CN100342035C - Method for testing receptor mutation of epidermkal growth factor in lung adenocarcinoma of non-smoker - Google Patents
Method for testing receptor mutation of epidermkal growth factor in lung adenocarcinoma of non-smoker Download PDFInfo
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- CN100342035C CN100342035C CNB200510049276XA CN200510049276A CN100342035C CN 100342035 C CN100342035 C CN 100342035C CN B200510049276X A CNB200510049276X A CN B200510049276XA CN 200510049276 A CN200510049276 A CN 200510049276A CN 100342035 C CN100342035 C CN 100342035C
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Abstract
The present invention provides a method for detecting the mutation of epidermal growth factor receptors (EGFR) in the lung adenocarcinoma of non-smokers, which is realized through the following steps: the genome DNA of the tumor is extracted, and a HotStarTaq Master Mix system is used for analyzing the mutation condition of the EGFR in the points of exons (18 to 21); in order to optimize result, the sequence is analyzed in a forward direction and a reverse direction. The method of the present invention discloses the phenomenon of mutation of the EGFR in the lung adenocarcinoma of non-smokers of China, and provides the basis for high-frequency mutation of extraterritorial exons of the structure of the tyrosine kinase (TK) of the EGFR and for using tyrosine kinase inhibitors (such as gefitinib and erlotinib) as treatment selection. The method of the present invention can be used for detecting the mutation of the epidermal growth factor receptor in the lung adenocarcinoma of non-smokers. The detection method of the present invention has the advantages of simple and convenient operation and easy popularization.
Description
Technical field
The invention belongs to biotechnology, relate to the detection method of EGF-R ELISA (EGFR) sudden change, relate in particular to the detection method of EGFR sudden change in non-smoker's adenocarcinoma of lung.
Background technology
Lung cancer is the first reason of cancer mortality in the world wide, and China also is the same, estimates that China in 2005 will have 429,000 people to die from lung cancer, and the next century, lung cancer mortality was with double
1At present, the main methods of treatment of progressive stage or recurrence lung cancer patient is a chemotherapy.Yet tyrosine kinase inhibitor (TKLs) is expected to change traditional therapy as the latest Progress of gefitinib and erlotinib, for lung cancer patient brings glad tidings.Gefitinib uses in America and Europe, Japan and Beijing nonsmall-cell lung cancer patient and shows clinical effectiveness, its efficient America and Europe 10%, Japan 26% and Beijing 36% of being respectively
2-4
Determine that recently the U.S. and Japanese excising have the somatic mutation of coding EGFR gene in the early stage lung tumor, its mutation frequency is respectively 8% and 26%
2-3The smoking number of times is less than " non-smoking " individuality of 100 times in life, and these sudden changes are more common, and with kinase inhibitor gefitinib (Iressa
)
5,6And erlotinib (Tarceba
TM)
7Treatment susceptibility relevant." non-smoking " adenocarcinoma of lung case of one group of U.S. has 7 examples (47%) EGFR sudden change in 15 examples
7Another group clinical experiment shows: after taking gefitinib and erlotinib, and the case of tool EGFR TK domain zone sudden change, 25 examples (81%) are effective in cure in 31 examples, and do not have the case of EGFR sudden change, and none example is effective in cure in 29 examples.China's patients with lung cancer EGFR mutation rate yet there are no report.For this reason, we detect the sudden change situation of non-smoking adenocarcinoma of lung patient's the EGFR in the China's Mainland especially, and compare with the immunohistochemistry detected result.
Summary of the invention
The detection method that the purpose of this invention is to provide non-smoker's adenocarcinoma of lung mesocuticle growth factor receptors (EGFR) sudden change, realize through the following steps:
Genomic dna is to extract from wax embedding block in the tumor tissues, phenol-chloroform method extracting DNA, measure DNA concentration then, 28 sites (28 of Tyrosylprotein kinase exons among the EGFR) are detected, primer sequence is seen SEQ ID NO:1-56, and pcr amplification adopts 25 μ l reaction systems, 10*buffer (Promega company product) 2.5 μ l, dATP, dCTP, each 0.5 μ l (final concentration 200 μ mol/L) of dGTP, dTTP, Mgcl
21.5 μ l (final concentration 1.5mmol/L), every primer 1 μ l (final concentration 500nmol/L), Taq enzyme (Promega company product) 0.2 μ l (1U), template DNA 1 μ l adds water to cumulative volume 25 μ l; Loop parameter: 95 ℃ of sex change 5 minutes, 95 ℃ of sex change 30 seconds, annealing temperature is respectively 58 ℃ of (exons l, 3,4,7-10,12-25,27,28), 56 ℃ (exons 2,5,6, and 26), 52 ℃ (exon11), 30 seconds time, 72 ℃ were extended 30 seconds, totally 30 circulations, last 72 ℃ were extended 4 ℃ of preservations 5 minutes.PCR product PCR Product Pre-Sequencing (Amersham Pharmacia Bioteth company) test kit purifying.Each exon is carried out positive and negative two-way sequencing, and sequencing analysis uses HotStarTaq Master Mix (Qiagen Hilden company, Germany) dna sequencing instrument to finish.
The inventive method provides Chinese non-smoking adenocarcinoma of lung to have the EGFR jumping phenomenon, and be Tyrosylprotein kinase (TK) the structural domain exon high frequency sudden change of EGFR, select to provide foundation as treatment for using tyrosine kinase inhibitor (as gefitinib and erlotinib).Detection method of the present invention is easy and simple to handle, is easy to promote.
Description of drawings
Fig. 1 is the sequential analysis of special EGFR exons 19 disappearances of the Chinese non-smoking adenocarcinoma of lung of an example.
Fig. 2 is tumour mesocuticle growth factor receptor gene sudden change position.
Embodiment
By detection method of the present invention, 5/8 routine lung cancer case detects the EGFR sudden change.See Table 1.EGFR sudden change instance analysis.
The table 1. adenocarcinoma of lung EGFR instance analysis that suddenlys change
| Case | Age | Sex | By stages | Histology | Smoking history | The EGFR mutation status |
| 1 2 3 4 5 6 7 8 | 41 53 55 51 70 44 60 64 | Woman woman woman woman woman woman woman man | T2N1M0 T1N0M0 T1N0M0 T2N1M0 T1N0M0 T3N1M0 T1N0M0 T2N0M0 | Gland cancer companion BAC gland cancer gland cancer companion BAC gland cancer gland cancer companion BAC mucinous adenocarcinoma gland cancer papillary adenocarcinoma | Do not have | Wild type wild type wild type delE746-A750 delE746-A750 delE746-A750insQP L858R L858R |
Clinical tumor by stages with nonsmall-cell lung cancer (NSCLC) standard of histological classification according to American Cancer Society (AJCC).Smoking history: smoking is less than 100 for not having in life.Amino acid change is observed in EGFR mutation status-attention.Del-deletion (disappearance); Ins-insertion (insertion).
The tumour pre-treatment
8 routine lung cancer cases of Xuan Zeing at random are the 2003-2004 of Zhejiang University Medical College The First Affiliated Hospital case of performing the operation in hospital.The male sex's 1 routine women's 7 examples among the patient, 55 years old mean age (41-70 year), be assessed as the non-smoker according to patient's medical history, complete tumor resection during operation, all tumours all are diagnosed as gland cancer through same high age and service seniority pathologist.
Immunohistochemistry
Section is through dewaxing to water 3%H
2O
2-methyl alcohol blocking-up endogenous peroxydase.Repaired 20 minutes with microwave in citrate buffer solution (pH6.0) the antigen retrieval liquid, be chilled to room temperature.The combination of sheep blood serum sealing non-specific antibody.One anti-EGFR (1: 100), 37 ℃, 60 minutes, it is anti-that the pH7.2 damping fluid is washed back dropping Envision two, and 37 ℃, 60 minutes.Damping fluid is washed back DAB colour developing 2～3 minutes, flowing water flushing color development stopping.Bush is plain dyes, and dehydration is transparent, sealing.To replace an anti-EGFR to establish negative control
The EGFR mutation analysis of lung tumor
Genomic dna is to extract from wax embedding block in the tumor tissues, and the PCR method is analyzed the EGFR sudden change, and the design of primers reference literature is paid special attention to the situation of EGFR in the sudden change of exon 18-21 place.Use HotStarTaq Master Mix system, carry out positive and negative both direction serial response, by two kinds of pcr amplifications independently dna fragmentation (isolates) determine all sudden changes, carry out sequential analysis (chromatograms) by two people, referring to Fig. 2, EGFR molecule dimer combines with Urogastron (EGF) part.Illustrate three parts--1. cell outskirt (contain two receptor sites: ligand L district and a furin-like district), 2. transmembrane domains and 3. cytoplasmic domain (containing catalysis kinases district (catalytic kinase)).The phosphorylation of tyrosine position (Y1068) is the target position of receptor activation, and the EGFR phosphorylation causes the signal transduction in its downstream and transcribes exciting son 3 (STAT3), and the product (AKT) of mitogen activated protein kinase (MAPK) and proto-oncogene c-akt is activated.Sudden change position when tumour takes place is represented with arrow all in the Tyrosylprotein kinase district.Wherein delE746-A750 and L858R are detected for the present invention.
The result
Immunohistochemistry result is that tumour cell 6 examples of 8 routine adenocarcinomas of lung are positive, the healthy tissues feminine gender.Relatively there is not dependency with EGFR sudden change detected result.
These wherein 5 routine tumours (62.5%) of organizing 8 routine adenocarcinomas of lung detect the EGFR sudden change, and the EGFR exon of its genomic dna is undergone mutation, 58 years old sudden change group mean age, no sudden change group 50 years old, the 1 routine male sex of being in 5 examples is organized in sudden change, and no sudden change is organized 3 examples and is the women, referring to table 2.As can be known: 4th, 5 examples are polynucleotide disappearance in exons 19 zones, the 6th example sudden change also occurs in exons 19, its disappearance is very peculiar, comprise the disappearance of 13 Nucleotide and the insertion of 4 Nucleotide, these changes have caused the insertion of disappearance, glutamine and 2 residues of proline(Pro) of amino acid E746-A750 (coding ELREA), referring to Fig. 1, Fig. 1 is the sequential analysis of special EGFR exons 19 disappearances of the Chinese non-smoking adenocarcinoma of lung of an example, sudden change relates to 5 amino-acid residues loses, and 2 residues insert (delE746-A750insQP).This routine tumor morphology shows as more mucus secretion, and oncocyte swims on the mucus lake, meets the diagnosis of mucinous adenocarcinoma.7th, the L858R of 8 routine tumour generation exon 2s 1 sudden change.Remaining 3 routine tumours (1-3 example) have the Wild type EGFR sequence.
This is that the Chinese non-smoking adenocarcinoma of lung of first report has the EGFR jumping phenomenon, and in this research, the EGFR sudden change takes place 5 examples in 8 examples, and its incidence is 62.5%.Though sample is less, these samples from the non-smoking lung cancer of large hospital case at random selection obtain, therefore have certain representativeness.Among the China nonsmall-cell lung cancer patient, non-smoking, smoking once and existing positive smoker's the equal no data of EGFR mutation rate, the financial resources that worthization is certain and manpower further investigation.
The Tyrosylprotein kinase of EGFR (TK) structural domain is undergone mutation in the adenocarcinoma of lung, all is positioned at exons 1 8-21.90% document is mentioned sudden change and is occurred in exons 19 and 21, is positioned at the polynucleotide framework disappearance of exons 19, often causes amino acid LREA deleted; And be positioned at the point mutation of exon 21, cause the leucine place of arginine in 858 sites (L858R).The mutation type that we find in the 4th, 5 examples and the 7th, 8 examples is identical with the type of the U.S. and Japan's report
5-7
This group has an example to be special EGFR exons 19 disappearances, and disappearance causes that 746 to 750 site amino-acid residues lose, and 2 residues of glutamine and proline(Pro) insert (delE746-A750insQP).The LREA sequence of contiguous 745 site lysine residues is for extremely important in conjunction with ATP, and EGFR exons 19 disappearances that sudden change causes have been lost the LREA sequence just.
Document prompting EGFR protein expression and tumour are irrelevant to the reaction of gefitinib treatment
8, there is the people in the xenograft model, to use gefitinib to experimentize, find that EGFRmRNA and EGFR protein level are also irrelevant
9, our detected result is shown EGFR protein expression and the no dependency of EGFR sudden change.
EGFR tyrosine kinase domain sudden change situation is relevant with the therapeutic response of tyrosine kinase inhibitor (TKIs), and the development of TKIs is very fast, gefitinib and erlotinib are the TKIs medicines of having used in the U.S., Europe etc. at present, China does not also have these two kinds of medicines of widespread use, and the non-smoking adenocarcinoma of lung patient of this group results suggest China may benefit from these medicines.This group detects and shows: Chinese non-smoking adenocarcinoma of lung EGFR mutation frequency height is a foundation of selecting gefitinib and erlotinib to use in China.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use to greatest extent.Therefore, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.
The partial reference document that the present invention relates to
1.Yang L，Parkin DM，Li LD，et al.Estimation and projection of the nationalprofile of cancer mortality in China：1991-2005.Br J Cancer 2004；90：2157-2166.
2.Kris M，Natale RB，Herbst R，et al.Efficacy of gefitinib，an inhibitor of theepidermal growth factor receptor tyrosine kinase，in symptomatic patients withnon-small cell lung cancer.JAMA 2003；290：2149-2158.
3.Fukuoka M，Yano S，Giaccone G，et al.Multi-institutional randomized phaseII trial of gefitinib for previously treated patients with advanced non-small-cell lungcancer.J Clin Oncol 2003；21：2237-2246.
4.Mu X-L，Li L-Y，Zhang X-T，Wang S-L，Wang M-Z.Evaluation of safetyand efficacy of gefitinib(′iressa′，zd1839)as monotherapy in a series of Chinesepatients with advanced non-small-cell lung cancer：experience from acompassionate-use programme.BMC Cancer 2004；4：51.
5.Lynch TJ，Bell DW，Sordella R，et al.Activating mutations in the epidermalgrowth factor receptor underlying responsiveness of non-small-cell lung cancer togefitinib.N Engl J Med 2004；350：2129-2139.
6.Paez JG，Janne PA，Lee JC，et al.EGFR mutations in lung cancer：correlation with clinical response to gefitinib therapy.Science 2004；304：1497-1500.
7.Pao W，Miller V，Zakowski M，et al.EGF receptor gene mutations arecommon in lung cancers from″never smokers″and are associated with sensitivityof tumors to gefitinib and erlotinib.Proc Natl Acad Sci USA 2004；101：13306-11.
8.Parra HS，Cavina R，Latteri F，et al.Analysis of epidermal growth factorreceptor expression as a predictive factor for response to gefitinib(′Iressa′，ZD1839)in non-small-cell lung cancer.Br J Cancer 2004；91：208-12.
9.Sirotnak F，Zakowski M，Miller V，et al.Efficacy of cytotoxic agents againsthuman tumor xenografts is markedly enhanced by co-administration of ZD1839(Iressa)，an inhibitor of EGFR tyrosine kinase.Clin Can Res 2000；6：4885-4892.
＜110〉Zhejiang University
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<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉match in the primer sequence of antisense strand, annealing temperature is 58 ℃
<400>54
ccttcactgt gtctgcaaat ctgc 24
<210>55
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉match in the primer sequence of sense strand, annealing temperature is 58 ℃
<400>55
cctgtcataa gtctccttgt tgag 24
<210>56
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉match in the primer sequence of antisense strand, annealing temperature is 58 ℃
<400>56
cagtctgtgg gtctaagagc taatg 25
Claims (2)
1. the detection method of non-smoker's adenocarcinoma of lung mesocuticle growth factor receptor mutations, realize through the following steps: genomic dna is to extract from wax embedding block in the tumor tissues, phenol-chloroform method extracting DNA, measure DNA concentration then, 28 sites are detected, primer sequence is seen SEQ ID NO:1-56, and pcr amplification adopts 25 μ l reaction systems, 10
*Damping fluid 2.5 μ l, dATP, dCTP, each 0.5 μ l of dGTP, dTTP, final concentration 200 μ mol/L, Mgcl
21.5 μ l, final concentration 1.5mmol/L, every primer 1 μ l, final concentration 500nmol/L, Taq enzyme 0.2 μ l, template DNA 1 μ l adds water to cumulative volume 25 μ l; Loop parameter: 95 ℃ of sex change 5 minutes, 95 ℃ of sex change 30 seconds, annealing temperature is respectively 58 ℃, 56 ℃, 52 ℃, and 30 seconds time, 72 ℃ were extended 30 seconds, totally 30 circulations, last 72 ℃ were extended 5 minutes, 4 ℃ of preservations, PCR product PCR Product Pre-Sequencing test kit purifying, each exon is carried out positive and negative two-way sequencing, and sequencing analysis uses HotStarTaq Master Mix dna sequencing instrument to finish.
2. the application of the detection method of non-smoker's adenocarcinoma of lung mesocuticle growth factor receptor mutations according to claim 1 in detecting non-smoker's adenocarcinoma of lung mesocuticle growth factor receptor mutations.
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| CN100342035C true CN100342035C (en) | 2007-10-10 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1073158A (en) * | 1991-12-12 | 1993-06-16 | 云南省香料研究开发中心 | The preparation method of beta-myrcene and device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1073158A (en) * | 1991-12-12 | 1993-06-16 | 云南省香料研究开发中心 | The preparation method of beta-myrcene and device |
Non-Patent Citations (2)
| Title |
|---|
| EGF receptor gene mutations are common in lung cancersfrom "never smokers" and are associated with sensitivity oftumors to gefitinib and erlotinib. Pao W et al,Proc Natl Acad Sci USA,Vol.101 2004 * |
| EGFR mutations in lung cancer:correlation with clinicalresponse to gefitinib therapy Paez JG etal,Science,Vol.304 2004 * |
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