of specificity, and ctDNA has genetics characteristic identical with primary tumo(u)r, and can
Detect tumour associated gene mutation.For example Taniguchi in 2011 et al. is detected and tumour in the ctDNA of patients with lung adenocarcinoma
Identical EGFR is organized to be mutated, and ctDNA mutation is related with the drug resistance of EGFR inhibitor, being put forward for the first time ctDNA can be applied to
Drug resistance process monitoring (Taniguchi K, et al. (2011) Quantitative detection of of EGFR targeted drug
EGFR mutations in circulating tumor DNA derived from lung
adenocarcinomas.Clin Cancer Res 17:7808-7815.).Spinndler et al. late suffer from by colorectal cancer
Find that its ctDNA level dramatically increases in person, and high ctDNA is related to the bad survival region of colorectal cancer patients, in ctDNA
EGFR mutation with tumor tissues EGFR mutation consistency be up to 85% (Spindler KL, et al. (2015)
Circulating free DNA as biomarker and source for mutation detection in
metastatic colorectal cancer.PLoS One 10:e0108247.).Using more sensitive abrupt climatic change side
Method (0.001%), Kidess et al. have found BRAF, EGFR, KKRAS present in metastatic colorectal cancer patients blood plasma ctDNA
It is up to 93% with the associated gene mutation consistency detected in PIKK3CA mutation and tissue, and in Partial tumors patient
It is detected in ctDNA in the not found associated gene mutation site of tumor tissues.Receive in hepatic metastases excision patient, ctDNA's
Level compares recurrence (Kidess E, the et al. (2015) predictive of tumour of change of serum C EA and iconography material earlier
Mutation profiling of tumor DNA from plasma and tumor tissue of colorectal
cancer patients with a novel,high-sensitivity multiplexed mutation detection
platform.Oncotarget 6:2549-2561.).The studies above shows the key gene of EGFR and its downstream passages, may
By being mutated or expanding the drug resistance caused to EGFR monoclonal antibody.
In recent years, along with the development of two generation sequencing technologies (Next-generation sequencing), we can be
Genomic level carries out the detection of hereditary variation, and targeting sequencing technologies then can reach highly sensitive to multiple target genes simultaneously
Detection, by increase sequencing depth can detecte gene mutation of the frequency 0.01%.In addition to this, BEAMing
(Bead,emulsion,amplification and magnetic method)、SCODA(sequence-specific
Synchronous coefficient of drag alteration) and the methods of digital pcr (digital PCR etc.) go out
Now make it possible to that more sensitively certain specific mutation site is detected and (reaches 0.001% or so).But these current skills
Art has the disadvantage in that (1) flux is lower: above-mentioned technology is only capable of being detected for the specific variant sites of some gene, and
Full genome screening can not be carried out for the more gene of whole gene, especially nucleotide number, it can not be more comprehensively to latent
Drug resistance factor researched and analysed, be unable to satisfy clinical demand；(2) higher cost: the relevant technologies are required to special instrument
Device, equipment and detection reagent, such as digital pcr technology then need related PCR instrument and relevant consumptive material.Currently, related skill
There has been no production domesticization, pertinent instruments and consumptive materials to be both needed to import for art, expensive；(3) unknown to the gene order of quasi- detection, at present
It is concerned only with the mutation of a small number of genes such as EGFR mostly.
Summary of the invention
It is limited for the targeted therapy effect prediction marker of current EGFR, the targeted therapy effect of EGFR cannot be predicted early
The problem of fruit, the present invention provide PTEN or/and BRAF or/and PIK3CA gene as EGFR monoclonal antibody class Drug-resistant
The application of marker.
In the present invention, inventor is to potential in the patient ctDNA for receiving the treatment of EGFR monoclonal antibody class drug targeting
Influence the drug resistant mutated gene of EGFR and carry out high-throughput, highly sensitive detection, trace analysis be not present before EGFR treatment and
The mutational site for occurring after secondary drug resistance and dramatically increasing with treatment clinical course its frequency of mutation, establishing includes RAS-
RAF-MAPK and relevant 10 key genes of two signal paths of PI3K-PTEN-AKT, including EGFR, HER2, MET, KRAS,
NRAS, BRAF, MEK1 (MAP2K1), PIK3CA, AKT1 and PTEN, ctDNA the spectrum of mutation, find in addition to RAS gene mutation,
PTEN, BRAF and PIK3CA gene mutation are related to clinically there is EGFR targeting drug resistance, and above three gene mutation site is situated between
Lead EGFR resistance mechanism.
Specifically, the present invention provides PTEN or/and BRAF or/and PIK3CA gene as EGFR monoclonal antibody class
The screening technique of the application of Drug-resistant marker, comprising the following steps:
Step 1, sample collection: collecting before the tumor tissues of fresh biopsy of tumor patient when making a definite diagnosis, treatment and EGFR is mono-
The periphery blood specimen of clonal antibody class drug administration treatment stage, separated plasma and peripheral white blood cells, to tumor tissues, periphery
Blood leukocytes and blood plasma ctDNA are extracted；
Step 2, target gene capture and library construction: to include EGFR, HER2, MET, KRAS, NRAS, BRAF, MEK1
(MAP2K1), 10 genes of PIK3CA, AKT1 and PTEN are as target gene, by building library kit and targeting sequence capturing
Kit, forms liquid phase and captures chip system, carries out enrichment and library construction to target gene；
Step 3, high-flux sequence: using both-end be sequenced, sequencing depth be 10000 × more than, sample mean sequencing data
Amount is no less than 2G raw data, average Q20 >=90%, and Q30 >=85% carries out conventional Quality Control to sequencing data, but not according to frequency
Rate carries out data filtering；
Step 4, it the bioinformatic analysis of DNA sequencing data: is carried out using human genome reference sequences GRCh37.p13
Sequence mapping compares the related gene group DNA of ctDNA and peripheral white blood cells to identify that somatic mutation, screening are being treated
The gene mutation and variation frequency information occurred afterwards；
Step 5, the screening of candidate resistant mutational site: the frequency of mutation of all target genes is ranked up, obtains four
Quantile, including Q1, Q2 and Q3 and interquartile-range IQR (Qd=Q3-Q1), to the frequency of mutation (Mf) of each gene with Mf-Q3 >
The condition of 1.5Qd is screened, and is over the course for the treatment of in gradual raised as final candidate gene using the frequency of mutation；
Step 6, clinical correlation resistant mutational site is determined: in conjunction with CT images clinical data and RECIST evaluation system
Investigate different treatment periods clinical therapeutic efficacy and secondary resistance occur timing node (occur progression of disease PD when
Between), analysis hereditary variation occurs and correlation in time occurs for clinical secondary event, and exclusion newly goes out after clinical drug-resistant
Existing low frequency mutant gene locus, it is final to determine that EGFR monoclonal antibody class clinical drug correlation resistant mutational site is
PTEN or/and BRAF or/and PIK3CA.
Specifically, in a specific embodiment of the invention, the EGFR monoclonal antibody class drug can be western appropriate former times
Monoclonal antibody or Victibix.
Specifically, PTEN or/and BRAF or/and PIK3CA gene are resistance to as the EGFR monoclonal antibody class drug of tumour
The application of medicine marker, the tumour are tumour or secondary EGFR high caused by primary EGFR high expression or unconventionality expression
Tumour caused by expression or unconventionality expression, can be colorectal cancer, lung cancer, breast cancer, nasopharyngeal carcinoma etc..
Further specifically, PTEN or/and BRAF or/and PIK3CA gene detecting kit are in detection tumour
Application in EGFR monoclonal antibody class Drug-resistant can be realized by above-mentioned detection kit to whether detected person produces
Raw EGFR monoclonal antibody class Drug-resistant, and then instruct later period medication and adjustment therapeutic scheme.
Further specifically, the reagent for reducing PTEN or/and BRAF or/and PIK3CA expression improves tumour in preparation
To the application in the preparation of EGFR monoclonal antibody class drug susceptibility.The reduction PTEN or/and BRAF or/and
PIK3CA expression reagent can be able to suppress PTEN or/and BRAF or/and PIK3CA expression nucleic acid molecules, including but
It is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), siRNA (siRNA) or short hairpin RNA (shRNA), it can also
To be the nucleic acid construct comprising the above-mentioned inhibition PTEN or/and BRAF or/and PIK3CA nucleic acid molecules expressed, such as slow disease
Poisonous carrier.
The present invention can refer to clinically to provide clinic using the precision treatment that targeting EGFR monoclonal antibody treats tumor patient
It leads.According to the technique and scheme of the present invention, using PTEN, BRAF and PIK3CA gene as EGFR monoclonal antibody class Drug-resistant mark
It is secondary resistance to find that the appearance in the gene genetic mutational site that this three kinds lead to secondary resistance is found than clinical imageology for will object
Medicine situation has done sth. in advance about 13 weeks, according to the medical expense in clinical average 1.5 ten thousand yuan/week, and the dynamic detection expense of related gene
Carried out once according to every 6 weeks, carrying out 8 calculating according to 1 year, (western appropriate former times drug resistance often started drug resistance occur at 6 months or so, can
8 testing times can be not achieved), every time carry out sequencing and data analysis spend in 4000 yuan or so calculating, it is only necessary to 3.2 ten thousand yuan,
By it is an average of at least be every patient save ten thousand yuan of about 13 × 1.5-3.2=16.3 unnecessary medical expense and strive for for patient
Longer clinical treatment optimizes window phase, for improving advanced colorectal cancer patient clinical therapeutic effect, improving its existence
Prognosis reduces unnecessary medical treatment cost with important value.
Detailed description of the invention
Fig. 1 is the frequency of mutation spectrogram of 5 patients, 10 gene locis in the ctDNA before and after western appropriate former times treats drug resistance.
Fig. 2 be DNA in gene mutation frequency (KRAS, PTEN, BRAF and PIK3CA), serologic marker object (CEA and
CA19-9) the Relationship Between Dynamic Change figure between imaging evaluation index, SD: stable disease, PR: part alleviate, PD: disease into
Exhibition.
Specific embodiment
Below with reference to embodiment and attached drawing, invention is further described in detail.Experiment as used in the following examples
Method and apparatus is conventional method in that art and equipment unless otherwise specified.Material as used in the following examples, reagent
Deng being commercially available unless otherwise specified.
The present invention builds library kit by Roche company Kapa and NimbleGenSeqCap EZ library targets sequence
Capture kit build library, at the same improve two generations sequencing depth reach 10000 × sequencing receive EGFR monoclonal antibody west to 20
The drug resistant mutated gene of potential impact EGFR carries out high-throughput, highly sensitive detection in the patient ctDNA of appropriate former times targeted therapy.
Investigated be not present before EGFR chemotherapy and after secondary drug resistance occur and with treatment clinical course, its frequency of mutation significantly increases
The mutational site added finds that in addition to RAS gene mutation, PTEN, BRAF and PIK3CA gene mutation may be clinically to occur
EGFR targets drug resistant other reasons, and new gene mutational site may mediate EGFR drug resistance new mechanism, and said gene can substitute
Tumor tissues carry out the detection of dynamic specific gene variation to predict going out for the secondary resistance after EGFR targeted therapy
It is existing, so that clinically EGFR monoclonal antibody medicine uses for guidance, reduces medical expense, improves therapeutic effect.It is very important that invention
People has found the measurement to related gene loci mutation status, relative to traditional clinical molecular mark in terms of predicting western appropriate former times drug resistance
Will object has better advantage, and the appearance of above-mentioned specific gene variation is done sth. in advance than clinical imageology discovery secondary resistance situation
About 13 weeks.
The clinical research of 1.EGFR monoclonal antibody (Cetuximab) list medicine or combination chemotherapy advanced colorectal cancer patient
The retrospective study scheme designed and implemented.
(1) researching and designing: the 20 advanced metastatic colorectal cancers accepted for medical treatment based on Ruijin Hospital are carried out relevant clinical and ground
Study carefully.
(2) tested crowd: oncogene detect KRAS NRAS the 12nd, 13,61 codons be wild type, through clinical disease
The methods of reason, iconography are diagnosed as metastatic colorectal cancer patients.
(3) it therapeutic scheme: selects western appropriate former times list medicine or western appropriate former times to combine by clinician according to the state of an illness of patient and routinizes
Treatment scheme, drug dose and period use standard clinical usage.
(4) therapeutic evaluation: the CT examination of progress in every 6 weeks during treatment assesses the size variation of all tumor focus,
Evaluating curative effect according to RECIST standard is complete incidence graph (CR), part alleviation (PR), stable disease (SD) or progression of disease
(PD).For drug resistant patient occurring being defined as initial drug-resistant in therapeutic evaluation for the first time in the 6th week, and do not occur it is drug resistant then fixed
Justice is secondary drug resistance.Every 6 weeks chemical examination serology tumor markers simultaneously, the reference index as state of an illness dynamic change.Record is suffered from
Optimum curative effect time, disease developing time (PFS) and total life span (OS) of person etc..
(5) the fresh biopsy tumor tissues (liquid nitrogen cryopreservation) when making a definite diagnosis and the peripheral blood mark before treatment collection of specimens: are collected
This, separated plasma and peripheral white blood cells, -80 DEG C of refrigerators freeze.Before the treatment, every 4 to 6 week and generation drug resistance in therapeutic process
Afterwards, the blood plasma of series of time-points is acquired.And it is fresh swollen before collection treatment, when curative effect is best, after drug resistance in the conceived case
Tumor tissue.
2. developing gene mutation site detection screening technology relevant to western appropriate former times secondary resistance and scheme in ctDNA:
(1) it utilizes targeted capture and combines two generation sequencing technologies to whole exon regions of 10 key genes such as EGFR
Carry out deep sequencing analysis.
(a) DNA is extracted: using the DNA extraction kit of QIAGEN company, to tumor tissues, peripheral white blood cells and blood
Slurry ctDNA is extracted.Plasma DNA need to use QIAamp Circulating Nucleic Acid Kit, be enriched with 200bp
DNA fragmentation below utilizes Qubit quantitative detection dissociative DNA concentration.Specifically: the extracting of peripheral blood ctDNA uses Qiagen
The QIAseqcfDNA All-in-One Kit of company carries out the extracting of ctDNA to 2ml colorectal cancer patients plasma specimen, uses
After Qubit quantifies double-strand ctDNA, 100ng ctDNA is taken to carry out building library.Concrete operation step is according to QIAseqcfDNA
All-in-One Kit (article No. 180015) is operated.
(b) target gene capture and library construction: by document screening, we are secondary resistance to 10 potential impact EGFR
The gene of medicine as target gene, including EGFR, HER2, MET, KRAS, NRAS, BRAF, MEK1 (MAP2K1), PIK3CA,
AKT1 and PTEN builds library kit by Roche company Kapa and customized NimbleGenSeqCap EZ library targets sequence
Column capture kit forms relevant EGFR_Resistance_Panel liquid phase capture chip system, carries out to target gene rich
Collection and library construction.Specifically:
(b.1) it carries out building library using KAPA Library Preparation Kit.It is right first according to related kit
DNA uses magnetic beads for purifying, carries out DNA end-filling later, after DNA 3 '-holds progress A tailing, carries out connector connection and segment
The segment of size screening 150-500bp is purified, later using LM-PCR to DNA purified product progress PCR amplification, and
Quality inspection is carried out on 2100 instrument of Agilent.
(b.2) 10 gene regions of NimbleGenSeqCap EZ library targeted capture target are used.We to
Roche company has customized the targeted capture kit of 10 genes based on NimbleGenSeqCap EZ library platform,
Thermal denaturation, chip hybridization and hybridization magnetic bead are carried out to the DNA after building library referring to its specification to clean, and are finally used again
LM-PCR is expanded and is purified to purified product.Gene sequencing library after obtaining targeted capture, is sequenced.
(c) high-flux sequence: being sequenced using the Miseq system of Illumina microarray dataset, 10000 × sequencing above
Depth (both-end sequencing), sample mean sequencing data amount is no less than 2G raw data, average Q20 >=90%, and Q30 >=85% is right
Sequencing data carries out conventional Quality Control, is more than 10% sequence and the reads of low quality base including transition joint sequence, N content
Deng, but data filtering is not carried out according to frequency.
The bioinformatic analysis of (2) two generation sequencing datas and site screening.
(a) bioinformatic analysis of DNA sequencing data: sequence is carried out using human genome reference sequences GRCh37.p13
Mapping, compares the related gene group DNA of ctDNA and peripheral white blood cells to identify somatic mutation, using GATK and
The gene mutation (mediating secondary drug resistance reason) and variation frequency information that Varscan software screening method occurs after the treatment.Tool
Body are as follows: utilize human genome reference sequences GRCh37.p13, sequence mapping is carried out to sequencing data using BWA software, is compared
CtDNA and the related gene group DNA of peripheral white blood cells are to identify somatic mutation.Using samtoolsIn conjunction with GATK to sequencing
Quality is corrected and is analyzed, and the gene occurred after the treatment using MUTECT the and Varscan software screening method of GATK software is prominent
Become (mediating secondary drug resistance reason) and variation frequency information, and gene note is carried out to mutational site using ANNOVAR software
It releases, deletes not in the variant sites of 10 gene regions, synonymous variant sites and low quality variant sites.
(b) screening and its analysis with clinical resistance state of potential resistant mutational site: the mutation to all genes
Frequency sequence, obtains quartile (Q1, Q2 and Q3) and interquartile-range IQR (Qd=Q3-Q1).To the frequency of mutation of each gene
(Mf) it is screened with the condition of Mf-Q3 > 1.5Qd, obtained gene continues next round screening, to select the frequency of mutation
Over the course for the treatment of in gradual raised as final candidate gene.
In terms of data analysis, the analysis method that uses for classical gene compare (BWA), Quality estimation (FastQC),
Sequence (Samtools), quality correction and abrupt climatic change (GATK) and functional annotation (ANNOVAR), by mutational site
The function and potential influence for analyzing to judge genetic mutation.To identifying the frequency of mutation for carrying out all genes in data analysis
Sequence, obtains quartile (Q1, Q2 and Q3) and interquartile-range IQR (Qd=Q3-Q1).To the frequency of mutation (Mf) of each gene
It is screened with the condition of Mf-Q3 > 1.5Qd, obtained gene continues next round screening, so that the frequency of mutation be selected to control
In gradual raised as final candidate gene during treating, so as to it is more accurate to potential drug resistance site into
Row screening.
Fig. 1 is the frequency of mutation spectrogram of 5 patients, 10 gene locis in the ctDNA before and after western appropriate former times treats drug resistance.
It was found that the variation of KRAS, PTEN, PIK3CA and western appropriate former times drug resistance are closely related, it may be as clinical western appropriate former times drug resistant molecule mark
Will object.
(3) the function and structure Bioinformatics Prediction and clinical correlation in candidate gene mutational site.
(a) the biological function prediction in mutational site: it is in adjoint treatment using online software PolyPhen2.0 and SIFT
The existing gradual increased mutation of frequency carries out function prediction, and two methods of removal think harmless mutation.
(b) to the two generations key gene site mutation that detects of sequencing the frequency in different treatment periods dynamic change into
Row analysis is paid close attention to those and is not present before the treatment with treatment early stage, and adjoint treatment process newly sends out mutation and the frequency of mutation
The hereditary variation feature gradually increased.Different treatment periods are investigated in conjunction with CT images clinical data and RECIST evaluation system
The timing node (time for progression of disease PD occur) that clinical therapeutic efficacy and secondary resistance occur.Analysis hereditary variation goes out
Correlation in time now occurs with clinical secondary event, excludes the emerging low frequency mutated gene position after clinical drug-resistant
Point, to find potential to instruct clinical for advanced colorectal cancer patient to carry out western appropriate former times chemotherapy is secondary alone or in combination
The drug resistant genetic mutation molecular marker of property.
Fig. 2 be DNA in gene mutation frequency (KRAS, PTEN, BRAF and PIK3CA), serologic marker object (CEA and
CA19-9) the Relationship Between Dynamic Change figure between imaging evaluation index.Fig. 2 illustrates relative to traditional tumor molecular marker
The mutation (such as KRAS, PTEN, BRAF and PIK3CA) of related gene has more sensitive in CEA and CA19-9, blood plasma ctDNA
Drug resistance prediction efficiency.
The mutation for being currently known KRAS gene is to cause the advanced colorectal cancer patient of EGFR monoclonal antibody single therapy drug resistant
Main cause, and clinically find occur the mutation of KRAS in the only patient of 38% secondary resistance, therefore other keys
The mutation of gene may also lead to EGFR monoclonal antibody secondary resistance, multiple genes of the present invention to EGFR downstream signaling pathway
The mutation of (10 genes such as KRAS, NRAS, BRAF and PIK3CA) carries out screening, and discovery PTEN, BRAF and PIK3CA are to cause
The mutant gene locus of secondary resistance, and using the clinical value for investigating related mutation in perspective crowd, for clinic
The upper accurate treatment for carrying out EGFR monoclonal antibody provides guidance.
Claims (10)
- The application of 1.PTEN or/and BRAF or/and PIK3CA gene as EGFR monoclonal antibody class Drug-resistant marker.
- The application of 2.PTEN or/and BRAF or/and PIK3CA gene as EGFR monoclonal antibody class Drug-resistant marker Screening technique, which comprises the following steps:Step 1, sample collection: collecting before the tumor tissues of fresh biopsy of tumor patient when making a definite diagnosis, treatment and EGFR monoclonal The periphery blood specimen of antibody class drug administration treatment stage, separated plasma and peripheral white blood cells, it is white to tumor tissues, peripheral blood Cell and blood plasma ctDNA are extracted；Step 2, target gene capture and library construction: to include EGFR, HER2, MET, KRAS, NRAS, BRAF, MEK1 (MAP2K1), 10 genes of PIK3CA, AKT1 and PTEN are as target gene, by building library kit and targeting sequence capturing Kit, forms liquid phase and captures chip system, carries out enrichment and library construction to target gene；Step 3, high-flux sequence: using both-end be sequenced, sequencing depth be 10000 × more than, sample mean sequencing data amount is not Less than 2G raw data, average Q20 >=90%, Q30 >=85% carries out conventional Quality Control to sequencing data, but not according to frequency into Row data filtering；Step 4, the bioinformatic analysis of DNA sequencing data: sequence is carried out using human genome reference sequences GRCh37.p13 Mapping compares the related gene group DNA of ctDNA and peripheral white blood cells to identify that somatic mutation, screening go out after the treatment Existing gene mutation and variation frequency information；Step 5, the screening of candidate resistant mutational site: the frequency of mutation of all target genes is ranked up, quartile is obtained Number, including Q1, Q2 and Q3 and interquartile-range IQR Qd carry out the frequency of mutation Mf of each gene with the condition of Mf-Q3 > 1.5Qd Screening, is in gradual raised as final candidate gene using the frequency of mutation over the course for the treatment of；Step 6, it determines clinical correlation resistant mutational site: being investigated in conjunction with CT images clinical data and RECIST evaluation system The timing node that the clinical therapeutic efficacy and secondary resistance in different treatment periods occur, analysis hereditary variation occurs and clinic Correlation in time occurs for secondary event, excludes the emerging low frequency mutant gene locus after clinical drug-resistant, finally Determine that EGFR monoclonal antibody class clinical drug correlation resistant mutational site is PTEN or/and BRAF or/and PIK3CA.
- 3. application according to claim 1 or 2 or method, which is characterized in that the EGFR monoclonal antibody class drug It can be Cetuximab or Victibix.
- 4. application according to claim 1, which is characterized in that PTEN or/and BRAF or/and PIK3CA gene are as swollen The application of the EGFR monoclonal antibody class Drug-resistant marker of tumor, the tumour are primary EGFR high expression or exception table Up to tumour caused by caused tumour or secondary EGFR high expression or unconventionality expression.
- 5. method according to claim 2 or 4 or application, which is characterized in that the tumour be colorectal cancer, lung cancer, Breast cancer or nasopharyngeal carcinoma.
- 6.PTEN or/and BRAF or/and PIK3CA gene detecting kit are in the EGFR monoclonal antibody class medicine for detecting tumour Application in object drug resistance.
- 7. the reagent for reducing PTEN or/and BRAF or/and PIK3CA expression improves tumour to EGFR monoclonal antibody in preparation Application in the preparation of class drug susceptibility.
- 8. application according to claim 7, which is characterized in that the reduction PTEN or/and BRAF or/and PIK3CA The reagent of expression is the nucleic acid molecules for inhibiting PTEN or/and BRAF or/and PIK3CA expression.
- 9. application according to claim 8, which is characterized in that the nucleic acid molecules are antisense oligonucleotides, double-strand RNA, siRNA or short hairpin RNA.
- 10. according to any application of claim 7 to 9, which is characterized in that the reduction PTEN or/and BRAF or/ Reagent with PIK3CA expression is the nucleic acid structure comprising inhibiting the nucleic acid molecules of PTEN or/and BRAF or/and PIK3CA expression Build body.
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