CN101400804A - Gene expression markers for colorectal cancer prognosis - Google Patents

Abstract

A method of predicting clinical outcome in a subject diagnosed with colorectal cancer comprising determining evidence of the expression of one or more predictive RNA transcripts or their expression products in a biological sample of cancer cells obtained from the subject.

Description

The gene expression markers that is used for the colorectal carcinoma prognosis

Cross-reference to related applications

This is the non-provisional application of submitting to according to 37C.F.R.1.53 (b), require the provisional application serial number 60/758 of submission on January 11st, 2006 according to 35U.S.C. § 119 (e), 392, the provisional application serial number of submitting on May 12nd, 2,006 60/800, the provisional application serial number 60/810 that on May 31st, 277 and 2006 submitted to, 077 right of priority, all priority documents all are hereby incorporated by in full with it.

Invention field

The invention provides gene and gene sets that its expression level can be used for predicting the colorectal carcinoma result.

Background of invention

At the U.S. and European Union colorectal carcinoma is the relevant cause of the death of cancer of position of being number two, and accounts for 10% of all cancer associated deaths.Although the colorectal carcinoma and the rectum cancer can be represented same or analogous disease on molecular level, because anatomical problem is very complicated at the operation of the rectum cancer.May be because of this reason, the local relapse of the rectum cancer be apparently higher than colorectal carcinoma, so methods of treatment is obviously different.In annual new diagnosis about 100, the 000 routine colorectal carcinomas of the U.S., wherein about 65% is diagnosed as II/III phase colorectal carcinoma as discussed below.

The improvement of colorectum cancerous diagnose relates to the state of progress that utilizes the criteria classification standard to estimate cancer.Two kinds of categorizing systems have been widely used in colorectal carcinoma: Du Ke Shi (Duck) of improvement or Astler-Coller be system's (A-D phase) (Astler VB, Coller FA., Ann Surg 1954 by stages; 139:846-52), and recently the TNM that is developed by american cancer joint committee system's (I-IV phase) (AJCC CancerStaging Manual, the 6th edition, Springer-Verlag, New York, 2002) by stages.The measurement that primary tumor all passes colon or rectum parietal layer arrival adjacent organs, lymphoglandula and distal site diffusion in these two kinds of systems is applied to estimate tumour progression.The evaluation of the risk of recurrence of colorectal carcinoma and treatment decision-making is at present mainly based on tumor stage.

The II phase colorectal carcinoma that the new diagnosis of about 33,000 examples is arranged in the U.S. every year.Nearly all these patients treat by ocal resection, and in addition, about 40% patient adopts at present and treats based on the chemotherapy of 5 FU 5 fluorouracil (5-FU).The decision-making that whether gives auxiliary chemotherapy is not simple and direct.For the II phase colorectal carcinoma patient of independent use operative treatment, five year survival rate is about 80%.In this class crowd, adopt the standard assisting therapy of 5-FU+ formyl tetrahydrofolic acid (folinic acid) to confirm only to have the absolute of 2-4% to be benefited, and show serious toxicity, comprise up to 1% the chemotherapy rate of being poisoned to death.So, in the patient who accepts the poisonous substance treatment in a large number, only there is minority to be benefited.

The test of energy prognosis should be a significant benefit to the treatment decision-making of guidance to these patients behind II phase colorectal carcinoma corrective surgery.

Compare with the II phase, chemotherapeutical benefit is more obvious in III phase colorectal carcinoma.The annual diagnosis has the auxiliary chemotherapy of significant proportion acceptance based on 5-FU among 31, the 000 routine patients that III phase colorectal carcinoma is arranged, depend on the concrete therapeutic regimen that is adopted, and is benefited to about 18-24% at the absolute of 5-FU+ formyl tetrahydrofolic acid under this background.(5-FU+ formyl tetrahydrofolic acid or 5-FU+ formyl tetrahydrofolic acid+oxaliplatin) handled in III phase colorectal carcinoma patient's current standard medical care chemotherapy is moderate effective, has realized the improvement of five year survival rate from about 50% (only undergoing surgery) to about 65% (5-FU+ formyl tetrahydrofolic acid) or 70% (5-FU+ formyl tetrahydrofolic acid+oxaliplatin).Use separately the 5-FU+ formyl tetrahydrofolic acid or with the oxaliplatin combination therapy with some disadvantageous side effects, comprise that about 1% treatment patient is poisoned to death.In addition, the III phase colorectal carcinoma patient who treats for independent use operation, 3 years survival rates are about 47%, and can't determine whether III phase patient subgroup exists and be similar to the viewed risk of recurrence to II phase patient.

Based on molecular marker but not use separately tumor stage quantitatively the test of risk of recurrence can be used for identifying and need not the III phase patient subgroup that assisting therapy just can obtain acceptable result.

Though exist some for example owing to the caused difference of arrangement difference of draining lymph node, rectal neoplasm is by stages still based on carrying out with the similar by stages standard of colon tumor.As a result of, about their progress situation, II/III phase rectal neoplasm has the relevant of appropriateness with II/III phase colon tumor.As mentioned above, have the difference aspect local relapse and other prognosis between the rectum cancer and colorectal carcinoma, these differences may come from and be difficult to realize that rectal neoplasm excises entirely.However, still not about the noticeable evidence of difference between the colorectal carcinoma of corresponding tumour characterization of molecules and the rectum cancer.Should have to be similar in essence to the prognosis of rectum cancer test described effectiveness is tested in the prognosis of colorectal carcinoma, and identical prognostic marker might be equally applicable to this two kinds of cancer types.

In addition, need safer undoubtedly and more efficient drug is used for treatment of colon cancer.The chemotherapy that is used for colorectal carcinoma at present is based on the rough relatively method of the medicine of using common interference somatoblast propagation.Recently clinical study has confirmed to develop the feasibility of the improvement medicine of understanding based on the detailed molecular of particular cancers type and hypotype.For example, HER2 (ERBB2) gene is amplified and HER2 albumen is crossed and to be expressed in the mammary cancer subgroup;

(Genentech, Inc.) (a kind of exploitation is used for the medicine of target HER2) only indicate and be used for those and be higher than normal HER2 copy number by having of being confirmed of fluorescence in situ hybridization (FISH) or by the patient that high-level HER2 expresses that has that immunohistochemistry confirmed.It expresses the gene relevant with clinical effectiveness among the human cancer patient is the valuable source that is used for medical compounds screening and further drug development active target thing selection.

Molecular targeted agents is such as HERCEPTIN (Genentech Inc.) can be developed and commercialization with the diagnostic test that can identify the patient that may benefit from this medicine; An aspect of this test is to identify that those do not take any treatment may have the patient of positive findings except that operation.For example, when undergoing surgery treatment separately, 80% II phase colorectal carcinoma patient survived 5 years or more of a specified duration.Identify to belong to more that not take the gene marker of the patient among 20% crowd that its cancer of extra treatment will recur be useful in drug development, for example can be used for screening the patient who is contained in the clinical trial.

Summary of the invention

In one aspect, the present invention relates to a kind of predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, one or more list in table 1A-B in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 2A-B, 3A-B, 4A-B, 5A-B, the predictability rna transcription thing in 6 and/or 7 or the expression level of their expression product, wherein: (a) one or more list in table 1A, 2A, 3A, the evidence that the expression of gene among 4A and/or the 5A or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 2B, 3B, 4B and/or the 5B, indicate the possibility of positive clinical effectiveness to raise.The possibility of positive clinical effectiveness is predicted can be reduced if contain, and then behind described excision described patient is further treated.Further contain this treatment and be chemotherapy and/or radiotherapy.

Can be for example according to recurrence-free interval (Recurrence-Free Interval, RFI), total lifetime (OverallSurvival, OS), disease free survival phase (Disease-Free Survival, DFS) or do not have long distance recurrence interval (Distant Recurrence-Free Interval, DRFI) clinical effectiveness of expression the inventive method.

In one embodiment, cancer is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, one or more list in table 1A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 5A, the predictability rna transcription thing among 1B and/or the 5B or the expression level of their expression product, wherein: (a) evidence that raises of one or more expression of listing in gene among table 1A or the 5A or corresponding expression product, indicate the predicted meeting of described RFI shorter; (b) evidence that raises of one or more expression of listing in gene among table 1B or the 5B or corresponding expression product indicates the predicted meeting of described RFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du's Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 2A and/or the 2B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2A, indicate the predicted meeting of described OS shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2B, indicate the predicted meeting of described OS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 3A and/or the 3B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3A, indicate the predicted meeting of described DFS shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3B, indicate the predicted meeting of described DFS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 4A and/or the 4B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4A, indicate the predicted meeting of described DRFI shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4B, indicate the predicted meeting of described DRFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, one or more list in table 1.2A-B in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 2.2A-B, 3.2A-B, 4.2A-B, 5.2A-B, 6.2 and/or the evidence of the expression level of predictability rna transcription thing in 7.2 or their expression product, wherein: (a) one or more list in table 1.2A, 2.2A, 3.2A, 4.2A and/or the evidence that raises of the expression of gene among the 5.2A or corresponding expression product, indicate the possibility of positive clinical effectiveness to reduce; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2B, 2.2B, 3.2B, 4.2B and/or the 5.2B, indicate the possibility of positive clinical effectiveness to raise.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, one or more list in table 1.2A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 1.2B, 5.2A and/or the expression level of predictability rna transcription thing among the 5.2B or their expression product, wherein: (a) evidence that raises of one or more expression of listing in gene among table 1.2A or the 5.2A or corresponding expression product, indicate the predicted meeting of described RFI shorter; (b) evidence that raises of one or more expression of listing in gene among table 1.2B or the 5.2B or corresponding expression product indicates the predicted meeting of described RFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du's Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 2.2A and/or the 2.2B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2.2A, indicate the predicted meeting of described OS shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2.2B, indicate the predicted meeting of described OS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 3.2A and/or the 3.2B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3.2A, indicate the predicted meeting of described DFS shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3.2B, indicate the predicted meeting of described DFS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 4.2A and/or the 4.2B or the expression level of their expression product, wherein: (a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4.2A, indicate the predicted meeting of described DRFI shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4.2B, indicate the predicted meeting of described DRFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, one or more list in table 1A-B in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 1.2A-B, 2A-B, 2.2A-B, 3A-B, 3.2A-B, 4A-B, 4.2A-B, 5A-B, 5.2A-B, 6,6.2, the evidence of the predictability rna transcription thing in 7 and/or 7.2 or the expression level of their expression product, wherein: (a) one or more list in table 1A, 1.2A, 2A, 2.2A, 3A, 3.2A, 4A, 4.2A, the evidence that the expression of gene among 5A and/or the 5.2A or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression of listing in gene among table 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or the 5.2B or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, one or more list in table 1A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 1.2A, 1B, 1.2B, 5A, 5.2A, the predictability rna transcription thing among 5B and/or the 5.2B or the expression level of their expression product, wherein: (a) one or more list in table 1A, 1.2A, the evidence that the expression of gene among 5A and/or the 5.2A or corresponding expression product raises indicates the predicted meeting of described RFI shorter; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 1.2B, 5B and/or the 5.2B, indicate the predicted meeting of described RFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du's Ke Shi C (III phase) colorectal carcinoma, one or more list in table 2A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 2.2A, the predictability rna transcription thing among 2B and/or the 2.2B or the expression level of their expression product, wherein: (a) evidence that raises of one or more expression of listing in gene among table 2A and/or the 2.2A or corresponding expression product, indicate the predicted meeting of described OS shorter; (b) evidence that raises of one or more expression of listing in gene among table 2B and/or the 2.2B or corresponding expression product indicates the predicted meeting of described OS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, one or more list in table 3A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 3.2A, the predictability rna transcription thing among 3B and/or the 3.2B or the expression level of their expression product, wherein: (a) evidence that raises of one or more expression of listing in gene among table 3A and/or the 3.2A or corresponding expression product, indicate the predicted meeting of described DFS shorter; (b) evidence that raises of one or more expression of listing in gene among table 3B and/or the 3.2B or corresponding expression product indicates the predicted meeting of described DFS longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, one or more list in table 4A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 4.2A, the predictability rna transcription thing among 4B and/or the 4.2B or the expression level of their expression product, wherein: (a) evidence that raises of one or more expression of listing in gene among table 4A and/or the 4.2A or corresponding expression product, indicate the predicted meeting of described DRFI shorter; (b) evidence that raises of one or more expression of listing in gene among table 4B and/or the 4.2B or corresponding expression product indicates the predicted meeting of described DRFI longer.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprise and determine that one or more are selected from ALCAM in described experimenter's the biological sample that comprises cancer cells in acquisition, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1, WIF, CAPG, CD28, CDC20, CKS1B, DKK1, the predictability rna transcription thing of HSD17B2 and MMP7 or the expression level of their expression product, wherein: (a) one or more are selected from ALCAM, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, the evidence that the expression of the gene of TP53BP1 and WIF or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

In one aspect of the method, the present invention relates to a kind of predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi C (III phase) colorectal carcinoma, comprise and determine that one or more are selected from CAPG in described experimenter's the biological sample that comprises cancer cells in acquisition, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7, SIR2, ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, the predictability rna transcription thing of TP53BP1 and WIF or the expression level of their expression product, wherein: (a) one or more are selected from CAPG, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, the evidence that the expression of the gene of MMP7 and SIR2 or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

For all aspects of the inventive method, can be for example method by genetic expression preface type analysis (geneexpression profiling) obtain determining of one or more expression of gene levels.The method of this genetic expression preface type analysis can be the method for PCR-based for example.

For all aspects of the present invention, the expression of gene level can with respect to one or more with reference to the expression level of gene (reference gene) or their expression product by stdn (normalize).

For all aspects of the present invention, the experimenter preferably is people patient.

For all aspects of the present invention, this method can further comprise the evidence of the expression level of determining at least two kinds of described genes or their expression product.Further contain the evidence that method of the present invention can further comprise the expression level of determining at least three kinds of described genes or their expression product.Also contain the evidence that method of the present invention can further comprise the expression level of determining at least four kinds of described genes or their expression product.Also contain the evidence that method of the present invention can further comprise the expression level of determining at least five kinds of described genes or their expression product.

For all aspects of the present invention, this method can further comprise the step that generates the report that gathers described prediction.

For all aspects of the present invention, each increment of containing for one or more predictability rna transcription things or their expression product level increases, and the patient just is accredited as the increment that shows clinical effectiveness to be increased.

For all aspects of the present invention, more than the determining of expression level can take place once.For all aspects of the present invention, the definite of expression level can betide the patient before carrying out any treatment behind the excision.

One different aspect in, the present invention relates to portion and comprise the report that diagnosis has the prediction clinical effectiveness of experimenter behind the excision of described cancer of colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more list in table 1A-B in described experimenter's the biological sample that comprises cancer cells, 2A-B, 3A-B, 4A-B, 5A-B, the predictability rna transcription thing in 6 and/or 7 or the expression level of their expression product, wherein: (a) one or more list in table 1A, 2A, 3A, the evidence that the expression of gene among 4A and/or the 5A or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 2B, 3B, 4B and/or the 5B, indicate the possibility of positive clinical effectiveness to raise.Can be for example according to recurrence-free interval (RFI), total lifetime (OS), disease free survival phase (DFS) or there is not the clinical effectiveness of long distance recurrence interval (DRFI) expression report of the present invention.In one embodiment, cancer is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.The prediction of clinical effectiveness can comprise estimation to experimenter's specific clinical the possibility of result and maybe can comprise based on described estimation the experimenter is classified into risk group (risk group).

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise that one or more list in table 1.2A-B in described experimenter's the biological sample that comprises cancer cells based on being included in acquisition, 2.2A-B, 3.2A-B, 4.2A-B, 5.2A-B, 6.2 and/or the prediction of the clinical effectiveness of the information of the expression level of predictability rna transcription thing in 7.2 or their expression product, wherein: (a) one or more list in table 1.2A, 2.2A, 3.2A, 4.2A and/or the evidence that raises of the expression of gene among the 5.2A or corresponding expression product, indicate the possibility of positive clinical effectiveness to reduce; (b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2B, 2.2B, 3.2B, 4.2B and/or the 5.2B, indicate the possibility of positive clinical effectiveness to raise.Can be for example according to recurrence-free interval (RFI), total lifetime (OS), disease free survival phase (DFS) or there is not the clinical effectiveness of long distance recurrence interval (DRFI) expression report of the present invention.In one embodiment, cancer is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.The prediction of clinical effectiveness can comprise estimation to experimenter's specific clinical the possibility of result and maybe can comprise based on described estimation the experimenter is classified into risk group.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise that one or more list in table 1A-B in described experimenter's the biological sample that comprises cancer cells based on being included in acquisition, 1.2A-B, 2A-B, 2.2A-B, 3A-B, 3.2A-B, 4A-B, 4.2A-B, 5A-B, 5.2A-B, 6,6.2, the prediction of the clinical effectiveness of the information of the predictability rna transcription thing in 7 and/or 7.2 or the expression level of their expression product, wherein: (a) one or more list in table 1A, 1.2A, 2A, 2.2A, 3A, 3.2A, 4A, 4.2A, the evidence that the expression of gene among 5A and/or the 5.2A or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression of listing in gene among table 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or the 5.2B or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise estimation to experimenter's specific clinical the possibility of result and maybe can comprise based on described estimation the experimenter is classified into risk group.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprise that one or more are selected from ALCAM in described experimenter's the biological sample that comprises cancer cells based on being included in acquisition, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1, WIF, CAPG, CD28, CDC20, CKS1B, DKK1, the prediction of the clinical effectiveness of the information of the predictability rna transcription thing of HSD17B2 and MMP7 or the expression level of their expression product, wherein: (a) one or more are selected from ALCAM, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, the evidence that the expression of the gene of TP53BP1 and WIF or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise estimation to experimenter's specific clinical the possibility of result and maybe can comprise based on described estimation the experimenter is classified into risk group.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi C (III phase) colorectal carcinoma, comprise that one or more are selected from CAPG in described experimenter's the biological sample that comprises cancer cells based on being included in acquisition, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7, SIR2, ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, the prediction of the clinical effectiveness of the information of the predictability rna transcription thing of TP53BP1 and WIF or the expression level of their expression product, wherein: (a) one or more are selected from CAPG, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, the evidence that the expression of the gene of MMP7 and SIR2 or corresponding expression product raises indicates the possibility of positive clinical effectiveness to reduce; (b) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise estimation to experimenter's specific clinical the possibility of result and maybe can comprise based on described estimation the experimenter is classified into risk group.

One different aspect in, the present invention relates to a kind of test kit, comprise be suitable for carrying out the inventive method with the next item down or multinomial: (1) extracts damping fluid/reagent and experimental program; (2) reverse transcription damping fluid/reagent and experimental program; (3) qPCR damping fluid/reagent and experimental program.This test kit can comprise data retrieval (retrieval) and analysis software.

The accompanying drawing summary

The shown dendrogram of Fig. 1 has been showed in single argument Cox ratio venture analysis (univariate Coxproportional hazards analysis) the expression cluster (clustering) with 142 kinds of genes (table 1.2A and 1.2B) of recurrence-free interval statistics significant correlation.This cluster analysis used unweighted to group mean count act of union (unweighted pair-group average amalgamation method) and with 1 Pearson (Pearson) r as distance measuring location (measure).The identity of specific gene indicates along the x axle in the cluster of being concerned about.

Detailed Description Of The Invention

A. definition

Unless otherwise prescribed, technology used herein and scientific terminology have with the present invention under the field The identical implication of common understanding of those of ordinary skill. " the Dictionary of of Singleton etc. Microbiology and Molecular Biology " the 2nd edition (J.Wiley ﹠ Sons, New York, NY 1994) and " Advanced Organic Chemistry Reactions, the Mechanisms and of March Structure " the 4th edition (NY 1992 for John Wiley ﹠ Sons, New York) offer this area skill The general guide of employed many terms among art personnel the application.

Those skilled in the art will be appreciated that many described herein those of being similar to or being equivalent to Method and material can be used for enforcement of the present invention. In fact, the present invention is confined to absolutely not described side Method and material. For the purposes of the present invention, following term is defined as follows.

When using in this article, term " tumour " refers to no matter be all pernicious or optimum superfluous natural disposition Growth of Cells and propagation, and the front property of all cancers and cancerous cells and tissue.

Term " cancer " and " carcinous " are pointed out or describe in the mammal usually thin with what do not regulated Intracellular growth is the physiological status of feature. The example of cancer includes but not limited to breast cancer, oophoroma, colon Cancer, lung cancer, prostate cancer, hepatocellular carcinoma, cancer of the stomach, cancer of pancreas, cervix cancer, liver cancer, carcinoma of urinary bladder, The urinary tract cancer, thyroid cancer, kidney, cancer knurl, melanoma and the cancer of the brain.

" pathology " of cancer comprises the phenomenon of all harm patient healths. This include but not limited to unusual or Uncontrolled Growth of Cells, transfer, interference adjacent cells work orderly, discharge with abnormal level Cell factor or other secretion property product, inhibition or exacerbate inflammation or immunological response, neoplasia (neoplasia), precancer (premalignancy), malignant tumour (malignancy), invade around or far away End tissue or organ (such as lymph node) etc.

Term " colorectal cancer " uses with broad sense and refers to that (1) drawn by large intestine and/or rectum epithelial cell All stadium that rise and all types of cancer and/or (2) invasion and attack large intestine and/or rectum internal layer (lining) All stadium and all types of cancer. In the staging system that is used for the colorectal cancer classification, knot Intestines and rectum are treated as an organ.

Tumour, tubercle, transfer (TNM) Staging System according to american cancer joint committee (AJCC) (" the AJCC Cancer Staging Manual " the 6th edition that Greene etc. compile, New York, NY: Springer; 2002), each stadium of colorectal cancer is defined as follows:

Tumour: T1: tumour is invaded submucosa (submucosa); T2: tumour is invaded muscularis propria (muscularis propria); T3: tumour is passed muscularis propria and is invaded subserosa (subserose), or invades Enter pericolic tissue or paraproctium; T4: tumour is directly invaded other organs or structure and/or is worn The hole.

Tubercle: N0: do not have regional lymphatic metastasis; In N1:1-3 the regional lymph node transfer is arranged; In N2:4 or the more regional lymph nodes transfer is arranged.

Shift: M0: no far-end shifts; M1: have far-end to shift.

Stadium grouping: I phase: T1 N0 M0; T2 N0 M0; The II phase: T3 N0 M0; T4 N0 M0; The III phase: any T, N1-2; M0; The IV phase: any T, any N, M1.

According to the Du Ke Staging System of improvement, each stadium of colorectal cancer is defined as follows:

The A phase: tumour penetrates intestinal mucosa but does not further penetrate; The B phase: tumour penetrates and runs through the intestines wall Muscularis propria; The C phase: tumour penetrates but does not run through the muscularis propria of intestines wall, has knot in lymph node The pathology evidence of the intestines carcinoma of the rectum; Or tumour penetrates and runs through the muscularis propria of intestines wall, deposits in lymph node Pathology evidence in cancer; The D phase: tumour has diffused out lymph node and has entered other organs, such as liver, Lung or bone.

Prognostic Factors is that those natural histories (natural history) with colorectal cancer are relevant, in a single day They develop into the variable that colorectal cancer just affects patient's recurrence rate and result. With worse prognosis Related clinical parameter comprises that for example lymph node is got involved and senior tumour. Prognostic Factors usually is used for the patient Be categorized as the subgroup with different baseline risks of recurrence.

Term " prognosis " is used in reference to the prediction of the possibility of the dead or progress that is attributable to cancer in this article, The Preventive diffusion and the resistance to the action of a drug that comprise ND (such as colon cancer).

Term " prediction " is used in reference in this article that the patient will have the spy behind the excision of primary tumor Decide the possibility of clinical effectiveness (no matter being positive or negative). By being that any particular patient is selected The treatment formula that is fit to, Forecasting Methodology of the present invention can be used for making the treatment decision-making clinically. The present invention Forecasting Methodology advantageously reply in the therapeutic scheme (such as surgical intervention) at prediction patient possibility and be Valuable instrument. This prediction can comprise Prognostic Factors.

Term " positive clinical effectiveness " refers to be improved any measuring of status of patient, comprises this area Middle normally used those are measured, such as the increase of recurrence-free interval (RFI) duration, Overall survival (OS) time increases, (DFS) time DFS phase increases, when no long distance recurrence interval (DRFI) is lasting Between increase etc. The rising of positive clinical effectiveness possibility is corresponding to the reduction of cancer return possibility.

Term " classification of risks " refers to that the experimenter can experience specific clinical result's risk level or prediction. Be subjected to Examination person can be divided into risk group or classifies at risk level based on Forecasting Methodology of the present invention. " risk group " It is one group of experimenter or individuality that has similar risk level for the specific clinical result.

Term " for a long time " existence is used in reference at least 3 years in this article, the more preferably existence at least 5 years.

Term " recurrence-free interval (RFI) " is used for pointing to for the first time colon cancer recurrence (on inspection in this article The primary cancer second time as initial event) or do not have the recurrence evidence Post-dead duration year number.

Term " Overall survival (OS) " is used in reference to the Post-dead duration from operation to any reason in this article Year number.

Term " DFS phase (DFS) " is used for pointing to colon cancer recurrence or any reason in this article Post-dead duration year number.

Term " no long distance recurs interval (DRFI) " is used in reference to from operation in this article to dissecting for the first time The time of long distance cancer return on (year number).

Depend on and to examine or the definition of irrespective event, in practice the meter of listed measured value above Calculation can be different with research.

Term " microarray " refers to the array element that can hybridize, and the preferred polynucleotide probe is positioned on the matrix Ordered arrangement.

Term " polynucleotides " is often referred to any polybribonucleotide when using with odd number or plural number Or polydeoxyribonucleotide, it can be RNA or the DNA of unmodified, or the RNA that modifies Or DNA. So, for instance, polynucleotides as defined herein include but not limited to strand and Double-stranded DNA, the DNA that comprises strand and double stranded region, strand and double-stranded RNA and comprise strand and two The RNA of sequence and comprise DNA and the hybrid molecule of RNA, DNA wherein and RNA can be Strand or more generally double-stranded or comprise strand and double stranded region. In addition, when using in this article, art Three sequences that language " polynucleotides " refers to comprise RNA or DNA or comprises simultaneously RNA and DNA. This Chain in the zone can be from identical molecule or different molecules. This zone can comprise one or more institutes State the whole of molecule, but more typically only be a zone of some described molecules. The branch in triple helix district One of son is oligonucleotides normally. Term " polynucleotides " clearly comprises cDNA. This term comprises and containing DNA (comprising cDNA) and RNA that the base of one or more modifications is arranged. So, have because of stable The DNA of property or other reasons and adorned main chain or RNA are the " multinuclears that mean of this term herein Thuja acid ". In addition, the base (such as the base of tritiate) that comprises rare bases (such as inosine) or modify DNA or RNA be included in the scope of term " polynucleotides " as defined herein. General and All chemistry, enzyme and/or the metabolism modification of the polynucleotides of unmodified contained in speech, term " polynucleotides " Form, and the change of virus and the distinctive DNA of cell (comprising simple cell and complex cell) and RNA Form.

Term " oligonucleotides " refers to the polynucleotides of relatively lacking, and includes but not limited to strand deoxyribose nuclear Thuja acid, strand or double-stranded ribonucleotide, RNA:DNA heterocomplex and double-stranded DNA. Oligonucleotides (such as the ssDNA probe oligonucleotides) is usually synthetic by chemical method, for example uses commercially available automatic Change oligonucleotide synthesizer. Yet, can prepare oligonucleotides by multiple additive method, comprise external The technology of recombinant DNA mediation and by expressible dna in the Cell and organism body.

Term " gene of differential expression ", " gene expression of difference " and their interchangeable the making of synonym With, refer to that with respect to its expression in normal or contrast experimenter (particularly cancer is all ill for it Such as colon cancer) expression among the experimenter is activated to the gene of higher or lower level. This term also comprises Its expression in the different phase of same disease is activated to the gene of higher or lower level. Also should manage What separate is that the gene of differential expression can be activated or suppresses at nucleic acid level or protein level, maybe can advance The row alternative splicing is to produce different polypeptide products. Such difference can be by for example mRNA level, many The change of the surface expression of peptide, secretion or other distribution (partitioning) and being confirmed. The base of difference Because expressing the expression can comprise between two or more genes of comparison or their gene outcome, or relatively Expression ratio between two or more genes or their gene outcome, or even comparison same gene Two kinds of different elaboration products, its between normal subjects and ill (particularly cancer) experimenter or Between the different phase of same disease, be different. Differential expression is included in for example normal and diseased cells Between or at the iuntercellular that has experienced various disease event or disease stage, gene or its expression product are in the time Or cellular expression has in graphic quantitative and difference qualitatively. For the purposes of the present invention, when normally And among the ill experimenter between the expression of given gene or in ill experimenter's various disease developing stage In exist at least about 2 times, preferably at least about 4 times, more preferably at least about 6 times, most preferably at least about 10 times During difference, just think to have " gene expression of difference ".

" cross and express " about the term of rna transcription thing and be used in reference to by to reference mRNA level standard The transcript level of measuring can be all measured transcript or spies in the sample with reference to mRNA Fixed mRNA reference set.

Phrase " gene magnification " refers to form gene or the gene sheet of multicopy in specific cells or clone The process of section. Duplicate field (section of the DNA of amplification) is commonly referred to " amplicon ". Generally speaking, The amount of the mRNA that produces (mRNA), i.e. the level of gene expression is also pressed by expressed specific base Because the ratio of the copy number that forms increases.

" stringency " of hybridization reaction determined by those of ordinary skills easily, and usually got Certainly calculate by rule of thumb in probe length, wash temperature and salinity. Generally speaking, long probe needs Higher temperature is used for correct annealing, and short probe then needs lower temperature. When complementary strand is deposited When being to be lower than in the environment of their melting temperatures, the energy that denatured DNA is annealed is again depended in hybridization usually Power. Expectation homology degree between probe and the sequence that can hybridize is more high, just can use more high relatively Temperature. As a result of, release, higher relative temperature trends towards making reaction condition stricter, and more Low temperature is then more not strict. For the subsidiary details of the stringency of hybridization reaction and explain referring to " the Current Protocols in Molecular Biology " of Ausubel etc., Wiley Interscience Publishers, (1995).

As defined herein, " stringent condition " or " high stringent condition " is generally: (1) uses low ionic strength and high temperature for washing, 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate for example, 50 ℃; (2) during hybridizing, use denaturing agent, such as methane amide, for example, 50% (v/v) methane amide and 0.1% bovine serum albumin(BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone/have 750mM sodium-chlor, the 50mM sodium phosphate buffer pH6.5 of 75mM Trisodium Citrate, 42 ℃, or (3) use 50% methane amide, 5x SSC (0.75M NaCl, 0.075M Trisodium Citrate), 50mM sodium phosphate (pH6.8), 0.1% trisodium phosphate, 5x DenhardtShi solution, the salmon sperm DNA of supersound process (50 μ g/ml), 0.1%SDS and 10% sulfuric acid dextran, 42 ℃, wash in 0.2x SSC (sodium chloride/sodium citrate) and 50% methane amide at 42 ℃, then 55 ℃ of high strict washings of forming by the 0.1x SSC that contains EDTA.

" medium stringent condition " can be defined as " Molecular Cloning:ALaboratory Manual " (the New York:Cold Spring Harbor Press as Sambrook etc., 1989) described, and comprise and use compare with mentioned above those undemanding washing soln and hybridization conditions (for example temperature, ionic strength and %SDS).An example of medium stringent condition is to contain: 20% methane amide, 5x SSC (150mM NaCl, the 15mM trisodium citrate), be incubated overnight in 37 ℃ in the solution of the salmon sperm DNA of the shearing of 50mM sodium phosphate (pH7.6), 5xDenhardtShi solution, 10% sulfuric acid dextran and 20mg/ml sex change, in 1x SSC, wash filter membrane in about 37-50 ℃ then.Those skilled in the art will be appreciated that how to come attemperation, ionic strength etc. according to the needs that adapt to such as factors such as probe length.

In the context of the present invention, list in " at least a ", " at least two kinds " in any specific gene set, any or any and all combinations that " at least five kinds " isogenic implication means listed gene.

Term " tubercle feminine gender " cancer such as " tubercle feminine gender " colorectal carcinoma, is used in reference to the cancer that is not diffused into lymphoglandula in this article.

Term " montage " and " RNA montage " are used interchangeably, and refer to remove intron and connect exon to produce the RNA processing of ripe mRNA, and wherein ripe mRNA has the continuous programming code sequence and moves into eukaryotic tenuigenin.

In theory, term " exon " refers to be embodied in any split gene section (B.Lewin.Genes IV, Cell Press, Cambridge Mass.1990) in the mature rna product.In theory, term " intron " refer to be transcribed but exon montage by will being arranged in its either side to any DNA section of being removed from transcript together.In operation, exon sequence appears in the mRNA sequence of gene, and gene limits by numbering referring to SEQ ID.In operation, intron sequences is the intervening sequence that is arranged in the genomic dna of gene, and its both sides are exon sequence and have GT and AG montage consensus sequence on their 5 ' and 3 ' border.

Term " expression cluster " is used in reference in this article when showing one group of gene that similar expression is graphic when stipulating in the sample that the patient organizes research.When using in this article, the gene when research in from the sample of II phase and/or III phase colon and/or rectal cancer patient in the same expression cluster shows that similarly expression is graphic.

B.1 general description

Unless otherwise stated, enforcement of the present invention will be adopted conventional Protocols in Molecular Biology (comprising recombinant technology), microbiological technique, cytobiology technology and Measurement for Biochemistry, and these technology all are within the art technology scope.Such technology is explained in following document fully, such as " Molecular Cloning:A Laboratory Manual ", the 2nd edition (Sambrook etc., 1989); " Oligonucleotide Synthesis " (M.J.Gait compiles 1984); " Animal Cell Culture " (R.I.Freshney compiles 1987); " Methods in Enzymology " (Academic Press, Inc.); " Handbook of Experimental Immunology ", the 4th edition (D.M.Weir ﹠amp; C.C.Blackwell compiles Blackwell Science Inc., 1987); " Gene Transfer Vectors forMammalian Cells " (J.M.Miller ﹠amp; M.P.Calos compiles 1987); " Current Protocols inMolecular Biology " (F.M.Ausubel etc. compile 1987); And " PCR:The PolymeraseChain Reaction ", (Mullis etc. compile 1994).

Based on the evidence of rna transcription thing differential expression in normal cell and cancer cells, the invention provides the prognosis gene marker that is used for colorectal carcinoma.Therefore, one specific aspect in, the invention provides the prognosis gene marker of II phase and/or III phase colorectal carcinoma, comprise specificity prognosis II phase or III phase disease result mark and this two interim have prognostic value, this two interim and/or in the tumour progression degree mark of basic difference in the reflection tumour cell.Prognostic marker provided by the present invention and relevant information make the doctor make wiser treatment conclusion and need customize the colorectal carcinoma treatment at individual patients and are able to possibility, make the treatment benefit reach maximization thus and make the patient be exposed to unnecessary treatment to reduce to minimum, any major benefit is not provided in this unnecessary treatment and because toxic side effect carries serious risk usually.

The destruction of the normal function of the various physiological processs that comprise propagation, apoptosis, blood vessel generation and invade relates to the pathology of cancer.Dysfunction is characterized fully to the Relative Contribution of the pathology of particular cancers type in particular physiological process.Any physiological process all combines the contribution of the expressed several genes product of the various kinds of cell that relates to this process.For example, tumour cell is invaded and to be infiltrated the proteinic influence that the recycle system all is subjected to the different cell characteristics of a collection of mediation in adjacent healthy tissues and the tumour cell, described cell characteristic comprise adhesion, the tumour cell of adhesive aggregation, tumour cell and normal cell between tumour cell and reticular tissue at first change its form then migration pass the ability of surrounding tissue and the tumour cell degraded ability of reticular tissue structure on every side.

Multiple analyte genetic expression test can be measured several important physiological processs or be constituted one or more expression of gene levels that each involved in the cell characteristic.In some situation, the forecasting power of this test and its effectiveness therefore can be improved by using the expression values that indivedual genes are obtained be calculated as follows score, be somebody's turn to do the indivedual expression of gene values of proportion by subtraction and result height correlation more.For example, the quantitative score (recurrence score) of calculating predicting recurrence possibility is recorded in common unsettled U.S. Patent application (publication number 20050048542) in estrogen receptor positive, tubercle negative breast cancer.The equation that is used to calculate such recurrence score can be with the gene grouping so that make the predictor of recurrence score reach maximum.Can based on being carried out gene to the understanding of the contribution of physiological function or formation cell characteristic (all as discussed above), they divide into groups to small part.In addition, grouping information can be beneficial to the contribution of the various expression values of mathematics weighting to the recurrence score.The weighting of representing physiological process or constituting the gene group of cell characteristic can reflect the contribution to cancer pathology and clinical effectiveness of this process or feature.Thereby, one important aspect in, the present invention also provides the concrete grouping of the prognosis gene of being identified herein, with indivedual genes of this genes identified or at random combination compare, above-mentionedly be grouped into more reliable and more effective prediction of result device.

In addition, based on the mensuration of recurrence score, can select with the patient be subdivided into have any specific recur the subgroup of score value, wherein all patients with given range value can classify and belong to specific risk group.So, selected value can limit the patient's subgroup that has greater or lesser risk respectively.

The effectiveness of gene marker in prediction colorectal carcinoma result may not be to have only this mark just to have.Have to the graphic optional mark of the closely similar expression of fc-specific test FC mark and can replace this test mark, or can test at this and use outside mark and influence very little the macro-forecast effectiveness of this test.The close similar expression of two kinds of genes is graphic to be derived from two kinds of genes and involves same particular procedure and/or be under the common adjusting control in colon tumor cell.The present invention clearly comprises and contains so alternative gene or gene set purposes in the method for the invention.

Can predict II phase and/or III phase colon and/or rectum cancer clinical effectiveness, prognostic marker provided by the present invention and relevant information can be used for developing the medicine that is used for the treatment of II phase and/or the III phase colon and/or the rectum cancer.

Can predict II phase and/or III phase colon and/or rectum cancer clinical effectiveness, prognostic marker provided by the present invention and relevant information also can be used for screening and be contained in the testing drug compound to the patient in the clinical trial of the effect of II phase and/or III phase colon and/or rectal cancer patient treatment.Particularly, this prognostic marker can be used on collection patient's in clinical trial the sample, and test result used with patient result so that determining and whole patient group or other subgroups compares, which patient's subgroup more likely or the more impossible response that demonstrates medicine.

Can predict II phase and/or III phase colon and/or rectum cancer clinical effectiveness, prognostic marker provided by the present invention and relevant information can be used as the standard that comprises of clinical trial.For example, the possibility that the patient that only undergos surgery if test result is pointed out can have bad clinical effectiveness is higher, then the patient more likely is included in the clinical trial, if and test result points out if the patient that only undergos surgery can have the possibility of bad clinical effectiveness lowlyer, then the patient can not be included in the clinical trial.

In a specific embodiment, prognostic marker and relevant information are used for design or produce regulatory gene transcript or its expression product level or active reagent.Described reagent can include but not limited to sense-rna, little inhibitory RNA, ribozyme, mono-clonal or polyclonal antibody.

In a further embodiment, described gene or its transcript, or more specifically, the expression product of described transcript is used for (screening) assay method to identify medical compounds, and wherein said medical compounds is used to develop the medicine of treatment II phase and/or the III phase colon and/or the rectum cancer.

In each embodiment of the present invention, can utilize various technological methods to measure disclosed expression of gene level, comprise but do not limit RT-PCR, microarray, serial analysis of gene expression (Serial Analysisof Gene Expression, SAGE) with (the Massively ParallelSignature Sequencing that checks order by extensive parallel signature, MPSS) gene expression analysis, it will go through in hereinafter.In specific embodiment, can measure every kind of expression of gene level with respect to the different characteristics (comprising exon, intron, protein epitope and protein active) of gene expression product.In other embodiments, the horizontal deducibility of expression of gene is for example inferred the pattern analysis that methylates from gene promoter from the gene structure analysis.

B.2 genetic expression preface type analysis

The method of genetic expression preface type analysis comprises the method analyzed based on multi-nucleotide hybrid, based on the method for polynucleotide order-checking, and based on the method for protein science.Become known for the method that mRNA in the sample expresses quantitative the most normal use in this area and comprise Northern trace and in situ hybridization (Parker ﹠amp; Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNA enzyme protection assay method (Hod, Biotechniques 13:852-854 (1992)); And the method for PCR-based, such as RT-polymerase chain reaction (RT-PCR) (Weis etc., Trends in Genetics 8:263-264 (1992)).Perhaps, can adopt the energy recognition sequence specific double-strand body antibody of (comprise DNA duplex, RNA duplex, reach DNA-RNA heterozygosis duplex or DNA-protein duplex).Comprise serial analysis of gene expression (SAGE) and the gene expression analysis by extensive parallel signature order-checking (MPSS) based on the exemplary process of gene expression analysis of order-checking.

A. reverse transcriptase PCR (RT-PCR)

In above listed technology, the sensitiveest and the most flexibly quantivative approach be RT-PCR, it can be used for measuring the mRNA level in the various samples.It is graphic that the result can be used between the comparative sample set genetic expression of (for example between healthy tissues and the tumor tissues and carrying out the patient of pharmacological agent or do not carry out between the patient of pharmacological agent).

The first step is from target sample separation mRNA.Parent material is typically respectively from people's tumour or tumor cell line and corresponding healthy tissues or the isolating total RNA of clone.So, can be from multiple primary tumor isolation of RNA, comprise tumour or tumor cell lines such as mammary gland, lung, colon, prostate gland, brain, liver, kidney, pancreas, spleen, thymus gland, testis, ovary, uterus, and from the merging DNA of healthy donors.If the source of mRNA is a primary tumor, mRNA can extract from the paraffin embedding of for example refrigerated or filing and the tissue sample of fixing (for example formalin fixed) so.

The universal method that is used to extract mRNA is well-known in the art, is disclosed in the molecular biological standard textbook, comprises people such as Ausubel, " Current Protocols of Molecular Biology ", John Wiley and Sons, 1997.Be used for being disclosed in for example Rupp and Locker, Lab Invest.56:A67 (1987) from the method for paraffin-embedded tissue extraction RNA; De Andr é s et al., Bio Techniques18:42044 (1995).Particularly, RNA separates purification kit, damping fluid group and the proteolytic enzyme can use from commercial manufacturers such as Qiagen and carries out according to the specification sheets of manufacturers.For example, the total RNA from culturing cell can use Qiagen RNeasy micro-column to separate.Other commercialization RNA separating kit comprises MasterPure Global DNA and RNA purification kit (EPICENTRE

, Madison, WI) and paraffin mass RNA separating kit (Ambion, Inc.).Total RNA from tissue sample can use RNA Stat-60 (Tel-Test) to separate.Can separate by for example cesium chloride density gradient centrifugation from the RNA of tumour preparation.

Because the template that RNA can not serve as PCR, the first step of carrying out genetic expression preface type analysis by RT-PCR is that RNA template reverse transcription is become cDNA, then is its exponential amplification in the PCR reaction.Two kinds of the most frequently used reversed transcriptive enzymes are bird myeloblastic leukemia viral reverse transcriptase (AMV-RT) and Moloney (Moloney) murine leukemia virus reverse transcriptase (MMLV-RT).The reverse transcription step is typically used Auele Specific Primer, sexamer or the initiation of oligo-dT primer at random, depends on circumstances and the target of expressing the preface type analysis.For example, the RNA of extraction can use GeneAmp RNA PCR test kit (Perkin Elmer, CA USA) follow the specification sheets reverse transcription of manufacturers.Deutero-cDNA can be used for follow-up PCR reaction as template then.

Although the PCR step can be used multiple heat-staple DNA dependent dna-polymerases, typically adopt the Taq archaeal dna polymerase, it has 5 '-3 ' nuclease and lacks 3 '-5 ' correction endonuclease enzymic activity.So, TaqMan

PCR typically utilize 5 of Taq or Tth polysaccharase '-nuclease comes hydrolysis and its target amplicon bonded hybridization probe, but can use have equivalence 5 ' nuclease any enzyme.Use two kinds of Oligonucleolide primers to generate amplicon, be typically PCR reaction.The third oligonucleotide or probe design are used to detect the nucleotide sequence between preceding two kinds of PCR primers.Probe is that the TaqDNA polysaccharase is inextensible and with report fluorescence dye and cancellation fluorochrome label.Two kinds of dyestuffs as they on probe the position near the time, any laser induced emission from the report dyestuff is subjected to the cancellation of quencher dyes.During amplified reaction, the Taq archaeal dna polymerase cuts probe in template dependency mode.The gained probe fragment dissociates in solution, no longer is subjected to the effect of the quenching effect of second fluorophore from the signal of discharge report dyestuff.Whenever a synthetic new molecule just discharges the report dyestuff of a molecule, and the detection of cancellation report dyestuff does not provide the basis that data are quantitatively set forth.

TaqMan

But RT-PCR commodity in use equipment carries out, such as for example ABI PRISM 7700 Sequence detection system ^TM(Perkin-Elmer-Applied Biosystems, Foster City, CA, USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).In a preferred embodiment, on the real-time quantitative PCR device, move 5 ' nuclease rules, such as ABI PRISM7700

Sequence detection system ^TMThis system is made up of thermal cycler, laser (device), charge coupled device (CCD), photographic camera and computer.This system on thermal cycler with 96 well format amplifications sample.In amplification procedure,, and detect at the CCD place by the laser induced fluorescent signal in all 96 holes of fiber optic cables real-time collecting.This system comprises the software that is used for operational outfit and analytical data.

5 '-nuclease assay method data are expressed as Ct at first, or cycle threshold (threshold cycle).Just as discussed above, at each record fluorescent value and the amount of the product of amplification when representing to amplified reaction this cycle period.The point that records the fluorescent signal of statistical significance first is cycle threshold (Ct).

Minimum for influence wrong and the sample room variation is reduced to, use the internal standard thing to carry out RT-PCR usually.Ideal internal standard thing is expressed with constant level between different tissues, is not tested the influence of processing.The most frequent RNA that is used for the genetic expression style standardsization is the mRNA of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P-Actin muscle.

A kind of up-to-date variation of RT-PCR technology is a real-time quantitative PCR, and it (is TaqMan by the fluorescence generation probe of double-tagging

Probe) measures the accumulation of PCR product.PCR in real time and quantitative competitive PCR (wherein using the internal competition thing of every kind of target sequence to carry out stdn) and quantitative comparison PCR (stdn gene that comprises in the use sample or housekeeping gene are for the usefulness of RT-PCR) are compatible.More details are consulted for example Held et al., Genome Research 6:986-994 (1996).

The many parts of journal articles of delivering have provided the step of using fixed, paraffin-embedded tissue to be used for the representative solution of genetic expression preface type analysis as the RNA source, comprise mRNA separation, purifying, primer extension and amplification (T.E.Godfrey et al. for example, J.Molec.Diagnostics 2:84-91 (2000); K.Specht et al., Am.J.Pathol.158:419-29 (2001)).In brief, a kind of exemplary process is by the about 10 micron thickness section beginning that cuts out paraffin-embedded neoplasmic tissue sample.Then, extract RNA, and remove deproteinize and DNA.After analyzing RNA concentration, can comprise that where necessary RNA repairs and/or amplification step, and use gene specific promotor reverse transcription RNA, follow by RT-PCR.

The b.MassARRAY system

At Sequenom, Inc. (San Diego, CA) in Kai Fa the genetic expression preface type analysis method based on MassARRAY, after isolation of RNA and reverse transcription, in the cDNA that is obtained, mix synthetic dna molecular (competition thing), mate in its cDNA district at all positions except that single base and target, and serve as the internal standard thing.This cDNA/ competition thing mixture is carried out the pcr amplification shrimp alkaline phosphotase (SAP) of going forward side by side behind the performing PCR handle, it causes the dephosphorylation of residual nucleus thuja acid.Behind the alkaline phosphatase enzyme-deactivating, the PCR product from competition thing and cDNA is carried out primer extension, it produces for competition thing-and distinct a large amount of signals of cDNA-deutero-PCR product (mass signal).Behind the purifying, these products are assigned on the chip array, this chip array is pre-loaded to have the auxiliary laser desorption ionisation flight time mass spectrum of matrix of use (MALDI-TOF MS) to analyze desirable ingredients.Quantitative by the ratio of analyzing peak area in the mass spectrum that is produced then to the cDNA that exists in the reaction.More details are referring to for example Dingand Cantor, Proc.Natl.Acad.Sci.USA 100:3059-3064 (2003).

C. the method for other PCR-based

The technology of more PCR-based comprises for example mRNA differential display mRNA (Liang and Pardee, Science257:967-971 (1992)); Amplified fragment length polymorphism (iAFLP) (Kawamoto et al., GenomeRes.12:1305-1312 (1999)); BeadArray ^TMTechnology (Illumina, San Diego, CA; Oliphant et al,, Discovery of Markers for Disease (Biotechniques supplementary issue), in June, 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); Be used to detect the pearl array (BADGE) of genetic expression, microballoon (the Luminex Corp. of commodity in use Luminex 100 LabMAP systems and polychrome coding in the genetic expression rapid test method, Austin, TX) (Yang et al., Genome Res.11:1888-1898 (2001)); And high the covering expressed preface type analysis (HiCEP) (Fukumura et al., Nucl.Acids.Res.31 (16) e94 (2003)).

D. microarray

Differential gene expression also can use microarray technology to identify or verify.So, can use microarray technology in fresh or paraffin-embedded tumor tissues, to measure colorectal carcinoma Expression of Related Genes preface type.In this method, coating (plate) or arrangement (array) polynucleotide sequence interested (comprising cDNA and oligonucleotide) on the microchip substrate.The sequence of arrangement is hybridized with the specificity DNA probing needle from cells of interest or tissue.As in the RT-PCR method, the source of mRNA is typically from people's tumour or tumor cell line and corresponding healthy tissues or the isolating total RNA of clone.So, RNA can separate from multiple primary tumor or tumor cell line.If the source of mRNA is a primary tumor, mRNA can extract from the paraffin embedding of for example refrigerated or filing and the tissue sample of fixing (for example formalin fixed) so, and described tissue sample is conventional preparation and preservation in the daily clinical practice.

In a specific embodiments of microarray technology, the cDNA of pcr amplification clone's inset is applied to substrate with closely spaced array.Preferably, at least 10,000 kind of nucleotide sequence is applied to substrate.The microarray gene that is fixed on the microchip with every kind of 10,000 compositions is suitable for hybridize under stringent condition.Fluorescently-labeled cDNA probe mixes fluorescent nucleotide can the reverse transcription by the RNA that extracts from tissue of interest and generates.Each the DNA spot on mark cDNA probe and array that is applied to chip is hybridized with specificity.The strict cleaning with behind the probe of removing non-specific binding is by the confocal laser microscopy or by other detection method such as CCD photographic camera scanning chip.The abundance of quantitatively allowing the assessment corresponding mRNA of the hybridization of every kind of array composition.Rely on dual coloured fluorescence, separately the cDNA probe and the array paired cross of mark that will generate from two kinds of RNA sources.So measure relative abundance simultaneously from the transcript in two kinds of sources of specifying gene corresponding to every kind.The hybridization of miniaturization scale provides the facility and the rapid evaluation of a large amount of genetic expression patterns.These class methods demonstrated desired sensitivity of rare transcript that detection expresses with each cell minority copy and reproducible detection expression level (Schena et al., Proc.Natl.Acad.Sci.USA 93 (2): 106-149 (1996)) at least about 2 times of differences.But microarray analysis commodity in use equipment is followed the scheme of manufacturers and is carried out, such as the microarray technology that uses Affymetrix GENCHIP technology or Incyte.

The microarray method of developing for large scale analysis genetic expression makes the cancer classification in might systematic search kinds of tumors type and the molecular marker of prediction of result.

E. genetic expression successive analysis (SAGE)

Genetic expression successive analysis (SAGE) is to allow simultaneously and a large amount of genetic transcription things of quantitative analysis and do not need to provide for every kind of transcript a kind of method of indivedual hybridization probes.At first, generate a kind of short sequence label (about 10-14bp) comprise the information that is enough to a kind of transcript of unique evaluation, obtain if this label is a unique position in every kind of transcript.Then, many transcripts are coupled together to form length, continuous molecule, they can check order, with the identity (identity) that discloses multiple label simultaneously.Any transcript group's expression pattern can be by measuring individual tag abundance and identify that the gene corresponding with every kind of label comes qualitative assessment.More details are referring to for example Velculescu et al., Science 270:484-487 (1995); Velculescu et al., Cell 88:243-51 (1997).

F. the gene expression analysis that is undertaken by massive parallel signature order-checking (MPSS)

By Brenner et al., this method of Nature Biotechnology 18:630-634 (2000) record is a kind of sequence measurement of joining together based on the signature order-checking of gel and the millions of body outer clone of template on the 5 micron diameter microballons that separate not.At first, the microballon storehouse by body outer clone constructed dna template.Then, in the groove that flows, (be typically greater than 3 x 10 with high-density ⁶Microballon/cm ²) assembling contains the planar array of template microballon.Use does not require the signature sequence measurement based on fluorescence that dna fragmentation is separated, and analyzes the free-end of the template of cloning on each microballon simultaneously.This method has demonstrated in single operation simultaneously and provides accurately from the number in yeast cDNA library in 100,000 gene signature sequence.

G. immunohistochemical methods

The immunohistochemical methods method also is applicable to the expression level that detects prognostic marker of the present invention.So, use every kind of specific antibody of mark or antiserum(antisera), preferred polyclonal antiserum, most preferably monoclonal antibody detects expression.Antibody can self detect by direct traget antibody, for example uses radioactively labelled substance, fluorescent marker, hapten-marked thing such as vitamin H or enzyme such as horseradish peroxidase or alkaline phosphatase.Perhaps, unmarked one anti-with through mark two anti-associating uses, comprise anti-specific antiserum(antisera), polyclonal antiserum or a monoclonal antibody.Immunohistochemical methods scheme and test kit are well-known in the art, and can obtain by commercial sources.

H. proteomics

Term " protein group " is defined as and puts exist in the sample (for example tissue, organism or cell culture) proteinic all sometime.Proteomics comprises the global change (being also referred to as " marking protein group ") of protein expression in the study sample etc.Proteomics comprises the following steps: that typically (1) by two-dimensional gel electrophoresis (2-D PAGE) with the range protein in the sample separately; (2) identify the range protein that from gel, reclaims, for example by mass spectrum or N-end sequencing; And (3) use the bioinformatic analysis data.Proteomics method is that the useful of other genetic expression preface type analysis method replenished, and can use separately or unites other method, is used to detect the product of prognostic marker of the present invention.

I. promoter methylation analysis

Multiple being used for rna transcription thing (gene expression analysis) or their protein translation product quantitative methods has been discussed in this article.Also can be according to inferring the expression of gene level, such as the methylation state of for example gene promoter and other regulatory element and the acetylize state of histone about the information of chromatin Structure.

Particularly, the influence of the methylation state of promotor is subjected to the expression of gene level of this promotor adjusting.The abnormal methylation of specific gene promotor relates to expressing regulates, such as the silence of for example tumor suppressor gene.So, the inspection to the gene promoter methylation state can be used as the direct quantized surrogate of rna level.

Designed the method that several are used to measure the methylation state of specific DNA element, comprise methylation status of PTEN promoter (Herman J.G.et al. (1996) Methylation-specific PCR:a novel PCRassay for methylation status of CpG islands.Proc.Natl Acad.Sci.USA.93,9821-9826) and the hydrosulphite dna sequencing (Frommer M.et al. (1992) A genomicsequencing protocol that yields a positive display of 5-methylcytosine residues inindividual DNA strands.Proc.Natl Acad.Sci.USA.89,1827-1831).Recently, based on the technology of microarray be used to characterize the promoter methylation state (Chen C.M. (2003) Methylation target array for rapid analysis of CpG island hypermethylation inmultiple tissue genomes.Am.J.Pathol.163,37-45).

J. the coexpression of gene

Another aspect of the present invention is that identified gene is expressed cluster.Use statistical analysis known in the art to analyze expression data, but identified gene is expressed cluster thus, comprises based on the dependency of Pearson correlation coefficient and analyzes (Pearson K.and Lee A. (1902) Biometrika 2,357) in pairs.

In one embodiment, the expression cluster of identifying herein comprises that BGN, CALD1, COL1A1, COL1A2, SPARC, VIM and other are known as stroma cell institute and mainly synthesize and relate to the gene of reinventing extracellular matrix.This expression cluster is referred to herein as extracellular matrix to be reinvented/the matrix cluster.

In another embodiment, the expression cluster of Jian Dinging comprises the gene of ANXA2, KLK6, KLK10, LAMA3, LAMC2, MASPIN, SLPI and other coding epithelial cell secretory product herein, wherein to be mainly epithelial cell secreted for great majority, but it is secreted also to can be other cell type.This expression cluster is referred to herein as epithelium/secretion cluster.

In another embodiment, the expression cluster of Jian Dinging comprises DUSP1, EGR1, EGR3, FOS, NR4A1, RHOB and other it transcribes the gene of early stage rise after some stimulates in cellular exposure herein.Multiple stimulation triggers transcribing of early stage response gene, for example is exposed to somatomedin, and it can make cell increase their mobility and the ability of their transhipment nutrient such as glucose fast.This expression cluster is referred to herein as replys cluster in early days.

In another embodiment, the expression cluster of Jian Dinging comprises MCP1, CD68, CTSB, OPN and other coding proteinic gene relevant with immune cell usually herein.This expression cluster is referred to herein as immune cluster.

In another embodiment, the expression cluster of Jian Dinging comprises that CCNE2, CDC20, SKP2, CHK1, BRCA1, CSEL1 and other involve the gene of cell proliferation and Cycle Regulation herein.This expression cluster is referred to herein as propagation/cell cycle cluster.

The generality of k.mRNA separation, purifying and amplification is described

The many parts of journal articles of delivering have provided the step of using fixed, paraffin-embedded tissue to be used for the representative solution of genetic expression preface type analysis as the RNA source, comprise mRNA separation, purifying, primer extension and amplification (T.E.Godfrey et al. for example, J.Molec.Diagnostics 2:84-91 (2000); K.Specht et al., Am.J.Pathol.158:419-29 (2001)).In brief, a kind of exemplary process is by the about 10 micron thickness section beginning that cuts out paraffin-embedded neoplasmic tissue sample.Then, extract RNA, and remove deproteinize and DNA.After analyzing RNA concentration, can comprise that where necessary RNA repairs and/or amplification step, and use gene specific promotor reverse transcription RNA, follow by RT-PCR.At last, based on the characteristic genetic expression pattern of identifying in the check tumor sample, according to the prediction possibility of cancer return, analytical data is to identify the optimum handling option useful to the patient.

L. colorectal carcinoma gene sets, the gene polyadenylation signal sequence of being measured, and the clinical application of gene expression data

An importance of the present invention is that the expression that utilizes some gene of colon cancer tissue to be measured provides prognosis information.For this purpose, must proofread and correct the difference of the quality variation of the amount of the RNA that (stdn) measured and employed RNA.Therefore, some stdn gene is measured and mixed to this assay method usually, comprises well-known housekeeping gene, such as the expression of GAPDH and Cyp1.Perhaps, can come stdn (global criteria method) based on the signal average or the intermediate value (Ct) of all determined genes or its large-scale inferior collection.For each gene, existing amount in the amount of measured standardized patient tumors mRNA and the colon cancer tissue reference set is compared in turn.The number of colon cancer tissue (N) should be enough high consistent basically to guarantee different reference set (generally) behavior performance in this reference set.If meet this condition, the relative quantity of the gene that the identity that is present in the single colon cancer tissue in the specific collection so just can remarkably influenced not be measured.Generally speaking, the colon cancer tissue reference set by at least about 30, preferably form at least about 40 different FPE colon cancer tissue samples.Unless indication is arranged in addition, the tumour/patient's of each mRNA/ test normalized expression level should be expressed as the percentage ratio of the expression level that records in reference set.More particularly, the reference set of tumour number enough high (for example 40) distribution that produced each mRNA kind standardization.Measured level drops on a certain hundredths in this scope in specific tumors sample to be analyzed, and it can be measured by method well-known in the art.Below, unless indication is arranged in addition, when mentioning the expression of gene level, although always do not stipulate clearly, but supposition is the normalized expression with respect to reference set.

M. based on the PCR primer of intron and the design of probe

According to one aspect of the present invention, PCR primer and probe are based on the intron sequences design that exists in the gene to be amplified.Thereby the first step of primer/probe design is to describe intragenic intron sequences.This can be undertaken by the available software of the public, such as by Kent, and W.J., the DNA BLAT software or the BLAST software of Genome Res.12 (4): 656-64 (2002) exploitation comprise its variation.Subsequent step is followed PCR primer and the probe design method of setting up fully.

For fear of non-specific signal, importantly when design primer and probe, cover the tumor-necrosis factor glycoproteins in (mask) intron.This can be easy to by using Repeat Masker program to realize, this program can be passed through the online acquisition of Baylor College Medicine (Baylor College of Medicine), and it is at repeat element library screening dna sequence dna and return the search sequence of wherein having covered repeat element.Can use any commercialization or the intron sequences by the obtainable primer of other approach public/after the probe design bag will be covered to be used to design primer and probe sequence then, such as Primer Express (Applied Biosystems); MGBassay-by-design (Applied Biosystems); (Steve Rozen and Helen J.Skaletsky (2000) Primer3 uses for general user and biologist programmer on the World Wide Web Primer3.At Krawetz S, in " Bioinformatics Methods and Protocols:Methods inMolecular Biology " that Misener S compiles, Humana Press, Totowa, N.J., 365-386 page or leaf).

The greatest factor of considering in the PCR design of primers comprise primer length, melting temperature(Tm) (meltingtemperature, Tm), G/C content, specificity, complementary primer sequence and 3 '-end sequence.Generally speaking, best long usually 17-30 the base of PCR primer comprises about 20-80% such as for example about 50-60%G+C base.Typical preferred Tm between 50 and 80 ℃, for example about 50-70 ℃.

About the further guidance of PCR primer and probe design referring to for example Dieffenbach C.W.etal., " General Concepts for PCR Primer Design " (universal of PCR design of primers), at " PCR Primer, A Laboratory Manual " in, Cold Spring Harbor LaboratoryPress, New York, 1995, the 133-155 pages or leaves; Innis and Gelfand, " Optimization ofPCRs " (optimization of PCR), in " PCR Protocols, A Guide to Methods and Applications ", CRC Press, London, 1994, the 5-11 pages or leaves; And Plasterer, T.N., Primerselect:Primerand probe design.Methods Mol.Biol.70:520-527 (1997) clearly is collected herein by reference its complete disclosure.

N. test kit of the present invention

The material that is used for method of the present invention is suitable for preparing the test kit that produces according to well-known rules.So, the invention provides the test kit that comprises reagent, described reagent can comprise and being used for being used to predict the quantitative gene specific of disclosed expression of gene or the gene Selection probe and/or the primer of prognosis result or treatment response.This type of test kit can randomly comprise and be used for extracting the reagent of RNA and/or being used for the reagent that RNA increases from tumor sample (particularly fixed, paraffin-embedded tissue sample).In addition, test kit can randomly comprise reagent and differentiate description or label or operation instruction, about they application in the method for the invention.Test kit can comprise container (comprising the microtiter plate that is applicable to automatization execution the inventive method), each container is equipped with one or more all ingredients that are used for the inventive method (existing with spissated form usually), comprises ready-formed microarray for example, damping fluid, suitable Nucleotide triphosphoric acid (for example dATP, dCTP, dGTP and dTTP; Or rATP, rCTP, rGTP and UTP), reversed transcriptive enzyme, archaeal dna polymerase, RNA polymerase and one or more probes of the present invention and primer (for example poly (T) or the random primer that are connected to the promotor that RNA polymerase reacts of suitable length).Be used to estimate or quantitatively the mathematical algorithm of prognosis or information of forecasting also suitably be the possible composition of test kit.

O. report of the present invention

When being implemented when being used for the commercialization diagnostic purpose, method of the present invention produces the report or the summary of a normalized expression level about one or more selected genes usually.Method of the present invention will produce a report that diagnosis has experimenter's prediction of clinical effectiveness behind the excision of described cancer of colorectal carcinoma that comprises.Method of the present invention and report can further comprise report is stored in the database.Perhaps, this method can further generate a record and padding data in this record in experimenter's database.In one embodiment, be reported as the report of paper spare, in another embodiment, be reported as the sense of hearing (auditory) report, in another embodiment, be reported as electronical record.Contain this report and be provided for doctor and/or patient.The reception of this report can further comprise sets up with the server that comprises data and report that network is connected and requested data and report from this server.

Method provided by the invention also can be on the whole or partly automatization carry out.

Also can implement all aspects of the present invention, make outside disclosed gene and/or replace disclosed gene, in prognosis or prediction experiment, comprise a limited number of other gene with disclosed gene co-expressing, for example by high Pearson (Pearson) gene that relation conefficient confirmed.

Described the present invention, will more easily understand the present invention by reference the following example, described embodiment provides as example, and the part intention limits the present invention by any way.

Embodiment

Explore the genome tumour for one and express preface type and research with dependency between Du Ke Shi B of colectomy treatment and the Ke Shi C patient's that shuts out the recurrence possibility

The main purpose of this research be to be determined at 757 kinds of amplicons identifying among the table B each expression and accept colectomy (operation) but do not carry out the chemotherapeutical II phase and III phase colorectal carcinoma patient's clinical effectiveness between whether have significant dependency.

Research and design

This is an exploratory study, be used to comfortable nearly 400 only accept colectomy (operation) or Du Ke Shi B (II phase) of the back bacille Calmette-Guerin vaccine (BCG) that undergos surgery and perform the operation and Ke Shi C (III phase) patient that shuts out in U.S.'s mammary gland and the plan of intestinal surgery assisting therapy (National Surgical Adjuvant Breastand Bowel Project NSABP) studies tissue and the result data of C-01 and C-02.

Comprise standard

The patient adds NSABP research C-01: " estimating immunotherapy after surgical operation of liver cancer and the chemotherapeutical clinical testing of postoperative whole body (A Clinical Trial To Evaluate PostoperativeImmunotherapy And Postoperative Systemic Chemotherapy In The ManagementOf Resectable Colon Cancer) in resectable colon cancer management " or NSABP research C-02: " estimating the scheme (A Protocol To Evaluate ThePostoperative Portal Vein Infusion Of 5-Flourouracil And Heparin InAdenocarcinoma Of The Colon) of postoperative 5 FU 5 fluorouracil and heparin portal vein infusion in adenocarcinoma of colon ". As seen the details of C-01 and C-02 are positioned at the NSABP website of following URL:

http://www.nsabp.pitt.edu/NSABP_Protocols.htm#treatment％20closed

To be merged into a sample sets from only the undergoing surgery and performing the operation of NSABP C01+postoperative BCG group and from the tissue sample of the group that only undergos surgery of NSABPC02.

Exclusion standard

If be suitable for following one or multinomial, patient's eliminating that then will add NSABP research C-01 or NSABP research C-02 is outside research of the present invention:

The tumor mass that in the NSABP archives, does not have the initial stage diagnosis.

When by phenodin and eosin (H﹠amp; When E) the slide glass inspection is estimated, tumour deficiency in the zone.

Self-organization section RNA deficiency that reclaim, that be used for the RT-PCR analysis (＜700ng).

In 1943 patients that add NSABP research C-01 or NSABP research C-02, after using exclusion standard 270 patient's samples to be arranged be obtainable and be used for the disclosed gene expression research of this paper.These 270 parts total population statistics and the Clinical symptoms that comprise sample are similar to initial NSABP in conjunction with group.

Gene sets (Gene Panel)

Select 761 kinds of genes to be used for expression analysis, comprise 7 kinds with reference to gene.These genes are listed in Table A with being used for qRT-PCR with the primer of mensuration expression level and the sequence of probe.

Experiment material and method

Use TaqMan

RT-PCR is used as expression of gene with reference to gene, this TaqMan to 750 kinds of cancer related genes of every patient's quantitative evaluation and 7 kinds of appointments RT-PCR is single-threaded to carry out, each reaction input 1ng RNA.

Data analysing method

Reference standardization

For the stdn alien influence, with respect to 6 kinds of cycle threshold (C that obtain by RT-PCR with reference to the expression average stdn of the set of gene _T) observed value.Usually in the scope of 0-15, wherein the increase of 1 unit has reflected 2 times of increases of RNA quantity to the expression observed value of gained reference standardization usually.

Study group and initial NSABP study population's comparison

For the study group of current valuable tissue block and initial NSABP C-01 and C-02 study population, we have compared clinical and distribution demographic variable.In distributing, this does not have significant clinically difference.

Univariate analysis

For in 757 kinds of amplicons in the research each, we use the Cox proportional hazard model to check dependency between genetic expression and the recurrence-free interval (RFI).With the check item of likelihood ratio as significance,statistical.Use the method (Benjamini of Benjamini and Hochberg, Y.and Hochberg, Y. (1995) .Controlling the false discovery rate:a practical and powerful approach tomultiple testing.J.R.Statist.Soc.B57,289-300) reach based on the method for sampling again and arrange (Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarraysapplied to the ionizing radiation response.Proc Natl Acad Sci USA, 98:5116-5121; Storey JD, Tibshirani R (2001) Estimating false discovery ratesunder dependence, with applications to DNA microarrays.Stanford:StanfordUniversity, Department of Statistics; Report number: technical report 2001-28; Korn EL, Troendle J, McShane L, Simon R (2001) Controlling the number of falsediscoveries:Application to high-dimensional genomic data. technical report 003.2001.National Cancer Institute) in gained p value set to estimate wrong discovery rate (false discoveryrate).For each selectable terminal point: no long distance recurrence interval (DRFI), total lifetime (OS) and disease free survival phase (DFS) repeat all analyses.

Multivariate analysis

For research in 757 kinds of amplicons in each, we use the Cox proportional hazard model to check dependency between genetic expression and the RFI, control the influence of other standard clinical concomitant variable (comprise tumor-localizing, type of surgery, tumor grade, the lymphatic node number that detects, and the number of positive lymphatic node) simultaneously.To only comprise (simplification) model of standard clinical concomitant variable and comprise that the difference of the log-likelihood of (complete) model that standard clinical concomitant variable gene mentation is expressed is used as the check item of significance,statistical.

Nonlinear analysis

For in 757 kinds of amplicons in the research each, other funtcional relationship between we use several different methods research genetic expressions and recur.For each amplicon, we use 2DOF (DF) natural spline the Cox proportional hazard model of RFI to be fitted to function (the Stone C of genetic expression, Koo C. (1985) In Proceedings of the Statistical Computing Section ASA.Washington, DC, 45-48).By being carried out the 2DF likelihood ratio test, model estimates significance,statistical.Also study funtcional relationship (GrayRJ (1992) Flexible methods for analyzing survival data using splines as the pattern of (level and smooth) halter strap residual error (Martingale residuals) of the single argument Cox proportional hazard model derivation of the RFI of the strictly linear function of genetic expression by inspection, withapplications to breast cancer prognosis.Journal of the American StatisticalAsssociation, 87:942-951; Gray RJ (1994) Spline-based tests in survival analysis.Biometrics, 50:640-652; Gray RJ (1990) Some diagnostic methods for Coxregression models through hazard smoothing.Biometrics, 46:93-102).In addition, to and be used to detect (the Lin D that departs from from the accumulation of the halter strap residual error of each identical Cox proportional hazard model apart from linear, Wei L, Ying Z. (1993) Checking the Cox Model with Cumulative Sumsof Martingale-Based Residuals.Vol.80, No.3,557-572).

With interaction by stages

We have determined whether to exist the significant difference relation between genetic expression and RFI in II phase and III phase patient.For in 757 kinds of amplicons each, we checked (simplification) proportional hazard model that is used for genetic expression and tumor stage and based on genetic expression, tumor stage, and their interactional (complete) proportional hazard model between have the hypothesis of significant difference.To simplify and the difference of the log-likelihood of complete model is used as the check item of significance,statistical.

Table A has shown qRT-PCR probe and the primer sequence that is included in all genes in the described research of embodiment.

Table B has shown the target amplicon that is included in all genes in the described research of embodiment.

Single order analysis and research result

The reference gene sets that is used for the single order analysis is CLTC, FZD6, NEDD8, RPLPO, RPS13, UBB, UBC.

Table 1A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted shorter recurrence-free interval (RFI).

Table 1B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted longer recurrence-free interval (RFI).

Table 2A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the total survival rate (OS) that reduces.

Table 2B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the total survival rate (OS) that raises.

Table 3A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the disease free survival rate (DFS) that reduces.

Table 3B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the disease free survival rate (DFS) that raises.

Table 4A has shown that based on the venture analysis of single argument ratio expressing for those its raises has predicted the association that shorter no long distance recurs the gene of interval (DRFI).

Table 4B has shown that based on the venture analysis of single argument ratio expressing for those its raises has predicted the association that longer no long distance recurs the gene of interval (DRFI).

Table 5A has shown the multivariate analysis that is included in particular demographic and the Clinical symptoms of the patient in this analysiss based on control, expresses the gene that shorter recurrence-free interval (RFI) predicted in rising for those its, related between genetic expression and RFI.

Table 5B has shown the multivariate analysis that is included in particular demographic and the Clinical symptoms of the patient in this analysiss based on control, expresses the gene that longer recurrence-free interval (RFI) predicted in rising for those its, related between genetic expression and RFI.

Table 6 shown based on non-linear ratio's venture analysis of using the 2DOF natural spline, identifies related gene between genetic expression and clinical effectiveness.

Table 7 has shown the gene of all displayings with interact neoplasm staging (p value＜0.05).

Table 1A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of RFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 1A

Table 1B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of RFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 1B

Table 2A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of OS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 2A

Table 2B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of OS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 2B

Table 3A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DFS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 3A

Table 3B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DFS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 3B

Table 4A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DRFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 4A

Table 4B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DRFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 4B

Table 5A has shown that control is included in particular demographic and the Clinical symptoms of the patient in this analysis, related between genetic expression and RFI.Listed its expression relevant with RFI (p＜0.1) and in the multivariate analysis that comprises following variable, show risk than all genes of 1: the tubercle of tumor-localizing, operation, tumor grade, check, and positive nodular number.

Table 5A

Table 5B has shown that control is included in particular demographic and the Clinical symptoms of the patient in this analysis, related between genetic expression and RFI.Listed its expression relevant with RFI (p＜0.1) and in the multivariate analysis that comprises following variable, shown risk than all genes of＜1: the tubercle of tumor-localizing, operation, tumor grade, check, and positive nodular number.

Table 5B

Table 6 shown based on non-linear ratio's venture analysis of using the 2DOF natural spline, related between genetic expression and clinical effectiveness.List all and shown the gene that in combined I I phase (Du Ke Shi B) and III phase (Ke Shi C shuts out) patient, has departed from the exact linear relationship (p＜0.05) of RFI.In this research in the whole expression values range of observation, relation between genetic expression and the RFI is also non-constant, for example genetic expression raises and may prolong relevantly with the RIF time length in the range of observation of a part, then shortens relevant with the RFI time length in the range of observation of different piece.

Table 6

Table 7 has shown that all show the gene with interact neoplasm staging (p value＜0.05).The proportional hazard model that uses RFI is to the data modeling, with genetic expression, neoplasm staging, and their interaction as predictor.

Table 7

The second order analysis result of study

The reference gene sets that is used for second order analysis is ATP5E, CLTC, GPX1, NEDD8, PGK1, UBB.

Table 1.2A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted shorter recurrence-free interval (RFI).

Table 1.2B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted longer recurrence-free interval (RFI).

Table 2.2A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the total survival rate (OS) that reduces.

Table 2.2B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the total survival rate (OS) that raises.

Table 3.2A has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the disease free survival rate (DFS) that reduces.

Table 3.2B has shown based on the venture analysis of single argument ratio, the association of the gene that expressing for those its raises has predicted the disease free survival rate (DFS) that raises.

Table 4.2A has shown that based on the venture analysis of single argument ratio expressing for those its raises has predicted the association that shorter no long distance recurs the gene of interval (DRFI).

Table 4.2B has shown that based on the venture analysis of single argument ratio expressing for those its raises has predicted the association that longer no long distance recurs the gene of interval (DRFI).

Table 5.2A has shown the multivariate analysis that is included in particular demographic and the Clinical symptoms of the patient in this analysiss based on control, expresses the gene that shorter recurrence-free interval (RFI) predicted in rising for those its, related between genetic expression and RFI.

Table 5.2B has shown the multivariate analysis that is included in particular demographic and the Clinical symptoms of the patient in this analysiss based on control, expresses the gene that longer recurrence-free interval (RFI) predicted in rising for those its, related between genetic expression and RFI.

Table 6.2 shown based on non-linear ratio's venture analysis of using the 2DOF natural spline, identifies related gene between genetic expression and clinical effectiveness.

Table 7.2 has shown the gene of all displayings with interact neoplasm staging (p value＜0.05).

Table 1.2A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of RFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 1.2B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of RFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 2.2A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of OS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 2.2B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of OS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 3.2A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DFS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 3.2B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DFS, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 4.2A shown for those confirmed risk than 1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DRFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 4.2B shown for those and confirmed risk than＜1.0 and the gene of p＜0.1, related between clinical effectiveness and genetic expression.Use the tolerance of DRFI, the regression analysis of single argument Cox ratio risk is applied to combined I I phase (Du Ke Shi B) and III phase (Du Ke Shi C) patient as clinical effectiveness.

Table 5.2A has shown that control is included in particular demographic and the Clinical symptoms of the patient in this analysis, related between genetic expression and RFI.Listed its expression relevant with RFI (p＜0.1) and in the multivariate analysis that comprises following variable, show risk than all genes of 1: tumor-localizing, operation time, tumor grade, treatment plan (C-01 or C-02), BCG treatment (be or not), and following patient's classification according to lymph node status: the tubercle of 0 positive tubercle and＜12 checks, 0 positive tubercle and 〉=tubercle of 12 checks, a 1-3 positive tubercle and 〉=4 positive tubercles.

Table 5.2B has shown that control is included in particular demographic and the Clinical symptoms of the patient in this analysis, related between genetic expression and RFI.Listed its expression relevant with RFI (p＜0.1) and in the multivariate analysis that comprises following variable, shown risk than all genes of＜1: tumor locus, operation time, tumor grade, treatment plan (C-01 or C-02), BCG treatment (be or not), and following patient's classification according to lymph node status: the tubercle of 0 positive tubercle and＜12 checks, 0 positive tubercle and 〉=tubercle of 12 checks, a 1-3 positive tubercle and 〉=4 positive tubercles.

Table 6.2 shown based on non-linear ratio's venture analysis of using the 2DOF natural spline, related between genetic expression and clinical effectiveness.List all and shown the gene that in combined I I phase (Du Ke Shi B) and III phase (Ke Shi C shuts out) patient, has departed from the exact linear relationship (p＜0.05) of RFI.In this research in the whole expression values range of observation, relation between genetic expression and the RFI is also non-constant, for example genetic expression raises and may prolong relevantly with the RIF time length in the range of observation of a part, then shortens relevant with the RFI time length in the range of observation of different piece.

Table 7.2 has shown the gene of all displayings with interact neoplasm staging (p value＜0.1).The proportional hazard model that uses RFI is to the data modeling, with genetic expression, neoplasm staging, and their interaction as predictor.To have 0 positive tubercle but＜the nodular patient of 12 checks gets rid of from these are analyzed.

Claims (44)

1. a predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise determine obtaining in described experimenter's the biological sample that comprises cancer cells the evidence that one or more list in the expression level of predictability rna transcription thing among table 1A-B, 2A-B, 3A-B, 4A-B, the 5A-B, 6 and/or 7 or their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1A, 2A, 3A, 4A and/or the 5A, indicate the possibility of positive clinical effectiveness to reduce; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 2B, 3B, 4B and/or the 5B, indicate the possibility of positive clinical effectiveness to raise.

2. the process of claim 1 wherein the described experimenter patient that behaves.

3. the method for claim 2 wherein obtains the evidence of described expression level by the method for genetic expression preface type analysis.

4. the method for claim 3, wherein said method is the method for PCR-based.

5. the method for claim 4, wherein said expression level with respect to one or more with reference to the expression level of gene or their expression product by stdn.

6. the method for claim 2 is wherein according to recurrence-free interval (RFI), total lifetime (OS), disease free survival phase (DFS) or do not have long distance recurrence interval (DRFI) and represent described clinical effectiveness.

7. the method for claim 2, wherein said cancer is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

8. the method for claim 7, wherein said cancer is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

9. the method for claim 2 comprises the evidence of the expression level of determining at least two kinds of described genes or their expression product.

10. the method for claim 2 comprises the evidence of the expression level of determining at least three kinds of described genes or their expression product.

11. the method for claim 2 comprises the evidence of the expression level of determining at least four kinds of described genes or their expression product.

12. the method for claim 2 comprises the evidence of the expression level of determining at least five kinds of described genes or their expression product.

13. the method for claim 2 further comprises the step that generates the report that gathers described prediction.

14. a predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 1A and the 1B or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 1A or the 5A or corresponding expression product indicates the predicted meeting of described RFI shorter; With

(b) evidence that raises of one or more expression of listing in gene among table 1B or the 5B or corresponding expression product indicates the predicted meeting of described RFI longer.

15. the method for claim 14, the wherein said experimenter patient that behaves.

16. the method for claim 15 wherein obtains the evidence of described expression level by the method for genetic expression preface type analysis.

17. the method for claim 16, wherein said method is based on the method for PCR.

18. the method for claim 17 if the predicted meeting of wherein described RFI is shorter, is then further treated described patient behind described excision.

19. the method for claim 18, wherein said further treatment is chemotherapy and/or radiotherapy.

20. the method for claim 15 further comprises the step that generates the report that gathers described prediction.

21. the method for claim 15 comprises the evidence of the expression level of determining at least two kinds of described genes or their expression product.

22. the method for claim 15 comprises the evidence of the expression level of determining at least three kinds of described genes or their expression product.

23. the method for claim 15 comprises the evidence of the expression level of determining at least four kinds of described genes or their expression product.

24. the method for claim 15 comprises the evidence of the expression level of determining at least five kinds of described genes or their expression product.

25. a predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 2A and the 2B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2A, indicate the predicted meeting of described OS shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2B, indicate the predicted meeting of described OS longer.

26. a predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 3A and the 3B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3A, indicate the predicted meeting of described DFS shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3B, indicate the predicted meeting of described DFS longer.

27. a predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 4A and the 4B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4A, indicate the predicted meeting of described DRFI shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4B, indicate the predicted meeting of described DRFI longer.

28. a predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprise and determine that one or more are selected from ALCAM in described experimenter's the biological sample that comprises cancer cells in acquisition, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1, WIF, CAPG, CD28, CDC20, CKS1B, DKK1, the predictability rna transcription thing of HSD17B2 and MMP7 or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

29. a predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi C (III phase) colorectal carcinoma, comprise and determine that one or more are selected from CAPG in described experimenter's the biological sample that comprises cancer cells in acquisition, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7, SIR2, ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, the predictability rna transcription thing of TP53BP1 and WIF or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7 and SIR2 or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

30. a predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise determine obtaining in described experimenter's the biological sample that comprises cancer cells the evidence that one or more list in the expression level of predictability rna transcription thing among table 1.2A-B, 2.2A-B, 3.2A-B, 4.2A-B, the 5.2A-B, 6.2 and/or 7.2 or their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2A, 2.2A, 3.2A, 4.2A and/or the 5.2A, indicate the possibility of positive clinical effectiveness to reduce; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2B, 2.2B, 3.2B, 4.2B and/or the 5.2B, indicate the possibility of positive clinical effectiveness to raise.

31. a predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 1.2A, 1.2B, 5.2A and the 5.2B or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 1.2A or the 5.2A or corresponding expression product indicates the predicted meeting of described RFI shorter; With

(b) evidence that raises of one or more expression of listing in gene among table 1.2B or the 5.2B or corresponding expression product indicates the predicted meeting of described RFI longer.

32. a predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 2.2A and the 2.2B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2.2A, indicate the predicted meeting of described OS shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 2.2B, indicate the predicted meeting of described OS longer.

33. a predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 3.2A and the 3.2B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3.2A, indicate the predicted meeting of described DFS shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 3.2B, indicate the predicted meeting of described DFS longer.

34. a predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 4.2A and the 4.2B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4.2A, indicate the predicted meeting of described DRFI shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among the 4.2B, indicate the predicted meeting of described DRFI longer.

35. a predictive diagnosis has the method for experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise determine obtaining in described experimenter's the biological sample that comprises cancer cells the evidence that one or more list in the expression level of predictability rna transcription thing among table 1A-B, 1.2A-B, 2A-B, 2.2A-B, 3A-B, 3.2A-B, 4A-B, 4.2A-B, 5A-B, the 5.2A-B, 6,6.2,7 and/or 7.2 or their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 1A, 1.2A, 2A, 2.2A, 3A, 3.2A, 4A, 4.2A, 5A and/or the 5.2A or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression of listing in gene among table 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or the 5.2B or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

36. a predictive diagnosis has the method for experimenter's recurrence-free interval (RFI) time length behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 1A, 1.2A, 1B, 1.2B, 5A, 5.2A, 5B and the 5.2B or the expression level of their expression product, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1A, 1.2A, 5A or the 5.2A, indicate the predicted meeting of described RFI shorter; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 1.2B, 5B or the 5.2B, indicate the predicted meeting of described RFI longer.

37. a predictive diagnosis has the method for experimenter total lifetime (OS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 2A, 2.2A, 2B and the 2.2B or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 2A or the 2.2A or corresponding expression product indicates the predicted meeting of described OS shorter; With

(b) evidence that raises of one or more expression of listing in gene among table 2B or the 2.2B or corresponding expression product indicates the predicted meeting of described OS longer.

38. a predictive diagnosis has the method for experimenter's disease free survival phase (DFS) behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 3A, 3.2A, 3B and the 3.2B or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 3A or the 3.2A or corresponding expression product indicates the predicted meeting of described DFS shorter; With

(b) evidence that raises of one or more expression of listing in gene among table 3B or the 3.2B or corresponding expression product indicates the predicted meeting of described DFS longer.

39. a predictive diagnosis has the method for experimenter's no long distance recurrence (DRFI) time length interval behind the excision of described cancer of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in the predictability rna transcription thing among table 4A, 4.2A, 4B and the 4.2B or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression of listing in gene among table 4A or the 4.2A or corresponding expression product indicates the predicted meeting of described DRFI shorter; With

(b) evidence that raises of one or more expression of listing in gene among table 4B or the 4.2B or corresponding expression product indicates the predicted meeting of described DRFI longer.

40. a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more list in the predictability rna transcription thing among table 1A-B, 2A-B, 3A-B, 4A-B, the 5A-B, 6 and/or 7 or the expression level of their expression product in described experimenter's the biological sample that comprises cancer cells, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1A, 2A, 3A, 4A and/or the 5A, indicate the possibility of positive clinical effectiveness to reduce; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1B, 2B, 3B, 4B and/or the 5B, indicate the possibility of positive clinical effectiveness to raise.

41. a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more list in the predictability rna transcription thing among table 1.2A-B, 2.2A-B, 3.2A-B, 4.2A-B, the 5.2A-B, 6.2 and/or 7.2 or the expression level of their expression product in described experimenter's the biological sample that comprises cancer cells, wherein

(a) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2A, 2.2A, 3.2A, 4.2A and/or the 5.2A, indicate the possibility of positive clinical effectiveness to reduce; With

(b) one or more list in gene or the expression of the corresponding expression product evidence that raise of table among 1.2B, 2.2B, 3.2B, 4.2B and/or the 5.2B, indicate the possibility of positive clinical effectiveness to raise.

42. a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more list in the predictability rna transcription thing among table 1A-B, 1.2A-B, 2A-B, 2.2A-B, 3A-B, 3.2A-B, 4A-B, 4.2A-B, 5A-B, the 5.2A-B, 6,6.2,7 and/or 7.2 or the expression level of their expression product in described experimenter's the biological sample that comprises cancer cells, wherein

(a) evidence that raises of one or more expression of listing in gene among table 1A, 1.2A, 2A, 2.2A, 3A, 3.2A, 4A, 4.2A, 5A and/or the 5.2A or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression of listing in gene among table 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or the 5.2B or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

43. a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more are selected from ALCAM in described experimenter's the biological sample that comprises cancer cells, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1, WIF, CAPG, CD28, CDC20, CKS1B, DKK1, the predictability rna transcription thing of HSD17B2 and MMP7 or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDH11, CENPE, CLTC, CYR61, EMR3, ICAM2, LOX, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SIR2, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

44. a predictive diagnosis has the report of experimenter's clinical effectiveness behind the excision of described cancer of Du Ke Shi C (III phase) colorectal carcinoma, comprise prediction based on the clinical effectiveness of following information, described information is included in acquisition, and one or more are selected from CAPG in described experimenter's the biological sample that comprises cancer cells, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7, SIR2, ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, the predictability rna transcription thing of TP53BP1 and WIF or the expression level of their expression product, wherein

(a) evidence that raises of one or more expression that are selected from the gene of CAPG, CD28, CKS1B, CYR61, DKK1, HSD17B2, LOX, MMP7 and SIR2 or corresponding expression product indicates the possibility of positive clinical effectiveness to reduce; With

(b) evidence that raises of one or more expression that are selected from the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or corresponding expression product indicates the possibility of positive clinical effectiveness to raise.

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