CN1829793B - Mark gene having differential expression between respondent and non-respondent of erbB receptor tyrosine kinase inhibitor - Google Patents

Abstract

The invention relates to a set of isolated marker genes comprising at least one gene identified as having differential expression as between patients who are responders and non responders to an erbB receptor tyrosine kinase inhibitor; said gene set comprising one or more genes selected from at least the group consisting of the (51) genes listed herein including gene-specific oligonucleotides derived from said genes; and uses of sauch sets in diagnostic applications.

Description

Between the respondent of erbB receptor tyrosine kinase inhibitors and non-responder, has marker gene of differential expression and uses thereof

Technical field

The present invention relates to a kind of test kit of using in a kind of personalized cancer treatment method and the said method, whether this method adopts a group echo genome chemotherapeutic to be produced with the prediction patient and replys.

Specifically, this method prediction patient replying to the erbB receptor tyrosine kinase inhibitors.More concrete is; This method is utilized in the level that has a group echo gene of differential expression between respondent and the non-responder of erbB receptor tyrosine kinase inhibitors; Be related to the patient that those are suffered from separately or part receives the cancer of erbB receptor tyrosine kinase regulation and control, especially suffer from the for example patient of gland cancer of nonsmall-cell lung cancer in late period (NSCLC).

Background of invention

Lung cancer is the main reason of cancer mortality, also is a main global health problem therefore.In this disease of treatment, mainly rely on chemotherapy, because great majority have suffered from the local late period 3 (44%) or the disease [1] in transitivity stage 4 (32%) when diagnosis.Yet the result of large-scale transfer analysis has disclosed with the assisted care of the best and has compared, and only nonsmall-cell lung cancer in late period (NSCLC) survival time of patients median has been prolonged about 6 week [2] based on the chemotherapy of platinum.

At nearest 10 years; Many new cytotoxic agents have been developed; Comprise taxol (paclitaxel), Docetaxel (docetaxel), gemcitabine (gemcitabine) and vinorelbine (vinorelbine), and multiple choices are provided for the lung cancer patients with terminal.Yet, to compare with treatment based on Platinol, every kind of therapy only provides moderate survival effect [3] [4].Recently, developed the new treat-ment of the limitation of being devoted to overcome conventional cytotoxic agent, this method comprises many molecular targeted dose of [5] [6].

Recent years, find that some growth factor Tyrosylprotein kinase is extremely important in the transmission of the biochemical signals that initiator cell duplicates.They are large proteins of crossing over cytolemma; And has an ectodomain and an intracellular portion that is used for binding growth factor such as Urogastron (EGF); This intracellular portion plays kinase whose effect, thereby makes the tyrosine amino acid phosphorylation in the protein influence cell proliferation.

Known various receptor tyrosine kinase classification based on the growth factor family that combines different receptor tyrosine kinases (Wilks, Advances in Cancer Research, 1993,60,43-73).These classification comprise I receptoroid Tyrosylprotein kinase, and it comprises the EGF family of receptor tyrosine kinase.This comprises the acceptor of acceptor, TGF α (being also referred to as TGFA), amphiregulin (being also referred to as AREG), β-tunicin, the heparin that combines EGF, table adjusting albumen (epiregulin) and the neuregulin (neuregulins) (comprising NRG-1, NRG-2, NRG-3 and NRG-4) of ligands, EGF.More concrete is; These acceptors comprise that those have the acceptor, II receptoroid Tyrosylprotein kinase and the III receptoroid Tyrosylprotein kinase that are called as erbB1 (EGFR), erbB2 (Neu, Her2) and erbB4 (Her4) and do not have the erbB3 (her3) in functional structure territory of functional kinase domain; The II receptoroid Tyrosylprotein kinase Regular Insulin family for example Regular Insulin and IGFI acceptor and the Regular Insulin associated receptor that comprise receptor tyrosine kinase wherein, III receptoroid Tyrosylprotein kinase comprises the hematoblastic growth factor of coming from of receptor tyrosine kinase (PDGF) family such as PDGF α, PDGF β and colony-stimulating factor 1 (CSF1) acceptor.

The erbB family of the receptor tyrosine kinase of the known EGFR of comprising, erbB2, erbB3 and erbB4 usually participates in driving the propagation and the survival (commentary is shown in Olayioye etc., EMBOJ., 2000,19,3159) of tumour cell.Its generable a kind of mechanism is the over-expresses of said acceptor on protein level, and this is the result of gene amplification normally.At many common human cancers, for example comprise gland cancer nonsmall-cell lung cancer in late period (NSCLC) (Cerny etc., Brit.J.Cancer, 1986,54,265; Reubi etc., Int.J.Cancer, 1990,45,269; Rusch etc., Cancer Research, 1993,53,2379; Brabenderetal, Clin.CancerRes., 2001,7,1850) and other lung cancer (Hendler etc., Cancer Cells, 1989,7,347) in all observed this point (commentary is shown in Klapper etc., Adv.CancerRes., 2000,77,25).

The result that one or more above-mentioned acceptor mistakes are regulated is, generally believes that many tumours become clinically more promptly to spread thereby cause more difficult patient being carried out Prognosis (Brabender etc., Clin.Cancer Res., 2001,7,1850; Ross etc., CancerInvestigation, 2001,19,554, Yu etc., Bioessays, 2000,22.7,673).Except these clinical discoveries, a lot of clinical preceding information show that the erbB family of receptor tyrosine kinase participates in transformation.In addition; Many preclinical studies are verified can induce antiproliferative effect (commentary is shown in Mendelsohn etc., Oncogene, 2000 through knock out one or more erbB activity with micromolecular inhibitor, dominant negative or inhibiting antibody; 19,6550).

Therefore, have realized that now the suppressor factor of these receptor tyrosine kinases should can be used as a kind of selective depressant of the propagation of mammalian cancer cell (.Science such as Yaish, 1988; 242,933, Kolibaba etc.; Biochimica et Biophysica Acta; 1997,133, F217-F248; Al-Obeidi etc., 2000, Oncogene, 19,5690-5701; Mendelsohn etc., 2000, Oncogene.19.6550-6565).Except these clinical datas in advance; Adopt anti-EGFR and erbB2 inhibiting antibody (being followed successively by c-225 and Herceptin (trastuzumab)) result of study proof its effectively (Mendelsohn etc. is seen in commentary clinically for treatment of selected solid tumor; 2000; Oncogene, 19,6550-6565).

Many micromolecular inhibitors of the erbB family of receptor tyrosine kinase are known, especially the suppressor factor of EGFR and erbB2 receptor tyrosine kinase.For example; European Patent Application No. 0566226 discloses some with International Patent Application WO 96/33980 with WO 97/30034 has a substituent quinazoline derivant of anilino to have the EGFR receptor tyrosine kinase activity in the 4-position, and they are the cancerous tissue inhibition of proliferation agent that comprise prostate cancer.J R Woodburn etc. are at Proc.Amer.Assoc.Cancer Research; 1997; 38,633 and Pharmacol.Ther., 1999; 82, disclosing compound N-(3-chloro-4-fluorophenyl)-7-methoxyl group-6-(3-morpholino propoxy-) quinazoline-4-amine among the 241-250 is a kind of effective EGFR tyrosine kinase inhibitor.Utilize Code Number ZD1839 and chemical abstracts registry no 184475-35-2, this compound also is known as Iressa (registered trademark), ZD1939 (name that the U.S. adopts).Hereinafter, this compound is called ZD1939.Recently ZD1939 has got the Green Light in Japan and has been used to treat nonsmall-cell lung cancer (NSCLC) that can't treat with operation or recurrence, and goes through in platinum and Docetaxel (docetaxel) chemotherapy failure back to treat as being used to treat the list of suffering from transitivity NSCLC patient in local late period in the U.S..

In addition from International Patent Application WO 96/30347 known some have the quinazoline derivant of a substituent structurally associated of anilino that EGFR tyrosine-kinase enzyme inhibition activity is also arranged in the 4-position.WO 99/55683 discloses compound N-(3-ethynyl phenyl)-6; 7-two (2-methoxy ethoxy) quinazoline-4-amine or its pharmacy acceptable salt (interrelate with Code Number CP 358774 and OSI-774, hereinafter is represented with Code Number OSI-774) are a kind of EGFR TKI.

Can know further that from International Patent Application WO 97/38983 some other the quinazoline derivant that a substituent structurally associated of anilino is arranged in the 4-position also has EGFR tyrosine-kinase enzyme inhibition activity.At J.Med.Chem.; 1999; 42; Disclosing compound 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholino propoxy-) quinazoline-4-amine (interrelate with Code Number PD 183805 and CI 1033, hereinafter is represented with Code Number CI1033) among 1803-1815 and the WO 00/31048 is a kind of EGFR TKI.

Can know further that from International Patent Application WO 97/02266 some has the Hete rocyclic derivatives of a substituent structurally associated of anilino that EGFR tyrosine-kinase enzyme inhibition activity is also arranged in the 4-position.For example compound 4-[(1R)-1-styroyl amino]-6-(4-hydroxy phenyl)-7H-pyrrolo-[2,3-d] pyrimidine (interrelate with Code Number PKI-166, CGP 75166 and CGP 59326, hereinafter is represented with Code Number PKI-166) is a kind of EGFR TKI.

From European Patent Application No. 0787722 and International Patent Application WO 98/50038, WO99/09016 and WO 99/24037, further can know some other has the quinazoline derivant of a substituent structurally associated of anilino that EGFR tyrosine-kinase enzyme inhibition activity is also arranged in the 4-position.For example compound N-[4-(3-bromobenzene amido) quinazoline-4-yl] fourth-2-alkynyl amide (interrelate with Code Number CL-387785 and EKB-785, hereinafter is represented with Code Number CL-387785) is a kind of EGFR TKI.

From Nature Medicine, 2000,6, further can know some in 1024-1028 and the U.S. Patent number 6,002,008 other has the quinazoline derivant of a substituent structurally associated of anilino that EGFR tyrosine-kinase enzyme inhibition activity is also arranged in the 4-position.For example compound 4-(3-chloro-4-fluoroanilino)-3-cyanic acid-6-(4-dimethylamino fourth-2 (E)-alkene carboxamido-group)-7-ethoxyquinoline (hereinafter is represented with Code Number EKB-569) is a kind of EGFR TKI.

What from WO 99/35146 and WO 01/04111, also can learn is that some quinazoline derivant is the suppressor factor of one or more erbB receptor tyrosine kinases.For example, compound N-f3-chloro-4-[(3-fluoro benzyl) oxygen] phenyl }-6-[5-({ [2-(methylsulfonyl) ethyl] amino } methyl)-2-furyl] quinazoline-4-amine (also be expressed as lapatinib or hereinafter represent with Code Number GW2016) just is considered to the suppressor factor of EGF and erbB2 receptor tyrosine kinase.NovartisAE788 is another kind of suitable inhibitor compound.Thereby can also suppress the outer part of born of the same parents with the antibody of suitable anti-erbB acceptor and be attached to inhibition erbB receptor tyrosine kinase on the acceptor.For example utilize the antibody Herceptin (trastuzumab) [HerceptinTM] and the anti-erbbl antibody Cetuximab (cetuximab) [C225] of anti-erbB 2.Verified in clinical, use this inhibiting antibody help treating selected solid tumor (commentary is shown in Mendelsohn etc., 2000, Oncogene, 19,6550-6565).

As stated, ZD1939 is the Orally active suppressor factor of a kind of EGF-R ELISA-Tyrosylprotein kinase (EGFR-TK), and it has blocked the signal transduction pathway [7] of responsible driving propagation, invasion and attack and cancer cells existence.In clinical study, observed the quick improvement of effective antitumour effect and NSCLC relevant symptoms and quality of life to the unresponsive registration patient who suffers from advanced NSCLC based on the chemotherapy of platinum.In double blinding II phase single therapy test (IDEAL 1 test) at random; It is 18.4% (95%CI:11.0-25.9%) that ZD1939 is used the chemotherapeutical the 2nd or the 3rd tumor response rate that line obtained of the advanced NSCLC of opposing; In IDEAL 2 test, be 11.8% (95%CI:6.2-19.7%) [8], [27], [28] with its use tumor response rate that the chemotherapeutical the 3rd or the 4th line of advanced NSCLC obtained of opposing.

And in these tests, the treatment of this medicine has obtained high disease control rate (being 54.4% among the IDEAL 1, is 42.2% among the IDEAL 2) and general overall doing well,improving rate (being 40.3% among the IDEAL 1, is 43.1% among the IDEAL 2).

When with for the reacting phase of the cytotoxic agent of routine than the time, these results are likely, but true remaining among the registration patient in this research has half not obtain effective treatment approximately, illness does not improve.And this pharmacological agent is prone to the nonresponder is had side effects, and comprises life-threatening spinoff such as interstitial pneumonia [11].

The patient has nothing in common with each other to the reaction of different chemical therapy, therefore just needs to find to predict the most suitable certain the concrete patient's of any therapeutic modality method.

More and more evidences prompting patient maybe be relevant with patient's genetic map to replying of producing of multiple medicine, influence for example to certain medicine reply genicly confirm can be used for personalized regimen to be provided to the patient.This personalized regimen has been for the patient provides the possibility that makes result of treatment best, for example reduced simultaneously and selectable treat-ment or the poorer relevant spinoff of treat-ment of effect.Therefore need a kind of method that can predict the patient to the reaction of medicine.

Summary of the invention

Experiment showed, that some cancer can predict through genetic expression the susceptibility of chemotherapeutic, therefore can confirm suitability through the related levels of measuring some gene in the patient tissue with these chemotherapeutic treatment cancer patientss.

Therefore, the invention provides one group of isolating marker gene, comprise that at least one is accredited as the gene that between the patient, has differential expression, said patient is to a kind of respondent of erbB receptor tyrosine kinase inhibitors and non-responder; Said isolating genome comprises one or more genes that are selected from least in the group that is made up of 51 listed in the table 4 that comprises the specific oligonucleotide that is derived from said gene genes.In the table 4, provided the accession number of these genes in the GeneBank DB.What one skilled in the art will appreciate that sequence representative in giving accession number only is the example of gene order related in this form; Comprise selectable sequence, allelotrope or other variation of the sequence that contains the sequence error correction, montage sudden change etc. is also included within the definition with the represented gene of these names.In most preferred embodiments, related sequence is the concrete sequence of listing in detail and stating among listed sequence and the table 4a in the accession number.

On the other hand, the present invention also provides one group of isolating marker gene, comprises that at least one is accredited as the gene that between the patient, has differential expression, and said patient is to a kind of respondent of erbB receptor tyrosine kinase inhibitors and non-responder; Said isolating genome comprises one or more genes that are selected from least in the group that is made up of 51 listed in the table 4 that comprises the specific oligonucleotide that is derived from said gene genes.

Thereby the present invention brings into play the more efficiently purposes of various existing medicines through treatment and each patient are adapted, and the raising of feasible improvement prediction and consequent cancer patients's quality of life becomes possibility.

Preferred group is at least one in listed preceding 40 genes or a plurality of in the table 4.

More preferred group is at least one in listed preceding 20 genes or a plurality of in the table 4.

More preferred group is at least one in listed preceding 12 genes or a plurality of in the table 4.

More preferred group is at least one in listed preceding 5 genes or a plurality of in the table 4.

Preferred especially group is listed preceding 12 genes among the table 4a, just FLJ22662 (for example GenBank NM024829), AREG (for example GenBank BC009799), CORO1C (for example GenBank NM-014325), AVEN (for example GenBankBC010488), DUSP3 (for example GenBankNM004090, DJ473B4 (for example GenBankAI026836), PHLDA2 (for example GenBankBU500509), RBM7 (for example GenBank NM016090), EST (GenBank BX092512), OSMR (for example GenBankAI436027), GCLC (for example GenBankAI971137), COL4A3BP (for example GenBank BQ024877).

Preferably, said suppressor factor is selected from ZD1939, OSI-774, PKI-166, EKB-569, GW2016, CI-1033 and a kind of anti-erbB antibody such as Herceptin (trastuzumab) and Cetuximab (cetuximab).

Most preferably, said suppressor factor is a ZD1939.

The present invention is particularly useful for predicting at those to suffer from separately or part receives replying aforementioned chemotherapeutic among patient or the patient crowd of cancer of erbB receptor tyrosine kinase regulation and control.These cancers comprise; For example non-solid tumor such as white blood disease, multiple myeloma or lymphoma and solid tumor such as bile duct, bone, bladder, brain/CNS, mammary gland, colorectum, uterine cervix, uterine endometrium, stomach, head and neck, lung, liver, muscle, neurone, esophagus, ovary, pancreas, pleura/peritonaeum, prostate gland, kidney, skin, testis, Tiroidina, uterus and vaginal orifice tumour.

The present invention be particularly useful for identifying to for example as above the such chemotherapeutic of the erbB receptor tyrosine kinase inhibitors of definition produce the NSCLC that suffers from that replys, especially comprise the patient of the advanced NSCLC of gland cancer in late period.

The present invention be tested and appraised NSCLC's " individual cancer collection of illustrative plates " thus confirming which kind of tumour can produce ZD1939 reply, aspect the such cancer therapy of NSCLC for example especially advanced NSCLC sizable advantage is being provided.This comprises can't treat the patient of other so any treat-ment treatment of line and for example chemotherapy with article one.

The present invention is effective especially like the patient based on the NSCLC of the chemotherapy of platinum treatment for using chemotherapy before the treatment.

The present invention is for accepting chemotherapy before the treatment as also effective especially based on the patient of local late period (IIIB stage) of the chemotherapy of platinum treatment or transitivity NSCLC (IV stage).

The present invention also provide a kind of prediction suffer from cancer for example among patients with lung cancer or the patient crowd to using the especially erbB receptor tyrosine kinase inhibitors responsiveness of treating of chemotherapeutic, comprise the differential expression of the group echo gene in the genome that relatively is selected from as above definition.

The evaluation of preferably expressing through gene expression atlas, the RT-PCR (reverse transcription-polymerase chain reaction) of array that is based on oligonucleotide that said gene expression atlas adopts or any type of array based on cDNA; The successive analysis (SAGE) of PCR in real time, in situ hybridization, RNA blotting, genetic expression for example Velculescu etc. at Science 270 (5235): describe among the 484-487, or differentially expressed.These can be at for example Sambrook etc. with the details of other method, 1989, Molecular Cloning:A Laboratory Manual) in find.Microarray analysis is preferably used in this evaluation.

As selection, or in addition, immunohistochemical analysis is also used in said evaluation.

On the other hand; The present invention also provides a kind of and has been used to predict to suffer from cancer patients or patient crowd to using the especially test kit of the responsiveness of erbB receptor tyrosine kinase inhibitors treatment of chemotherapeutic, comprises the as above marker gene group of definition on the suitable supporting dielectric.Preferably, marker gene be connected on the solid support material or the film as nitrocellulose on, or on nylon or plastic film or the slide glass.

This test kit preferably contains a kind of microarray.

The detailed Description Of The Invention that comprises preferred implementation

To carry out more detailed explanation and elaboration to the present invention through following embodiment, said embodiment is used to help those skilled in the art to realize the present invention, but in no case is to limit scope of the present invention.

Be described in more detail in the face of key elements more of the present invention down.

According to the content of embodiment described here, " isolating marker gene group " is meant that here one group can be used for the gene of replying classification or sorting out according to patient of the present invention.

" differential expression " gene is with the higher level or the gene of low expression level more between respondent or non-responder group.

" respondent/non-responder "

Reply classification as follows according to the target tumor of International Union Against Cancer/World Health Organization (UICC/WHO) standard: thoroughness is replied (CR): in all valuable damages, do not have residual tumor; Part is replied (PR): residual tumour is arranged in the total amount of measurable damage, show as because chemotherapeutic inducing, reduced 50% or more and do not have a new damage with baseline; Stable disease (SD): residual tumour is arranged, do not meet the requirement of CR; And PD (PD): residual tumour shows as with baseline in the total amount of measurable damage has increased by 25% or more or new damage occurs.

The present invention is that CR or PR are particularly effective for definite patient.

" the ErbB acceptor inhibitor includes but are not limited to the erbB receptor tyrosine kinase inhibitors "

This family comprises EGF, erbB2 (HER), erbB3 (noticing that erbB3 does not have functional type kinases zone) and the erbB4 that partly describes like background of invention of the present invention.

" gene specific oligonucleotide " meaning is that these oligonucleotide are distinctive for each gene, so that this gene of the unique identification of the fragment of said gene for example.Advantageously, a gene specific oligonucleotide length is preferably about 15 to 30 oligonucleotide between 5 to 50 Nucleotide, most preferably be about 23 oligonucleotide.

" array or microarray "

In many textbooks and document, described array technique and various relevant with it technology prevailingly and used.The gene array technique is particularly suited for embodiment of the present invention.The method for preparing microarray is well known in the art.These methods comprise Lemieux etc., (1998), and MolecularBreeding 4; 277-289, Schena and Davis.Parallel Analysis withBiological Chips.in PCR Methods Manual (eds.M.Innis, D.Gelfand; J.Sninsky), Schena and Davis, (1999); Genes, Genomes and Chips.In dna microarray: A Practical Approach (ed.M.Schena), OxfordUniversity Press, Oxford, UK, 1999), The Chipping Forecast (NatureGenetics special issue; January 1999 Supplement), MarkSchena (Ed.), Microarray Biochip Technology; (Eaton PublishingCompany), Cortes, 2000; The Scientist 14 [17]: 25, and Gwynne and Page, microarray analysis: the next revolution in molecular biology; Science, 1999August 6; With Eakins and Chu, 1999, Trends in Biotechnology, 17,217-218.

Said technology has description in PCT/US01/10063 and US 2,002 090979 and reference thereof.

Supplier comprises Affymetrix (California) and Clontech Laboratories (California).

The main application of array technique comprises the evaluation (nucleotide sequence/nucleotide sequence sudden change) of sequence and measures the expression level (abundance) of nucleotide sequence.Gene expression atlas array technique capable of using also can combine to utilize protein science technology (Celis etc., 2000, FEBS Lett, 480 (1): 2-16; Lockhart and Winzeler, 2000, Nature 405 (6788): 827-836; Khan etc., 1999,20 (2): 223-9).Other application of array technique also is as known in the art; For example, the discovery of nucleotide sequence, cancer research (Marx, 2000, Science 289:1670-1672; Scherf, etc., 2000, Nat Genet; 24 (3): 236-44; Ross etc., 2000, NatGenet.2000 Mar; 24 (3): 227-35), snp analysis (Wang etc., 1998; Science, 280 (5366): 1077-82), drug discovery, pharmacology genomics (pharmacogenomics), medical diagnosis on disease (for example, utilize the microfluid device: Chemical & Engineering News; February 22,1999, and 77 (8): 27-36), toxicology (Rockett and Dix (2000); Xenobiotica, 30 (2): 155-77; Afshari etc., 1999, CancerResl; 59 (19): 4759-60) with toxicity genomics (toxicogenomics) (heterozygote of functioning gene group and molecular toxicology).The target of toxicity genomics is to find out the toxic reaction of Toxic and be exposed to the dependency (Nuwaysir between the change of nucleotide sequence collection of illustrative plates of object of this Toxic; Deng (1999); Molecular Carcinonucleotidesequencesis, 24:153-159).

Generally speaking, through spatially separating the member in the library, any library can be arranged in a kind of array with neat mode.Be suitable for comprising nucleic acid library, peptide, polypeptide and protein library as the example in library of lining up array, and comprising the library of molecule arbitrarily ligand library for example.

Therefore, mention " library " part, it comprises the library of array format.

The member in library is fixed to usually or is immobilized on the solid phase, and preferred immobilization is on a kind of solid substrate, with the diffusion and the mixing of restriction sample.Especially, these libraries can be immobilized on the solid phase of blocky plane, and it matrix that comprises film and atresia is plastic and glass for example.

In addition, the mode of (just with reference to or lead to a certain specific sample) is arranged sample so that demarcate.Be typically, in grid forms with these sample point sample maculations.In order to realize this purpose, can make amendment to general detection system.For example, can array be fixed on the surface of microplate, both can be many parts of samples in a hole, also can be a kind of sample in each hole.

In addition, solid substrate can be a film, for example Nitrocellulose or nylon membrane (film that for example, is used for Blot experiment).Alternative substrate comprises glass or based on the substrate of silicon-dioxide.Therefore, through the approach well known of any appropriate, for example the bottom that sample is fixed to hole wall or hole perhaps is fixed on the film surface through coulombic interaction or through chemical coupling.Can also use other arrangement and fixing means, for example pipetting, drippage-contact, piezoelectricity mode, ink jet and foam jet technology, electrostatic interaction etc.With regard to the chip based on silicon, photolithography capable of using is arranged sample and be fixed on the chip.Can be arranged on the solid substrate through forming " spot "; This can be through manually or utilize animatronics to fix sample.Generally speaking, array can be called as VLA row or microarray, and difference is the size of substance spot.The VLA row contain size usually and are about 300 microns or bigger substance spot, just can form images easily through existing gel and trace scanner.Substance spot size diameter in the microarray is usually less than 200 microns, and this array contains thousands of spots usually.Therefore, microarray possibly need specialized animatronics and imaging device, and this possibly need customized.By Cortese, 2000, Tlae Scientist 14 [11]: briefly described the method for using instrument in 26 commentaries done.

Be used to produce existing in the prior art description of technology in the immobilization library of dna molecular.Generally speaking, technological method has the earliest described how to prepare the single stranded nucleic acid molecule library, the various arrangements of adopting a plurality of discontinuous site of masking technique on solid substrate for example to set up sequence.US5,837,832 have described a kind of modification method that is fixed on siliceous suprabasil DNA array that is used to produce, and it is based on the ultralarge scale integration technology.Especially, US 5,837, and 832 have described a kind of strategy that is referred to as " tiling " that is positioned at specific probe combinations on the site of confirming in suprabasil space that is used to prepare, and it can be used for producing immobilized DNA library of the present invention.US 5,837, and 832 also provide the reference of operable early stage technology.

In order to help to detect, can be with as fluorescence, noclilucence, phosphorescence, radioactivity reporter molecule, coming target-marking and probe by simply detected reporter molecule arbitrarily.At Shalon etc., 1996, Genome Res 6 (7): the mark that discloses probe and target among the 639-45.

The material ideal ground that is adopted in the inventive method is the material that is suitable for preparing test kit.Generally comprise a cover specification sheets.

Conventional recombinant DNA method technology

Except as otherwise noted, the present invention has adopted conventional chemistry, molecular biology, microbiology, recombinant DNA and immunological technique, and these technology are in those of ordinary skills' limit of power.These technology have explanation in document.Referring to for example J.Sambrook, E.F.Fritsch, and T.Maniatis, 1989, Molecular Cloning:A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, (1995 and periodic supplements such as F. M.; Current Protocolsin Molecular Biology, ch.9,13, and 16, John Wiley & Sons, New York, N.Y.); B.Roe, J.Crabtree, and A.Kahn, 1996, DNA Isolation andSequencing:Essential Techniques, John Wiley & Sons; M.J.Gait (Editor), 1984, Oligonucleotide Synthesis:A Practical Approach, IrlPress; And D.M.J.Lilley and J.E.Dahlberg; 1992; Methods ofEnzymology:DNA Structure Part A:Synthesis and Physical Analysisof DNA Methods in Enzymology, these regular files of Academic Press. are all incorporated this paper into as a reference.

In an embodiment of the present invention, adopt the cDNA microarray system screening of 27,648 genes of a kind of expression can be the one group gene of advanced NSCLC prediction to the responsiveness of ZD1939.The statistical study of expression map has been identified the gene of 12 differential expressions between to the respondent of ZD1939 and non-responder.A kind of drug responses points-scoring system based on these genetic expressions has successfully been predicted replying treatment with gefitinib.

Description of drawings

Fig. 1: the figure of the laser of four kinds of typical adenocarcinomas of lung of graphic extension-microbeam micro-dissection.

Up demonstration be the sample before dissecting; Following line display be cancer cell (H.E. strain X 100) through dissecting.What TBB showed is to change the segmental bronchus examination of living tissue; What LN represented is lymphoglandula.

Fig. 2: set up a kind of points-scoring system that is used to predict treatment with gefitinib efficient.

A., the different predicting branch has appearred when the quantity of distinguishing gene changes.Distinguish the numbering of genomic numbering (from 5 to 51) corresponding to the gene of selecting from the top by the ranking compositor tabulation of table 4.The value of classification scoring (CS) shows that more greatly two components leave well more.

B. adopted the grade clustering of 17 " study " cases of 51 candidate genes being used for ZD1939-susceptibility (left side) and 12 predicted genes of finally selecting for GRS.What dendrogram was represented is the paralogy of expression pattern between the one case; The longer expression of branch difference is big more.These two groups are the most clearly separated by the 12-genome.

C. the synoptic diagram of non-responder and respondent's difference and based on " the check case " of GRS check.Red rhombus representes to learn the prediction branch of PR case, and blue rhombus representes to learn the PD case.The pink colour trilateral representes not to be used to set up the check PR case of GRS, and blue trilateral representes to check the PD case.Yellow triangle table is shown in the check SD case that whole 4 months viewing duration keeps the SD state, and green trilateral representes that once the point sometime in this research was accredited as SD and demonstrated progressive check case in treatment beginning back in three to four months.

Fig. 3: with the affirmation of sxemiquantitative RT-PCR and immunohistochemical analysis to GRS.

A. the representative image of the sxemiquantitative RT-PCR of the RNA of PR and PD group being analyzed.OSMR and GCLC gene are crossed in the non-responder and are expressed (PD).Control the integrity of each cDNA template through the amplification of ACTB.

B. use anti-AREG antibody (X 200) that the fiberscope that obtains from same PD patient (No.LC21) is carried out the histogenic immunity chemical staining through segmental bronchus (lung) examination of living tissue (TBB) and the bioptic representative sample of lymphoglandula (LN).

C. the antibody (X 200) with anti-other four predictive marker (TGFA, ADAM9, CD9 and OSMR) carries out the histogenic immunity chemical staining to the representative sample from PD patient (No.LC21).

Fig. 4: the TGFA serum-concentration in 5 PR that measure by ELISA, 10 SD and 20 the PD gland cancer cases.The average serum level of TGFA is represented by black streaking: being 19.0 ± 2.8pg/ml (mean+/-standard error) in PD patient, is 13.9 ± 1.9pg/ml in SD patient, is 12.8 ± 1.4pg/ml in PR patient.

Fig. 5. excretory AREG is to the anti-apoptotic effect of ZD1939-susceptibility PC-9 cell.

A. the AREG transcript through sxemiquantitative RT-PCR inspection clone PC-9, NCI-H358 and-expression among the H522.

B. from NCI-H358 or-the PC-9 cell cultivated the substratum that is supplemented with 10%FCS, serum free medium or the serum-free condition substratum that the culture of H522 cell obtains.Each substratum all once replaced to a kind of substratum at 48 hours time point; After adding the ZD1939 that concentration is 0.5 or 1.0 μ M 72 hours, detect through MTT and to measure cell viability.This test has been done three times.The Y axle representes to cultivate the relative MTT value (there is not the MTT of ZD1939 in the MTT/ of the ZD1939 of existence 0.5 or 1.0 μ M) of the cell in different substratum.

C. with autocrine form excretory AREG to of the influence of NSCLC cell to the resistance of ZD1939.When cultivating beginning, with the PC-9 cell inoculation in the substratum of the AREG of ZD1939 that contains 1.0 μ M and reorganization albumen (final concentration is 1-100ng/ml); After 72 hours, use through three multiple MTT and measure the viability (Blue Streak) that detects cell.The Y axle is represented the relative MTT value (MTT/ under the AREG of each concentration does not have the MTT of AREG) of cell.Also studied under the ZD1939 existence condition that does not have 1.0 μ M AREG to the influence of the viability of NSCLC cell.One PC-9 cell joined contain reorganization AREG albumen but do not have in the substratum of ZD1939; After 72 hours, use through three multiple MTT and measure the viability (red) that detects cell.

Fig. 6: the two regulin in PD and PR patient's the section are expressed the immunohistochemical analysis that carries out.

Material and method

Patient and tissue samples

Comprise the II phase clinical study of multiple center trial, to probe into the main biological factor that produces clinical antitumous effect and to study former disadvantageous drug reaction (ADR) and the pharmacokinetics of ZD1839 of suffering among the nonsmall-cell lung cancer patient that chemotherapy does not have success of accepting with 250mg dosage every day.Main purpose is exactly to illustrate the gene expression atlas that can confirm the antitumous effect that ZD1939 is possible in advance.Research is initial, adopts and has measured sample-sized as the research of basic principle up to now ^12,13Because the response rate to ZD1939 has been lower than 20% in suffering from the patient of lung cancer ^8-10,, detected about 50 patients in order to obtain the study case of above-mentioned detection.Those local patients with terminal (IIIB stage) or transitivity NSCLCs are admitted in this test the patient that one or more conventional chemotherapy regimens produce opposing.The standard of including is that (1) age is greater than 20 years old (2) performance status (PS) 0-2, liver that (3) are enough and renal function test.All patients receive treatment in the Tokushima university or the Kinki University hospital of Japan, every day an oral 250mg ZD1939.This treatment lasts till carry out of patient owing to (1) disease always, and (2) intolerable virulence or (3) are recalled promise and broken away from this research.

Behind begin treatment, whenever just target tumor is replied and evaluate according to the standard of International Union Against Cancer/World Health Organization (UICC/WHO) general introduction at a distance from 4 weeks.That replys is classified as follows: reply (CR) fully, in all valuable damages, do not have residual tumor; Part is replied (PR): residual tumour is arranged in the total amount of measurable damage, show as because chemotherapeutic inducing, reduced 50% or more and do not have a new damage with baseline; Stable disease (SD): residual tumour is arranged, do not meet the requirement of CR; And PD (PD): residual tumour shows as with baseline in the total amount of measurable damage has increased by 25% or more or new damage occurs.All valuable damages all are (maximum diameter of measurable damage and the sum of products of its longest vertical line) that adopts the technology identical with baseline such as common X-ray, CT or MRI to measure with two-dimensional approach.

When treatment finished (or withdrawal) in 4 months, always reply according to the best of every patient of following definition evaluation: CR is spaced apart at least 28 days the patient that CR requires that meets between two continuous detection time points; PR, between two continuous detection time points be spaced apart at least 28 days be judged as PR or better patient; SD, between two continuous detection time points be spaced apart at least 28 days be judged as SD or better but do not meet the patient of CR or PR.Judge for the first time of SD case should the first time lesion detection time point or this after (after the random sampling 28 days); PD, the first time lesion detection time point or this before be confirmed as patient's (after random sampling 28 days) of PD; The unknown does not meet the patient that the disease the best that increases the weight of is replied, after baseline (before the random sampling) and before carry out all dbjective states all be unknown.

Before treatment with gefitinib, through written agreement through segmental bronchus (lung) (TBB), skin or biopsy of lymph node take out tumor sample.Obtained the permission of logic from the Ethics Committee of each institute.Freezing at once biopsy sample is embedded in the TissueTek OCT substratum that (Sakura, Tokyo Japan), store down at 80 ℃.All samples have all been carried out microscopical determination, and the sample of having selected at the very start to come from 28 patients (17 study cases and 11 check cases) is further to analyze, and these samples all contain enough cancer cell to be used for the expression map analysis.In order to verify this prognoses system, the combination at random (blinded set) that comes from the sample of 5 new cases of registering (4 PD and 1 SD) also is added in 11 check cases.These patients' clinical and histology information is summarized among the table 1-3.

Micro-dissection

Consider cancer cell and various from a kind of tumour to submission the difference of parenchyma on ratio to the another kind, micro-dissection is a kind of necessary means that on the cDNA array, obtains accurate gene expression atlas.Therefore, we dye to the thick frozen section of 8 μ m with phenodin and eosin, and use μ CUT Laser Microbeam micro-dissection system (Molecular Machines &Industries AG, Glattbrugg, Switzerland) ¹⁴Optionally collect cancer cell.In this system, tissue slice is positioned on a kind of thin supportive polyethylene film, and this film will be downcut with this target tissue; Pulse-ultraviolet ray (UV) narrow beam laser focusing is along downcutting cancer cell at the observed route of selecting in advance of image display.This material that will extract never directly is exposed under the laser but is only surrounded by laser; Unlike other LMM system, thereby this makes the cell that downcuts not have to be able under the radiating condition recover to continue survival.Adopt this system, the small region that we can the sharp separation tissue, and from tissue slice, isolate unicellular (Fig. 1).

RNA extracts and based on the RNA amplification of T7

With the mini test kit of RNeasy with do not contain the DNA enzyme reagent kit of RNA enzyme (QIAGEN, Hilden Germany) extract total RNA according to the scheme that the manufacturer provides from each micro-dissection crowd of cancer cell.As stated, total RNAs is carried out the RNA amplification based on T7 ¹⁵Carry out the aRNA (through the RNA of amplification) (＞100,000 times) that two round-robin amplifications obtain 40-200 μ g from every increment this.As the contrast probe, amplify normal people's lung poly (A) with the same manner ⁺RNA (BD Biosciences Clontech, Palo Alto, CA andBIOCHAIN, Hayward, CA, USA).In the presence of Cy5-dCTP and Cy3-dCTP respectively with the aRNA aliquot sample in each sample and the control group (2-5 μ g) reverse transcription.

The cDNA microarray

Our " extensively reaching genome-scope " cDNA microarray system contains 27,648 cDNAs that from the UniGene DB of NCBI (National Center for Biotechnology Information), select ¹⁵The detection of the making of said microarray, hybridization, cleaning and strength of signal all was described in the past ¹⁵In order to make the mRNA amount stdn in tumour and the control group; With the Cy5/Cy3 ratio of every kind of genetic expression adjust to make the average Cy5/Cy3 ratio of 52 house-keeping genes equal 1. we utilize variance analysis that cutoff is distributed to each microarray slide: the Cy5/Cy3 ratio that calculates gene as follows: (1) if Cy5 (cancer sample) is lower than cutoff level, and the Cy5/Cy3 ratio of this gene just is replaced by 2.5% in the Cy5/Cy3 ratio of other gene that its Cy5 and Cy3 be higher than the cut-out level so; (2) if Cy3 (check sample) is lower than cutoff level, the Cy5/Cy3 ratio of this gene just is replaced by 97.5% in the Cy5/Cy3 ratio of other gene that its Cy5 and Cy3 be higher than the cut-out level so; (3) if Cy5 and Cy3 are lower than cutoff level, the Cy5/Cy3 ratio of this gene is just empty so.

Be used to predict the extraction of the gene that ZD1939 is replied

For find maybe with the gene relevant to the susceptibility of ZD1939, compared the various measuring results of about 27,648 genes between two groups of patients, in these two groups wherein one group be classified as to the ZD1939 respondent (PR), another component is non-responder (PD).For the dimension of the number of the effective gene that reduces between two types, to there are differences, we have only extracted the gene that satisfies following two standards: 1) each group at least 60% in strength of signal be higher than cutoff level and 2) | MED _PR-MED _PD| 1, and wherein the MED indication is calculated and the median of acquisition from the relative expression's ratio to functional transformation in every group.Use random permutation then and test the ability that each gene is distinguished two types (PR and PD) of measuring; From through relative expression's ratiometer of every kind of gene two groups of number conversion is calculated MV (μ) and standard deviation (σ).Every kind of gene distinguish that the branch value defined is following:

DS＝(μPR-μPD)/(σPR+σPD)。

For in each group each to all with sample random permutation 1000 times.Because the DS data set of every kind of gene all demonstrates normal distribution, so we calculate the p value that a kind of user of being used for defines grouping.

The calculating of drug reaction score value

We reply scoring (GRS) according to the ZD1939 that preceding method calculates reflection candidate predicted gene expression level ^16-18Each gene (gi) votes for respondent (PR) or non-responder (PD) and depends on expression level in the sample (xi) and whether approach one group or the average expression level of another group in the standard samples.The quantity of poll (deviation that has vi) reflected the MV of expression level and two classification in the sample:

Vi＝|xi-(μ _PR+μ _PD)/2|。

We have added up poll and have obtained support respondent (V _PR) and non-responder (V _PD) aggregate votes, and calculate the GRS value as follows:

GRS=((V _PR-V _PD)/(V _PR+ V _PD)) * 100, wherein the GRS value has reflected the boundary of the triumph in respondent or non-responder's direction.The scope of GRS value is-100 to 100; The absolute value of GRS is high more, and forecasting reliability is strong more.

The cross-validation of scoring and to the evaluation of prognoses system

Prediction score through all samples of elimination of the last one method acquisition in the method, removes a kind of sample each time from the sample group; Use the displacement p value and the MV of two classification of remaining every kind of gene of sample calculation.Predict to such an extent that assign to predict the drug reaction of the sample that stays through calculating.Originally all to come again above-mentioned steps for every increment ^16-17

In order to estimate the safety of prognoses system, we adopt respondent and non-responder's the GRS value in each assortment of genes to calculate " classification score " as follows (CS):

CS＝(μGRSpr-μGRSpd)/(σGRSpr+σGRSpd) ¹⁷。

The big more explanation of CS value is opened two components good more by prognoses system.

Hierarchical clustering

The software (" Cluster " and " TreeView ") that the M.Eisen (http://genome-www5.stanford.edu/MicroArray/SMD/restech.html) that our utilization can obtain from network writes is produced the diagram of microarray sequence data and is produced the dendrogram of hierarchical clustering.Before using clustering algorithm, the fluorescence ratio of each spot is at first carried out number conversion, for the data of every kind of sample, be that central row is removed experimental error then with the median.

Sxemiquantitative RT-PCR analyzes

Few (dT) 12-18 primer of use and SuperScript II reversed transcriptive enzyme (Invitrogen, Carlsbad, CA, USA) rt hybridizes to the aliquot sample that comes from each sample and the identical aRNA of normal control lung (5.0 μ g) on the microarray slide.Use group down to be specific to be used to the synthetic primer of preceding 12 genes of setting up GRS or use beta-actin (ACTB) Auele Specific Primer to carry out sxemiquantitative RT-PCR experiment as a kind of internal contrast:

FLJ22662,5 '-GCCATAAGTGGTCCCACAGT-3 ' and 5 '-GTCTTCTAGTCCGTCATCTCCCT-3 '; Amphiregulin (AREG) 5 '-CCATAGCTGCCTTTATGTCTGC-3 ' and 5 '-CTTTTTACCTTCGTGCACCTTT-3 '; Coronin, ABP matter, 1C (CORO1C), 5 '-TAATCTGCTGAGGACCTTTTGTC-3 ' and 5 '-TAATTCACTGTCCTCTTCTGGGA-3 '; Apoptosis, the active supressor (AVEN) of aspartic acid specificity cysteine protease, 5 '-GCTCACAGCAGTAAATGCCTA-3 ' and 5 '-TGCTATGCTGTAAACACTGGCTA-3 '; Dual specificity phosphatase enzyme 3 (DUSP), 5 '-GGATCCTTTATTGGTGGTAGAGC-3 ' and 5 '-CCAGAGTGACCCTGAAGATAAAT-3 '; DJ473B4,5 '-ACCTGATTCTCTAGGTGCAGTTT-3 ' and 5 '-GTCGTTTCAACCAGGTAGTTTTG-3 '; Thrombocyte white corpuscle C kinase substrate homology appearance zone, the A of family, member 2 (PHLDA2), 5 '-GGGCGCCTTAAGTTATTGGA-3 ' and 5 '-GGATGGTAGAAAAGCAAACTGG-3 '; RNA binding motif albumen 7 (RBM7), 5 '-TGTAAATGGAGATTGTACAGGTTG-3 ' and 5 '-AGGAACAGTACAAATGCTGTGGT-3 '; BX092512 (EST), 5 '-GCACTCCTTGAAGGTACACTAAC-3 ' and 5 '-ATTTGTATTCACTCAGCCATGC-3 '; Oncostatin M acceptor (OSMR), 5 '-ACCCAACTTCAAAACTAGGACTC-3 ' and S '-ACAGCTTGATGTCCTTTCTATGC-3 '; L-glutamic acid-halfcystine ligase enzyme, catalytic subunit (GCLC), 5 '-TCATGAAAGGCACTGAGTTTTG-3 ' and 5 '-GTTAGCTGAAGCAGCTITATTGC-3 '; Collagen, IV type, α 3 conjugated protein (COL4A3BP), 5 '-ATATGCACAATCCTGGAAGTGA-3 ' and 5 '-TGCCTTACTAGCATTACCACCAT-3 '; ACTB, 5 '-GAGGTGATAGCATTGCTTTCG-3 ' and 5 '-CAAGTCAGTGTACAGGTAAGG3 '.To cycle number PCR has been reacted and carried out optimizing to guarantee that production concentration is in the logarithmic phase of amplification.We have carried out phosphorescence imaging quantitative analysis (Molecular Imager FX Bio-Rad Laboratories; Hercules; CA USA), and has carried out quantitative comparison with the band intensity of RT-PCR and the Cy5/Cy3 ratio of the standardized genetic expression in the microarray data.

Carry out RT-PCR with filter out nearest report as the sudden change the complete area (encircling to the activation ring) of the 709-870 bit codon of a kind of EGFR of mutantional hotspot from p-; Three groups of primers have wherein been adopted: fragment 1,5 '-TCTTACACCCAGTGGAGAAGC-3 ' and 5 '-GTCTTTGTGTTCCCGGACAT-3 '; Fragment 2,5 '-ACTATGTCCGGGAACACAAA-3 ' and 5 '-TTCCGTCATATGGCTTGG-3 '; Fragment 3,5 '-CGTCGCTATCAAGGAATTAAGAG-3 ' and 5 '-GTAGCTCCAGACATCACTCTGGT-3 '.Through direct sequencing analysis through 19 patients' of treatment with gefitinib RT-PCR product.

Immunohistochemical analysis

For the AREG of confirm to encode EGFR and other ERBB member's part and transforminggrowthfactor-(TGFA) albumen and other 3 known and relevant being used to of EGFR signal transmission predict that (a kind of separating joins albumen and regional 9 (ADAM9) of metalloprotease, CD9 antigen (p24) and differential expression OSMR) for the respondent of ZD1939 with to candidate's mark of non-responder; (DakoCytomation is GlostrupDenmark) to being dyeed through the clinical tissue section of segmental bronchus (lung) examination of living tissue (TBB) and the acquisition of lymphoglandula biopsy by fiberscope with ENVISION+ test kit/HRP for we.Behind endogenous peroxydase and protein capping, add anti-people AREG polyclonal antibody (Neo Markers, Fremont, CA rapidly; USA), anti-people TGFA monoclonal antibody (Calbiochem, Darmstadt, Germany), anti-people ADAM9 monoclonal antibody (R & D Systems Inc.Minneapolis; MN, USA), anti-people CD9 monoclonal antibody (Novocastra Laboratories Ltd, Newcastle upon Tyne; UK) or anti-people OSMR monoclonal antibody (Santa CruzBiotechnology, Inc., Santa Cruz; CA, USA), then with through the anti-rabbit of HRP mark or anti-mouse IgG as SA.Add matrix-chromogen then, use phenodin the sample counterstaining.

Select the viral tissue samples that comes from 11 patients and be used for immunohistochemical analysis.By three of the anticipatory knowledge that does not have clinical repeating data independently the researchist through scoring intensity the positive sxemiquantitative ground of immunostaining is evaluated as shortage or male.Have only and think just that when the reviewer is defined as the positive independently these cases are male.

ELISA

From having accepted to obtain serum the independently group that the same scheme of basis and this clinical study of the hospital of Hiroshima University of Japan forms with the lung-ADC patient (5 PR, 10 SD and 20 PD) of treatment with gefitinib by 35.All patients serums obtain the patient and agree behind begin treatment, whenever to obtain at a distance from 4 weeks down when diagnosis, and are stored in-80 ℃.Through adopting a kind of commercially available enzyme detection kit (TGF-alpha ELISA test kit: OncogeneRsearch Products, San Diego, CA, the TGFA level of ELISA detection serum USA).

External treatment with gefitinib and AREG-autocrine are measured

People NSCLC (gland cancer) clone PC-9, NCI-H358 and NCI-H522 are from American type culture collection (ATCC; Rockville, MD USA) buys.In order to detect the expression of AREG in these NSCLC (gland cancer) cell, total RNA reverse transcription of each clone is become strand cDNA with few (dT) 12-18 primer and Superscript II (Invitrogen).Carry out sxemiquantitative ThermoScript II-PCR (RT-PCR) by the mode of former description ¹⁴ZD1939 (4-(3-chloro-4-fluoroanilino)-7-methyl-6-(3-morpholino propoxy-) quinazoline: ZD1839; Iressa) be by AstraZeneca Pharmaceuticals (Macclesfield; UK) provide, it is a kind of suppressor factor of epidermal growth factor recipient tyrosine kinase.Concentration with 10mM is dissolved in DMSO with this medicine, and is kept at-20 ℃.

We have carried out flow cytometry to confirm the susceptibility of lung adenocarcinoma cell system to treatment with gefitinib.With 5 * 10 ⁵The concentration of cell/100-mm petridish tiles cell, and in suitable serum free medium, handles with the ZD1939 of 1.0 μ M.After processing,, be collected among the PBS in 70% cold ethanol fixing 30 minutes with cell tryptase protease digestion 72 hours.(MO USA) after the processing, dyes with third ingot of the iodate among the PBS of 50 μ g/ml (Sigma-Aldrich Co.) pair cell for Sigma-Aldrich Co., St.Louis with 100 μ g/ml rnases.On Becton Dickinson FACScan, carry out flow cytometry, with ModFit software (Verity Software House, Inc., Topsham, ME, USA) analysis.From at least 20, confirmed nuclear percentage ratio and the sum of G1 subage of G0/G1, S and the G2/M phase of cell cycle in the cell of 000 no gate.

For whether the effect of studying AREG is the effect of the performance autocrine anti-apoptotic factor in the lung adenocarcinoma cell that ZD1939 is handled, we have carried out following mensuration.At first before ZD1939 is handled, in serum free medium, cultivate the ZD1939 susceptibility PC-9 cell at least 8 hours of not expressing AREG.Then with these cells with 0.5 or 1.0 μ M ZD1939s serum free medium or be supplemented with the substratum of 10%FCS or the serum-free condition substratum from 72 hours cultures of the cell (NCI-H358 or NCI-H522) of expressing AREG, collected in cultivated 72 hours.Every kind of substratum is all changed once with the same medium that contains ZD1939 at 48 hours time points.In order to detect the reaction of each clone to ZD1939, (WAKO, Osaka, MTT Japan) detect and estimate viability through adopting cell counting test kit (Cell Counting Kits).

In order to confirm the influence of AREG to the autocrine of ZD1939 resistance NSCLC cell; We are containing the reorganization AREG albumen (Genzyme-Techne that 1.0 μ M ZD1939s and final concentration are 1-100ng/ml; Minneapolis, MN cultivated the PC-9 cell 72 hours in serum free medium USA).Detect the viability of estimating cell with MTT.Also through do not have at serum-free ZD1939 only contain cultivate in the reorganization AREG proteic substratum PC-9 cell estimated AREG self to the NSCLC cell the influence of viability.Carrying out MTT in the above described manner detects.

The result

To replying of treatment with gefitinib

In the patient of 53 these tests of participation, 46 tumour is diagnosed as gland cancer (86.8%); 5 squamous cell carcinomas (9.4%) by name; Two large cell carcinomas (3.8%) by name.15 patients have realized that PR and nobody demonstrate CR; 17 patients are classified as SD, and 19 are classified as PD.There are 2 patients' clinical response data to obtain.The tumor response rate (CR+PR/CR+PR+SD+PD) of this treatment is 29.4%, and disease control rate is (CR+PR+SD/CR+PR+SD+PD) 62.8% (table 1).

Tumor sample is collected from 43 patients.The sample that comes from these 43 patients contains enough tumour cell quantity with the expression map on the cDNA microarray that is used to analyze us.The quantity that being suitable for of being assert carried out the sample of further microarray analysis is 8 PR, 7 SD, 13 PD (table 2).With 17 in 28 samples as study case (7 PR, 10 PD) analyze, with 11 samples as the check case (1 PR, 3 PD, 7 SD) to set up a kind of prediction points-scoring system of predicting the treatment with gefitinib effect.In order further to confirm the validity of this prognoses system, obtained other one group of combination at random (blinded set) that comes from the sample of 5 initiate detection cases (4 PD and 1 SD), at last it is joined in above-mentioned 11 check cases originally.

With evaluation to the relevant gene of the susceptibility of ZD1939

We attempt to be extracted in 7 patients in the PR group (being defined as the respondent) through 27,648 expression of gene levels relatively tumour and PD organize the gene of differential expression between 10 patients' the tumour of (being defined as the non-responder).(table 2,3)

We carry out the random permutation check to distinguish two kinds of subclass by the tumor response definition, identify displacement p value and are lower than 51 genes (table 4) of 0.001.In the non-responder, 40 expression of gene higher level some, other 11 expression of gene levels are lower.

A kind of foundation of predicting the prediction points-scoring system of treatment with gefitinib effect

Based on above-mentioned 51 the expression of gene collection of illustrative plates of selecting, we attempt to set up a kind of prediction points-scoring system of predicting the treatment with gefitinib effect.The prediction score is referred to as ZD1939 and replys score (GRS), calculates (referring to the method part) according to preceding method.The quantity of the candidate gene of distinguishing for the best of confirming to be used to provide two groups, we based on the significance of their displacement p value these 51 genes have been carried out arranging according to the order of sequence and through elimination of the last one method of inspection (leave-one-out test) begin to successively decrease from the bottom of the tabulation of arrangement according to the order of sequence 1 (51,50,49,48 or the like) calculated the prediction score.We calculate a kind of classification score (CS), this be before us definition be used to estimate a kind of standard that every group of gene distinguished the ability of two kinds ¹⁷

Shown in Fig. 2 A, we have obtained the different predicting score when the quantity of difference gene changes.When we adopted 12 genes in our the candidate gene tabulation to count the score, we had obtained best CS, this means that respondent and non-responder are able to best differentiation.

Adopt all 51 genes or the only hierarchical clustering analysis of 12 genes, according to ZD1939 reply with wherein one group in all 17 case branch to two groups (Fig. 2, B).When we use these 12 genes to carry out bunch analyzing, can the most clearly be distinguished for these two groups.At last, we have set up a kind of medicine of numerical value-reply-scoring algorithm, and it can be used to predict the susceptibility of individual NSCLC to ZD1939 clinically based on these 12 expression of gene levels of being selected.

In order to confirm the validity of this prognoses system, we have studied and these 17 " study case " irrelevant fully other 8 (" checks ") NSCLC cases (1 PR, 7 PD) that are used to set up said system.We check the gene expression atlas in each sample in these samples, calculate GRS according to these 12 difference expression of gene levelers then.Shown in Fig. 2 C, the score that is obtained by the GRS system in the case all 8 " checks " is with consistent to the clinical response of ZD1939.

The GRS value of patient in tumor response of suffering from SD

According to the prediction points-scoring system of top foundation, calculate 8 detection-SD patients' GRS value.Though these values are distributed in-83.0 (being predicted as the non-responder) widely to 61.6 (respondents); The score that whole viewing duration maintains the patient of SD state possibly be higher than that certain time point in this research is identified as SD but in treatment beginning back 3 or demonstrate in 4 months the progressive patient of disease score (Fig. 2, C).Gene expression atlas between the patient who distinguishes over the patient who suffers from PR in the tumor response and suffer from PD (not having SD) sets up although the GRS system is based on, and these results show that GRS is suitable for by the patient replying of ZD1939 being divided into each group with SD patient.

The checking of GRS validity being carried out with sxemiquantitative RT-PCR

In order to confirm the differential expression of preceding 12 predicted genes between PR and PD case, connect (5 PR and 7 PD) (Fig. 3 A, table 5A) to the value of the expression values that obtains from microarray and the sxemiquantitative RT-PCR gained of the RNA that from same patient, obtains.The Spearman rank correlation property of all these 12 genes is positive correlation, and to have 7 in this 12 gene be significantly positively related.

The immunohistochemistry checking of GRS validity

In order to verify that predicted protein is marked at the differential expression between PR and PD case; We have carried out immunohistochemical staining with five kinds of different anti-AREG, TGFA, ADAM9, CD9 and OSMR antibody; It is relevant to be known that AREG, TGFA, ADAM9, CD9 and OSMR and part-EGFR signal transmit, and their displacement p-value is all less than 0.01.At first we with these 5 kinds of antibody to dyeing by the paired tumor tissue section that from same patient, obtains through segmental bronchus (lung) examination of living tissue and biopsy of lymph node.The protein expression of in 3 different patients, not observing these 5 marks exist difference between the patient (Fig. 3, B).We also in 11 NSCLC samples (5 PR and 6 PD) with 5 kinds of marks microarray data is verified.Its result and microarray data consistent (Fig. 3 C, table 5B).

The serum level of TGFA

In order further to estimate the feasibility of this prognoses system in the routine clinical situation, we detect TGFA albumen in collection separately is used for the serum sample that is obtained by 5 PR, 10 SD and 20 PD patients that serologic test do not join microarray analysis with ELISA.In PD patient, the serum level of TGFA is 19.0 ± 2.8pg/ml (mean+SD), in SD patient, is 13.9 ± 1.9pg/m, is 12.8 ± 1.4pg/ml (Fig. 4) in PR patient.When during as cutoff, from 20 parts of serum samples that PD patient obtains, having 12 parts to be rendered as the TGFA positive with 16.0pg/ml, all are negative from the serum sample that PD patient obtains.

External ZD1939 is handled and the AREG autocrine detects

In the non-responder, a kind of part AREG of EGFR and other ERBB member has obtained significant the expression excessively and in the respondent, can't (almost not have) to detect in the non-responder.When secreting with the autocrine form, whether caused the resistance of NSCLC to treatment with gefitinib in order to study AREG albumen, we have carried out following biological analysis.We have gone out the expression of AREG mRNA in lung-gland cell system NCI-H358 and-H522 through the RT-PCR experimental identification at the beginning, and in PC-9, do not express (Fig. 5, A).Then, we have carried out flow cytometry handling the PC-9 cell with the ZD1939 of 1.0 μ M after 72 hours, and discovery is compared ZD1939 with undressed cell (6%) and improved nuclear percentage of G1 phase (24%) (data not shown).This result shows that ZD1939 possibly induce apoptosis in the PC-9 cell.

Then serum free medium or from exist or lacking the NCI-H358 that grows under the 0.5 or 1.0 μ M ZD1939 conditions or-after cultivating the serum-free condition substratum that the H522 cell obtains; We have analyzed the viability of PC-9 cell, and wherein the PC-9 cell is responsive and do not express AREG to ZD1939.Shown in Fig. 5 B, the viability that is incubated at the PC-9 cell in the conditioned medium that contains ZD1939 of serum-free is eager to excel than the PC-9 cell that is grown in the serum free medium with identical ZD1939 concentration.Because reported the provider of ZD1939 in the past, the antitumous effect of ZD1939 reduces in the presence of 10%FCS, this shows that this detection should be suitable for ZD1939 dosage and active quantitative measurment.

In order to study with autocrine form excretory AREG whether suppressed the apoptosis through the NSCLC of treatment with gefitinib cell, we are to cultivate the PC-9 cell in the reorganization AREG albumen of 1-100ng/ml and the serum free medium that contains or do not contain 1.0 μ M ZD1939s containing final concentration.With only comparing with the PC-9 cell that 1.0 μ M ZD1939s are cultivated, the viability of the PC-9 cell of cultivating with AREG albumen and 1.0 μ M ZD1939s with AREG-dosage-dependent mode be improved (Fig. 5, C).On the other hand, the reorganization AREG separately to the viability of PC-9 cell do not exert an influence (Fig. 5, C).This observations has shown that obviously AREG suppresses by ZD1939 inductive apoptosis, but himself does not influence the viability of cell.Immunostaining to AREG is presented among Fig. 6.

Discuss

A large amount of evidences has been supported following viewpoint, promptly the molecule in the autocrine approach of EGFR with some for the formation of cancer with to carry out vital process relevant, these processes comprise that cell proliferation, blood vessel take place and shift diffusion ⁵Therefore, the therapeutic blocking-up to the specific signal transmission possibly be a kind of promising strategy of cancer treatment.ZD1939, a kind of synthetic anilinoquinazoline, it has suppressed tyrosine kinase activity through the binding site with the intracellular region territory of Triphosaden competition tyrosine kinase receptor.In testing in the II phase, with 2nd, 3rd or 4th the line single therapy of ZD1939 as advanced NSCLC, the tumor response rate has reached nearly 20% ^8-10, be superior to the conventional response rate that cytotoxic agent obtained.In IDEAL 1, the response rate among patient's the multivariate analysis prompting women is higher than the male sex possibly, the response rate among the gland cancer patient is higher than squamous cell carcinoma patient (diversity ratio is respectively 2.7 and 3.5) ⁹The nearest effectively individual gland cancer that more possibly suffer from the bronchioloalveolar hypotype of research prompting ZD1939, and will never be smoker's (diversity ratio is respectively 13.5 and 4.2) ¹⁹Here the higher response rate of putting down in writing in the clinical trial of being reported (29.4%) possibly reflect that gland cancer patient's ratio (46 gland cancer, 5 squamous cell carcinomas and 2 large cell carcinomas) is higher than the ratio of the case in other research.The clinical pathology determinative that comprises the ZD1939 susceptibility of bronchioalveolar carcinoma (BAC) characteristic has predictability to a certain extent ^9,10,19,20Yet report in the past and our observation obviously demonstrate does not have the factor can ideally predict NSCLC replying treatment with gefitinib.Therefore the method that is used in advance distinguishing from the non-responder respondent can make the application of ZD1939 clinical settings obtain the more concern more.

The statistical analysis of the gene expression atlas through the advanced NSCLC that obtains from microarray data, we identify 12 with the gene relevant to the susceptibility of ZD1939.We have introduced a kind of being based on and have replied group and demonstrate the most significantly prediction points-scoring system of 12 expression of gene of expression level difference in the group with non-replying.This group gene is from the expression map of adenocarcinoma of lung, to elect; Yet the GRS system successfully classifies according to them our 8 " check " PR and PD case to the clinical response of ZD1939, and one of them is a squamous cell carcinoma.In addition, this system possibly be divided into two groups with intermediary tumor response (SD), and one group of representative is successfully maintaining the patient of tumour-static effect for a long time, and another group representative fails to reach the patient of this effect.

In fact, we need use the general bottom line invasion property technology of each hospital to predict the chemosensitivity of each tumour, because seldom there is the advanced NSCLC patient to be suitable for the candidate as ocal resection.Therefore we attempt setting up a kind of prognoses system, and it just needs the cancerous tissue that can pass through for example flexible (photoconduction) fiberoptic bronchoscopy acquisition of some amount.Through verifying each step of this method, we can accurately draw little genetic expression to the biopsy specimen of 1mm.Confirm corresponding microarray results through 12 sxemiquantitative RT-PCR that demonstrate the gene of significant difference, thereby set up the GRS system.And; We have verified the validity of the antibody of the biomarker (AREG, TGFA, ADAM9, CD9 and OSMR) that possible respondent and non-responder is distinguished to 5 kinds of different being used in TBB and biopsy of lymph node sample, these five kinds of biomarkers are all relevant with part-EGFR signal transmission.In addition, we can detect serum TG FA albumen through ELISA in lung-ADC patient.To these further assessments that are used for clinical mark is necessary, and the finite population of the gene that forecasting institute needs should finally make the laboratory to diagnose out the validity of treatment with gefitinib for a NSCLC patient in advance through the ordinary method the immunohistochemical analysis that utilizes serologic test, PCR experiment or biopsy specimen as blood.

According to our understanding, this is first about the report of the gene expression atlas of " late period " lung cancer that can't excise, although reported the collection of illustrative plates of the excision sample of " in early days " lung cancer.Yet suffering from after diagnosing 70% tumour is arranged among the patient of NSCLC approximately has been local late period or transport property, and this makes their produce resistance to the therapeutic modality of routine.Therefore the listed gene of this paper should help to disclose the molecule mechanism of lung cancer progress, and possibly be drug development potential target.

ZD1939 is to be developed out as a kind of " selectivity " suppressor factor of EGFR-TK; Yet be all not find the EGFR activation levels and get in touch in vivo or external to having clearly between the replying of ZD1939 ^7,23In clinical trial, ZD1939 is more effective to gland cancer comparison squamous cell carcinoma ^9,10Although the frequency that the mistake of EGFR is expressed in the gland cancer is lower ²⁴Therefore, identifying which kind of tumour is that the suitable target of this treatment is extremely important.In our analysis of use clinical sample, the difference of the EGFR protein expression between respondent and non-responder is inapparent on statistics.On the other hand, the two-ways regulation factor (AREG) of two kinds of all encode EGFR and other ERBB members' part and transforming growth factor-alpha (TGFA) have obtained remarkable cross to express and in replying, can't (almost not have) to detect and (be respectively p=0.0000000000093 and 0.0095 in the non-responder; Table 4).

These parts and EGFR autocrine loop are unarguable for the growth of lung carcinoma cell and the importance of existence ^24-26, solve seldom but AREG but gone back in the formation of cancer and ongoing effect.Yet it is relevant with NSCLC patient's the lost of life that some evidence lines point out crossing of AREG to express ²⁴And, reported that recently the anti-apoptotic of AREG in human lung carcinoma cell is active ²⁵For whether the anti-apoptotic activity of studying AREG has induced the resistance of NSCLC cell to treatment with gefitinib, we adopt the NSCLC clone PC-9 that does not still express AREG to ZD1939 susceptibility to carry out biological detection.We find that ZD1939 has been reduced by the autocrine of AREG the anti-tumor activity of PC-9 significantly.It is all complicated by significantly that the growth factor signal that the complicacy of part, dimerization mating partner, effector and path downstream is mediated by EGFR although this evidence is showed consumingly is delivered in each step ²⁶But AREG causes cancer progression and to a kind of important acvator in the ligand-receptor autocrine growth path of the resistance of ZD1939.

Several kinds of key elements relevant with the EGFR-TK path are present in our tabulation of difference expression gene.For example, the gene of coding dual specificity phosphatase enzyme 3 (DUSP3), ADAM9, CD9 and OSMR has significantly been expressed (being respectively p=0.0000000000094,0.01,0.000022 and 0.0000011) in the non-responder.The DUSP3 gene is through regulating the transmission of EGFR signal to the dephosphorylate effect of mitogen activated protein kinase (MAPK); The mitogen activity protein kinase of dephosphorylate is the regulatory factor that a kind of important signal conducts, and ADAM9 has participated in the activation that the EGFR signal transmits through the extracellular region (ectodomain) of the proHB-EGF that comes off (pro HB-EGF like growth factor) ²⁸The interaction of physical property takes place in CD9 and the TGFA that strides film.The expression of CD9 has reduced by growth factor and PMA inductive consumingly strides the proteolyze conversion of film to soluble T GFA, and has greatly strengthened TGFA inductive EGFR activation ²⁹It is reported that OSMR is structurally relevant with ERBB2 in breast cancer cell.Although proposed other target molecule of ZD1939, the transmission of our result of study prompting EGFR signal be at least with to one of in the relevant significant process of replying of this medicine.

Because ZD1939 can be induced the apoptosis of some tumour cells in vivo, other active molecule of anti-apoptotic that has as AREG has the resistance of the tumour of helping to this medicine.Be known that AVEN (apoptosis, aspartic acid specific cysteine proteinase inhibitors) has strengthened the anti-apoptotic activity of Bcl-xL, suppresses the aspartic acid specificity cysteine protease activity that Apaf-1 regulates ³¹, it expresses (p=0.00000000042) specifically in our non-responder.On the other hand, the mechanism of adjusting transport of drug also should influence the resistance of medicine.GCLC (L-glutamic acid-halfcystine ligase enzyme, the subunit of catalytic) has vital role in the cell detoxifcation of the anticarcinogen as Platinol, VP and Zorubicin ³², it is crossed in our non-responder's group and expresses (p=0.00000012).Because these genes are in our tumor experiment group (panel) and to the chemotherapeutical negative correlation (just these expression of gene are high more, and are just strong more to the resistance of ZD1939) of replying, they maybe be relevant with the mechanism that causes resistance to produce.Should also be noted that it is unknown in our candidate's predicted gene almost half function being arranged.Therefore need further research more to clearly reveal the biology incident to the basis of replying of ZD1939 as NSCLC.

In a word; We have identified 51 genes of differential expression significantly between to the respondent of ZD1939 and non-responder in people's lung cancer; And set up a kind of points-scoring system based on 12 expression of gene patterns wherein, be used to predict various tumours replying to this medicine.Although the clinical case that adopts bigger group further verifies it is necessary; But the data that the present invention appeared can produce for the valuable understanding as the molecular events on signal suppressing strategy basis, and provide about the important information to each NSCLC patient's treatment with gefitinib through detecting one group of gene with high predictive value.

Reference

1.Fossella; F.V.; Deng; Randomized phase III trial of docetaxelversus vinorelbine or if osfamide in patients with advanced non-small-cell lung cancer previously treated with platinum-containingchemotherapy regimens.The TAX320 Non-Small Cell Lung CancerStudy Group.J Clin Oncol, 2000.18 (12): p.2354-62.

2.Non-small?Cell?Lung?Cancer?Collaborative?Group.Chemotherapy?in?non-small?cell?lung?cancer：a?meta-analysis?usingupdated?data?on?individual?patients?from?52?randomised?clinical?trials.Bmj，1995.311(7010)：p.899-909.

3.Schiller, J.H., etc., Comparison offour chemotherapy regimensfor advanced non-small-cell lung cancer.N Engl J Med, 2002.346 (2): p.92-8.

4.Kelly; K.; Deng; Rartdomized phase III trial of paclitaxel pluscarboplatin versusvinorelbine plus cisplatin in the treatment ofpatientswith advanced non--small-cell lung cancer:a Southwest Oncology Group trial J Clin Oncol, 2001.19 (13): p.3210-8.

5.Baselga，J.，Why?the?epidermal?gromth?factor?receptor?Sherationale?for?cancer?therapy.Oncologist，2002.7?Suppl?4：p.2-8.

6.Traxler，P.，Tyrosine?kinases?as?targets?in?cancer?therapy-successes?and?failures.Expert?Opin?Ther?Targets，2003.7(2)：p.215-34.

7.Wakeling; A.E.; Deng, ZD1839 (Iressa): an orally activeinhibitor ofepidermal growth factor siguali7lg with potentialfor cancertherapy.Cancer Res, 2002.62 (20): p.5749-54.

8.Herbst，R.?S.，Dose-comparative?monotherapy?trials?of?ZD1839in?previously?treated?non-small?cell?lung?cancer?patients.Semin?Oncol，2003.30(1?Suppl1)：p.30-8.

9.Fukuoka; M.; Deng, Finalresults from a phase#trial of ZD1839 (' Iressa ') for patients wirh advanced non-small cell lung cancer (IDEAL1J.Pro Am Soc Clin.Oncol 2002.21; 298a (A1188).

10.Kris, MG., etc., A phase IItrial of ZD1839 (' Iressa ' J inadvanced non-small cell

11.Inoue, A., etc., Severe acute intersitial pneumonia and ZD1939.Lancet，2003.361(9352)：p.137-9.

12.Bohm, M., etc., Microbeam MOMeNT.hon-contact lasermicrodissection of membrane-mounted native tissue.Am J Pathol, 1997.151 (1): p.63-7.

13.Okabe; H.; Deng; Genome-wide analysis of gene expression inhuman hepatocellular carcinomas using cDNA microarray.identification of genes involved in viral carcinogenesis and tumorprogression.Cancer Res, 2001.61 (5): p.2129-37.

14.Kitahara，O.，etal.，Alterationsofgeiie?expression?duringcolorectat?carcinogenesis?revealed?by?cDNA?microarrays?after?laser-capture?microdissection?of?tumor?tissues?and?normal?epithelia.CancerRes，2001.61(9)：p.3544-9.

15.Golub, T.R., etc., Molecular classificationof cancer:classdiscovery andclass prediction by gene expression monitoring.Science, 1999.286 (5439): p.531-7.

16.MacDonald; T.J.; Deng, Expression profiling ofmedulloblastoma.PDGFRA and the RAS/MAPK pathway astherapeutic targets for metastatic disease.Nat Genet, 2001.29 (2): p.143-52.

17.Kaneta.Y.; Deng; Predictionof sensitivity to STI571 amongchronic myeloid leukemia patients by genome-wide cDNA microarrayanalysis.Jpn J Cancer Res 2002.93, p.849-856.

18.Pavelic, K., etc., Evidence for a role of EGF receptor in theprogression ofhuman lung carcinoma.Anticancer Res, 1993.13 (4): p.1133-7.

19.Kikuchi; T.; Deng; Expression profiles of non-small cell lungcancers on cDNA microarrays:Identifi8cation of genes for predictionof lymph-node metastasis and sensitivity to anti-cancer drugs.Oncogene, 2003.22 (14): p.2192-205.

20.Heighway, J., etc., Expressionprof ling of primary non-smallcell lung cancer for target identification.Oncogene, 2002.21 (50): p.7749-63.

21.Beer, D.G., etc., Gene-expression profiles predict survival ofpatients with lung adenocarcinoma.Nat Med, 2002.8 (8): p.816-24.

22.Miura; K.; Deng; Laser capture microdissection andmicroarray expression analysisof lung adenocarcinoma reveals tobaccosmoking-and prognosis-related molecular profiles.Cancer Res, 2002.62 (11): p.3244-50.

23.Moasser; M.M.; Deng; The tyrosine kinase inhibitor ZD1839 (" Iressa) inhibits HER2-driven signaling and suppresses the growthofHER2-overexpressing tumor cells.Cancer Res, 2001.61 (19): p.7184-8.

24.Rusch; V.; Deng; Overexpression of the epidermal growthfactorreceptor andits ligand transforming growth factor alpha is frequent inresectable non-small cell lung cancer but does not predict tumorprogression.Clin Cancer Res, 1997.3 (4): p.515-22.

25.Fontanini; G.; Deng; Evaluation of epidermal growth factor-related growthfactors and receptors and of neoangiogenesis incompletely resected stage I-IIIA noiz-small-celllungcancer:amphiregulin and microvessel count are independentprognostic indicators of survival.Clin Cancer Res, 1998.4 (1): p.241-9.

26.Brundage，M.D.，D.Davies，and?W.J.Mackillop，Prognosticfactors?in?non-small?cell?lung?cancer：a?decade?of?progress.Chest，2002.122(3)：p.1037-57.

27.Hurbin; A.; Deng; Inhibition of apoptosis by amphiregulin via aninsulin-like growth factor-1 receptor-dependent pathway in non-smallcell lung cancer cell lines.J Biol Chem, 2002.277 (51): 28.Yarden p.49127-33., Y.and M.X.Sliwkowski; Untangling the ErbBsignalling network. Nat Rev Mol Cell Biol, 2001.2 (2): p.127-37.

29.Prenzel, N., etc., EGF receptortransactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.Nature 1999.402 (6764): 884-8

30.Nelson, Chau, etc., Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-l.Molec Cell 2000.6:p.31-41.

31.Tipnis; SR.; Deng; Overexpression of the regulatory subunit ofr-glutamylcysteine synthetase in Hela cells increases r-glutamylcysteine synthetase activity and confers drug resistance.Biochem J 1999.337, p.559-566.

Table 1: the characteristic of baseline patient and the summary sheet of replying

Table 2: the quantity of the case that is suitable for analyzing and their best totally replying

(1) the study case is used to develop GRS, is used to verify and detect case

(2) combination at random (blindedset) (4PD and 1SD) that from 5 cases of newly including, obtains another sample afterwards also joins these 11 and detects in the cases.

Table 4: be used for 51 candidate genes (*) that the respondent (PR) of ZD1939 and non-responder (PD) are distinguished

The dependency of table 5A:cDNA microarray data and sxemiquantitative RT-PCR

The dependency of all 12 genes all is positive, and wherein 7 in 12 genes are significantly positively related.

Table 5B: the result of immunohistochemical staining

Claims (18)

1. an isolating marker gene group comprises being accredited as the gene that between the patient, has differential expression, and said patient is to a kind of respondent of erbB receptor tyrosine kinase inhibitors and non-responder; Said genome comprises all listed in the table 4 of the present invention preceding 12 genes simultaneously, and said 12 genes are: gene FLJ22662, its false albuminoid FLJ22662 that encodes; AREG, its amphiregulin of encoding; CORO1C, its coding coronin by name, ABP matter, the albumen of 1C; AVEN, its Codocyte apoptosis, aspartic acid specific cysteine proteinase inhibitors; DUSP3, its coding dual specificity phosphatase enzyme 3; Gene DJ473B4, its false albuminoid dJ473B4 that encodes; PHLDA2, its coding thrombocyte white corpuscle C kinase substrate homology spline structure territory A member of family 2; RBM7, its coding RNA binding motif albumen 7; GenBank number is the EST of BX092512; OSMR, its coding oncostatin m receptor; GCLC, its L-glutamic acid-halfcystine ligase enzyme catalytic subunit of encoding; COL4A3BP, its coding collagen by name, the IV type, α 3 protein-bonded albumen, or respectively be the specific oligonucleotide of the said gene of unique identification.

2. according to the genome of claim 1, wherein said gene is included in the sequence of listing among the table 4a.

3. according to the genome of claim 1, wherein said genome comprises the gene specific oligonucleotide, and said oligonucleotide comprises 5 to 50 oligonucleotide of listed sequence in the table 4.

4. according to each genome among the claim 1-3, wherein said suppressor factor is selected from ZD1939, OSI-774, PKI-166, EKB-569, GW2016 and CI-1033.

5. according to the genome of claim 4, wherein said suppressor factor is a ZD1939.

6. according to the genome of claim 1 or 2, wherein said suppressor factor is a kind of anti-erbB antibody.

7. according to the genome of claim 6, wherein said antibody is Herceptin or Cetuximab.

Among the claim 1-7 all marker gene in the genome of each definition in the purposes of preparation in the diagnostic kit; Wherein said diagnostic kit is used for predicting through the differential expression of the marker gene in the genome that relatively is selected from each definition of claim 1-7 the responsiveness of the treatment of among cancer patients or the patient crowd use erbB receptor tyrosine kinase inhibitors being carried out, and perhaps is used for the selection meeting and the erbB receptor tyrosine kinase inhibitors is produced patient or the patient crowd who replys.

9. according to Claim 8 purposes, wherein patient's responsiveness is that generation by the drug responses scoring embodies.

10. according to Claim 8 or 9 purposes, wherein saidly relatively carry out through microarray assay.

11. according to Claim 8 or 9 purposes, wherein saidly relatively carry out through immunohistochemistry.

12. according to the purposes of claim 11, said purposes comprises the differential expression that detects amphiregulin.

13. according to Claim 8 or 9 purposes, wherein said suppressor factor such as claim 4 to 7 in each definition.

14. diagnostic kit; It is used for the differential expression through the marker gene in the genome that relatively is selected from each definition of claim 1-7; The responsiveness of the treatment of among prediction cancer patients or the patient crowd use erbB receptor tyrosine kinase inhibitors being carried out perhaps is used for selection meeting patient that generation is replied to the erbB receptor tyrosine kinase inhibitors or patient crowd's method; Wherein said diagnostic kit comprises a kind of marker gene group that is positioned on the suitable supporting dielectric, and this combination is selected from as in the claim 1 to 3 each defined group.

15. according to the test kit of claim 14, it comprises a kind of microarray.

16. by claim 1 definition and list in isolated gene sequence in the group that preceding 12 genes of table 4 of the present invention constitute in the purposes of measuring the expression level of said gene the tissue samples that obtains from NSCLC patient, described gene comprises the specific oligonucleotide of the said gene of unique identification.

17. diagnostic kit; The instrument that comprises the expression level of isolated gene sequence the tissue samples that obtains from NSCLC patient that is used for confirming the group that preceding 12 genes of being selected from claim 1 definition and listing in table 4 of the present invention constitute; Described gene comprises the specific oligonucleotide of the said gene of unique identification, and this test kit comprises a kind of support material, superincumbent each gene of binding that comprises each defined isolating marker gene group in the claim 1 to 3.

18. each defined genome carries out clinical trial with the purposes in the diagnostic kit of the effect of the restraining effect of measuring the erbB receptor tyrosine kinase or suppressor factor or validity in preparation in the claim 1 to 3, is included in the level relatively of measuring the genomic expression that defines in each like claim 1 to 3 among patient or the patient crowd.

Images