CN101400804B - 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

Gene expression markers for 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), according to 35U.S.C. § 119 (e), require the provisional application serial number 60/758 of submitting on January 11st, 2006,392, the provisional application serial number 60/800 of submitting on May 12nd, 2006, the provisional application serial number 60/810 that on May 31st, 277 and 2006 submits to, 077 right of priority, all priority documents are all hereby incorporated by full with it.

Invention field

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

Background of invention

At the U.S. and European Union colorectal carcinoma, be the relevant cause of the death of cancer of position of being number two, account for 10% of all cancer associated deaths.Although colorectal carcinoma and the rectum cancer can represent same or analogous disease on molecular level, because anatomical problem is very complicated for 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 for the treatment of is obviously different.In about 100, the 000 routine colorectal carcinomas of the annual new diagnosis of the U.S., wherein approximately 65% be diagnosed as II/III phase colorectal carcinoma as discussed below.

The improvement of colorectum cancerous diagnose relates to the state of progress that utilizes criteria classification standard to evaluate cancer.Two kinds of categorizing systems have been widely used in colorectal carcinoma: Du Ke Shi (Duck) of improvement or Astler-Coller Staging System (A-D phase) (Astler VB, Coller FA., Ann Surg1954; 139:846-52), and the TNM Staging System of recently being developed by american cancer joint committee (I-IV phase) (AJCC CancerStaging Manual, the 6th edition, Springer-Verlag, New York, 2002).The measurement that these two kinds of systems are all passed primary tumor colon or rectum parietal layer arrival adjacent organs, lymphoglandula and distal site diffusion is applied to evaluate 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 has every year the new diagnosis of about 33,000 example in the U.S..Nearly all these patients treat by ocal resection, and in addition, approximately 40% patient adopts the chemotherapy based on 5 FU 5 fluorouracil (5-FU) to treat at present.The decision-making that whether gives auxiliary chemotherapy is not simple and direct.For the II phase colorectal cancer patients 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 there be being definitely benefited of 2-4%, and show serious toxicity, comprise up to 1% the chemotherapy rate of being poisoned to death.So, in the patient who accepts in a large number poisonous substance treatment, only there is minority to be benefited.

After the operation of II phase colorectal cancer patients, the test of energy prognosis should be a significant benefit to the treatment decision-making of instructing these patients.

Compare with the II phase, in III phase colorectal carcinoma, chemotherapeutical benefit is more obvious.Annual diagnosis in 31, the 000 routine patients that have III phase colorectal carcinoma has significant proportion to accept the auxiliary chemotherapy based on 5-FU, depends on adopted concrete therapeutic regimen, and under this background, 5-FU+ formyl tetrahydrofolic acid is definitely benefited as 18-24% left and right.(5-FU+ formyl tetrahydrofolic acid or 5-FU+ formyl tetrahydrofolic acid+oxaliplatin) processed in the current standard medical care chemotherapy of III phase colorectal cancer patients is moderate effective, has realized the improvement of five year survival rate from approximately 50% (only performing the operation) to approximately 65% (5-FU+ formyl tetrahydrofolic acid) or 70% (5-FU+ formyl tetrahydrofolic acid+oxaliplatin).Use separately 5-FU+ formyl tetrahydrofolic acid or treat with some disadvantageous side effects with oxaliplatin, comprising that about 1% treatment patient is poisoned to death.In addition, the III phase colorectal cancer patients for the treatment of for independent use operation, within 3 years, survival rate is approximately 47%, and cannot 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 the III phase patient subgroup that just can obtain acceptable result without assisting therapy.

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

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

(Genentech, Inc.) (a kind of exploitation for target HER2 medicine) only indicates having of confirming by fluorescence in situ hybridization (FISH) for those higher than normal HER2 copy number or the patient with high-level HER2 expression that confirms by immunohistochemistry.It expresses the gene relevant to clinical effectiveness in human cancer patient is the valuable source of selecting for the target thing of medical compounds screening and further drug development activity.

Molecular targeted agents such as

(Genentech, Inc.) can be developed and commercialization together with identifying the patient's that may benefit from this medicine diagnostic test; An aspect of this test is to identify that those do not take any treatment may have the patient of positive findings except operation.For example, when carrying out operative treatment separately, 80% II phase colorectal cancer patients existence 5 years or more of a specified duration.Identify more to belong to that not take its cancer of extra treatment be useful by the gene marker of the patient among 20% crowd of recurrence in drug development, for example, can be used for screening and be contained in the patient in 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 after 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, 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 the gene in 4A and/or 5A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1B, 2B, 3B, 4B and/or 5B or the evidence that the expression of expression product raises accordingly, indicate the possibility of positive clinical effectiveness to raise.If contained, the possibility of positive clinical effectiveness is predicted can be reduced, and after described excision, described patient is further treated.Further contain this treatment for chemotherapy and/or radiotherapy.

Can be for example according to recurrence-free interval (Recurrence-Free Interval, RFI), Overall survival (OverallSurvival, OS), disease free survival phase (Disease-Free Survival, DFS) or without long distance recur the clinical effectiveness that interval (Distant Recurrence-Free Interval, DRFI) represents 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 after 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, predictability rna transcription thing in 1B and/or 5B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 1A or 5A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described RFI shorter, (b) one or more list in the gene of showing in 1B or 5B or the evidence that the expression of expression product raises accordingly, 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's Overall survival (OS) after 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 predictability rna transcription thing in table 2A and/or 2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described OS shorter; (b) one or more list in the gene of showing in 2B or the evidence that the expression of expression product raises accordingly, 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) after 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 predictability rna transcription thing in table 3A and/or 3B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 3A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DFS shorter; (b) one or more list in the gene of showing in 3B or the evidence that the expression of expression product raises accordingly, 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 experimenter of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma after the excision of described cancer, without long distance, to recur the method for (DRFI) time length interval, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in predictability rna transcription thing in table 4A and/or 4B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 4A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DRFI shorter, (b) one or more list in the gene of showing in 4B or the evidence that the expression of expression product raises accordingly, 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 after 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, the evidence of the predictability rna transcription thing in 6.2 and/or 7.2 or the expression level of their expression product, wherein: (a) one or more list in table 1.2A, 2.2A, 3.2A, the evidence that the expression of the gene in 4.2A and/or 5.2A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1.2B, 2.2B, 3.2B, 4.2B and/or 5.2B or the evidence that the expression of expression product raises accordingly, 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 after 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, predictability rna transcription thing in 5.2A and/or 5.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 1.2A or 5.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described RFI shorter, (b) one or more list in the gene of showing in 1.2B or 5.2B or the evidence that the expression of expression product raises accordingly, 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's Overall survival (OS) after 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 predictability rna transcription thing in table 2.2A and/or 2.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 2.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described OS shorter; (b) one or more list in the gene of showing in 2.2B or the evidence that the expression of expression product raises accordingly, 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) after 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 predictability rna transcription thing in table 3.2A and/or 3.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 3.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DFS shorter, (b) one or more list in the gene of showing in 3.2B or the evidence that the expression of expression product raises accordingly, 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 experimenter of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma after the excision of described cancer, without long distance, to recur the method for (DRFI) time length interval, comprise that determine obtaining in described experimenter's the biological sample that comprises cancer cells one or more lists in predictability rna transcription thing in table 4.2A and/or 4.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 4.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DRFI shorter, (b) one or more list in the gene of showing in 4.2B or the evidence that the expression of expression product raises accordingly, 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 after 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 the gene in 5A and/or 5.2A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or 5.2B or the evidence that the expression of expression product raises accordingly, 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 after 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, predictability rna transcription thing in 5B and/or 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 the gene in 5A and/or 5.2A or corresponding expression product raises, indicate the predicted meeting of described RFI shorter, (b) one or more list in the gene of showing in 1B, 1.2B, 5B and/or 5.2B or the evidence that the expression of expression product raises accordingly, 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's Overall survival (OS) after 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 2A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 2.2A, predictability rna transcription thing in 2B and/or 2.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 2A and/or 2.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described OS shorter, (b) one or more list in the gene of showing in 2B and/or 2.2B or the evidence that the expression of expression product raises accordingly, 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) after 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, predictability rna transcription thing in 3B and/or 3.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 3A and/or 3.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DFS shorter, (b) one or more list in the gene of showing in 3B and/or 3.2B or the evidence that the expression of expression product raises accordingly, 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 experimenter of Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma after the excision of described cancer, without long distance, to recur the method for (DRFI) time length interval, one or more list in table 4A in described experimenter's the biological sample that comprises cancer cells to comprise definite acquisition, 4.2A, predictability rna transcription thing in 4B and/or 4.2B or the expression level of their expression product, wherein: (a) one or more list in the gene of showing in 4A and/or 4.2A or the evidence that the expression of expression product raises accordingly, indicate the predicted meeting of described DRFI shorter, (b) one or more list in the gene of showing in 4B and/or 4.2B or the evidence that the expression of expression product raises accordingly, 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 after the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprises and determines 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) one or more are selected from the evidence of the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or the expression rising of corresponding expression product, 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 clinical effectiveness after 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, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more are selected from the evidence of the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or the expression rising of corresponding expression product, indicate the possibility of positive clinical effectiveness to raise.

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

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

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

For all aspects of the present invention, the 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, the 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 patient is just accredited as and shows that the increment of clinical effectiveness increases.

For all aspects of the present invention, more than the determining of expression level can occur once.For all aspects of the present invention, expression level determine that can betide patient carries out any treatment after excision before.

One different aspect in, the present invention relates to the report that portion comprises experimenter that diagnosis has the colorectal carcinoma prediction clinical effectiveness after the excision of described cancer, comprise the prediction of the clinical effectiveness based on following information, described information is included in acquisition, and in described experimenter's the biological sample that comprises cancer cells, one or more list in table 1A-B, 2A-B, 3A-B, 4A-B, 5A-B, 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 the gene in 4A and/or 5A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1B, 2B, 3B, 4B and/or 5B or the evidence that the expression of expression product raises accordingly, indicate the possibility of positive clinical effectiveness to raise.Can be for example according to recurrence-free interval (RFI), Overall survival (OS), disease free survival phase (DFS) or represent the clinical effectiveness of report of the present invention without long distance recurrence interval (DRFI).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 and maybe can comprise based on described estimation experimenter is classified into risk group (risk group) the estimation of experimenter's specific clinical the possibility of result.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness after 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, the prediction of the clinical effectiveness of the information of the predictability rna transcription thing in 6.2 and/or 7.2 or the expression level of their expression product, wherein: (a) one or more list in table 1.2A, 2.2A, 3.2A, the evidence that the expression of the gene in 4.2A and/or 5.2A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1.2B, 2.2B, 3.2B, 4.2B and/or 5.2B or the evidence that the expression of expression product raises accordingly, indicate the possibility of positive clinical effectiveness to raise.Can be for example according to recurrence-free interval (RFI), Overall survival (OS), disease free survival phase (DFS) or represent the clinical effectiveness of report of the present invention without long distance recurrence interval (DRFI).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 and maybe can comprise based on described estimation experimenter is classified into risk group the estimation of experimenter's specific clinical the possibility of result.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness after 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 the gene in 5A and/or 5.2A or corresponding expression product raises, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more list in the gene of showing in 1B, 1.2B, 2B, 2.2B, 3B, 3.2B, 4B, 4.2B, 5B and/or 5.2B or the evidence that the expression of expression product raises accordingly, indicate the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise and maybe can comprise based on described estimation experimenter is classified into risk group the estimation of experimenter's specific clinical the possibility of result.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness after the excision of described cancer of Du Ke Shi B (II phase) colorectal carcinoma, comprises 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) one or more are selected from the evidence of the gene of CAPG, CD28, CDC20, CKS1B, DKK1, HSD17B2 and MMP7 or the expression rising of corresponding expression product, indicate the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise and maybe can comprise based on described estimation experimenter is classified into risk group the estimation of experimenter's specific clinical the possibility of result.

In one aspect of the method, the present invention relates to a predictive diagnosis has the report of experimenter's clinical effectiveness after 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, indicate the possibility of positive clinical effectiveness to reduce, (b) one or more are selected from the evidence of the gene of ALCAM, CD24, CDC20, CDH11, CENPE, CLTC, EMR3, ICAM2, MADH2, MGAT5, MT3, NUFIP1, PRDX6, SOS1, STAT5B, TFF3, TMSB4X, TP53BP1 and WIF or the expression rising of corresponding expression product, indicate the possibility of positive clinical effectiveness to raise.The prediction of clinical effectiveness can comprise and maybe can comprise based on described estimation experimenter is classified into risk group the estimation of experimenter's specific clinical the possibility of result.

One different aspect in, the present invention relates to a kind of test kit, comprise and be suitable for carrying out the following one or more of the inventive method: (1) Extraction buffer/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.

Accompanying drawing summary

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

Detailed Description Of The Invention

A. definition

Unless otherwise prescribed, technology used herein has the implication identical with those skilled in the art's common understanding with scientific terminology.The 2nd edition (J.Wiley & Sons of < < Dictionary ofMicrobiology and Molecular Biology > > of Singleton etc., New York, NY1994) and the < < Advanced Organic Chemistry Reactions of March, the 4th edition (John Wiley & Sons of Mechanisms andStructure > >, New York, NY1992) offer the general guide of the many terms that use in those skilled in the art the application.

Those skilled in the art will be appreciated that many method and materials that are similar to or are equivalent to described herein those can be used for enforcement of the present invention.In fact, the present invention is confined to absolutely not described 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 pernicious or optimum all neoplastic cell growths and propagation, and the front property of all cancers and cancerous cells and tissue.

Term " cancer " and " carcinous " point out or describe in Mammals conventionally to take the physiological status that not modulated Growth of Cells is feature.The example of cancer includes but not limited to mammary cancer, ovarian cancer, colorectal carcinoma, lung cancer, prostate cancer, hepatocellular carcinoma, cancer of the stomach, carcinoma of the pancreas, cervical cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid carcinoma, 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 abnormal or uncontrolled Growth of Cells, transfer, interference adjacent cells work orderly, with the abnormal level release cells factor or other secretion property product, inhibition or exacerbate inflammation or immunological response, tumorigenesis (neoplasia), precancer (premalignancy), malignant tumour (malignancy), invade around or remote organization or organ (such as lymphoglandula) etc.

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

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

Tumour: T1: tumour is invaded submucosa (submucosa); T2: tumour is invaded muscularis propria (muscularis propria); T3: tumour is invaded subserosa (subserose) through muscularis propria, or invade pericolic tissue or paraproctium; T4: tumour is directly invaded other organs or structure and/or perforation.

Tubercle: NO: without regional nodus lymphoideus transferring rate; In N1:1-3 regional lymphoglandula, there is transfer; In N2:4 or more regional lymphoglandula, there is transfer.

Shift: MO: shift without far-end; M1: have far-end to shift.

Stadium grouping: I phase: T1 NO MO; T2 NO MO; The II phase: T3 NO MO; T4 NO MO; The III phase: any T, N1-2; MO; The IV phase: any T, any N, M1.

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

The A phase: tumour penetrates intestinal mucosa but further do not penetrate; The B phase: tumour penetrates and run through the muscularis propria of intestines wall; The C phase: tumour penetrates but do not run through the muscularis propria of intestines wall, has the pathology evidence of colorectal carcinoma in lymphoglandula; Or tumour penetrates and run through the muscularis propria of intestines wall, in lymphoglandula, there is the pathology evidence of cancer; The D phase: tumour has diffused out lymphoglandula and entered other organs, such as liver, lung or bone.

Prognostic Factors be those to the natural history (natural history) of colorectal carcinoma once relevant they develop into the variable that colorectal carcinoma just affects patient's recurrence rate and result.Associated with worse prognosis clinical parameter comprises that for example lymphoglandula is got involved and senior tumour.Prognostic Factors is usually for being categorized as patient 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, comprises Preventive diffusion and the resistance of neoplastic disease (such as colorectal carcinoma).

Term " prediction " is used in reference to the possibility that after the excision of primary tumor patient will have specific clinical result (no matter being positive or negative) in this article.By selecting optimal treatment formula for any particular patient, Forecasting Methodology of the present invention can be clinically for making treatment decision-making.Forecasting Methodology of the present invention is valuable instrument in prediction patient possibility is advantageously replied treatment plan (such as surgical intervention).This prediction can comprise Prognostic Factors.

Term " positive clinical effectiveness " refers to be improved on any measuring of status of patient, comprise that in this area, normally used those are measured, such as recurrence-free interval (RFI) time length increase, Overall survival (OS) time increases, (DFS) time disease free survival phase increases, increase etc. without long distance recurrence (DRFI) time length interval.The rising of positive clinical effectiveness possibility is corresponding to the reduction of cancer return possibility.

Term " classification of risks " refers to that experimenter can experience risk level or the prediction of specific clinical result.Experimenter can be divided into risk group or classify on risk level based on Forecasting Methodology of the present invention." risk group " is one group of experimenter or individuality for specific clinical result with similar risk level.

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

Term " recurrence-free interval (RFI) " is in this article for pointing to the recurrence of the colorectal carcinoma for the first time primary cancer for the second time of initial event (on inspection as) or without the dead year time number of recurrence evidence.

Term " Overall survival (OS) " is used in reference to the dead year time number from operation to any reason in this article.

Term " disease free survival phase (DFS) " is in this article for pointing to the dead year time number of colorectal carcinoma recurrence or any reason.

Term " recurs interval (DRFI) without long distance " and is used in reference in this article from performing the operation for the first time the time of long distance cancer return (year number) anatomically.

Depend on and will examine or the definition of irrespective event, the calculating of listed observed value above can be different with research in practice.

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

Term " polynucleotide ", when using with odd number or plural number, is often referred to any polybribonucleotide or polydeoxyribonucleotide, and it can be RNA or the DNA of unmodified, or the RNA or the DNA that modify.So, for instance, polynucleotide as defined herein include but not limited to strand and double-stranded DNA, comprise the DNA of strand and double stranded region, strand and double-stranded RNA and comprise strand and the RNA of double stranded region and comprise DNA and the hybrid molecule of RNA, and DNA wherein and RNA can be strands or more generally double-stranded or comprise strand and double stranded region.In addition, when using in this article, three sequences that term " polynucleotide " refers to comprise RNA or DNA or comprises RNA and DNA simultaneously.Chain in this region can be from identical molecule or different molecules.This region can comprise the whole of one or more described molecules, but is only more typically a region of molecule described in some.One of the molecule in triple helix district is oligonucleotide normally.Term " polynucleotide " clearly comprises cDNA.This term comprises DNA (comprising cDNA) and the RNA of the base that contains one or more modifications.So, the DNA or the RNA that have because of stability or the adorned main chain of other reasons are " polynucleotide " that this term means herein.DNA or the RNA of the base (such as the base of tritiate) that in addition, comprises rare base (such as inosine) or modification are included in the scope of term " polynucleotide " as defined herein.Generally speaking, all chemistry, enzyme and/or the metabolism modified forms of the polynucleotide of unmodified contained in term " polynucleotide ", and the chemical species of virus and the distinctive DNA of cell (comprising simple cell and complex cell) and RNA.

Term " oligonucleotide " refers to relatively short polynucleotide, includes but not limited to strand deoxyribonucleotide, strand or double-stranded ribonucleotide, RNA:DNA heterocomplex and double-stranded DNA.Oligonucleotide (such as ssDNA probe oligonucleotide) synthesizes by chemical process conventionally, for example, use commercially available automated oligonucleotide synthesizer.Yet, can prepare oligonucleotide by multiple additive method, comprise the technology of extracorporeal recombinant DNA mediation and pass through expressible dna in Cell and organism body.

Term " gene of differential expression ", " genetic expression of difference " and their synonym are used interchangeably; finger is expression in normal or contrast experimenter with respect to it; its expression in ill (particularly cancer, such as colorectal carcinoma) experimenter is activated to the gene of higher or lower level.This term also comprises that its expression in the different steps of same disease is activated to the gene of higher or lower level.The gene that it is to be further understood that differential expression can be activated or suppress on nucleic acid level or protein level, maybe can carry out alternative splicing to produce different polypeptide products.Such difference can be by for example mRNA level, polypeptide surface expression, secretion or other distribute the change of (partitioning) to be confirmed.The genetic expression of difference can comprise the expression between two or more genes of comparison or their gene product, or the expression ratio between two or more genes of comparison or their gene product, or two kinds of different processed products of same gene even relatively, it is different between the different steps between normal subjects and ill (particularly cancer) experimenter or in same disease.Differential expression is for example included between normal and diseased cells or at the iuntercellular that has experienced various disease event or disease stage, and gene or its expression product have quantitative and difference qualitatively in graphic at time or cell expressing.For the purposes of the present invention, in the various disease developmental stage between the expression of given gene in normal and ill experimenter or ill experimenter, exist at least about 2 times, preferably at least about 4 times, more preferably at least about 6 times, during most preferably at least about 10 times of difference, just think and have " genetic expression of difference ".

About the term of rna transcription thing, " crossing expression " and be used in reference to by the transcript level to measuring with reference to mRNA level standard, can be all measured transcripts or specific mRNA reference set in sample with reference to mRNA.

Phrase " gene amplification " refers to form the gene of multiple copied or the process of gene fragment in specific cells or clone.Duplicate field (section of the DNA of amplification) is commonly referred to " amplicon ".Generally speaking, the amount of the messenger RNA(mRNA) producing (mRNA), i.e. the level of genetic expression, also increases in the ratio of the copy number being formed by expressed specific gene.

" severity " of hybridization easily determined by those of ordinary skills, and conventionally depend on probe length, wash temperature and salt concn calculate by rule of thumb.Generally speaking, longer probe needs higher temperature for correct annealing, and shorter probe needs lower temperature.In the time of in complementary strand is present in lower than the environment of they melting temperature(Tm)s, the ability that denatured DNA is annealed is again depended in hybridization conventionally.Expectation homology degree between probe and the sequence that can hybridize is higher, just can use higher relative temperature.As a result of, release, higher relative temperature trends towards making reaction conditions stricter, and lower temperature is more not strict.For the subsidiary details of the severity of hybridization and explain the < < CurrentProtocols in Molecular Biology > > referring to Ausubel etc., Wiley IntersciencePublishers, (1995).

As defined herein, " stringent condition " or " high stringent condition " is generally: (1) is used 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) are used 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 ℃, at 42 ℃, in 0.2x SSC (sodium chloride/sodium citrate) and 50% methane amide, wash, then at 55 ℃ of height that formed by the 0.1x SSC that contains EDTA, strictly wash.

" medium stringent condition " can be stipulated as < < Molecular Cloning:ALaboratory Manual > > (the New York:Cold Spring Harbor Press of 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, 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, then in about 37-50 ℃, in 1x SSC, wash filter membrane.Those skilled in the art will be appreciated that how according to the needs that adapt to such as factors such as probe length, to regulate temperature, ionic strength etc.

In the context of the present invention, list in " at least one ", " 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 is negative " cancer, such as " tubercle is negative " 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 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 mature rna product.In theory, term " intron " refer to transcribed but by by the exon montage that is 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 is by numbering to limit 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 on their 5 ' and 3 ' border, have GT and AG montage consensus sequence.

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

B.1 general description

Unless otherwise stated, enforcement of the present invention will adopt conventional Protocols in Molecular Biology (comprising recombinant technology), microbiological technique, cytobiology technology and Measurement for Biochemistry, and these technology are all within art technology scope.Such technology is explained fully in following document, 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 & C.C.Blackwell compiles Blackwell Science Inc., 1987); " Gene Transfer Vectors forMammalian Cells " (J.M.Miller & M.P Calos compiles 1987); " Current Protocols inMolecular Biology " (F.M.Ausubel etc. compile 1987); And " PCR:The PolymeraseChain Reaction ", (Mullis etc. compile 1994).

Evidence based on rna transcription thing differential expression in normal cell and cancer cells, the invention provides the prognosis gene marker 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 tumour progression degree, reflect tumour cell in the mark of basic difference.Prognostic marker provided by the present invention and relevant information make doctor make wiser treatment conclusion and need to customize colorectal carcinoma treatment for individual patients and are able to possibility, make thus to treat benefit and reach and maximize and make patient to be exposed to unnecessary treatment to reduce to minimum, this unnecessary treatment does not provide any major benefit and because toxic side effect carries serious danger conventionally.

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.In particular physiological process, dysfunction is characterized fully to the Relative Contribution of the pathology of particular cancers type.Any physiological process all combines the contribution that relates to the expressed several genes product of the various kinds of cell of this process.For example, tumour cell is invaded and in adjacent healthy tissues and tumour cell, is infiltrated the impact that the recycle system is all subject to the protein of the different cell characteristics of a collection of mediation, described cell characteristic comprise adhesion, the tumour cell of adhesive aggregation, tumour cell and normal cell between tumour cell and reticular tissue first change its form then migration through the ability of connective tissue around the ability of surrounding tissue and tumour cell degraded.

Multiple analyte genetic expression test can be measured several important physiological processs or be formed the expression level of each one or more gene that involve in 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 to be calculated as follows score, are somebody's turn to do to obtain the expression values of the indivedual genes of proportion by subtraction and result height correlation more.For example, the quantitative score (recurrence score) of calculating predicting recurrence possibility in estrogen receptor positive, tubercle negative breast cancer is recorded in common unsettled U.S. Patent application (publication number 20050048542).Can be by Genotypic subgroup to make the predictor that recurs score reach maximum for calculating the equation of such recurrence score.Can be at least partly based on they are carried out to Genotypic subgroup to the understanding of the contribution of physiological function or formation cell characteristic (as discussed above all).In addition, grouping information can be beneficial to the contribution of the various expression values of mathematics weighting to recurrence score.The weighting that represents the gene group of physiological process or formation cell characteristic can reflect this process or the contribution of feature to cancer pathology and clinical effectiveness.Thereby, one important aspect in, the present invention also provides the concrete grouping of the prognosis gene identified herein, indivedual genes or the random combine of the gene of identifying with this are compared, and are above-mentionedly grouped into more reliable and more effective prediction of result device.

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

The effectiveness of gene marker in prediction colorectal carcinoma result may not only have this mark just to have.There is the graphic optional mark of the expression closely similar to fc-specific test FC mark and can replace this test mark, or can test use and very little on the macro-forecast effectiveness impact of this test outside mark at this.The close similar expression of two kinds of genes is graphic derives from that two kinds of genes involve same particular procedure in colon tumor cell and/or under common adjusting is controlled.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 the medicine that exploitation is used for the treatment of II phase and/or 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 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 the sample of collecting patient in clinical trial, and test result is used together with patient's result to determine and to compare with whole patient's group or other subgroups, which patient's subgroup more likely or the more impossible response demonstrating 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, if if test result points out only to carry out patient with operation, can to have the possibility of bad clinical effectiveness higher, patient is more likely included in clinical trial, if and if test result point out only to carry out patient with operation can to have the possibility of bad clinical effectiveness lower, patient can not be included in clinical trial.

In a specific embodiment, prognostic marker and relevant information are for designing 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 for (screening) assay method to identify medical compounds, and wherein said medical compounds is for developing the medicine for the treatment of II phase and/or III phase colon and/or the rectum cancer.

In each embodiment of the present invention, can utilize various technological methods to measure the expression level of disclosed gene, 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 discuss in detail in below.In specific embodiment, can with respect to the different characteristics of gene expression product, measure (comprising exon, intron, protein epitope and protein active) expression level of every kind of gene.In other embodiments, the expression level deducibility of gene from gene structure analysis, for example, is inferred the Pattern analysis that methylates from gene promoter.

B.2 genetic expression order type analysis

The method of genetic expression order type analysis comprises method, method and the method based on protein science based on polynucleotide order-checking of analyzing based on multi-nucleotide hybrid.The quantitative method of the most often using of mrna expression in sample that becomes known in this area comprises Northern trace and in situ hybridization (Parker & 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)).Or, can adopt can recognition sequence specific double-strand body the antibody of (comprising DNA duplex, RNA duplex and DNA-RNA heteroduplex body or DNA-protein duplex).The exemplary process of the gene expression analysis based on order-checking comprises the gene expression analysis of serial analysis of gene expression (SAGE) and check order by extensive parallel signature (MPSS).

A. reverse transcriptase PCR (RT-PCR)

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

The first step is from target sample separation mRNA.Parent material is typically respectively from total RNA of people's tumour or tumor cell line and corresponding healthy tissues or clone separation.So, can be from multiple primary tumor isolation of RNA, comprise tumour or the 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 primary tumor, mRNA can for example, extract from for example tissue sample of paraffin embedding freezing or filing and fixing (formalin is fixed) so.

For extracting the universal method of mRNA, be well-known in the art, be disclosed in molecular biological standard textbook, comprise the people such as Ausubel, < < Current Protocols of Molecular Biology > >, John Wiley and Sons, 1997.For extract the method for RNA from paraffin-embedded tissue, be disclosed in for example Rupp and Locker, Lab Invest.56:A67 (1987); De Andr é s et al., Bio Techniques18:42044 (1995).Particularly, RNA separation can be used purification kit, damping fluid group and proteolytic enzyme from commercial manufacturers such as Qiagen to carry out according to the specification sheets of manufacturers.For example, the total RNA from culturing cell can be used Qiagen RNeasy micro-column separated.Other commercialization RNA separating kit comprises global DNA and RNA purification kit (

madison, WI) and paraffin mass RNA separating kit (Ambion, Inc.).Total RNA from tissue sample can be used RNA Stat-60 (Tel-Test) separation.The RNA preparing from tumour can come separated by for example cesium chloride density gradient centrifugation.

Due to the template that RNA can not serve as PCR, the first step of carrying out genetic expression order type analysis by RT-PCR is that RNA template reverse transcription is become to cDNA, is then its exponential amplification in 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).Reverse transcription step is typically used Auele Specific Primer, random sexamer or oligo-dT primer to cause, and depends on circumstances and the target of expressing order type analysis.For example, the RNA of extraction can be used GeneAmp RNA PCR test kit (Perkin Elmer, CA, USA) to follow the specification sheets reverse transcription of manufacturers.Then can be using derivative cDNA as template for follow-up PCR reaction.

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

pCR typically utilizes 5 of Taq or Tth polysaccharase '-nuclease to be hydrolyzed the hybridization probe of being combined with its target amplicon, but can use have equivalence 5 ' nuclease any enzyme.With two kinds of Oligonucleolide primers, generate amplicon, be typically PCR reaction.The third oligonucleotide or probe design are for detection of the nucleotide sequence between first two PCR primer.Probe is that TaqDNA polysaccharase is inextensible and with report fluorescence dye and cancellation fluorochrome label.Two kinds of dyestuffs as them on probe position near time, any laser induced transmitting from report dyestuff is subject to the cancellation of quencher dyes.During amplified reaction, Taq archaeal dna polymerase cuts probe in template dependency mode.Gained probe fragment dissociates in solution, is no longer subject to the effect of the quenching effect of the second fluorophore from the signal of discharge report dyestuff.Often 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 quantitative is set forth.

rT-PCR can carry out by commodity in use equipment, such as for example ABI PRISM

sequence detection system TM (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).In a preferred embodiment, on real-time quantitative PCR device, move 5 ' nuclease rules, such as ABI PRISM

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

5 '-nuclease assay method data are expressed as Ct at first, or cycle threshold (threshold cycle).As discussed above, each cycle period record fluorescent value and represent in amplified reaction this time amplification the amount of product.First record is cycle threshold (Ct) to the point that has the fluorescent signal of statistical significance.

Minimum for impact wrong and sample room variation is down to, conventionally use internal standard thing to carry out RT-PCR.Desirable internal standard thing is expressed with constant level between different tissues, the impact that not processed by experiment.RNA for genetic expression style standards is the mRNA of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P-Actin muscle the most frequently.

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

probe) measure the accumulation of PCR product.PCR in real time and quantitative competitive PCR (wherein with the internal competition thing of every kind of target sequence, carrying out stdn) and quantitative comparison PCR (the stdn gene comprising in use sample or housekeeping gene are for RT-PCR's) 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 and have used setup action RNA that fix, paraffin-embedded source for the step of the representative solution of genetic expression order type analysis, 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 started by the approximately 10 micron thickness sections that cut out paraffin-embedded neoplasmic tissue sample.Then, extract RNA, and except deproteinize and DNA.Analyze after 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.

B.MassARRAY system

At Sequenom, Inc. (San Diego, CA) in the genetic expression order type analysis method based on MassARRAY of exploitation, after isolation of RNA reverse transcription, in obtained cDNA, mix synthetic DNA molecular (competition thing), it mates with the cDNA district of target in all positions except single base, and serves as internal standard thing.This cDNA/ competition thing mixture is carried out to the pcr amplification shrimp alkaline phosphotase (SAP) after performing PCR of going forward side by side and process, it causes the dephosphorylation of residual nucleus thuja acid.After alkaline phosphatase enzyme-deactivating, the PCR product from competition thing and cDNA is carried out to primer extension, it produces for competition thing-and derivative distinct a large amount of signals of PCR product (mass signal) of cDNA-.After purifying, these products are assigned on chip array, this chip array is pre-loaded has the laser desorption ionisation flight time mass spectrum (MALDI-TOF MS) of use Matrix-assisted to analyze needed composition.Then in the mass spectrum producing by analysis, the ratio of peak area is quantitative to the cDNA existing in reaction.More details are referring to for example Dingand Cantor, Proc.Natl.Acad.Sci.USA100:3059-3064 (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)); Pearl array (BADGE) for detection of genetic expression, the microballoon of commodity in use Luminex100LabMAP system and multi-coloured codes (Luminex Corp. in genetic expression rapid test method, Austin, TX) (Yang et al., Genome Res.11:1888-1898 (2001)); And high covering expressed order type analysis (HiCEP) (Fukumura et al., Nucl.Acids.Res.31 (16) e94 (2003)).

D. microarray

Differential gene expression also can be identified or verify with microarray technology.So, can use microarray technology in fresh or paraffin-embedded tumor tissues, to measure the expression order type of colorectal carcinoma genes involved.In this method, coating (plate) or arrangement (array) polynucleotide sequence interested (comprising cDNA and oligonucleotide) on microchip substrate.Then make the sequence of arranging hybridize with the specificity DNA probing needle from cells of interest or tissue.As in RT-PCR method, the source of mRNA is typically from total RNA of people's tumour or tumor cell line and corresponding healthy tissues or clone separation.So, RNA can be separated from multiple primary tumor or tumor cell line.If the source of mRNA is primary tumor, mRNA can for example, extract from for example tissue sample of paraffin embedding freezing or filing and fixing (formalin is fixed) so, and described tissue sample is that in daily clinical practice, routine is prepared and preserves.

In a specific embodiments of microarray technology, the cDNA clone's of pcr amplification 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 being fixed on 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.Strictly clean to remove after the probe of non-specific binding, by confocal laser microscopy or by other detection method such as CCD photographic camera scanning chip.The abundance of quantitatively allowing assessment corresponding mRNA of the hybridization of every kind of array composition.Rely on dual coloured fluorescence, by separately cDNA probe and the array paired cross of mark that generate from two kinds of RNA sources.So measure the relative abundance from the transcript in two kinds of sources corresponding to every kind of appointment gene simultaneously.The hybridization of miniaturization scale provides lots of genes to express facility and the rapid evaluation of pattern.These class methods demonstrated detect the desired sensitivity of rare transcript expressed with each cell minority copy and reproducible detection expression level at least about 2 times of differences (Schena et al., Proc.Natl.Acad.Sci.USA93 (2): 106-149 (1996)).Microarray analysis can commodity in use equipment be 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 cancer classification in systematic search kinds of tumors type likely and the molecular marker of prediction of result.

E. serial analysis of gene expression (SAGE)

Serial analysis of gene expression (SAGE) is a kind of method of allowing while and quantitative analysis lots of genes transcript and not needing to provide for every kind of transcript indivedual hybridization probes.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, if this label is to obtain from a unique position in every kind of transcript.Then, many transcripts are coupled together to form long, continuous molecule, they can check order, to disclose the identity (identity) of multiple label simultaneously.Any transcript group's expression pattern can be by measuring the abundance of distinguishing label and identifying that the gene corresponding with every kind of label carrys out qualitative assessment.More details are referring to for example Velculescu et al., Science270:484-487 (1995); Velculescu et al., Cell88:243-51 (1997).

F. by the massive parallel gene expression analysis that order-checking (MPSS) carries out of signing

By Brenner et al., this method that Nature Biotechnology 18:630-634 (2000) records is by a kind of sequence measurement that the body outer clone on the 5 micron diameter microballons that separate is joined together with millions of template of the order-checking of the signature based on gel not.First, by the microballon storehouse of body outer clone constructed dna template.Then, in the groove that flows, with high-density, (be typically and be greater than 3x10 ⁶microballon/cm ²) assembling is containing 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 the tens of thousands gene signature sequence from yeast cDNA library.

G. immunohistochemical methods

ImmunohistochemistryMethods Methods is also applicable to detect the expression level of 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, use radioactively labelled substance, fluorescent marker, hapten-marked thing such as vitamin H or enzyme such as horseradish peroxidase or alkaline phosphatase.Or unmarked primary antibodie is combined use with through mark two is anti-, comprises the specific antiserum(antisera) of primary antibodie, polyclonal antiserum or 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 sometime all of the middle protein existing of sample (for example tissue, organism or cell culture).Proteomics comprises the global change (also referred to as " expression proteomics ") of protein expression in study sample etc.Proteomics typically comprises the following steps: that (1) by two-dimensional gel electrophoresis (2-D PAGE) by the range protein in sample separately; (2) identify the range protein reclaiming from gel, for example, by mass spectrum or N-end sequencing; And (3) are used bioinformatic analysis data.Proteomics method is that the useful of other genetic expression order type analysis method supplemented, and can use separately or combine other method, for detection of the product of prognostic marker of the present invention.

I. promoter methylation analysis

Discussed in this article multiple for to rna transcription thing (gene expression analysis) or their the quantitative method of protein translation product.Also can be according to infer the expression level of gene about the information of chromatin Structure, such as for example gene promoter and the methylation state of other regulatory element and the acetylize state of histone.

Particularly, the impact of the methylation state of promotor is subject to the expression level of the gene of this promotor adjusting.The abnormal methylation of specific gene promotor relates to expresses adjusting, such as the silence of for example tumor suppressor gene.So, the inspection of gene promoter methylation state be can be used as to the surrogate that rna level directly quantizes.

Several have been designed for measuring the method for 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) with 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, technology based on microarray is for characterizing 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.With statistical analysis known in the art, analyze expression data, can express cluster by identified gene thus, comprise that the dependency based on Pearson correlation coefficient is analyzed in pairs (Pearson K.and Lee A. (1902) Biometrika2,357).

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

In another embodiment, the expression cluster of identifying herein comprises the gene of ANXA2, KLK6, KLK10, LAMA3, LAMC2, MASPIN, SLPI and other coding epithelial cells product, 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 identifying herein comprises and DUSP1, EGR1, EGR3, FOS, NR4A1, RHOB and other is exposed to some at cell it transcribes the gene of early stage rise after stimulating.Multiple stimulation triggers transcribing of early stage response gene, for example, be exposed to somatomedin, and it can make cell increase fast their mobility and the ability of their transhipment nutrient such as glucose.This expression cluster is referred to herein as replys cluster in early days.

In another embodiment, the expression cluster of identifying herein comprises MCP1, CD68, CTSB, OPN and other coding gene of the protein relevant to immune cell conventionally.This expression cluster is referred to herein as immune cluster.

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

K.mRNA is separated, the generality of purification and amplification is described

The many parts of journal articles of delivering have provided and have used setup action RNA that fix, paraffin-embedded source for the step of the representative solution of genetic expression order type analysis, comprise mRNA separation, purifying, primer extension and amplification (T.E.Godfrey et al. for example, J.Molec.Diagnostics2:84-91 (2000); K.Spechtetal., Am.J.Pathol.158:419-29 (2001)).In brief, a kind of exemplary process is started by the approximately 10 micron thickness sections that cut out paraffin-embedded neoplasmic tissue sample.Then, extract RNA, and except deproteinize and DNA.Analyze after 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.Finally, the characteristic genetic expression pattern based on identifying in checked tumor sample, according to the prediction possibility of cancer return, analytical data is to identify the optimum handling option useful to patient.

1. colorectal carcinoma gene sets, the gene polyadenylation signal sequence of measuring 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 measure provides prognosis information.For this object, must proofread and correct the amount of the RNA that (stdn) measure and the difference of the quality variation of the RNA that uses.Therefore, some stdn gene is measured and mixed to this assay method conventionally, comprises well-known housekeeping gene, such as the expression of GAPDH and Cyp1.Or, can signal average or intermediate value (Ct) based on all determined genes or its large-scale sub-collection carry out stdn (global criteria method).For each gene, existing amount in the amount of measured standardized patient tumors mRNA and colon cancer tissue reference set is compared in turn.In this reference set, the number of colon cancer tissue (N) should be enough high substantially consistent to guarantee different reference set (generally) behavior performance.If meet this condition, the relative quantity of the gene that the identity that is present in so the single colon cancer tissue in specific collection just can remarkably influenced not be measured.Generally speaking, colon cancer tissue reference set by least about 30, preferably at least about 40 different FPE colon cancer tissue samples, form.Unless otherwise directed, the tumour/patient's of each mRNA/ test normalized expression level should be expressed as the percentage ratio of the expression level recording in reference set.More particularly, the tumour number distribution that enough reference set of high (for example 40) has produced each mRNA kind standardization.In specific tumors sample to be analyzed, measured level drops on a certain hundredths within the scope of this, and it can be measured by method well-known in the art.Below, unless otherwise directed, while mentioning the expression level of gene, although always do not stipulate clearly, but supposition is the normalized expression with respect to reference set.

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

According to one aspect of the present invention, PCR primer and probe are that the intron sequences based on existing in gene to be amplified designs.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., 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 completely.

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 realizing by Repeat Masker program, this program can be passed through Baylor College Medicine (Baylor College of Medicine) and obtain online, and it is for repeat element library screening DNA sequence dna and return to the search sequence of wherein having covered repeat element.Then can use any commercialization or by the obtainable primer/probe design of other approach public bag by the intron sequences after covering for designing primer and probe sequence, such as Primer Express (Applied Biosystems); MGBassay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J.Skaletsky (2000) Primer3 on World Wide Web for general user and biologist programmer.At Krawetz S, in the < < Bioinformatics Methods and Protocols:Methods inMolecular Biology > > that Misener S compiles, Humana Press, Totowa, N.J., 365-386 page).

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

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, in A Laboratory Manual > >, Cold Spring Harbor LaboratoryPress, New York, 1995, the 133-155 pages; Innis and Gelfand, " Optimization ofPCRs " (optimization of PCR), at < < PCR Protocols, in A Guide to Methods and Applications > >, CRC Press, London, 1994, the 5-11 pages; And Plasterer, T.N., Primerselect:Primerand probe design.Methods Mol.Biol.70:520-527 (1997), is clearly collected herein by reference its complete disclosure.

N. test kit of the present invention

Material for method of the present invention is suitable for the test kit that preparation produces according to well-known rules.So, the invention provides the test kit that comprises reagent, described reagent can comprise for to for predicting quantitative gene specific or gene Selection probe and/or the primer of expression of the disclosed gene of prognosis result or treatment response.This type of test kit optionally comprises for extracting the reagent of RNA and/or the reagent increasing for RNA from tumor sample (particularly fixing, paraffin-embedded tissue sample).In addition, test kit optionally comprises reagent and differentiates 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 automated execution the inventive method), each container is equipped with one or more all ingredients for the inventive method (conventionally existing with the form concentrating), comprises for example prefabricated microarray, 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).For evaluate or quantitatively the mathematical algorithm of prognosis or information of forecasting be suitably also the possible composition of test kit.

O. report of the present invention

When being implemented for commercialization diagnostic purpose, method of the present invention produces report or the summary of a normalized expression level about one or more selected genes conventionally.Method of the present invention will produce a report that diagnosis has experimenter's prediction of clinical effectiveness after the excision of described cancer of colorectal carcinoma that comprises.Method of the present invention and report can further comprise report is stored in database.Or the 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 part, 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, at prognosis or prediction experiment, comprise a limited number of other gene with disclosed gene co-expressing, the gene for example confirming by high Pearson (Pearson) relation conefficient.

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

Embodiment

Explore the research that genome tumour is expressed dependency between order type and Du Ke Shi B treating by colectomy and Du Ke Shi C patient's recurrence possibility for one

The main purpose of this research be to be determined at 757 kinds of amplicons identifying in table B each expression and accept colectomy (operation) but do not carry out the chemotherapeutical II phase and the clinical effectiveness of III phase colorectal cancer patients between whether there is significant dependency.

Research and design

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

comprise standard

Patients join NSABP study C-01: "Evaluation of postoperative immunotherapy clinical trials and postoperative systemic chemotherapy in resectable colon cancer management (A, Clinical, Trial, To, Evaluate, PostoperativeImmunotherapy, And, Postoperative, Systemic, Chemotherapy, In, The, ManagementOf, Resectable, Colon, Cancer) "or NSABP study C-02:" colon adenocarcinoma were evaluated in 5 - fluorouracil and portal vein infusion of heparin solution (A, Protocol, To, Evaluate, ThePostoperative, Portal, Vein, Infusion, Of ,5-Flourouracil, And, Heparin, InAdenocarcinoma, Of, The, Colon) ".The details of C-01 and C-02 are positioned at the NSABP website of following URL as seen:

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

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

exclusion standard

If applicable following one or more, will add the patient of NSABP research C-01 or NSABP research C-02 to get rid of outside research of the present invention:

The tumor mass that there is no Newly diagnosed in NSABP archives.

When evaluating by phenodin and eosin (H & E) slide glass inspection, in region, tumour is not enough.

Self-organization section RNA that reclaim, that analyze for RT-PCR not enough (<700ng).

In 1943 patients that add NSABP research C-01 or NSABP research C-02, it is obtainable and for disclosed herein gene expression research after application exclusion standard, having 270 patient's samples.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 for expression analysis, comprise that 7 kinds with reference to gene.These genes with for qRT-PCR, to measure together with the primer of expression level and the sequence of probe, list in Table A.

Experiment material and method

Use

rT-PCR is used as the expression with reference to the gene of gene to 750 kinds of cancer related genes of every patient's quantitative evaluation and 7 kinds of appointments, should

rT-PCR is single-threaded to carry out, every secondary response input 1ng RNA.

Data analysing method

reference standard

For 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.The expression observed value of gained reference standard is conventionally in the scope of 0-15, and wherein the increase of 1 unit has reflected 2 times of increases of RNA quantity conventionally.

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 this distributes without significant difference clinically.

univariate analysis

For research in 757 kinds of amplicons in each, we check the dependency between genetic expression and recurrence-free interval (RFI) with Cox proportional hazard model.Check item by likelihood ratio as significance,statistical.Method (the Benjamini of application 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) and 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, Kom 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 evaluate false discovery rate (false discoveryrate).For each selectable terminal: repeat all analyses without long distance recurrence interval (DRFI), Overall survival (OS) and disease free survival phase (DFS).

multivariate analysis

For research in 757 kinds of amplicons in each, we check the dependency between genetic expression and RFI with Cox proportional hazard model, control the impact of (comprising tumor-localizing, type of surgery, tumor grade, the lymphatic node number detecting and the number of positive lymphatic node) of other standard clinical concomitant variable 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 each in 757 kinds of amplicons in research, our other funtcional relationship between using several different methods research genetic expressions and recurring.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 to 2DF likelihood ratio test, model evaluates significance,statistical.The pattern of (level and smooth) halter strap residual error (Martingale residuals) also deriving as the single argument Cox proportional hazard model of the RFI of the strictly linear function of genetic expression by inspection carrys out Study Function relation (GrayRJ (1992) Flexible methods for analyzing survival data using splines, 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, by the accumulation of the halter strap residual error from each identical Cox proportional hazard model with for detection of (the Lin D that departs from apart from linear, Wei L, Ying Z. (1993) Checking the Cox Model with Cumulative SumsofMartingale-Based Residuals.Vol.80, No.3,557-572).

With interaction by stages

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

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

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

Single order analysis and research result

Reference gene sets for single order analysis is CLTC, FZD6, NEDD8, RPLPO, RPS13, UBB, UBC.

Table 1A has shown based on the analysis of single argument Proportional hazards, 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 analysis of single argument Proportional hazards, 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 analysis of single argument Proportional hazards, the association of the gene that expressing for those its raises has predicted the overall survival (OS) reducing.

Table 2B has shown based on the analysis of single argument Proportional hazards, the association of the gene that expressing for those its raises has predicted the overall survival (OS) raising.

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

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

Table 4A has shown based on the analysis of single argument Proportional hazards, expresses the association that the shorter gene without long distance recurrence interval (DRFI) has been predicted in rising for those its.

Table 4B has shown based on the analysis of single argument Proportional hazards, expresses the association that the longer gene without long distance recurrence interval (DRFI) has been predicted in rising for those its.

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, associated 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, associated between genetic expression and RFI.

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

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

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

Table 1A

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

Table 1B

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

Table 2A

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

Table 2B

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

Table 3A

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

Table 3B

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

Table 4A

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

Table 4B

Table 5A shown and controlled particular demographic and the Clinical symptoms that is included in the patient in this analysis, associated between genetic expression and RFI.Having listed it expresses relevant to RFI (p<0.1) and in the multivariate analysis that comprises following variable, shows risk than all genes of >1: the tubercle of tumor-localizing, operation, tumor grade, check and the number of positive tubercle.

Table 5A

Table 5B shown and controlled particular demographic and the Clinical symptoms that is included in the patient in this analysis, associated between genetic expression and RFI.Having listed it expresses relevant to RFI (p<0.1) and in the multivariate analysis that comprises following variable, shows risk than all genes of <1: the tubercle of tumor-localizing, operation, tumor grade, check and the number of positive tubercle.

Table 5B

Table 6 has shown the non-linear ratio's venture analysis based on using 2DOF natural spline, associated between genetic expression and clinical effectiveness.All genes that departed from the exact linear relationship (p<0.05) of RFI that show have been listed in the II phase of combination (Du Ke Shi B) and III phase (Ke Shi C shuts out) patients.In this research in whole expression values range of observation, relation between genetic expression and RFI is also non-constant, for example in a part of range of observation, genetic expression raises and may extend relevantly with the RIF time length, in the range of observation of different piece, shortens relevant with the RFI time length.

Table 6

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

Table 7

Second order analysis result of study

Reference gene sets for second order analysis is ATP5E, CLTC, GPX1, NEDD8, PGK1, UBB.

Table 1.2A has shown based on the analysis of single argument Proportional hazards, 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 analysis of single argument Proportional hazards, 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 analysis of single argument Proportional hazards, the association of the gene that expressing for those its raises has predicted the overall survival (OS) reducing.

Table 2.2B has shown based on the analysis of single argument Proportional hazards, the association of the gene that expressing for those its raises has predicted the overall survival (OS) raising.

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

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

Table 4.2A has shown based on the analysis of single argument Proportional hazards, expresses the association that the shorter gene without long distance recurrence interval (DRFI) has been predicted in rising for those its.

Table 4.2B has shown based on the analysis of single argument Proportional hazards, expresses the association that the longer gene without long distance recurrence interval (DRFI) has been predicted in rising for those its.

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

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

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

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

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

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

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

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

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

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

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

Table 5.2A shown and controlled particular demographic and the Clinical symptoms that is included in the patient in this analysis, associated between genetic expression and RFI.Having listed it expresses relevant to RFI (p<0.1) and in the multivariate analysis that comprises following variable, shows risk than all genes of >1: tumor-localizing, the operation time, tumor grade, treatment plan (C-01 or C-02), BCG treatment (be or no), and as follows according to patient's classification of lymph node status: the tubercle of 0 positive tubercle and <12 check, the tubercle of 0 positive tubercle and >=12 checks, 1-3 positive tubercle, with >=4 positive tubercles.

Table 5.2B shown and controlled particular demographic and the Clinical symptoms that is included in the patient in this analysis, associated between genetic expression and RFI.Having listed it expresses relevant to RFI (p<0.1) and in the multivariate analysis that comprises following variable, shows risk than all genes of <1: tumor locus, the operation time, tumor grade, treatment plan (C-01 or C-02), BCG treatment (be or no), and as follows according to patient's classification of lymph node status: the tubercle of 0 positive tubercle and <12 check, the tubercle of 0 positive tubercle and >=12 checks, 1-3 positive tubercle, with >=4 positive tubercles.

Table 6.2 has shown the non-linear ratio's venture analysis based on using 2DOF natural spline, associated between genetic expression and clinical effectiveness.All genes that departed from the exact linear relationship (p<0.05) of RFI that show have been listed in the II phase of combination (Du Ke Shi B) and III phase (Ke Shi C shuts out) patients.In this research in whole expression values range of observation, relation between genetic expression and RFI is also non-constant, for example in a part of range of observation, genetic expression raises and may extend relevantly with the RIF time length, in the range of observation of different piece, shortens relevant with the RFI time length.

Table 7.2 has shown the gene of interact all displayings and neoplasm staging (p value <0.1).Use the proportional hazard model of RFI to data modeling, using genetic expression, neoplasm staging and their interaction as predictor.0 positive tubercle will be there is but patient's eliminating from these are analyzed of <12 the tubercle of checking.

Claims (13)

1. for gene specific or gene Selection probe and/or the purposes of primer in the reagent of people experimenter's clinical effectiveness that has colorectal carcinoma for the preparation of predictive diagnosis of BGN gene, wherein said prediction comprises determines the rna transcription thing of BGN or the normalized expression level of its expression product in described people experimenter's the biological sample that comprises cancer cells that obtain, and according to described normalized expression level, be the possibility of described people experimenter's Predict masculine gender clinical effectiveness, the positive clinical effectiveness possibility negative correlation of the rna transcription thing of BGN or the normalized expression of its expression product and rising wherein.

2. the purposes of claim 1, wherein said colorectal carcinoma is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

3. the purposes of claim 1, wherein said colorectal carcinoma is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

4. the purposes of claim 1, wherein according to recurrence-free interval (RFI), Overall survival (OS), disease free survival phase (DFS) or represent described clinical effectiveness without long distance recurrence interval (DRFI).

5. the purposes in the reagent of possibility of people experimenter's colorectum cancer recurrence that has colorectal carcinoma for the preparation of predictive diagnosis for the gene specific of BGN gene or gene Selection probe and/or primer, wherein said prediction comprises determines the rna transcription thing of BGN or the normalized expression level of its expression product in described people experimenter's the biological sample that comprises cancer cells that obtain, and be the possibility that described people experimenter predicts colorectum cancer recurrence according to described normalized expression level, the wherein colorectum cancer recurrence possibility positive correlation of the rna transcription thing of BGN or the normalized expression of its expression product and rising.

6. the purposes of claim 5, wherein said colorectal carcinoma is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

7. the purposes of claim 5, wherein said colorectal carcinoma is Du Ke Shi B (II phase) or Du Ke Shi C (III phase) colorectal carcinoma.

8. the purposes of claim 5 if wherein the possibility of described colorectum cancer recurrence raises, is further treated described patient after excision.

9. the purposes of claim 8, wherein said further treatment comprises chemotherapy

10. the purposes of claim 8, wherein said further treatment comprises radiotherapy.

The purposes of 11. claim 1-10 any one, the normalized expression level of the rna transcription thing of wherein said BGN is to use the method based on polymerase chain reaction to determine.

The purposes of 12. claim 1-11 any one, wherein said normalized expression level with respect at least one expression level with reference to the rna transcription thing of gene by stdn.

The purposes of 13. claim 1-12 any one, wherein said prediction further comprises the step that generates the report that gathers described prediction.

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