CN107022602A - A kind of method of detection ALK genetic recombination - Google Patents
A kind of method of detection ALK genetic recombination Download PDFInfo
- Publication number
- CN107022602A CN107022602A CN201610074021.7A CN201610074021A CN107022602A CN 107022602 A CN107022602 A CN 107022602A CN 201610074021 A CN201610074021 A CN 201610074021A CN 107022602 A CN107022602 A CN 107022602A
- Authority
- CN
- China
- Prior art keywords
- sweyjawbu
- genes
- expression
- alk
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the measure of genetic recombination, a kind of assay method of ALK gene restructuring is related in particular to.
Description
Technical field
The present invention relates to the measure of genetic recombination, a kind of measure of ALK gene restructuring is related in particular to
Method.
Background technology
Hereditary change is frequently observed in cancer causes the constitutive activation of kinases.Morris in 1994
Find that the anaplastic lymphoma kinase (ALK) in primary cutaneous type (ALCL) is merged Deng first
To nuclear phosphoprotein (NPM).In past 20 years, it is found that substantial amounts of protein is fused to ALK and shape
Into function chimeric protein, such as in many different types of human cancers, including ALCL, diffusivity
Large B cell lymphoid tumor (DLBCL), inflammatory myofibroblastic tumor, the cancer of the esophagus, breast cancer, colon
Cancer, thyroid cancer, clear-cell carcinoma and non-small cell lung cancer (NSCLC) etc..These oncogenic fusion proteins with
Chromosome translocation or inversion are related.
In many cases, kinases of the growth and survival of tumour cell dependent on activation so that suppress
Its activity turns into a kind of effective anti-cancer therapies.There is the EGFR-TK suppression for cancer patient
The first oral ALK inhibitor of preparation (TKI), such as Pfizer companies(gram azoles replaces Buddhist nun).
For new diagnosis advanced NSCLC patients, the middle position of the Combination chemotherapy based on platinum class is always given birth to
It is 7.4-9.9 months to deposit the phase (OS), and the middle position OS of chemotherapy combined bevacizumab treatment is 12.5 months；
Middle position progression free survival phase (PFS) about 2.2-2.9 of second-line chemotherapy medicine such as pemetrexed and docetaxel
Individual month, survival rate was less than 20% within 5 years, treats not satisfactory.In contrast, with before without controlling
In the late period ALK positives NSCLC for the treatment of patient, gram azoles treats beautiful better than the line training of standard for Buddhist nun
Qu Saijia platinum-based chemotherapies, and it is related to bigger Lung Cancer Symptoms reduction and larger quality of life improvement.
Gram azoles must be based on that ALK is positive to clarify a diagnosis for the Clinical practice of Buddhist nun.
About 5-7% lung cancer tumor has ALK restructuring in the world.So to the evening newly diagnosed
Phase NSCLC suggestion tests ALK defrag status are considered as conventional clinical practice.However, plus
Put on airs, its ALK is determined only less than 1/3 patient in the advanced NSCLC patients of Japan and Germany
State, so as to the decision-making for first-line treatment, and the U.S. determines the trouble of its ALK state in contrast
Person exceedes half (www.kantarhealth.com/infographics/nsclc).From initial consulting to final
Test result, patient needs to wait 2-3 week, and this wait can be delayed the state of an illness of cancer patient
It is determined that starting with treatment.It is about 16 weeks that the median of IV phase patients with lung cancer, which does not treat life expectancy, because
This time should not be excessively spent in wait test result.Or, patient is always chosen in histology inspection
The chemotherapy of at least one course for the treatment of is carried out after looking into, ALK heterozygous state information is then waited in chemotherapy.
There are several detection ALK restructuring or the commercialization of heterozygosis experiment.One kind is that Abbott Laboratories Vysis ALK break
Separation FISH probe test is split, this is considered as goldstandard method.Other method includes Roche groups
The VENTANA ALK immunohistochemistries (IHC) of member Ventana Medical Systems companies
Determine, this method has been considered as substituting screening technique selecting the sample detected for ALK FISH.
Another method is the real-time PCR method in AmoyDx (Chinese Xiamen), and it only goes through to use in China
In detection patients with lung cancer ALK heterozygosis.Next generation's sequencing is provided detects multiple molecules in unitary determination
The ability of change, but it may need could to complete in several weeks experiment and data analysis.Although sequencing
Technological innovation is very promising, but the sensitiveness of this method, specificity and Clinical efficacy at present
Clinically without enough data.It has been reported that gram azoles replaces the clinical response of Buddhist nun's therapy in a few cases
It is lower inconsistent with diagnostic result.
Therefore, there is very big space at present and recombinate test to improve current ALK, so as to help patient
More suitable treatment.
The content of the invention
In one aspect, the method that ALK gene is recombinated in biological sample is determined the present invention relates to a kind of,
Methods described includes determining the expression of sweyjawbu genes in the biological sample, wherein
The sequence of sweyjawbu genes such as SEQ ID NO:Described in 1 or with SEQ ID NO:1 has at least
90% homogeneity.
In another aspect of the present invention, sequence and the SEQ ID NO of sweyjawbu genes:1 has
At least 95%, preferably 98%, more preferably 99% homogeneity.
In another aspect of the present invention, the expressions of sweyjawbu genes by determine RNA or
CDNA level is determined.
In another aspect of the present invention, the expression of sweyjawbu genes is determined by PCR,
It is preferred that quantitative fluorescent PCR, the sequence such as SEQ ID NO for the primer that the PCR is used:2 and 3 institutes
State.
In another aspect of the present invention, the expression of sweyjawbu genes determines the tumour from patient
Tissue, the tumor tissues are selected from surgery excision tissue, aspiration biopsy sample, hydrothorax sample, brain ridge
Liquid sample, blood sample and other body fluid samples.
In another aspect of the present invention, method of the invention further comprises in specimens
Sweyjawbu genes expression and patient's normal structure in sweyjawbu genes table
Compared up to level, the sweyjawbu gene expression doses wherein in tumor tissues are higher than normal structure
In sweyjawbu gene expression doses represent that the ALK gene restructuring of the patient tumors is positive.
In another aspect of the present invention, the expression of sweyjawbu genes is determined by Δ Ct values,
Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,CtHPRT1) it is CtESDAnd CtHPRT1
Geometric mean.In another aspect of the present invention, Δ Ct represents ALK gene restructuring less than 1.8
It is positive.
One aspect of the present invention is related to a kind of kit for being used to detect ALK gene restructuring, the examination
Agent box includes being used to determine the specificity examination of the expression of sweyjawbu genes in the biological sample
Agent, the wherein sequence of sweyjawbu genes such as SEQ ID NO:Described in 1 or with SEQ ID NO:1
With at least 90% homogeneity.In another aspect of the present invention, the sequence of sweyjawbu genes with
SEQ ID NO:1 have at least 95%, preferably 98%, more preferably 99% homogeneity.
In another aspect of the present invention, kit of the invention, which includes at least two parts, to be used to determine
The specific reagent of the expression of sweyjawbu genes, the specific reagent is respectively used to identical trouble
The tumor tissues and normal structure of person.In another aspect of the present invention, kit of the invention is also wrapped
Include the specific reagent of the expression for determining ESD and HPRT1 genes.
In another aspect of the present invention, kit of the invention also includes label, and the label has
Illustrate that the kit is used for the content for detecting ALK gene restructuring.In another aspect of the present invention,
The label of kit also has herein below：
1) illustrate that kit of the present invention is used for the expression of the sweyjawbu genes in specimens
Level is compared with the expression of the sweyjawbu genes in patient's normal structure, wherein tumour
Sweyjawbu gene expression doses in tissue are higher than the sweyjawbu gene expressions in normal structure
Level represents that the ALK gene restructuring of the patient tumors is positive；Or
2) illustrate that kit of the present invention is used for the Δ Ct values for determining the expression of sweyjawbu genes,
Wherein Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,CtHPRT1) it is CtESDWith
CtHPRT1Geometric mean, Δ Ct represents that ALK genes restructuring is positive less than 1.8.
One aspect of the present invention is related to whether a kind of detection patient meets ALK inhibitor medicaments treatment mark
Accurate method, methods described determines the ALK bases in the patient tumors including the use of the method for the present invention
Because of restructuring.If measurement result shows that the ALK gene restructuring of the patient tumors is positive, the patient
ALK inhibitor medicaments can be used to be treated.
These and other content of the present invention by the following drawings and embodiment in an illustrative manner
Explanation.
Brief description of the drawings
Fig. 1 shows sweyjawbu genes (upper row) and SNP site therein (lower to arrange), wherein Y
Represent that C or T, S represent that G or C, W represent that A or T, R represent A or G.Underscore is marked
Part correspond respectively to SEQ ID NO:5 ' primers and 3 ' primers shown in 2 or 3.
Fig. 2 shows the positive sweyjawbu genes between the negative lung cancer cell lines of ALK of ALK
Relative gene expresses the difference of (Ct) value, and Fig. 2A is the signal intensity for comparing probe 242964_at, figure
2B is that sweyjawbu genes Relative gene expresses (Ct) value.Probe 242964_at signal intensity comes from
Cancer Cell Line Encyclopedia, www.broadinstitute.org/ccle.PCR experiment is carried out
Four repetitions.
Fig. 3 shows the difference observed between the negative lung cancer of the ALK positive lung cancers and ALK of patient,
The Ct values (B) of the Ct values (A) of sweyjawbu genes, ALK transcripts 5' parts and 3' parts are compared,
And Ct value+ALK transcripts 5' parts and the Ct values (C) of 3' parts of sweyjawbu genes.Grind
Study carefully and include 30 adenocarcinomas of lung (brown+black), 4 large cell carcinomas (black), 20 squamous cell carcinoma (oranges
Color), 12 normal structures (grey).Confirm that 5 gland cancer and 1 large cell carcinoma are ALK by FISH
The positive, shows minimum Ct values.Generally speaking, CtsweyjawbuIt is positive negative with ALK in ALK
Notable difference is shown between sample, p value is equal to 4.59E-06.CtALK3'/5'In the ALK positives and ALK
Higher difference is shown between negative sample, p value is equal to 6.45E-18.By Ctsweyjawbu and
CtALK3'/5'The positive differences between ALK negative samples of highest ALK, p value are observed during addition
Equal to 5.99E-20.
Embodiment
Term " ALK gene " used herein refers to anaplastic lymphoma kinase or CD246, and it is
Phosphocarnic acid albumen (the NPM)-ALK initially identified in primary cutaneous type insulin receptor
Receptor tyrosine kinase (Morris etc. (1994), Science 263 in superfamily:1281-1284；Morris
Deng (1997), Oncogene 14:2175-2188).Subsequent research has identified diffusivity large B cell
Lymthoma, malignant histiocytosis, inflammatory myofibroblastic tumor sarcoma, esophageal squamous cell
ALK in cancer, breast cancer, colorectal cancer and non-small cell lung cancer subclass is recombinated (see Webb etc.
(2009), Expert Rev Anticancer Ther 9:331-356).ALK polypeptides are to match somebody with somebody comprising extracellular
The single transmembrane albumen of body land, membrane spaning domain and cytoplasmic kinase catalytic domain.ALK is by human body
Interior chromosome band 2p23 (Morris etc., (1994) Science 263:1281-1284；Shiota etc.,
(1994)Oncogene 9:Locus coding on 1567-1574).
Term " ALK gene restructuring " used herein refers to the adjacent of ALK gene and normal chromosomal
Portion fractures and with other connection breakings of chromosome, so as to cause exception, such as ALK genome
DNA cloning, protein are overexpressed and activation point mutation.
Term " sweyjawbu genes " used herein is a kind of non-coding RNA, and it is located at people 2
Number chromosome.The gene low abundance table in people reaches, and just corresponds to gene average expression amount in database
5.5%.7 GenBank accession number that the sequence origin of the gene comes from 5 cDNA clones are true
It is fixed.
Term " ESD genes " used herein refers to encode the serine for belonging to esterase D families hydrolysis
The gene of enzyme.The enzyme of the coded by said gene is active to various substrates, includes the sialic acid of O- acetylations,
The recycling of sialic acid may be participated in.The gene is used frequently as retinoblastoma and Wei Ersenshi
The genetic marker of disease.
Term " HPRT1 genes " used herein refers to coding hypoxanthine phosphoric acid nucleoside transferase 1
Gene, the enzyme is catalyzed time yellow fast by the transfer of the 5- phosphate groups of ribose 5-phosphate -1- pyrophosphoric acids
Purine is converted into inosinicacid and guanine is converted into guanosine 5-monophosphate, by fast in the generation of purine nucleotides
Purine remedial pathway plays central role.The mutation of the gene can cause Lai-Buddhist nun's syndrome or gout.
The amount of product produced by term " expression " used herein refers to the transcription of gene, translation.
Under regulation mechanism control, the process such as gene experience gene activation, transcription and translation, producing has life
The protein molecule of thing function.Due to the influence of the factors such as environment, same gene Different Individual,
May even have transcription product or the translation production of entirely different amount in the different tissues of same individual
Thing, i.e., with different expressions.The expression of usual gene typically passes through the genetic transcription
MRNA level is weighed.
Term " homogeneity " used herein refers in the case of two or more polynucleotide sequences,
Compare identical residue in the highest consistent degree time series for specifying comparison window.When using percentage,
Percentage of sequence identity refers to by being compared determination to two optimal comparison sequences in comparison window
Value, wherein for the optimal comparison of two sequences, (do not include addition with reference sequences or lack)
Compare, the polynucleotide sequence part in comparison window may include addition or missing (that is, room).Hundred
Divide than being calculated by following：Determine occur the position of identical nucleic acid base or amino acid residue in two sequences
Quantity is put to obtain the quantity of matched position, by the position in the quantity divided by comparison window of matched position
Sum, and result is multiplied by 100 to obtain Percentage of sequence identity.
Unless otherwise prescribed, provided herein is sequence identity/similarity refer to using GAP versions 10
And its equivalent programs, use the homogeneity percentage value of the nucleotide sequence of following gain of parameter：GAP
Starting point penalty is that 50, GAP extension point penalties are 3, uses nwsgapdna.cmp rating matrixs." etc.
Effect program " means for any two sequences in discussion, when the phase with being produced with GAP versions 10
Should compare compared to when, produce the comparison with identical nucleotides match and identical Percentage of sequence identity
Any sequence comparison program.
It was found by the inventors of the present invention that the inventive method can be used for the sweyjawbu with various SNP
Gene, such as the sweyjawbu genes with SNP shown in Fig. 1.Therefore, the present invention's
On one side, the sequence of the sweyjawbu genes in the present invention and SEQ ID NO:1 has at least
95%th, preferably at least 98%, more preferably at least 99% homogeneity.
Term " PCR " used herein refers to PCR, is by least one polymerase
Effect by specificity amplification primer produce target nucleic acid fragments multiple copies process.It is this to expand
Increase reaction be well known to a person skilled in the art.When amplification is the PCR carried out after reverse transcription reaction,
It is referred to as RT-PCR.
Term " real-time fluorescence quantitative PCR " used herein refers to the process of in pcr amplification reaction
In, product total amount after each PCR cycle is determined with fluorescent chemical, joins method by internal reference or outside
The method that quantitative analysis is carried out to the specific dna sequence in testing sample.PCR in each round circulation
Product amount recorded all in the form of fluorescence signal by the Systems for optical inspection of PCR instrument, a certain
When the intensity of fluorescence signal reaches threshold value set in advance in circulation, period now is referred to as Ct values
(Threshold Cycle).Ct values are inversely proportional with the template amount originated, that is, the pcr template amount originated is more,
Reach that the i.e. Ct values of period of threshold value are smaller.For example, those skilled in the art are it is understood that CtESDWith
CtHPRT1Represent that ESD and HPRT1 reaches the period of threshold value.
Term " Δ Ct values " used herein refers to the difference of Ct values.
Term " biological sample " used herein refers to any material from patient, and it may contain
The biomaterial of gene expression can be detected.Biological sample can be the blood of patient, serum, saliva,
The sample of tissue or circulating cells.This kind of biological sample can pass through well known by persons skilled in the art
What sampling mode is obtained, and for example blood sample can be obtained by drawing blood.Term used herein is " biological
Material " represents that any material of gene expression, such as nucleic acid or albumen, or its coded sequence can be detected.
Nucleic acid can be with especially RNA (ribonucleic acid) such as mRNA (mRNA).According to of the invention preferred
Embodiment, biomaterial is nucleic acid, even more preferably mRNA.From extraction from biological material life
Thing material can be by the way that well known to a person skilled in the art extract all schemes of nucleic acid or albumen come real
Apply.
Term " tumor tissues " used herein refers to the tissue for constituting the major part of tumour.Generally,
Tumor tissues include essence and interstitial two parts, and wherein tumor epithelial cell is tumour cell, with tissue
Source specificity.The interstitial of tumour acts to support and nutrition tumor epithelial cell do not have specificity, typically
It is made up of connective tissue and blood vessel, can also there is lymphatic vessel sometimes.Tumor tissues can be obtained from various sources,
Including but not limited to surgery excision tissue, aspiration biopsy sample, hydrothorax sample, CSF sample and its
Its body fluid sample.
The present invention method and kit can be used for various tumours, including but not limited to the cancer of form of ownership,
Melanoma, sarcoma, lymthoma and leukaemia, such as lung cancer, cancer of the esophagus, stomach cancer, cancer of pancreas, intestines
Cancer, kidney, carcinoma of urinary bladder, carcinoma of urethra, prostate cancer, liver cancer, osteocarcinoma, nervous system cancer, son
Palace cancer, oophoroma, leukemia, cutaneum carcinoma etc..
Term " normal structure " used herein refers to the nonneoplastic tissue with normal function and structure.
Those skilled in the art are it is understood that normal structure different in same individual can have different gene tables
Up to spectrum.Therefore, in one aspect of the invention, method of the invention or kit are used and tumor group
Knit same or like normal structure of originating.
Term " label " used herein refers to that any be associated to kit, offer kit is related
The material of information.Label may include in the commercial packing of kit, such as independent specification,
It can either be attached on the container in kit or directly print to the container, or can directly print
In the outer surface of the commercial packing of kit.Label can include any information related to kit, example
Such as the manufacturer of kit, purposes, application method, points for attention and/or on using such product
The information of warning.The present invention label can be any form, for example packaging, inset, printing material,
Multimedia, interconnected network address, bar code etc..
According to the purpose of the present invention, total serum IgE extract can by cracking be present in it is thin in blood sample
Born of the same parents are realized with discharging the step of nucleic acid that Patient cells are included.It is intended to citing, can be used such as special
Profit applies for WO00/05338 (it is on mictomagnetism and mechanical lysis), (it is on electricity by WO99/53304
Cracking), the cleavage method described in WO99/15321 (it is on mechanical lysis).Those skilled in the art
Other well known cleavage method, such as heat shock or osmotic shock or use chaotropic agent such as guanidine can be used
The chemical cracking (U.S. Patent number 5,234,809) of salt.It is also possible to provide extra step, for from
The other cell component seperated nuclear acids discharged in cleavage step.This generally makes it possible condensed nucleic acid.
It is intended to citing, can be used optionally by absorption or the covalent effect coated magnetic-particle of oligonucleotides
(in this respect, seeing U.S. Patent number 4,672,040 and U.S. Patent number 5,750,338), and thus can lead to
Cross the nucleic acid that washing step purifying is incorporated into these magnetic-particles.If necessary to then expand the nucleic acid,
This nucleic acid purification step is particularly advantageous.In patent application WO97/45202 and WO99/35500
In describe the particularly advantageous embodiment of these magnetic-particles.
Term used herein " specific reagent " refers to such reagent, when make its with it is as defined above
Biologic material contact when, can be combined with the material special to the target gene.It is intended to explanation,
When specific reagent and biologic material are core sources, specific reagent is set to be contacted with biologic material
Specific reagent can be made to hybridize with the material special to the target gene.Term " hybridization " refers to such mistake
Journey, i.e., during the process under suitable condition, two nucleotide fragments are with stable and special
Hydrogenbond is so as to form double-stranded complex.These hydrogen bonds are in complementary adenine (A) and thymidine
(T) between (or uracil (U)) base (this is referred to as A-T keys), or it is phonetic in complementary guanine (G) and born of the same parents
(this is referred to as G-C keys) is formed between pyridine (C) base.The hybridization of two nucleotide fragments can be complete
(being referred to as complementary nucleotide acid fragment or sequence), i.e., the double-stranded complex obtained in this crossover process is only wrapped
Key containing A-T and C-G keys.This hybridization can be (being referred to as abundant complementary nucleotide acid fragment or sequence) of part,
The double-stranded complex obtained both comprising the A-T keys and C-G keys for allowing to be formed double-strand, was also wrapped
Containing the base not combined with complementary base.Hybridization between two nucleotide fragments depends on being used
Condition of work, particularly stringency.Stringency is specifically defined as the base group of two nucleotide fragments
Into function and the extent of mismatch also between two nucleotide fragments define.Stringency is additionally depended on
Response parameter, for example, be present in ion concentration and type in hybridization solution, the property of denaturant and dense
Degree and/or hybridization temperature.All these data are known, and can be determined by those skilled in the art
Appropriate condition.In general, the length of the nucleotide fragments hybridized as needed, hybridization temperature is
About 20 DEG C to 70 DEG C, particularly 35 DEG C to 65 DEG C, in concentration about 0.5-1M salting liquid.
Sequence, or nucleotide fragments, or oligonucleotides, or polynucleotides are a series of by phosphoric acid ester bond
The nucleotides motif fitted together, is characterized as the native sequence nucleic acid containing information, and it can be with nucleosides
Acid fragment hybridizes, and the series may contain the monomer with different structure, and may be from natural acid
Molecule and/or obtained by Genetic Recombination and/or by chemical synthesis.Motif is the derivative of monomer, institute
The natural nucleotide that monomer can be nucleic acid is stated, its composed component is sugar, phosphate group and nitrogenous base；
In DNA, the sugar is deoxidation -2- ribose, in RNA, and the sugar is ribose；According to whether
It is related to DNA and RNA, nitrogenous base is selected from adenine, guanine, uracil, cytimidine and thymus gland
Pyrimidine；Alternatively, monomer is the nucleotides that at least one of three kinds of composed components are modified；It is intended to
Citing, modification can occur base level (have modification base for example inosine, methyl -5- deoxycytidines,
BrdU, dimethylamino -5-FU, diaminourea -2,6- purine, bromo- 5-FU are appointed
The what base for the modification that it can hybridize), or occur level (for example, at least one deoxyribose in sugar
Replace with polyamide (P.E.Nielsen et al., Science, 254,1497-1500 (1991) [3])), or
Occur (for example to replace with ester in phosphate group level, be chosen in particular from bisphosphate, alkyl phosphate in addition
With acyl phosphate and thiophosphate).
According to specific embodiment of the present invention, the specific reagent includes at least one hybridization probe,
Or at least one hybridization probe and at least one primer special to target gene, or at least one hybridization spy
Pin and two kinds of primers special to target gene.
Term " amplimer " used herein refers to nucleotide fragments, and it includes 5-100 nucleotides,
It is preferred that 15-30 nucleotides, it causes enzymatic polymerization such as enzymatic amplification reaction starting.Term " enzymatic
Amplified reaction " refers to the process of produce multicopy nucleotide fragments by the effect of at least one enzyme.So
Amplified reaction be well known to a person skilled in the art, and should be particularly mentioned that following technologies：PCR (polymerases
Chain reaction) (such as in U.S. Patent number 4,683,195, U.S. Patent number 4,683,202 and U.S. Patent number
Described in 4,800,159), LCR (ligase chain reaction) is (such as in patent application EP0201184
It is open), RCR (reparation chain reaction) (described in patent application WO 90/01069), 3SR is (autonomous
Sequence replicating) (patent application WO 90/06995), NASBA (amplification based on nucleotide sequence) (patent Shen
Please WO 91/02818), TMA (transcript mediated amplification) (U.S. Patent number 5,399,491) and RT-PCR.
When enzymatic amplification is PCR, specific reagent draws including the amplification of at least two target genes specifically
Thing, it allows to expand the special material of target gene.The material special to target gene, which is then preferably comprised, to be passed through
The complementary DNA (being referred to as the special cDNA of target gene) that mRNA of the reverse transcription from target gene is obtained
Or (it is referred to as target gene special by transcribing complementary RNA that the special cDNA of target gene obtains
cRNA).The PCR, referred to as RT-PCR completed after enzymatic amplification is reverse transcription reaction.
In one aspect of the invention, sequence such as SEQ ID can be used in method of the invention and kit
NO:Primer described in 2 and 3.
Term " hybridization probe " used herein refers to nucleotide fragments, and it includes at least five nucleosides
Acid, such as 5-100 nucleotides, particularly 10-75 nucleotides, such as 15-35 nucleotides and
60-70 nucleotides, its under prescribed conditions have hybrid specificities so as to the material special to target gene
Material forms hybridization complex.In the present invention, the material special to target gene can be included in from target
Nucleotide sequence (be referred to as target gene special mRNA) in the mRNA of gene, included in passing through
Nucleotide sequence in the complementary DNA that mRNA described in reverse transcription is obtained (is referred to as target gene special
CDNA) or in addition it is included in the complementation obtained by the transcription cDNA described above
Nucleotide sequence (being referred to as the special cRNA of target gene) in RNA.Hybridization probe, which can be included, is used for its inspection
The label of survey.Term " detection " refers to directly detect such as method of counting, or indirect detection, and it is by making
With the detection method of label.In the presence of many detection methods for being used to detect nucleic acid (see for example, Kricka etc.
People, ClinicalChemistry, 1999, no 45 (4), 453-458 pages, or Keller G.H. et al., DNA
Probes, 2nd Ed., Stockton Press, the 1993, the 5th and the 6th part, 173-249 pages).
Term " label " is the tracer for referring to produce the signal that can be detected.These tracers it is unrestricted
Property list include enzyme (its for example, by colorimetric, fluorescence or luminous produce detectable signal), for example
Horseradish peroxidase, alkaline phosphatase, 3- galactosidases, glucose-6-phosphate dehydrogenase (G6PD)；Hair
Color group, such as fluorescer, luminous agent or dye composition；Electron dense group, it can pass through electronics
Microscope detects or can be by the way that their characteristic electron such as electrical conductivity is by Amperometric or voltammetry or passes through
Impedance measurement is detected；Optically it can change such as diffraction, surface plasma body resonant vibration or contact angle,
Or by physics method such as atomic force spectrum, the group of the detection such as tunnel-effect；Geigers,
Such as 32P, 35S or 125I.
According to the purpose of the present invention, hybridization probe can be " detection " probe.In this case, label is used
" detection " probe is marked.Detection probe can be specially as Tyagi and Kramer (Naturebiotech,
1996,14:" molecular beacon " detection probe described by 303-308).These " molecular beacons " are in the hybridization phase
Between become fluorescer.They have stem-loop type structure and comprising fluorogen and " quenching " group.Special
The combination that ring sequence is complementary to target nucleic acid sequence causes stem to deploy and in appropriate wavelength excitation process
Send fluorescence signal.Detection probe can be specially comprising " colored according to NanoStringTM technology
" reporter probe " of the bar code of coding ".
In order to detect hybridization reaction, can be used directly (mix target sequence particular by by label) or
It is grounded the target sequence marked (specifically using detection probe as defined above).Especially, in hybridization step
The step of may being marked before rapid and/or cut target sequence, such as in enzymatic amplification course of reaction
Use the deoxyribonucleoside triphosphate of mark.The cutting can specifically pass through imidazoles or the work of manganese chloride
With completion.Can also according to the sandwich hybridization technology for example described in document WO 91/19812 by with
Detection probe hybridizes and target sequence is marked after amplification step.Described in application FR2780059
The method for optimizing of another specific labeling nucleic acid.
According to the preferred embodiment of the invention, detection probe includes fluorogen and quencher.According to this
Invention even more preferably embodiment, hybridization probe includes FAM (6- Fluoresceincarboxylic acids) at its 5 ' end
Or ROX (6- carboxy-X-rhodamines) fluorogens and its 3 ' end include quencher (Dabsyl).
Hybridization probe can also be " capture " probe.In this case, by any appropriate method, i.e.,
Either directly or indirectly, for example by covalent or absorption, " capture " probe is fixed or may be fixed on
On solid substrate.As solid substrate, synthetic material or natural material can be used (to be optionally chemistry to repair
Decorations), specially the polysaccharide such as such as paper of the material based on cellulose, cellulose derivative such as acetic acid
Cellulose and nitrocellulose or glucan, polymer, copolymer (are based particularly on styrene type monomer
), natural fiber such as cotton, and synthetic fibers such as nylon；Inorganic material such as silica,
Quartz, glass or ceramics；Latex；Magnetic-particle；Metal derivative, gel etc..Solid substrate can
For the form of microtiter plate, film (as described in application WO94/12670) or particle.May be used also
Some different capture probes can be fixed on base material, every kind of capture probe is special to a kind of target gene.
Specifically, it can be used biochip as base material, a large amount of probes can be fixed thereon.Term " biochip "
Refer to the solid substrate of small size, substantial amounts of capture probe can be attached in predetermined position.It is raw
The concept of thing chip (or DNA chip) can trace back to nineteen ninety for initial stage.It is based on multidisciplinary technology,
Incorporate microelectronics, nucleic acid chemistry, graphical analysis and information technology.Operation principle is based on molecular biosciences
Basis：The hybridization DNA of phenomenon, i.e., two and/or RNA sequence are by crying out the complementary pairing of base.
Use of the biochip method based on the capture probe being connected on solid substrate, direct or indirect terrestrial reference
Remember the target nucleic acid fragments sample effect of fluorescent dye in the capture probe.Capture probe is specifically fixed
Hybridization provides one bar relevant with target nucleic acid fragments specifically letter on base material or chip, and each
Breath.The information obtained is cumulative, make for example quantify one or more target genes expression into
For possibility.Then in order to analyze the expression of target gene, the base material for including a large amount of probes can be prepared, it is described
Probe corresponds to all or part for the target gene for being transcribed into mRNA.For the purposes of the present invention, art
Language " low density substrate " refers to the base material comprising less than 50 kinds of probes.For the purposes of the present invention, term
" Midst density base material " refers to comprising the base material from 50 kinds of probes to 10000 kinds of probes.In order to the present invention's
Purpose, term " high density substrate " refers to the base material comprising more than 10000 kinds of probes.
Then by the cDNA or cRNA that are specific to the target gene for needing to analyze and for example special capture
Probe hybridizes.After hybridization, washing base material or chip, and by with such as fluorescent dye type label knot
CDNA or cRNA/ the capture antibody complex of the high-affinity part display mark of conjunction.With for example sweeping
Instrument is retouched to read fluorescence and carry out fluorescence analysis by information technology.It is intended to explanation, it may be mentioned that by
The exploitation of Affymetrix companies, the DNA chip (" Accessing diagnosed for molecule
GeneticInformation with High-Density DNA arrays,, M.Chee et al.,
Science,1996,274,610-614。“Light-generated oligonucleotide arrays for
RapidDNAsequence analysis,, the Proc.Natl.Acad.Sc1. such as A.Caviani Pease
USA,1994,91,5022-5026).In the art, capture probe is typically small, about 25 nucleosides
Acid.In publication G.Ramsay, NatureBiotechnology, 1998, No.16,40-44 pages；F.Ginot,
Human Mutation, page 1997, No.10,1-10；J.Cheng et al., Molecular diagnosis,
1996, No.1 (3), 183-200 pages；T.Livache et al., NucleicAcids Research, 1994, No.
22 (15), 2915-2921 pages；J.Cheng et al., Nature Biotechnology, 1998, No.16,
541-546 pages or in U.S. Patent number 4,981,783, U.S. Patent number 5,700,637, U.S. Patent number
5,445,934th, biological core is given in U.S. Patent number 5,744,305 and U.S. Patent number 5,807,522
Piece others example.The principal character of solid substrate should be holding capture probe to target nucleic acid fragments
Hybridization characteristicses, and produce simultaneously to the minimum background noise of detection method.Fixation of the probe on base material
Three kinds of main Types can be divided into.
Firstly, there are the first technology, it is to deposit pre-synthesis probe.The connection of probe is led to
Cross directly transfer to complete, it is by micropipettor (micropipette) or microdot (microdot) or passes through
Ink discharge device.This technology allows connection magnitude range from several bases (5-10) to 60 relatively large bases
The probe of (impact system) extremely hundreds of bases (microdeposit method).
Impact system is the change of the method used ink-jet printer.It is based on can reach 4000 drops/
The speed of second promotes very small liquid ball (volume ＜ 1nl).Impact system be not related to release liquid system with
Any contact between the surface of liquid deposition thereon.Microdeposit method is characterised by will be tens of to hundreds of
The long probe of base is connected to the surface of glass slide.These probes generally extract from database and are warp
Amplification and purified Product Form.This technology makes it possible that production is referred to as the chip of microarray, institute
State chip and carry about 10,000 on the surface area (being referred to as identification region) for being slightly less than 4 square centimeters
DNA points.However, the purposes of nylon membrane (being referred to as " VLA arrange (macroarray) ") should not be forgotten, its with
Product (typically passes through after the density carrying diameter 0.5-1mm of maximum 25 points/square centimeter amplification
PCR).Many laboratories all use this very flexible technology.In the present invention, biochip is examined
Worry includes latter technique.However, such as patent application WO00/71750 and FR00/14896 situation
Under, the sample of certain volume can be deposited on to the bottom in each hole of microtiter plate, or according to another
Patent application FR00/14691, the droplet deposition that can separate certain amount each other is in same sterile training
Support ware bottom.
Second of technology referred to as fabricated in situ for probe to be connected to base material or chip.This technology is led
Cause directly produces short probe in chip surface.It is based on situ oli-gonucleotide synthesis and (is specifically shown in
WO89/10977 and WO90/03382) and based on oligonucleotide synthesizer method.It is along glass
Surface mobile response room, occurs nucleotides extension in the reative cell.
Finally, the third technology is referred to as optical lithography, and it is Affymetrix exploitations for biology
The method of chip.It is also fabricated in situ.Optical lithography comes from microprocessor technology.Pass through connection
Photo-labile (can be by photoactivation) chemical group modifies chip surface.Once by illumination, these group energy
React at 3 ' ends of enough and oligonucleotides.This surface is protected by using the lightshade cover for limiting shape, it is possible to
Optionally illumination and the therefore region of one of four kinds of nucleotides of activation expectation connection or other nucleotides.
Continuously allow to alternately protection/reaction cycle and therefore about tens of using different lightshade covers
Square micron (pm2) point on produce oligonucleotide probe.This resolution ratio allows at several square centimeters
(cm2) surface area on produce up to hundreds thousand of points.Optical lithography has the advantage that：It is parallel in batches,
It allows to only multiply N number of circulation generation N aggressiveness chips through 4.All these technologies can be used for this hair
It is bright.According to the preferred embodiment of the invention, step b) as defined above at least one is specific
Reagent includes at least one hybridization probe, and it is preferably fixed on base material.This base material is preferably as described above
Low, the high or Midst density base material of definition.
In order to improve the amount of target genetic stocks, it can hybridize in these on the base material comprising a large amount of probes and walk
Enzymatic amplification reactions steps as defined above are carried out before rapid.
Target gene expression can be completed really by any scheme well known by persons skilled in the art
It is fixed.Generally speaking, can be by detecting the mRNA transcribed in given time from target gene (mRNA)
To analyze the expression of target gene.
Present invention is preferably related to by being derived from target according to well known to a person skilled in the art the detection of any scheme
The mRNA of gene determines the expression of target gene.According to specific embodiment of the present invention, lead to
Cross and detect some different mRNA (every kind of mRNA is derived from a target gene), can determine simultaneously some
The expression of target gene.
The expression of target gene can be identified below：
1) after extraction from biological material total serum IgE, reverse transcription step is carried out as previously described, to obtain
With the various DNA (or cDNA) for the various mRNAs complementation that initially there are in biological sample；
2) specific amplification cDNA.In this case, the specific reagent used includes at least one
The individual foregoing amplimer special to target gene.Especially can be anti-by the amplification of PCR types
Or this stage should be carried out by any other suitable amplification technique；
3) expression of target gene is determined by quantitative cDNA.Especially can be full by using proceeding to
Quantizing range that the amplified reaction of sum is obtained quantifies cDNA.In view of different phase (reverse transcription, PCR
Deng) observe enzymatic effect change, can by simultaneously determine so-called housekeeping gene (its express exists
Similar in the patients of difference group) expression the expression of target gene of different groups of patients is standardized.It is logical
Cross and determine the ratio that expression of target gene and housekeeping gene are expressed, can correct any between different experiments
Variability.
The expression of target gene can also be identified below:
1) after extraction from biological material total serum IgE, reverse transcription step is carried out as previously described, to obtain
With the various DNA (or cDNA) for the various mRNAs complementation that initially there are in biological sample；
2) cDNA is fixed on film；
3) surveyed by the way that cDNA and the preceding mark hybridization probe special to target gene are hybridized
The expression of targeting gene.These hybridization techniques are that well known to a person skilled in the art we especially refer to
Be Northern traces.It is complementary in the mRNA with target gene especially when gene is weak expression
DNA the specific amplification stage after carry out this hybridization reaction.
In the present invention, the mRNA that can be transcribed by preset time expression is determined
The expression of sweyjawbu, ESD and HPRT1 gene.In this case, biomaterial is nucleic acid,
And specific reagent can be foregoing amplimer or hybridization probe.
In one aspect of the invention, in order to reduce or eliminate shadow of the interindividual variation for testing result
Ring, by the expression of the sweyjawbu genes in specimens and patient's normal structure
The expressions of sweyjawbu genes compare.Preferably, the present invention comes using with tumor tissues
The same or like normal structure in source.When the sweyjawbu gene expression doses in tumor tissues are higher than
During sweyjawbu gene expression doses in normal structure, it is believed that the tumour of the patient is ALK
Genetic recombination is positive.
In another aspect of the present invention, sweyjawbu bases directly can also be judged by Δ Ct values
The expression of cause.It is as described herein, Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,
CtHPRT1) it is CtESDAnd CtHPRT1Geometric mean., can when the Δ Ct of testing result is less than 1.8
Think that the tumour of the patient recombinates for ALK gene positive.
The present inventor is it was surprisingly found that the expression of sweyjawbu genes and turning for ALK
Height correlation is recorded, coefficient correlation reaches more than 0.9.The expression of sweyjawbu genes is positive in ALK
Highly raised in cell line.In different types of tumour cell, sweyjawbu rna expressions and ALK
Gene magnification or transposition state are related.The signal of 4 lung cancer cell lines can be for example extracted from data set
Intensity is used as example.Sweyjawbu genes only show positive expression in ALK positive cells, and
It is less than background level (Fig. 2A) in ALK negative cells.Real-time PCR experiments display that ALK is positive
Sweyjawbu rna expression levels between cell and ALK negative cells differ by more than 100 times of (figures
2B)。
Therefore, according to the present invention, by the expression of the sweyjawbu genes in specimens
Compared with the expression of the sweyjawbu genes in patient's normal structure.In tumor tissues
The sweyjawbu gene expression doses that sweyjawbu gene expression doses are higher than in normal structure are to represent
The ALK gene restructuring of the patient tumors is positive.For example, according to the present invention, in tumor tissues
Sweyjawbu gene expression doses can be higher than 2 of the sweyjawbu gene expression doses in normal structure
Times, now the Δ Ct values in tumor tissues are bigger by 1 than the Δ Ct values in normal structure.Similarly, tumour
Sweyjawbu gene expression doses in tissue can be higher than the sweyjawbu gene tables in normal structure
Up to horizontal 4 times, 8 times, 16 times, 32 times, 64 times or 128 times.That is, tumor tissues
In Δ Ct values it is bigger by 2 than Δ Ct values in normal structure, 3,4,5,6 or 7.
Embodiment
The tumor cell line of embodiment 1 and clinical sample
Use ALK positive cell lines NCI-H2228 (gland cancer, non-small cell lung cancer) and three ALK the moon
Property cell line, NCI-H1975 (gland cancer, non-small cell lung cancer), NCI-H460 (large cell carcinoma, it is non-small
Cell lung cancer) and NCI-H1703 (squamous cell carcinoma, non-small cell lung cancer).ATCC cells
NCI-H2228, NCI-H1975, NCI-H460 and NCI-H1703 be supplemented with 10%FBS and
The RPMI-1640 medium cultures of 2mM Glus.Cell culture and 6 cm dishes, are used
RNAprotect cell reagents box (Qiagen) collection and about 1 × 106Individual cell.With QIAshredder from
Stem (Qiagen) is homogenized after lysate, and RNA is extracted with RNeasy+ mini kits (Qiagen).
By the tumor samples of 54 Patients with Non-small-cell Lung (including 30 gland cancer, 4 large cell carcinomas and
20 squamous cell carcinomas), and 12 non-tumour lung tissues are for analyzing.Obtained from all participants
Obtain informed consent form.The research is ratified through Chinese Tumor Hispital Attached to Fudan Univ Ethics Committee.It is cold
The tumor tissues and normal structure mortar and pestle of jelly are ground under liquid nitrogen existence condition, are re-dissolved in TRIZOL
In.RNA is extracted with RNeasy+ mini kits (Qiagen).
By total serum IgE electrophoretic examinations RNA amount, ethidium bromide staining is then used.Use spectrophotometric
Meter determines absorbances of each RNA at 260nm and 280nm, obtains RNA amounts and purity.
The design of primers of embodiment 2
Use Beacon Designer softwares (Premier Biosoft, Palo Alto, California
State) design primer pair.By the sequence of primer and the known single nucleotide polymorphism of UCSC snp databases
(SNP) information compares.Primer and template plasmid with heterozygosis sub-district are by Jin Sirui Co., Ltds (south
Capital, China) synthesis.The plasmid of synthesis examines primer amplification efficiency as template.It is every in reaction tube
The final concentration of individual primer is equal to 0.3 μM.Primer pair causes effective amplification, and retaining single melting curve is used for
Further analysis.
Embodiment 3PCR is determined
To detect the template from cell line or tissue sample, QuantiTect Reverse Transcriptase kits are used
(Qiagen) cDNA is synthesized from 1 μ g RNA.Use QuantiFast SYBR Green PCR reagents
Box (Qiagen), carries out quantitative real-time PCR detections on ABI 7900HT instruments.At 95 DEG C 5 points
After the initial activation of clock, carry out 40 using 95 DEG C of 10 seconds and 60 DEG C of two step cycles of 30 seconds and follow
Ring, then carries out melting curve analysis.HPRT1 and ESD genes as gene expression reference gene.
The confirmation of the ALK defrag status of the patient of embodiment 4
According to guide, the ALK defrag status of selected patient test (Abbott) or IHC by FISH
(Roche) is tested to check.FISH positive cases are defined as two positive ALK reform patterns.It is positive
Case is defined as the break signal in 50 tumour cells more than 15% or the red letter of separation
Number.FISH negative cases are defined as in tumour cell the presence of the overlapping of red and green (yellowish),
Show that ALK is not recombinated.IHC dyeing is scored as follows jointly by two pathologists：More than 10%
In tumour cell, 0 (dye-free)；1 (weak, cytoplasm dyeing)；2 (appropriateness, homogeneous cytoplasm dyeing)；
3 (strong, granular cytoplasm dyeing).
The method of the present invention has been obtained and reference method identical ALK state outcomes (Fig. 3).Such as Fig. 3
It is shown, wherein Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,CtHPRT1) it is CtESD
And CtHPRT1Geometric mean.(5 gland cancer and 1 are big thin for the ALK positives that FISH confirms
Born of the same parents' cancer) show Δ Ct values less than 1.8.Therefore, method of the invention determines Δ Ct less than 1.8
To represent that ALK gene restructuring is positive.
The present invention incorporates 30 adenoma samples, including 5 FISH confirm the positive samples of ALK.
The ALK positives of sample are further confirmed by IHC researchs and AmoyDx PCR.ALK is positive
Δ Ct in patientsweyjawbuChanged according to the percentage of ALK positive cells in sample.In addition, grinding
Studying carefully also includes 4 large cell carcinomas and 20 squamous cell carcinomas.
Play-by-play not on sweyjawbu genes before making the present invention, gene work(in vivo
Can be unknown.Gene A LK is in No. 2 chromosome reverse strands, from sequence 30144451 to sequence
29415634 (NCBI in Augusts, 37,2010), and also to be located at No. 2 chromosomes anti-by gene sweyjawbu
To on chain, from 2 sequences 9414340 to sequence 29413859.On chromosome between the two genes
Only 1294bp distance.Sweyjawbu expression recombinates positive cell and ALK restructuring in ALK
Significant difference is shown between negative cells.
The fracture of ALK transcripts is simultaneously fused to another fusion partner and can drive ALK kinases
The strongly expressed of domain, and cause the uneven expression of ALK transcript 5' and 3' parts.According to before
Report, devise primer pair in research, the imbalance for checking ALK transcript 5' and 3' parts
Expression, positive is recombinated from ALK restructuring negative samples to efficiently differentiate ALK.In addition, plus
Cooperative effect can be provided by entering sweyjawbu differential expressions.
Those skilled in the art it is understood that description above only by property for example and not limitation mode
Illustrate the present invention.The various equivalents of all of the embodiments of the present invention are included in the present invention's
In the range of.
Claims (13)
1. a kind of determine the method that ALK gene is recombinated in biological sample, methods described includes determining the expression of sweyjawbu genes in the biological sample, the wherein sequence of sweyjawbu genes such as SEQ ID NO:Described in 1 or with SEQ ID NO:1 has at least 90% homogeneity.
2. the method described in claim 1, the wherein sequence of sweyjawbu genes and SEQ ID NO:1 have at least 95%, preferably at least 98%, more preferably at least 99% homogeneity.
3. the method described in claim 1, the wherein expression of sweyjawbu genes are determined by determining RNA or cDNA level.
4. the method described in claim 1, the expression of wherein sweyjawbu genes is determined by PCR, preferably quantitative fluorescent PCR, the sequence such as SEQ ID NO for the primer that the PCR is used:Described in 2 and 3.
5. the expression of the method described in claim 1, wherein sweyjawbu genes determines the tumor tissues from patient, the tumor tissues are selected from surgery excision tissue, aspiration biopsy sample, hydrothorax sample, CSF sample, blood sample and other body fluid samples.
6. the method described in claim 1, methods described further comprises comparing the expression of the sweyjawbu genes in specimens with the expression of the sweyjawbu genes in patient's normal structure, and the sweyjawbu gene expression doses that the sweyjawbu gene expression doses wherein in tumor tissues are higher than in normal structure represent that the ALK gene restructuring of the patient tumors is positive.
7. the method described in claim 1, the wherein expression of sweyjawbu genes are determined by Δ Ct values, Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,CtHPRT1) it is CtESDAnd CtHPRT1Geometric mean, Δ Ct represents that ALK genes restructuring is positive less than 1.8.
8. a kind of kit for being used to detect that ALK gene is recombinated in biological sample, the kit includes the specific reagent for being used to determine the expression of sweyjawbu genes in biological sample, the wherein sequence of sweyjawbu genes such as SEQ ID NO:Described in 1 or with SEQ ID NO:1 has at least 90% homogeneity.
9. the kit described in claim 8, wherein the kit includes at least two parts specific reagents for being used to determine the expression of sweyjawbu genes, the specific reagent is respectively used to the tumor tissues and normal structure of same patient.
10. the kit described in claim 8, wherein the kit also includes label, the label, which has, illustrates that the kit is used for the content for detecting ALK gene restructuring.
11. the kit described in claim 8, wherein the kit also includes label, the label, which has, illustrates that the kit is used for the content for detecting ALK gene restructuring.
12. the kit described in claim 11, wherein the label also has herein below：
1) illustrate that the kit is used to compare the expression of the sweyjawbu genes in specimens with the expression of the sweyjawbu genes in patient's normal structure, the sweyjawbu gene expression doses that the sweyjawbu gene expression doses wherein in tumor tissues are higher than in normal structure represent that the ALK gene restructuring of the patient tumors is positive；Or
2) illustrate that the kit is used for the Δ Ct values for determining the expression of sweyjawbu genes, wherein Δ Ct=Ctsweyjawbu-GM(CtESD,CtHPRT1), GM (CtESD,CtHPRT1) it is CtESDAnd CtHPRT1Geometric mean, Δ Ct represents that ALK genes restructuring is positive less than 1.8.
Detect whether patient meets the method that ALK inhibitor medicaments treat standard 13. a kind of, methods described determines the ALK gene restructuring in the patient tumors including the use of any one of claim 1-7 method, represents that ALK inhibitor medicaments can be used to be treated for the patient if the ALK gene restructuring of patient tumors is positive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610074021.7A CN107022602A (en) | 2016-02-02 | 2016-02-02 | A kind of method of detection ALK genetic recombination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610074021.7A CN107022602A (en) | 2016-02-02 | 2016-02-02 | A kind of method of detection ALK genetic recombination |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107022602A true CN107022602A (en) | 2017-08-08 |
Family
ID=59524557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610074021.7A Pending CN107022602A (en) | 2016-02-02 | 2016-02-02 | A kind of method of detection ALK genetic recombination |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107022602A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011140187A2 (en) * | 2010-05-04 | 2011-11-10 | University Of Rochester | Detecting chromosomal rearrangement |
| CN103397045A (en) * | 2007-04-13 | 2013-11-20 | 细胞信号技术公司 | ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor |
| CN105624275A (en) * | 2014-11-06 | 2016-06-01 | 上海市东方医院 | Applications of EIF4G1 in diagnosis and treatment of squamous cell carcinomas |
| US20190292529A1 (en) * | 2014-04-10 | 2019-09-26 | Memorial Sloan Kettering Cancer Center | Novel isoform of anaplastic lymphoma kinase and its uses |
-
2016
- 2016-02-02 CN CN201610074021.7A patent/CN107022602A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397045A (en) * | 2007-04-13 | 2013-11-20 | 细胞信号技术公司 | ALK (Anaplastic Lymphoma Kinase) for gene deletion and mutant in human solid tumor |
| WO2011140187A2 (en) * | 2010-05-04 | 2011-11-10 | University Of Rochester | Detecting chromosomal rearrangement |
| US20190292529A1 (en) * | 2014-04-10 | 2019-09-26 | Memorial Sloan Kettering Cancer Center | Novel isoform of anaplastic lymphoma kinase and its uses |
| CN105624275A (en) * | 2014-11-06 | 2016-06-01 | 上海市东方医院 | Applications of EIF4G1 in diagnosis and treatment of squamous cell carcinomas |
Non-Patent Citations (2)
| Title |
|---|
| HUI YU等: "Detection of ALK rearrangements in lung cancer patients using a homebrew PCR assay", 《ONCOTARGET》 * |
| KAI XUE等: "Sweyjawbu expression is a predictor of ALK rearrangement status in lymphoma", 《ONCOTARGET》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20120270228A1 (en) | Predictors of patient response to treatment with egf receptor inhibitors | |
| EP2121988B1 (en) | Prostate cancer survival and recurrence | |
| ES2311852T3 (en) | IDENTIFICATION OF A SPECIFIC MODEL OF THE ERBB2 GENE EXPRESSION IN CANCER OF BREAST | |
| KR101501826B1 (en) | Method for preparing prognosis prediction model of gastric cancer | |
| JP2013531485A (en) | Method and kit for determining prognosis of colorectal cancer | |
| KR20100120657A (en) | Molecular staging of stage ii and iii colon cancer and prognosis | |
| US8283122B2 (en) | Prediction of clinical outcome using gene expression profiling and artificial neural networks for patients with neuroblastoma | |
| CN102732636A (en) | Method and chip for detecting tumor cell EGFR (epidermal growth factor receptor) gene mutation | |
| CN103667490B (en) | Mass spectrographic preparation method of a kind of nucleic acid for detecting colorectal cancer K-ras transgenation and products thereof | |
| JP6337358B2 (en) | Methods and kits for determining in vitro the likelihood that an individual will suffer from colorectal cancer | |
| Bertucci et al. | Prognosis of breast cancer and gene expression profiling using DNA arrays | |
| TW201120449A (en) | Diagnostic methods for determining prognosis of non-small-cell lung cancer | |
| CN112852967A (en) | Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR | |
| US20180051342A1 (en) | Prostate cancer survival and recurrence | |
| JP2017169580A (en) | Novel complex mutation in epidermal growth factor receptor kinase domain | |
| JP5932778B2 (en) | Method and kit for distinguishing breast cancer from benign breast disease | |
| CN101457254B (en) | Gene chip and kit for liver cancer prognosis | |
| CN108660193A (en) | Mankind's LMNA-NTRK1 Gene Fusion abrupt climatic changes primer, probe and detection kit | |
| CN107022602A (en) | A kind of method of detection ALK genetic recombination | |
| US20090181397A1 (en) | Predictive and diagnostic methods for cancer | |
| US20110318732A1 (en) | Prediction of chemotherapeutic response via single-cell profiling of transcription site activation | |
| US20210277454A1 (en) | Atac-array for prediction of disease-free survival in pancreatic cancer | |
| Hu et al. | A highly sensitive and specific system for large-scale gene expression profiling | |
| Kocabey et al. | Ultrasensitive detection of cancer-associated nucleic acids and mutations by primer exchange reaction-based signal amplification and flow cytometry | |
| JP6356175B2 (en) | Method and kit for distinguishing breast cancer from benign breast disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170808 |