CN106148508A

CN106148508A - Method and kit for colorectal cancer prognosis

Info

Publication number: CN106148508A
Application number: CN201610262437.1A
Authority: CN
Inventors: 叶迅; 吴非; 徐清华; 刘芳; 孟夏; B·穆然
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2010-06-08
Filing date: 2010-06-08
Publication date: 2016-11-23
Anticipated expiration: 2030-06-08
Also published as: CN106148508B

Abstract

nullThe present invention relates to the method for colorectal cancer prognosis and kit，Especially method，It comprises: a) obtains peripheral blood sample and extracts total serum IgE from described blood sample，B) described total serum IgE and at least one reagent special at least one NK cytogene and not more than 25 kinds are made to contact the reagent that 25 kinds of NK cytogenes are special，C) determine the expression of described at least one NK cytogene and most 25 kinds of NK cytogenes to obtain the express spectra of patient，The express spectra to described patient for the NK cellular gene expression of the patient of the NK cellular gene expression d) using the patient of clinical classification prognosis preferably before and the prognosis being categorized as difference before is analyzed，If wherein the express spectra of described patient and the express spectra of the patient from the prognosis that clinical classification before is difference cluster，Then determine that described patient has poor prognosis，If the express spectra cluster of the patient with the express spectra of described patient and from the prognosis preferably of clinical classification before，Then determine the prognosis that described patient has had.

Description

Method and kit for colorectal cancer prognosis

The application is that filing date June 8, Application No. in 2010 are the 201080067290.1st, invention entitled " to be used for tying The divisional application of the application for a patent for invention of the method for intestines carcinoma of the rectum prognosis and kit ".

Invention field

The present invention relates to the prognosis of colorectal cancer, be particularly used for method and the kit of this cancer prognosis.

Background technology

Colorectal cancer (CRC), also referred to as colon cancer or colorectal cancer, be the 5th modal cancer forms in the U.S., It is the 4th common cancer in China and be the 3rd main cause causing cancer related mortality in Europe.The early detection of CRC is still It is so the significant challenge of publilc health.It is true that CRC is typically recoverable, when particularly the stage is diagnosed in early days.Not Some screening strategies are taked with country.Conventional CRC examination test include fecal occult blood test (FOBT), sigmoidoscopy, Colonoscopy, double-contrast barium examination or digital rectal examination.They all have the advantage that and limitation, but compliance is still below expected, main If the discomfort due to logistics (logistics) or patient.

Over several years, finding the blood marker for detection CRC in early days becomes focus, especially because its convenience.With When, very small amount of research supports the feasibility of the test based on blood, and its gene biological label showing in blood can Distinguish CRC patient and compare.These researchs, based on flow cytometry, which is counting and check the technology of microscopic particles such as cell (by they being suspended in liquid stream and being passed to electronic detecting device).

It has been found by the present inventors that difference expression gene is mainly related to activated immune cell and transport.Especially, they demonstrate,prove Natural killer cell tomorrow (NK cell) represents biomarker important in peripheral blood sample.They do not use the streaming of classics Cell technology, but in whole blood, determine difference expression gene.Determine gene expression dose by analyzing transcript in whole blood It is uncommon, because those skilled in the art generally recognize, when in the RNA mixture (total serum IgE) being diluted in complexity, do not having It is difficult for obtaining customizing messages in the case of having specific purification step.This method have the advantage that and it also avoids purifying RNA Step.

Therefore, the present invention relates to the method for determining colorectal cancer prognosis in the peripheral blood sample from patient, Described method includes:

A) obtain described peripheral blood sample and extract total serum IgE from described blood sample,

B) make described total serum IgE and at least one reagent special at least one NK cytogene and not more than 25 kinds to 25 Plant the special reagent contact of NK cytogene,

C) determine that the expression of described at least one NK cytogene and at most 25 kinds of NK cytogenes is described to obtain The express spectra of patient,

D) use the NK cellular gene expression of the patient of clinical classification prognosis preferably before and be categorized as the pre-of difference before After express spectra to described patient of the NK cellular gene expression of patient be analyzed, wherein

If the express spectra of-described patient clusters with the express spectra of the patient from the prognosis that clinical classification before is difference (cluster), it is determined that described patient has poor prognosis, and

If the express spectra of-described patient and the express spectra cluster of the patient from the prognosis preferably of clinical classification before, Then determine the prognosis that described patient has had.

Specifically, in above-mentioned steps b), total serum IgE and at least one reagent special at least one NK cytogene are made The not more than 25 kinds reagent contacts special to 25 kinds of NK cytogenes, described NK cytogene includes SEQ ID NO:1-12 institute The nucleotide sequence showing, wherein said at least one reagent is to special selected from following at least one NK cytogene:

(i) KLRB1 gene, it comprises the full length sequence shown in such as SEQ ID NO:1,

(ii) KLRC2 gene, it comprises such as SEQ ID NO:2, the full length sequence shown in 3 or 4,

(iii) KLRC3 gene, it comprises such as SEQ ID NO:5, the full length sequence shown in 6 or 7,

(iv) KLRD1 gene, it comprises such as SEQ ID NO:8, the 9th, the 10th, the full length sequence shown in 11 or 12, and

(v) KLRK1 gene, it comprises the full length sequence shown in such as SEQ ID NO:13, and

Determine the expression of described at least one NK cytogene to obtain the express spectra of patient in step c).

As described in detail in experimental data, the expression of at least one said gene is enough to prediction CRC risk Information.

In one embodiment, in step b), described total serum IgE is made and at least 5 kinds of NK cytogenes and not more than 25 Planting the special reagent contact of the combination of NK cytogene, wherein said reagent at least includes to following NK cytogene special Reagent:

(v) KLRK1, it comprises the full length sequence shown in such as SEQ ID NO:13,

Determine the expression of at least described 4 kinds of NK cytogenes to obtain the express spectra of patient in step c).

In addition, in step b), described total serum IgE and at least one reagent at least one target cell gene specific can be made Not more than 5 kinds to the contact of the reagent of 5 kinds of target cell gene specifics, described target cell gene includes SEQ ID NO:12-24 institute The nucleotide sequence showing, wherein said at least one reagent at least one selected from following target cell gene specific:

(i) GZMB gene, it comprises such as SEQ ID NO:14, the 15th, the full length sequence shown in 16 or 17,

(ii) CD247 gene, it comprises such as SEQ ID NO:18, the full length sequence shown in 19 or 20,

(iii) RRAS2 gene, it comprises the full length sequence shown in such as SEQ ID NO:21 or 22, and

(iv) SH2D1B gene, it comprises the full length sequence shown in such as SEQ ID NO:23 or 24, and

(v) LCK gene, it comprises such as SEQ ID NO:25, the 26th, the 27th, the 28th, the full length sequence shown in 29 or 30, and

Determine the expression of described at least one target cell gene to obtain the express spectra of patient in step c)；And In one embodiment, the reagent making described total serum IgE special with the combination to 5 kinds of target cell genes contacts, wherein said reagent To following target cell gene specific:

Described in determining in step c), the expression of at least 5 kinds of cytogenes is to obtain the express spectra of patient.

In another embodiment, in step b), make described total serum IgE with at least one at least one target further The special reagent of cytogene and the not more than 100 kinds reagent contacts to 100 kinds of target cell gene specifics, described target cell gene Including the nucleotide sequence shown in SEQ ID NO:25-59, wherein said at least one reagent is to selected from following at least one target Cytogene is special:

(i) MRPS6 gene, it comprises such as SEQ ID NO:31, the full length sequence shown in 32 or 33,

(ii) SPRY4 gene, it comprises the full length sequence shown in such as SEQ ID NO:34,

(iii) NEAT1 gene, it comprises the full length sequence shown in such as SEQ ID NO:35,

(iv) CYBB gene, it comprises the full length sequence shown in such as SEQ ID NO:36,

(v) DUSP2 gene, it comprises the full length sequence shown in such as SEQ ID NO:37,

(vi) PDEAD gene, it comprises the full length sequence shown in such as SEQ ID NO:38 or 39,

(vii) SH2D2A gene, it comprises such as SEQ ID NO:40, the full length sequence shown in 41 or 42,

(viii) INSR gene, it comprises the full length sequence shown in such as SEQ ID NO:43 or 44,

(ix) ITGAM gene, it comprises the full length sequence shown in such as SEQ ID NO:45,

(x) VCAN gene, it comprises such as SEQ ID NO:46, the 47th, the full length sequence shown in 48 or 49,

(xi) CD163 gene, it comprises the full length sequence shown in such as SEQ ID NO:50 or 51,

(xii) P2RY10 gene, it comprises the full length sequence shown in such as SEQ ID NO:52 or 53,

(xii) CD226 gene, it comprises the full length sequence shown in such as SEQ ID NO:54,

(xiii) MRPL10 gene, it comprises the full length sequence shown in such as SEQ ID NO:55 or 56,

(xiv) ITPRIPL2 gene, it comprises the full length sequence shown in such as SEQ ID NO:57,

(xv) CD2 gene, it comprises the full length sequence shown in such as SEQ ID NO:58, and

(xvi) NUDT16 gene, it comprises the full length sequence shown in such as SEQ ID NO:59, and

Determine the expression of described at least one target cell gene to obtain the express spectra of patient in step c).

Especially, in step b), described total serum IgE is made and at least 17 kinds of target cell genes and not more than 100 kinds target cells The combination special reagent contact of gene, wherein said reagent at least includes the reagent to following target cell gene specific:

Described in determining in step c), the expression of at least 17 kinds of target cell genes is to obtain the express spectra of patient.

More properly, in method as described above, at least one specific reagent described in step b) includes at least one Plant hybridization probe, specifically include at least one hybridization probe and at least one primer, and more specifically include at least one miscellaneous Hand over probe and two kinds of primers.

Total serum IgE includes that transfer RNA (tRNA), mRNA (mRNA) (are for example transcribed from target gene and from any other gene The mRNA transcribing) and rRNA.

Being intended to explanation, the extraction of total serum IgE can be by cracking the cell being present in blood sample to discharge Patient cells The step of the nucleic acid being comprised realizes.Be intended to citing, can use as patent application WO00/05338 (its with regard to mictomagnetism and Mechanical lysis), the cracking side described in WO 99/53304 (it is with regard to electric cracking), WO99/15321 (it is with regard to mechanical lysis) Method.Those skilled in the art can use cleavage method known to other, such as heat shock or osmotic shock or use chaotropic agent example Chemical cracking (U.S. Patent number 5,234,809) such as guanidinesalt.It is also possible to provide extra step, for from cleavage step Other cell component separation nucleic acid of release.This generally makes condensed nucleic acid be possibly realized.Be intended to citing, can use optionally by Absorption or the coated magnetic-particle of covalent effect oligonucleotides (in this respect, are shown in U.S. Patent number 4,672,040 and the U.S. The patent No. 5,750,338), and the nucleic acid being incorporated into these magnetic-particles thus can be purified by washing step.If need with The described nucleic acid of rear amplification, this nucleic acid purification step is particularly advantageous.At patent application WO-A-97/45202 and WO-A-99/ The particularly advantageous embodiment of these magnetic-particles is described in 35500.

Term " specific reagent " refers to such reagent, when making it contact with biologic material as defined above, Can combine with to the material that described target gene is special.It is intended to explanation, when specific reagent and biologic material are core sources, Making specific reagent contact with biologic material can make specific reagent hybridize with the material special to described target gene.Term " hybridization " refers to such process, and i.e. during described process under suitable condition, two nucleotide fragments are with stable and special Different Hydrogenbond thus form double-stranded complex.These hydrogen bonds (or are urinated phonetic at complementary adenine (A) and thymidine (T) Pyridine (U)) between base (this is referred to as A-T key), or between complementary guanine (G) and cytimidine (C) base, (this is referred to as G-C key) formed.The hybridization of two nucleotide fragments can be i.e. miscellaneous at this for completely (referred to as complementary nucleotide acid fragment or sequence) Double-stranded complex obtained in friendship process only comprises A-T key and C-G key.This hybridization can be (the referred to as fully complementary core of part Acid fragments or sequence), the double-stranded complex i.e. being obtained both comprises A-T key and the C-G key allowing to form double-strand, also wraps Containing the base not being combined with complementary base.Article two, used condition of work is depended in the hybridization between nucleotide fragments, special It is not stringency.Stringency is specifically defined as the function of the base composition of two nucleotide fragments and also by two nucleotides pieces Extent of mismatch definition between Duan.Stringency additionally depends on response parameter, the ion concentration being for example present in hybridization solution and Type, the character of denaturant and concentration and/or hybridization temperature.All these data are known, and can be by art technology Personnel determine suitable condition.In general, the length of the nucleotide fragments hybridizing as required, hybridization temperature is of about 20 DEG C To 70 DEG C, particularly 35 DEG C to 65 DEG C, in the salting liquid of concentration about 0.5-1M.Sequence, or nucleotide fragments, or few nucleosides Acid, or polynucleotides, be a series of nucleotides motifs being fitted together by phosphoric acid ester bond, be characterized as the natural nucleus containing information Acid sequence, it can hybridize with nucleotide fragments, and described series may be containing the monomer with different structure, and may be from natural Nucleic acid molecules and/or obtain by Genetic Recombination and/or by chemical synthesis.Motif is the derivative of monomer, and described monomer can For the natural nucleotide of nucleic acid, its composed component is sugar, phosphate group and nitrogenous base；In DNA, described sugar is deoxidation-2- Ribose, in RNA, described sugar is ribose；According to whether relate to DNA and RNA, nitrogenous base is phonetic selected from adenine, guanine, urine Pyridine, cytimidine and thymidine；Alternatively, monomer is the adorned nucleotides of at least one in three kinds of composed components；It is intended to Citing, modification can occur base level (have the base such as inosine of modification, methyl-5-deoxycytidine, BrdU, two The alkali of methylamino-5-FU, diaminourea-2,6-purine, bromo-5-FU or any other modification that can hybridize Base), or occur sugared level (for example, at least one deoxyribose replace with polyamide (P.E.Nielsen et al., Science, 254,1497-1500 (1991) [3])), or additionally occur (for example to replace with ester in phosphate group level, specifically select From bisphosphate, alkyl phosphate and acyl phosphate and thiophosphate).

According to specific embodiment of the present invention, described specific reagent includes at least one hybridization probe, or at least one Kind of hybridization probe and at least one primer special to target gene, or at least one hybridization probe and two kinds special to target gene Primer.

For the purposes of the present invention, term " amplimer " refers to nucleotide fragments, and it comprises 5-100 nucleotides, preferably 15-30 nucleotides, it makes the reaction of enzymatic polymerization such as enzymatic amplification initial.Term " enzymatic amplification reaction " refers to by extremely The effect of few a kind of enzyme produces the process of multicopy nucleotide fragments.Such amplified reaction is as well known to those skilled in the art , and it should be particularly mentioned following technology: PCR (PCR) (as at U.S. Patent number 4,683,195, U.S. Patent number 4,683,202 and U.S. Patent number 4, described in 800,159), LCR (ligase chain reaction) is (for example at patent application EP Disclosed in 0201 184), RCR (reparation chain reaction) (described in patent application WO 90/01069), (Self-sustained Sequence is multiple for 3SR System) (patent application WO 90/06995), NASBA (based on the amplification of nucleotide sequence) (patent application WO 91/02818), TMA (transcript mediated amplification) (U.S. Patent number 5,399,491) and RT-PCR.

When enzymatic amplification is PCR, specific reagent includes the special amplimer of at least two target gene, and it allows to expand Increase the special material of target gene.The material that target gene is special is then preferably comprised by reverse transcription from the mRNA of target gene The complementary DNA (referred to as target gene special cDNA) that obtains or by transcribing the complementary RNA (title that the special cDNA of target gene obtains Make the special cRNA of target gene).The PCR completing after enzymatic amplification is reverse transcription reaction, referred to as RT-PCR.

Term " hybridization probe " refers to nucleotide fragments, and it comprises at least 5 nucleotides, such as 5-100 nucleotides, spy Not being 10-75 nucleotides, such as 15-35 nucleotides and 60-70 nucleotides, it is special that it has hybridization under prescribed conditions Property thus form hybridization complex with the material special to target gene.In the present invention, bag can be to the material that target gene is special It the nucleotide sequence that is contained in the mRNA being derived from target gene (referred to as target gene special mRNA), is included in and passes through reverse transcription Nucleotide sequence (referred to as target gene special cDNA) or additionally logical for being included in the complementary DNA that described mRNA obtains Cross the nucleotide sequence (referred to as target gene special cRNA) transcribed in the complementary RNA that described cDNA described above obtains. Hybridization probe can comprise the label detecting for it.Term " detection " refers to directly detect such as method of counting, or indirectly examines Surveying, it is by using the detection method of label.Exist many for detect nucleic acid detection method (see, for example, Kricka etc. People, Clinical Chemistry, 1999, no 45 (4), 453-458 page, or Keller G.H. et al., DNA Probes, 2nd Ed., Stockton Press, the 1993, the 5th and the 6th part, 173-249 page).Term " label " refers to generation can The tracer of the signal being detected.The nonrestrictive list of these tracers include enzyme (its pass through such as colorimetric, fluorescence or The signal that luminous generation can detect that), such as horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose-6- Phosphate dehydrogenase；Chromophore, such as fluorescer, luminous agent or dye composition；Electron dense group, it can pass through electron microscopic Microscopy is surveyed and or can be detected by Amperometric or voltammetry or by impedance measurement by their characteristic electron such as electrical conductivity；Can lead to Cross the change of optical means such as diffraction, surface plasma body resonant vibration or contact angle, or pass through physics method such as atomic force spectrum, The group of the detection such as tunnel-effect；Geigers, for example³²P、³⁵S or¹²⁵I。

For the purposes of the present invention, hybridization probe can be " detection " probe.In this case, by label, " detection " is visited Pin is marked.Detection probe can be specially such as Tyagi and Kramer (Nature biotech, 1996,14:303-308) institute " molecular beacon " detection probe describing.These " molecular beacons " become fluorescer during hybridizing.They have stem-ring-like knot Structure simultaneously comprises fluorogen and " quencher " group.Special ring sequence be complementary to the combination of target nucleic acid sequence cause stem to launch and Suitable wavelength excitation process sends fluorescence signal.Detection probe can be specially according to NanoString^TMThe comprising of technology " reporter probe " of " bar code (barecode) of coloud coding ".

In order to detect hybridization reaction, can use and directly (mix target sequence particular by by label) or indirectly (have Body is to use detection probe as defined above) target sequence that marks.Especially, may be marked before hybridization step And/or the step of cutting target sequence, in enzymatic amplification course of reaction, for example use the deoxyribonucleoside triphosphate of mark.Institute State cutting specifically to be completed by the effect of imidazoles or manganese chloride.Also can be according to for example described in document WO 91/19812 Sandwich hybridization technology by with detection probe hybridization and after amplification step labels targets sequence.Application FR 2780059 retouches State the method for optimizing of another concrete labeling nucleic acid.

According to the preferred embodiment of the invention, detection probe comprises fluorogen and quencher.According to the present invention even more Preferred embodiment, it is glimmering that hybridization probe comprises FAM (6-Fluoresceincarboxylic acid) or ROX (6-carboxy-X-rhodamine) at its 5' end Light blob simultaneously comprises quencher (Dabsyl) at its 3' end.

Hybridization probe can be also " capture " probe.In this case, by any suitable method, i.e. directly or Ground connection, for example, pass through covalency or absorption, and " capture " probe is fixed or may be fixed on solid substrate.As solid-based Material, can use synthetic material or natural material (being optionally chemical modification), and specially polysaccharide is for example based on the material of cellulose Expecting such as paper, cellulose derivative such as cellulose acetate and celluloid or glucan, polymer, copolymer is (particularly Styrene-based type monomer), natural fiber such as cotton, and synthetic fibers such as nylon；Inorganic material such as silica, Quartz, glass or pottery；Latex；Magnetic-particle；Metal derivative, gel etc..Solid substrate can for microtiter plate, film (as Described in application WO-A-94/12670) or the form of particle.It is also possible to some different capture probes are fixed on base material On, every kind of capture probe is special to a kind of target gene.Specifically, biochip can be used as base material, it can be fixed a large amount of Probe.Term " biochip " refers to the solid substrate of small size, and substantial amounts of capture probe can be connected to it in predetermined position On.The concept of biochip (or DNA chip) can trace back to nineteen ninety for the initial stage.It incorporates micro-electricity based on multidisciplinary technology Son, nucleic acid chemistry, graphical analysis and information technology.Operation principle is based on the basis of molecular biology: hybridization phenomenon, i.e. two DNA and/or the pairing by base complementrity for the RNA sequence.Biochip method is based on the capture probe being connected on solid substrate Use, the target nucleic acid fragments sample effect of mark fluorescent dyestuff is in described capture probe directly or indirectly.Capture probe It is positioned specifically on base material or chip, and each hybridization provides relevant with a target nucleic acid fragments specifying information.Institute The information obtaining is cumulative, makes the expression of one or more target gene for example quantitative be possibly realized.Then in order to analyze The expression of target gene, can prepare the base material comprising a large amount of probe, and described probe is corresponding to being transcribed into target gene whole of mRNA Or part.For the purposes of the present invention, term " low density substrate " refers to comprise the base material less than 50 kinds of probes.For the present invention Purpose, term " Midst density base material " refers to comprise the base material from 50 kinds of probes to 10000 kinds of probes.Mesh for the present invention , term " high density substrate " refers to comprise the base material more than 10000 kinds of probes.

CDNA or cRNA of the target gene of analysis and for example special capture probe is needed to hybridize by being specific to subsequently.Hybridization Afterwards, base material or chip are washed, and by marking with high-affinity part display that for example fluorescent dye type label is combined CDNA or cRNA/ captures antibody complex.Read fluorescence with such as scanner and carry out fluorescence analysis by information technology.It is intended to Explanation, it may be mentioned that developed by Affymetrix company, for DNA chip (" the Accessing Genetic of molecular diagnosis Information with High-Density DNA arrays ", M.Chee et al., Science, 1996,274,610- 614." Light-generated oligonucleotide arrays for rapid DNA sequence analysis ", A.Caviani Pease et al., Proc.Natl.Acad.Sci.USA, 1994,91,5022-5026).In the art, capture Probe is typically small, about 25 nucleotides.At publication G.Ramsay, Nature Biotechnology, 1998, No.16, 40-44 page；F.Ginot, Human Mutation, 1997, No.10,1-10 page；J.Cheng et al., Molecular Diagnosis, 1996, No.1 (3), 183-200 page；T.Livache et al., Nucleic Acids Research, 1994, No.22 (15), 2915-2921 page；J.Cheng et al., Nature Biotechnology, 1998, No.16,541-546 page or In U.S. Patent number the 4,981,783rd, U.S. Patent number the 5,700,637th, U.S. Patent number the 5,445,934th, U.S. Patent number 5, 744,305 and U.S. Patent number 5,807,522 in give biochip other example.The principal character of solid substrate should be Keep the hybridization characteristics to target nucleic acid fragments for the capture probe, and produce the background noise minimum to detection method simultaneously.Probe Fixing on base material can divide into three kinds of main Types.

Firstly, there are the first technology, it is to deposit pre-synthesis probe.Connecting through of probe has directly been shifted Becoming, it passes through micropipettor (micropipette) or microdot (microdot) or passes through ink discharge device.This technology allows even Connect magnitude range from several bases (5-10) to relatively large 60 bases (impact system) to the probe of hundreds of bases (microdeposit method).

Impact system is the change to the method that ink-jet printer uses.It pushes away based on the speed can reach 4000 drops/sec Enter very little liquid ball (volume < 1nl).Impact system is not related to discharge between system and the liquid deposition surface thereon of liquid Any contact.

Microdeposit method is characterised by that the long probe by tens of to hundreds of bases is connected to the surface of glass slide.These probes Generally extract from database and for through amplification and purified Product Form.The chip that this technology makes production be referred to as microarray becomes May, described chip carries about 10,000 DNA points on the surface area (referred to as identifying region) be slightly less than 4 square centimeters.So And, should not forget the purposes of nylon membrane (referred to as " VLA arranges (macroarray) "), it is with the density of maximum 25 points/square centimeter Carry the product after the amplification (typically passing through PCR) of diameter 0.5mm to 1mm.Many laboratories all use this very flexibly Technology.In the present invention, biochip considers to include latter technique.But, such as patent application WO-A-00/71750 and FR In the case of 00/14896, the sample of certain volume can be deposited on the bottom in each hole of microtiter plate, or according to another Patent application FR 00/14691, can be by droplet deposition separated from one another for some bottom same sterile petri dish.

The second is used for that probe is connected to base material or the technology of chip is referred to as fabricated in situ.This technology causes in chip list Face directly produces short probe.It (is specifically shown in, patent application WO 89/10977 and WO 90/ based on situ oli-gonucleotide synthesis 03382) and based on oligonucleotide synthesizer method.It is along glass surface mobile response room, sends out in described reative cell Raw nucleotides extension.

Finally, the third technology is referred to as optical lithography, which is the method for biochip of Affymetrix exploitation. It is also fabricated in situ.Optical lithography comes from microprocessor technology.By connecting photo-labile (can be by photoactivation) chemistry base Chip surface is modified by group.Once by illumination, these groups can react with the 3' end of oligonucleotides.By with limiting shape Lightshade cover protects this surface, it is possible to optionally illumination therefore one of four kinds of nucleotides of activation expectation connection or other nucleosides The region of acid.Different lightshade covers is used continuously to allow to alternately to protect/reaction cycle therefore putting down about tens of Side's micron (μm²) point on produce oligonucleotide probe.This resolution ratio allows at several square centimeters of (cm²) surface area on Produce up to hundreds thousand of points.Optical lithography has an advantage in that batch is parallel, and it allows to only take advantage of N number of circulation to produce N through 4 Aggressiveness chip.All these technology can be used for the present invention.According to the preferred embodiment of the invention, step b) as defined above At least one specific reagent include at least one hybridization probe, it is preferably fixed on base material.This base material is preferably as above State the low, high of definition or Midst density base material.

In order to improve the amount of target genetic stocks, can carry out before these hybridization steps on the base material comprising a large amount of probe Enzymatic amplification reactions steps as defined above.

The determination of step c) target gene expression can be completed by any scheme well known by persons skilled in the art.Always For, the expression of target gene can be analyzed from target gene at the mRNA (mRNA) that given time is transcribed by detection.

Present invention is preferably related to well known to a person skilled in the art that the detection of any scheme is derived from target gene by basis MRNA determines the expression of target gene.According to specific embodiment of the present invention, (every by the some different mRNA of detection Plant mRNA and be derived from a target gene), the expression of some target genes can be determined simultaneously.

When specific reagent includes at least one amplimer, the expression water of target gene may be determined in the following manner Flat: 1) after whole blood extracts total serum IgE (comprising transfer RNA (tRNA), rRNA (rRNA) and mRNA (mRNA)), enter Row reverse transcription step is to obtain the complementary DNA (or cDNA) of described mRNA.It is intended to explanation, can use and can obtain from RNA fragment The reverse transcriptase of complementary DNA fragment carries out this reverse transcription reaction.Specifically can use from AMV (avian myeloblastosis virus) or The reverse transcriptase of MMLV (Moroni murine leukemia virus).As the more specific cDNA only needing acquisition mRNA, only comprising chest Carry out this reverse transcription step in the presence of the nucleotide fragments (poly T) of gland pyrimidine bases, described poly T by with mRNA Poly A sequence complementary and hybridize thus form poly T-poly A compound, it is then anti-as carried out by reverse transcriptase The initiation site of responsive transcription.Then obtain and be derived from the complementary cDNA (the special cDNA of target gene) of the mRNA of target gene and The cDNA (cDNA of non-target gene specific) complementary with the mRNA of the gene being derived from outside target gene.2) expansion that target gene is special is made Increase the primer cDNA special with target gene and the cDNA of non-target gene specific to contact.The special amplimer of target gene and target gene Predefining of special cDNA hybridization and specifically amplification cDNA (it is derived from the mRNA from target gene) known length Region.The cDNA of non-target gene specific is not amplified, thus obtains the special cDNA of a large amount of target gene.Mesh for the present invention , can indistinguishably quote " the special cDNA of target gene " or " being derived from the cDNA of the mRNA from target gene ".Can be especially by The amplified reaction of PCR type or carry out this step by the method for any other amplification technique as defined above.To PCR, also may be used By use is some, different amplimers (every pair of primers is special to a target gene) can be expanded some different simultaneously CDNA (each cDNA is special to different target genes): be referred to as multiplex amplification.3) by detection and quantitatively in above-mentioned steps 2) The special cDNA of the target gene of middle acquisition determines the expression of target gene.Can be at the special cDNA of target gene according to its size electrophoresis Carry out this detection after migration.Gel and medium for migrating can comprise ethidium bromide, thus after given transit time section, When placing the gel on UV (ultraviolet) platform, directly detect the special cDNA of target gene by the optical signal sending.Target base Because the amount of special cDNA is bigger, optical signal is stronger.These electrophoretic techniques are known to those skilled in the art.Also may be used The quantification range being obtained by entering to walk to saturated amplified reaction is used to detect the cDNA of simultaneously quantifying target gene specific.In order to examine Consider in different steps (reverse transcription, PCR etc.) can it is observed that the difference of enzyme efficiency, can be by determining " looking after the house " base simultaneously Expression because of the target gene expressing normalization difference group patient of (its expression is similar in difference group patient).By obtaining target base Because expressing the ratio expressed with house-keeping gene, i.e. by cDNA that the amount obtaining the special cDNA of target gene is special with house-keeping gene The ratio of amount, any change between correction different experiments.Those skilled in the art can be with specific reference to following publication: Bustin S A, J Mol Endocrinol, 2002,29:23-39；Giulietti A Methods, 2001,25:386- 401。

When specific reagent comprises at least one hybridization probe, the expression of target gene may be determined in the following manner: 1) After whole blood extracts total serum IgE, complete reverse transcription step as described above, thus obtain and the mRNA complementation being derived from target gene CDNA (the special cDNA of target gene) and the cDNA complementary with the mRNA of the gene being derived from outside target gene be (non-target gene specific cDNA).2) make all of cDNA contact with base material, described base material is fixed with to needing the target gene analyzing its expression special Capture probe, thus carry out the hybridization reaction between the special cDNA of target gene and capture probe, rather than target gene is special CDNA does not hybridizes with capture probe.Hybridization reaction can complete on the solid substrate including all materials as indicated above.Root According to the preferred embodiment of the invention, hybridization probe is fixed on base material.Preferably, base material is low, Gao Huo as defined above Midst density base material.The step being made up of the enzymatic amplification of the special cDNA of target gene as defined above can be before hybridization reaction Carry out, thus obtain the special cDNA of a large amount of target gene and improve the special cDNA of target gene and the capture special to target gene spy The possibility of pin hybridization.It was likely to before hybridization reaction be marked and/or cut target gene described above special The step of cDNA, for example, use the deoxyribonucleoside triphosphate of mark for amplified reaction.Described cutting specifically can pass through miaow The effect of azoles and manganese chloride completes.Also can after amplification step the cDNA of labels targets gene specific, such as according at document WO- Sandwich hybridization technology described in A-91/19812 is by the probe hybridization with mark.In application WO the 99/65926th, WO 01/ 44507th, WO the 01/44506th, WO the 02/090584th, WO 02/090319 describes other preferably mark and/or cut core The concrete grammar of acid.3) step being made up of detection hybridization reaction is carried out subsequently.Described detection can be by making base material (to target gene The cDNA hybridization special with target gene on it of special capture probe) contact with " detection " probe of mark, detection is by label The signal sending is carried out.When the special cDNA of target gene marks with label in advance, the signal that directly detection label sends.

When at least one specific reagent in step b) comprises at least one hybridization probe, it is also possible to following manner is true The expression of targeting gene: 1) after whole blood extracts total serum IgE, complete reverse transcription step as described above, thus obtain biology The cDNA of the mRNA of material.Use T7 polymerase carries out the polymerization of the complementary RNA of cDNA subsequently, and described T7 polymerase is in promoter The lower function of control simultaneously allows to obtain complementary RNA from DNA profiling.Obtain the cDNA's of the special mRNA of target gene subsequently The cRNA of the cDNA of the mRNA of cRNA and non-target gene specific.2) all of cRNA is made to contact with base material, fixing on described base material Have a capture probe special to needing to analyze its target gene expressed, thus carry out the special cRNA of target gene and capture probe it Between hybridization reaction, rather than the special cRNA of target gene not with capture probe hybridization.When needing to analyze some target genes simultaneously During expression, can fix some different capture probes on base material, each probe is special to a target gene.It is likely to miscellaneous The step being marked and/or cutting the special cRNA of target gene described above before handing over reaction.3) carry out subsequently by examining The step surveying hybridization reaction composition.Detection can by make base material (to the special capture probe of target gene on it special with target gene Different cRNA hybridization) contact with " detection " probe being marked with label, and detect and completed by the signal that label sends.When target base When marking with label in advance because of special cRNA, directly detect the signal that label sends.There is a large amount of spy when using hybridization on it During the biochip class base material of pin, cRNA is used to be particularly advantageous.

Present invention additionally comprises kit, it is for prognosis colorectal cancer in the peripheral blood sample from patient, described Kit includes at least one reagent special at least one NK cytogene and not more than 25 kinds to 25 kinds of NK cytogenes Special reagent, described NK cytogene at least includes the nucleotide sequence shown in SEQ ID NO:1-13, wherein said at least one Kind of reagent is at least one special selected from following NK cytogene:

V () KLRK1 gene, it comprises the full length sequence shown in such as SEQ ID NO:13.

In one embodiment, kit includes the combination of the reagent special to following NK cytogene:

In such embodiments, specific reagent can target some NK cytogenes but not more than 25 NK genes Combination.

In addition, kit can include at least one target cell gene and not more than 5 kinds target cell gene specifics at least A kind of reagent, described at least one target cell gene is selected from:

V () LCK gene, it comprises such as SEQ ID NO:25, the 26th, the 27th, the 28th, the full length sequence shown in 29 or 30.

Specifically, it includes 5 kinds of reagent to following target cell gene specific:

In such embodiments, specific reagent can target for example above-described some target cell genes but few Combination in 5 kinds of target cell genes.

In another embodiment, kit for example defined above can include at least one at least one target cell The reagent of gene specific and the at most 100 kinds reagent to 100 kinds of target cell gene specifics, described at least one target cell gene choosing From:

(xvi) NUDT16 gene, it comprises the full length sequence shown in such as SEQ ID NO:59.

And specifically, it includes 17 kinds of reagent to following 17 kinds of target cell gene specifics:

In such embodiments, specific reagent can target for example above-described some target cell genes but few Combination in 100 kinds of target cell genes.

As explained above, the specific reagent of described at least one includes at least one hybridization probe, specifically include to Few a kind of hybridization probe and at least one primer, and more specifically include at least one hybridization probe and two kinds of primers.

Finally, the present invention relates at least one reagent special at least one NK cytogene and not more than 25 kinds to 25 Planting purposes in preparing composition for the special reagent of NK cytogene, described composition is for imitating at the biology from patient Prognosis colorectal cancer in product, described NK cytogene comprises the nucleotide sequence shown in SEQ ID NO:1-12, wherein said extremely Few a kind of reagent is special at least one NK cytogene comprising selected from the arbitrary shown nucleotide sequence of SEQ ID NO:1-12 Different；

Particularly prepared by the reagent that the combination of at least 5 kinds of NK cytogenes and not more than 25 kinds of NK cytogenes is special Purposes in composition, described composition is for prognosis colorectal cancer in the biological sample from patient, wherein said Reagent is to comprising at least 5 kinds of NK cytogenes being respectively selected from the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7 and 8-12 It is special；

Particularly to purposes in preparing composition of the special reagent of combination of 10 kinds of target cell genes, described composition For prognosis colorectal cancer in the biological sample from patient, wherein said reagent is to comprising to be respectively selected from SEQ ID The target cell gene of the nucleotide sequence described in NO:1,2-4,5-7,8-12, the 13rd, 14-17,18-20,21-22,23-24 and 25-30 It is special；With

More particularly to the special reagent of the combination of 10 kinds of target cell genes and not more than 100 kinds of target genes in preparation group Purposes in compound, described composition is used for prognosis colorectal cancer, wherein said examination in the biological sample from patient Agent is to comprising to be respectively selected from SEQ ID NO:1,2-4,5-7,8-12, the 13rd, 14-17,18-20,21-22,23-24,25-30,31- 33rd, the 34th, the 35th, the 36th, the 37th, shown in 38-39,4-42,43-44, the 45th, 46-49,50-51,52-53, the 54th, 55-56, the 57th, 58 and 59 The target cell gene of nucleotide sequence is special；

Wherein, the specific reagent of described at least one include at least one hybridization probe, at least one hybridization probe and At least one primer or at least one hybridization probe and two kinds of primers.

Summary of drawings

Fig. 1 is the NK cell in the blood sample of colonoscopy negative control (CNC) and colorectal cancer (CRC) patient Score, and CRC sample is according to the distribution map of carcinoma stage.Circle represents CNC；Square frame, positive triangle, inverted triangle and rhombus are respectively Represent I phase, II phase, III phase and IV phase CRC.

Embodiment

I) materials and methods

1. patient and sample collection

This research has obtained the approval of local clinical research Ethics Committee.Obtain the written informed consent of all participants Book.

To CRC group, at Fudan University of China tumour hospital (FUCH) colorectum between in July, 2006 and in March, 2008 Surgery is that 119 colorectal cancer patients have been recruited in this research continuously.The tumour recommended according to International Union Against Cancer (UICC)- Lymph node-transfer system is to neoplasm staging.Patient is not had to accept preoperation radiotherapy or chemotherapy.This research eliminates suffers from heredity knot The intestines carcinoma of the rectum or the patient of inflammatory bowel disease (Crohn's disease or ulcerative colitis).To each patient, at Sigmoidoscope After at least one week of inspection, operation consent, collect 2.5ml peripheral blood into PAXgene^TMBlood rna pipe (PreAnalytiX GmbH, Hombrechtikon, CH) in, and according to manufacturer specification process.To control group, the community hospital's registration from District of Shanghai 101 confirm not carry the positive participant of FOBT test of any polyp or colorectal cancer symptom by colonoscopy.? The last week of colonoscopy inspection collects peripheral blood sample in PAXgene pipe.All participants' that this research includes is detailed Thin feature is given in Table 1.

Table 1: patient characteristic

2.RNA extracts and Microarray Experiments

Follow manufacturer specification PAXgene^TMBlood rna system (PreAnalytix) extracts total serum IgE.Pass through light splitting The amount of the photo densitometry total serum IgE in 260 nanometers for the photometer, at BioAnalyzer Agilent 2100 (Agilent Technologies, Palo Alto, CA, U.S.A.) upper use RNA 6000NanoKit quality of evaluation. Only analyze the sample of the RNA integrality numerical value having 7 to 10.Then by 50 nanogram total serum IgE reverse transcriptions and according to manufacture criterion Scheme uses Ribo-SPIA^TMThe WT-of technology (NuGEN Technologies Inc., San Carlos, CA, U.S.A.) Ovation^TMThe amplification of RNA amplification system linear is strand cDNA, and uses QIAquick^TMPCR purification kit (QIAGEN GmbH, Hilden, Germany) purified product.Subsequently with RQ1 without RNase DNA enzymatic (Promega Corp., Fitchburg, WI, U.S.A.) fragmentation 2 milligrams amplification and cDNA after purification, and pass through end with biotinylated deoxynucleoside triphosphate Transferase (Roche Diagnostics Corp., Indianapoli, IN, U.S.A.) andDNA marker tries Agent (Affymetrix Inc., Santa Clara, CA, U.S.A) marks.At hybrid heater 640 (Agilent Technologies) 50 DEG C 18 minutes in, the cDNA of mark is hybridized to HG U133Plus 2.0Array by 60 turns per minute (Affymetrix) on.HG U133Plus 2.0Array comprises 54,675 probe collection, which represent about 39, and 000 The people's gene characterizing well.After hybridization, use according to Affymetrix scheme EukGE-WS2v4Fluidics Station 450 (Affymetrix) washing microarray simultaneously dyes.WithScanner 3000(Affymetrix) Scanning microarray.

3. microarray data analysis

Affymetrix quality-controlling parameters suggestion difficulty action accomplishment control according to standard is analyzed.Based on evaluation criteria, I All of experiment all reached minimum quality requirement.By RMA (robust multi-chip is average) background correction, quantile normalizing Change and the smooth pretreatment Affymetrix that concludes of median expresses microarray [1].To have extreme signals intensity and (be less than 50 or high In 2 × 10¹⁴) probe collection filter.In order to reduce the possibility of batch effect, the expression market demand normalization after filtering is calculated Method CamBat¹¹.CamBat method (http://statistics.byu.edu/johnson/ComBat/) application parameter or non-ginseng Number experience bayesian framework is in data-oriented Central Regulation batch effect.By microarray significance analysis (SAM) at error detection Rate (FDR) is equal to 0.05 time and determines difference expression gene (DEG)¹².The R environment with Bioconductor storehouse is used to perform pre-place Reason and statistics step.Use Ingenuity Pathway Analysis software 8.5 version (Ingenuity Systems, Redwood City, CA, U.S.A) carry out gene ontology (Gene Ontology) and classical pathway (Canonical Pathway) analyze.

II) result

1. the feature of colorectal cancer and comparison patient population

The clinic of 119 colorectal cancer (CRC) patients and 101 colonoscopy negative controls (CNC) and demography become Amount is summarized in table 1.To CRC, after colonoscopy, confirmed the diagnosis of colorectal cancer by virologist.Step on from community hospital Selecting comparison in the FOBT positive patient of note, they are finally the moon at the colonoscopy that tumour hospital of Fudan University (FUCH) is carried out Property.Age and sex is balanced well between CRC and CNC group.

2. the qualification expressing genes different in colorectal cancer patients and colonoscopy negative control in peripheral blood

Inventor finds the difference expression gene having High Defferential between the two groups in 119 CRC and 101 CNC (DEG), CRC group (I, II, III and IV phase) is considered as overall.After suitable pretreatment, remain 20169 probes Collection carries out DEG analysis.Being equal to 0.05 at FDR, multiple change (FC) is more than 1.2, uses SAM to identify 327 DEG.

In this 327 DEG, find respectively 195 (59.6%) and 132 (40.36%) in CRC sample with higher More low expression level.The P value scope of T inspection is from 1.43 × 10^-25To 1.51 × 10^-01, 18 DEG have less than 6.27 × 10^-15T inspection P value and all correspond to the gene annotating very well: MRPS6, SPRY4, NEAT1, CYBB, DUSP2, PDE4D, SH2D2A, G (1-2) NSR, ITGAM, VCAN, CD163, P2RY10, CD226, MRPL10, ITPRIPL2, CD2 and NUDT16 (table 2).The highest multiple change (FC) value is 1.83 (having higher levels of NEAT1 in CRC) and 1.71 (has lower level in CRC HBG2), 26 (8%) and in 327 DEG have the FC value higher than 1.40.

For example, to SPRY4, (in CRC, more high expression level ranks the first, and T checks P value 4.04 × 10^-23, FC 1.79) and (in CRC, lower expression ranks the first MRPS6, and T checks P value 1.43 × 10^-25, FC 1.27) and the result observed.So Example represent the gene that between CRC and CNC patient significant difference is expressed.To SPRY4,101 CNC observed very Similar hybridization signal value, and the value of CRC is more various but has notable (p value 4.04 × 10 compared with CNC^-23) improve average Value (FC 1.78).To MRPS6, Liang Ge colony all show similar dispersiveness, notable (the p value 1.43 × 10 of CRC tool^-25) reduce Mean value (FC 1.27).

In front 18 DEG, it was observed that 4 film leukocyte marker things, show compared with CNC at the peripheral blood of CRC patient Middle different expression: lower level CD2 and CD226, it is respectively by T cell and mainly being expressed by NK cell；More Gao Shui Flat CD163 and CD11B (ITGAM), it is main by monocyte with many leucocytes relating to innate immune system respectively Express.What is interesting is equally in cytotoxic T lymphocyte and NK (NK) cell by the particle of GZMB gene code Enzyme B, the lower expression in CRC sample.Other gene is reported as INSR, SPRY4, DUSP2, PDE4D and ITPRIPL2 For a part for different signal paths, it is special that SH2D2A is reported as T cell.Report VCAN table in monocyte Reach, and its in CRC sample higher expression together with CD163 with ITGAM can with these patients compared with CNC outside Certain activation of circulating monocytic cell in all blood is related.

Being analyzed 327 DEG by using Ingenuity Pathway Analysis (IPA), it returns 321 The ID of individual positioning, it is suitable for explaining related biological function (Bio Fuctions) and classical pathway.For department of physiology System is grown and function (Physiological System Developmentand Function), it was observed that immunocyte transports (p value is from 1.44 × 10 for (Immune Cell Trafficking) tool high score^-12To 1.57 × 10^-02, including 50 kinds of molecules), its Cover the activation of panimmunity cell, migration, accumulation, interior stream, chemotaxis, cellular invasion, cell movement, chemical induction (chemoattraction), pre-swash (priming) and adhere to.For classical pathway it is interesting that natural killer cell signal turns Leading (Natural Killer Cell Signaling) is to have minimum P value (2.55 × 10^-05) one, it includes 10 bases Cause: CD247, KLRB1, KLRC2, KLRC3, KLRD1, KLRK1, LCK, PRKCH, RRAS2 and SH2D1D.5 NK cell-specifics Membrane receptor (KLRB1, KLRC2, KLRC3, KLRD1, KLRK1) relate to very strongly show the peripheral blood in CRC patient In in the specific NK cellular component difference of gene expression dose.In CRC, all of NK cellular gene expression reduces.Result is general It is set forth in following table 2 and 3.

Table 2: differential expression between negative comparison (CNC) Patient Sample A of colorectal cancer (CRC) and colonoscopy Front 18 genes (DEG)；Gene describes, the T inspection P value information related with multiple change

* from NetAffx^TMWith from Ingenuity PathwayThe gene of 8.5 editions describes

Table 3:NK cell score: the gene of selection, T inspection P value and multiple change related information

* from NetAffx^TMWith from Ingenuity PathwayThe gene of 8.5 editions describes

The gene related to this 10 NK cells, it was observed that lower expression in CRC group, prompting circulation NK cell number Mesh reduces, or such cell outflows to other organ-/ tissue compartment especially knub positions.Observe lower level GZMB Expression is also it is noted that represent the main matter occurring in CRC patient at cytotoxicity levels.

Ranking classical pathway the most front relates to φt cell receptor signal conduction (T Cell Receptor Signaling), elder generation Contact (Communication between Innate and Adaptive between nature and adaptive immunity cell Immune Cells) and t helper cell in iCOS-iCOSL signal conduction (iCOS-iCOSL Signaling in T Helper Cells), it is respectively provided with 9.08 × 10^-05、2.85×10^-04With 5.78 × 10^-04P value.

It is interesting that to 51 in the sample of 119 CRC patient, it was observed that the low NK cell less than front 1/4th Score, and in 101 CNC Patient Sample A only 4.Using so direct cutoff value, the performance of this differentiation can be expressed as The sensitivity of 43% and 96% specific.In addition, when according to tumour TNM phase (I phase, II phase, III phase or IV phase) differentiation CRC During patient, it is observed that this NK cell score is gradually lowered (Fig. 1) from the I phase to the IV phase in CRC patient.Mainly at CNC and Between II phase, III phase and IV phase CRC, and between I phase CRC and II-III and IV phase CRC, observe the difference of statistically significant Different.

This research shows the potentiality of biological marker in transcript profile discovery peripheral blood, and in colorectal cancer Immune response provides new understanding.In addition to preparing possible replacement/complementation to existing filtering mode, these results are also aobvious Show that the expression analysis to for example related to NK cell gene allows to distinguish the patient suffering from colorectal cancer, thus opened Open the gate of personalized medicine.

Bibliography

1.Irizarry RA,Hobbs B,Collin F,Beazer-Barclay YD,Antonellis KJ,Scherf U,Speed TP.Exploration,normalization,and summaries of high density oligonucleotide array probe level data.Biostatistics 2003；4:249-64)

2.Johnson WE,Li C,Rabinovic A.Adjusting batch effects in microarray expression data using empirical Bayes methods.Biostatistics 2007；8:118-27.

3.Tusher VG,Tibshirani R,Chu G.Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci U S A2001；98: 5116-21.

4.Team RDC.R:A Language and Environment for Statistical Computing.Vienna,Austria,2009.

Claims

1. method, it is for determining colorectal cancer prognosis in the peripheral blood sample from patient, and described method includes:

A) obtain described peripheral blood sample and from described blood sample, extract total serum IgE,

B) make described total serum IgE and at least one reagent special at least one NK cytogene and not more than 25 kinds to 25 kinds of NK The special reagent contact of cytogene,

C) determine the expression of described at least one NK cytogene and at most 25 kinds of NK cytogenes to obtain described patient Express spectra,

D) use the NK cellular gene expression of the patient of clinical classification prognosis preferably before and be categorized as poor prognosis before The express spectra to described patient for the NK cellular gene expression of patient is analyzed, wherein

If the express spectra of-described patient clusters with the express spectra of the patient from the prognosis that clinical classification before is difference, then really Fixed described patient has poor prognosis, and

If the express spectra of-described patient and the express spectra cluster of the patient from the prognosis preferably of clinical classification before, then really The prognosis that fixed described patient has had.

2. the process of claim 1 wherein and make described total serum IgE with at least one at least one NK cytogene in step b) Special reagent and the not more than 25 kinds reagent contacts special to 25 kinds of NK cytogenes, described NK cytogene includes SEQ ID Nucleotide sequence shown in NO:1-12, wherein said at least one reagent is to special selected from following at least one NK cytogene:

Determine the expression of described at least one NK cytogene to obtain the express spectra of described patient in step c).

3. the method for claim 2, wherein make in step b) described total serum IgE with at least 5 kinds of NK cytogenes and not more than The reagent that the combination of 25 kinds of NK cytogenes is special contacts, and wherein said reagent at least includes to following NK cytogene special Reagent:

(v) KLRK1, it comprises the full length sequence shown in such as SEQ ID NO:13,

4. the method for claim 3, wherein makes described total serum IgE thin at least one target with at least one in step b) further The reagent of born of the same parents' gene specific and the not more than 5 kinds reagent contacts to 5 kinds of target cell gene specifics, described target cell gene includes Nucleotide sequence shown in SEQ ID NO:12-24, wherein said at least one reagent is to selected from following at least one target cell Gene specific:

(v) LCK gene, it comprises such as SEQ ID NO:25, the 26th, the 27th, the 28th, full length sequence shown in 29 or 30, and

Determine the expression of described at least one target cell gene to obtain the express spectra of described patient in step c).

5. the method for claim 4, wherein makes described total serum IgE special with to combining of 5 kinds of target cell genes in step b) Reagent contacts, and wherein said reagent is to following target cell gene specific:

Described in determining in step c), the expression of at least 5 kinds of cytogenes is to obtain the express spectra of described patient.

6. the method for claim 5, wherein makes described total serum IgE thin at least one target with at least one in step b) further The reagent of born of the same parents' gene specific and the not more than 100 kinds reagent contacts to 100 kinds of target cell gene specifics, described target cell gene bag Including the nucleotide sequence shown in SEQ ID NO:25-59, wherein said at least one reagent is to thin selected from following at least one target Born of the same parents' gene specific:

7. the method for claim 6, wherein rapid b) in make described total serum IgE with at least 17 kinds of target cell genes and not more than The reagent that the combination of 100 kinds of target cell genes is special contacts, and wherein said reagent at least includes to following target cell gene specific Reagent:

Described in determining in step c), the expression of at least 17 kinds of target cell genes is to obtain the express spectra of described patient.

8. the method any one of aforementioned claim, wherein at least one specific reagent described in step b) includes at least A kind of hybridization probe.

9. the method for claim 8, wherein the specific reagent described in step b) includes at least one hybridization probe and at least A kind of primer.

10. the method for claim 7, wherein the specific reagent described in step b) includes at least one hybridization probe and two kinds Primer.

11. kits, it is for prognosis colorectal cancer in the peripheral blood sample from patient, and described kit includes at least A kind of reagent special at least one NK cytogene and the not more than 25 kinds reagent special to 25 kinds of NK cytogenes, described NK cytogene at least includes the nucleotide sequence shown in SEQ ID NO:1-13, and wherein said at least one reagent is at least one Special selected from following NK cytogene:

The kit of 12. claims 11, it includes the reagent special to following NK cytogene:

The kit of 13. claims 12, it includes at least one target cell gene and not more than 5 kinds target cell gene specifics At least one reagent, described at least one target cell gene is selected from:

The kit of 14. claims 13, it includes 5 kinds of reagent to following target cell gene specific:

The kit of 15. claims 14, it includes at least one reagent at least one target cell gene specific and at most 100 kinds of reagent to 100 kinds of target cell gene specifics, described target cell gene at least is selected from:

The kit of 16. claims 15, it includes 17 kinds of reagent to following 17 kinds of target cell gene specifics:

17. at least one reagent special at least one NK cytogene and not more than 25 kinds special to 25 kinds of NK cytogenes Purposes in preparing composition for the reagent, described composition is for prognosis colorectum in the biological sample from patient Cancer, described NK cytogene comprises the nucleotide sequence shown in SEQ ID NO:1-12, and wherein said at least one reagent is to comprising In SEQ ID NO:1-12, at least one NK cytogene of arbitrary shown nucleotide sequence is special.

Composition prepared by the special reagent that combines of 18. pairs at least 5 kinds NK cytogenes and not more than 25 kinds of NK cytogenes In purposes, described composition in the biological sample from patient prognosis colorectal cancer, wherein said reagent pair At least 5 kinds of NK cytogenes comprising to be respectively selected from the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7 and 8-12 are special 's.

Purposes in preparing composition for the special reagent of combination of 19. pairs of 10 kinds of target cell genes, described composition for Prognosis colorectal cancer in the biological sample of patient, wherein said reagent is to comprising to be respectively selected from SEQ ID NO:1,2- 4th, 5-7, the target cell gene of 8-12, the 13rd, the nucleotide sequence shown in 14-17,18-20,21-22,23-24 and 25-30 are special 's.

Use in preparing composition for the special reagent of the combination of 20. pairs of 10 kinds of target cell genes and not more than 100 kinds of target genes On the way, described composition is for prognosis colorectal cancer in the biological sample from patient, and wherein said reagent is to comprising point Xuan Zi SEQ ID NO:1,2-4,5-7,8-12, the 13rd, 14-17,18-20,21-22,23-24,25-30,31-33, the 34th, the 35th, 36th, the 37th, the nucleotide sequence shown in 38-39,4-42,43-44, the 45th, 46-49,50-51,52-53, the 54th, 55-56, the 57th, 58 and 59 Target cell gene be special.

Purposes any one of 21. claims 17-20, wherein said at least one specific reagent includes at least one hybridization Probe.

Purposes any one of 22. claims 17-20, wherein said at least one specific reagent includes at least one hybridization Probe and at least one primer.

Purposes any one of 23. claims 17-20, wherein said at least one specific reagent includes at least one hybridization Probe and two kinds of primers.