WO2005015236A2

WO2005015236A2 - A method for predicting the progression of adenocarcinoma

Info

Publication number: WO2005015236A2
Application number: PCT/EP2004/007936
Authority: WO
Inventors: Ruediger Rueger; Johannes Auer; Baerbel Porstmann; Martin Werner; Anja Wiese
Original assignee: Roche Diagnostics Gmbh; F. Hoffmann-La Roche Ag
Priority date: 2003-07-18
Filing date: 2004-07-16
Publication date: 2005-02-17
Also published as: WO2005015236A3

Abstract

The present invention is related to a method for predicting the progression of adenocarcinoma, in particular colorectal cancer, after surgery whereby the level of expression of one or several gene markers is compared to a sample serving as control. Another method according to the invention is related to identifying an agent which will inhibit the progression of adenocarcinoma. The invention is further related to kits and arrays comprising the marker genes according to the invention.

Description

A method for predicting the progression of adenocarcinoma

Field of the Invention

Background of the Invention

Colorectal cancer appears to be the second leading cause of cancer incidence and cancer death world-wide (OMED). Recent advances in prevention, screening, and therapy of colorectal cancers give rise to increased overall survival rate in the last decade. The likelihood of a recurrence is subjected to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal and distant metastases, respectively. Sporadic adenocarcinomas of the colon identified at early stages (I, II) are often cured by complete surgical resection without concomitant treatment of chemotherapy and radiation. However, resection of early-stage adenocarcinoma is associated with incidences of recurrence of 10 % in stage I (DUKES A) and 75 % in stage II (DUKES B). There is no apparent benefit of chemotherapy in stage II patients and therefore the application remains controversial. Additional to clinical and pathological parameters, there is a need to identify novel molecular, prognostic markers to establish accurate diagnoses of early colorectal cancer with poor prognosis and to select appropriate individuals within the group of high-risk patients, who will or will not benefit from adjuvant therapy.

Array technology facilitates the simultaneous analysis of the expression of thousands of genes simultaneously. Gene expression profiles from individual samples correlate with cancer subclasses (Golup, 1999; lizuka, 2002), pathological status (Van't Veer, 2002; Ramaswamy, 2003) and clinical outcome (Wigle et al., 2002). Previous studies of colorectal cancer have been successfully performed comparing normal and neoplastic tissue (Kitahara et al., 2001; Notterman et al, 2001; Lin et al., 2002), normal and cancerous epithelium (Alon et al., 1999; Williams et al., 2003; Zou et al., 2002) and different DUKES stages (Birkenkamp- Demtroder et al, 2002).

Despite these studies, there is still a need to provide further markers which allow of the evaluation of the progression of cancer, the early diagnosis, and for therapy planning, e.g. adjuvant therapy. Hence, it was an object of the present invention to provide new markers in order to allow the evaluation of the progression of adenocarcinoma, in particular early colorectal cancer.

Summary of the Invention

In order to identify molecular gene markers which correlate to progression in early colorectal adenocarcinomas, 18 cases were analyzed by oligonucleotide microarray representing approximately 12600 genes. The methods according to the present invention are useful for predicting the progression of adenocarcinoma, particularly of colorectal cancer, particularly after surgery has been performed.

Therefore, the invention is related to a method of evaluating the progression of cancer of a patient who is afflicted with an adenocarcinoma, the method comprising comparing:

a) the level of expression of one or several marker genes in a patient sample, and b) the level of expression of one or several of said marker genes in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma,

wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3 and a significant difference between the level of expression of one or several of said marker genes in the patient sample and the level of one or several of said marker genes in a sample from a control subject is an indication that the patient carries the risk of progression of cancer.

Further, the invention is related to a method of selecting a composition for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the method comprising: a) providing a sample comprising cancer cells from the patient; b) separately exposing aliquots of the sample in the presence of a plurality of test compositions; c) comparing expression of one or several marker genes in each of the aliquots; and d) selecting one of the test compositions which alters the level of expression of one or several of the marker genes in the aliquot containing that test composition relative to other test compositions; wherein at least one of said marker gene is selected from the group consisting of the marker genes listed in Table 3.

In another embodiment of the invention, a kit is provided for assessing whether a patient carries a risk of progression of adenocarcinoma, particularly colorectal cancer, the kit comprising reagents for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3.

In yet another embodiment, a kit is provided for assessing the suitability of each of a plurality of compounds for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the kit comprising:

a) the plurality of compounds; and b) a reagent for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3.

In a further embodiment of the invention, a method of deriving a candidate agent is provided, said method comprising:

(a) contacting a sample containing adenocarcinoma cells, preferably colorectal cancer cells, with said candidate agent;

(b) determining the level of expression of one or several marker genes in the sample contacted with the candidate agent and determining the level of expression of one or several of said marker genes in a sample not contacted with the candidate agent;

(c) observing the effect of the candidate agent by comparing the level of expression of one or several of said marker genes in the sample contacted with the candidate agent and the level of one or several of said marker genes in the sample not contacted with the candidate agent, (d) deriving said agent from said observed effect,

wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3 and

wherein an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of one of said marker genes in the sample contacted with the candidate agent and the level of expression of the same marker gene in the sample not contacted with the candidate agent is an indication of an effect of the candidate agent.

In yet another embodiment of the invention, a pharmaceutical preparation is provided comprising an agent according to the invention. In still another embodiment of the invention, the agent according to the invention is used for the preparation of a composition for the inhibition of progression of colorectal cancer.

In another embodiment of the invention, a method of producing a drug comprising the steps of the method according to the invention; and

(i) synthesizing the candidate agent identified in step (d) or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or

(ii) combining the drug candidate the candidate agent identified in step (d) or an analog or derivative thereof with a pharmaceutically acceptable carrier.

In all embodiments of the invention, preferably all of said marker genes are selected from Table 2, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. More preferably, all marker genes are selected from the marker genes analysed in Fig. 4 to 14.

As used herein and the claims, each of the following terms has the meaning associated with it in this section. By "array" is meant an arrangement of addressable locations on a device (see e.g. US 5,143,854; US 6,022,963; US 6,156,501; WO 90/15070; WO 92/10092). The locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats. The number of locations can range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site. Each location carries a nucleic acid as e.g. an "oligomeric compound", which can serve as a binding partner for a second nucleic acid, in particular a target nucleic acid. Methods for the manufacturing thereof are described in EP-A 0 476 014 and Hoheisel, J.D., TIBTECH 15 ((1997) 465-469; WO 89/10977; WO 89/11548; US 5,202,231; US 5,002,867; WO 93/17126). Further development has provided methods for making very large arrays of oligonucleotide probes in very small areas (US 5,143,854; WO 90/15070; WO 92/10092). Microfabricated arrays of large numbers of oligonucleotide probes, called "DNA chips" offer great promise for a wide variety of applications (see e.g. US 6,156,501 and US 6,022,963). According to the invention, a "solid phase" may be controlled pore glass (CPG), polystyrene or silica gel as used for oligonucleotide synthesis.

In the context of this invention, "hybridization" shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. s to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "marker gene" is meant to include a gene which is useful according to this invention for the evaluation the progression of adenocarcinoma, in particular colorectal cancer. It could be also termed adenocarcinoma marker gene or more specifically colorectal cancer marker gene.

The term "marker polynucleotide" is meant to include nucleotide transcript (hnRNA or mRNA) encoded by a marker gene, preferably a marker gene listed in Table 3 or Table 2, 6 and 7, or cDNA derived from the nucleotide transcript, or a segment of said transcript or cDNA. The term "marker protein" is meant to include protein or polypeptide encoded by a marker gene, preferably a marker gene listed in Table 3 or Table 2, 6 and 7, or a polypeptide or protein fragment comprising said marker protein.

The term "gene product" is meant to include marker polynucleotide and marker protein encoded by the referenced gene.

As used herein the term "polynucleotide" is synonymous with "nucleic acid."

Further a polynucleotide "corresponds to" another (a first) polynucleotide if it is related to the first polynucleotide by any of the following relationships: the second polynucleotide comprises the first polynucleotide and the second polynucleotide encodes a gene product; the second polynucleotide is the complement of the first polynucleotide and, the second polynucleotide is 5' or 3' to the first polynucleotide in cDNA, RNA, genomic DNA, or fragment of any of these polynucleotides. For example, a second polynucleotide may be a fragment of a gene that includes the first and second; polynucleotides. The first and second polynucleotides are related in that they are components of the gene coding for a gene product, such as a protein or antibody.

However, it is not necessary that the second polynucleotide comprises or overlaps with the first polynucleotide to be encompassed within the definition of "corresponding to" as used herein. For example, the first polynucleotide may be a segment of a 3' untranslated region of the second polynucleotide. The first and second polynucleotide may be fragments of a gene coding for a gene product. The second polynucleotide may be an exon of the gene while the first polynucleotide may be an intron of the gene.

The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker gene of the invention. Probes can either be synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, proteins, antibodies, organic monomers, RNA, DNA, and cDNA. However, the term „probe" preferably refers to synthetically or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (i.e., preferentially) to "target nucleic acids", i.e. marker polynucleotides according to the invention. A „probe" can be identified as a „capture probe" meaning that it "captures" the target nucleic acid so that it can be separated from undesirable materials which might obscure its detection. Once separation is accomplished, detection of the captured "target nucleic acid" can be achieved using a suitable procedure. „Capture probes" are often already attached to a solid phase. A specific example therefor is the microarray situation wherein a multitude of "capture probes" are attached to a "solid phase" which "capture" labeled cRNA or cDNA.

A "colon-associated" body fluid is a fluid which, when in the body of a patient, contacts or passes through colorectal cells or into which cells, nucleic acids or proteins shed from colorectal cells are capable of passing. Exemplary colon- associated body fluids include blood fluidsand lymph. Other suitable body fluids include urine.

The "normal" level of expression of a marker gene is the level of expression of the marker gene in colorectal cells or colon-associated body fluids of a subject, e.g a human, not afflicted with colorectal cancer.

"Over-expression" and "under-expression" of a marker gene refer to expression of the marker gene of a patient at a greater or lesser level, respectively, than the level of expression of the marker gene (e.g. at least 1.5-fold greater or 0J5-fold lesser level) in another sample or control sample.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.

A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of thetissue type corresponding to the promoter.

A "transcribed polynucleotide" is a polynucleotide (e.g an RNA, a cDNA, or an analog of one of an RNA or cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a gene, such as any of the marker genes of the invention, and normal post-transcriptional processing (e.g. splicing), if any, of the transcript.

"Complementary" refers to the broad concept of sequence complementarily between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of fortning specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. "Homologous" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC- 3' share 50% homology.

Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

A nucleic acid or protein is "fixed" to a substrate if it is covalently or non covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g standard saline citrate, pH 7.4) without a substantial fraction of the nucleic acid or protein dissociating from the substrate.

As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature.

Expression of a marker gene in a patient is "significantly" altered from the level of expression of the marker gene in a control subject (afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma) if the level of expression of the marker gene in a sample from the patient differs from the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 1.5, twice, and more preferably three, four, five or ten times that amount if overexpressed, or preferably at least 0.75, 0.5 or 0.25 if underexpressed. Expression of a marker gene in a patient is "significantly" higher than the level of expression of the marker gene in a control subject if the level of expression of the marker gene in a sample from the patient is greater than the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 1.5, twice, and more preferably three, four, five or ten times that amount. Alternately, expression of the marker gene in the patient can be considered "significantly" lower than the level of expression in a control subject if the level of expression in a sample from the patient is lower than the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 0.75, 0.5 or 0.25 that amount.

The progression of adenocarcinoma, in particular colorectal cancer is meant to include that adenocarcinoma, preferably colorectal cancer recurs or metastasizes. The progression of adenocarcinoma, in particular colorectal cancer, is "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g a probe, for specifically detecting a marker gene or peptide of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.

Detailed Description of the Invention

The invention relates to newly discovered correlations between expression of certain marker genes and the progression of adenocarcinoma.

In an embodiment of the invention, a method is provided of evaluating the progression of cancer of a patient who is afflicted with an adenocarcinoma, the method comprising comparing:

wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3 and a significant difference between the level of expression of one or several of said marker genes in the patient sample and the level of one or several of said marker genes in a sample from a control subject is an indication that the patient carries the risk of progression of cancer. Preferably, the adenocarcinoma is a colorectal cancer.

Preferably, several of said marker genes are selected from the group consisting of the marker genes listed in Table 3 or Table 2, 6 and 7. More preferably, all of said marker genes are selected from Table 2, 3, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. Most preferred, all marker genes are selected from the marker genes analysed in Fig. 4 to 14. The markers in Table 7a and c, or Table 7b and c are particularly suitable for the prediction of the occurrence of distant metastases, i.e. metastasis occurring at sites distant from the site where the primary tumor was surgically removed.

In another embodiment, the sample comprises cells obtained from the patient. Particularly, in one embodiment of the present invention, the patient sample is a colon tissue sample e.g. obtained by surgery or by biopsy. In another example, the patient sample is stool, urine or a colon-associated body fluid. Such fluids include more particularly blood fluids in particular serum, and lymph fluid which shall be regarded as a colon-associated body fluid. The sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g. fixation, storage, freezing, lysis, homogenization, DNA or RNA extraction, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the marker gene product in the sample.

Further, the invention provides a kit for assessing whether a patient carries a risk of progression of adenocarcinoma, particularly colorectal cancer, the kit comprising reagents for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3, preferably all of said marker genes are selected from Table 2, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. Most preferred, all marker genes are selected from the marker genes analysed in Fig. 4 to 14. In another embodiment, a kit for assessing the suitability of each of a plurality of compounds for inhibiting the progression of adenocarcinoma is provided, particularly colorectal cancer, in a patient, the kit comprising: a) the plurality of compounds; and b) a reagent for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3, preferably all of said marker genes are selected from Table 2, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. Most preferred, all marker genes are selected from the marker genes analysed in Fig. 4 to 14. The kit comprises a plurality of reagents, each of; which is capable of binding specifically with a protein or nucleic acid encoded by a marker gene of the invention. Suitable reagents for binding with a protein encoded by a marker gene of the invention include antibodies, antibody derivatives, antibody fragments, and the like. Additional reagents for specifically binding with a protein lo encoded by a marker gene include any natural ligands of the protein and derivatives of such ligands. Suitable reagents for binding with a nucleic acid encoded by a marker gene (e.g. an hnRNA, a spliced mRNA, a cDNA corresponding to the mRNA, or the like) include complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled 5 oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like. The kit of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit may comprise; fluids (e.g SSC buffer) suitable for binding an antibody with a protein with which it specifically binds or, for annealing complementary nucleic acids one or more sample compartments, instructional material which describes performance of a method of the invention, a sample of control cells, a sample of adenocarcinoma cells, and the like.

In the methods according to the invention, a significant difference comprises an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of one of said marker genes in the patient sample and the level of expression of the same marker gene in the sample from the control subject. Preferably, the difference is 1.5 to 20 fold, 1.5 to 200 fold or 1.5 to 2000 fold for overexpression. Preferably, the difference is 0.75 to 0.01 fold, 0.75 to 0.1 fold for underexpression. In another embodiment, the significant difference comprises an at least 2 fold difference or a less than 0.5 fold difference between the level of expression of one of said marker genes in the patient sample and the level of expression of the same marker gene in the sample from the control subject.

According to the present invention, the level of expression of a marker gene in a sample can be determined, for example, by detecting the level in the sample of: a protein encoded by the marker gene, or a polypeptide or a fragment comprising the protein (e.g using a reagent, such as an antibody, an antibody derivative, which binds specifically with the protein or a fragment thereof); a metabolite which is produced directly by catalysis or indirectly by; the protein encoded by the marker gene; and/or a polynucleotide (e.g. an mRNA, hnRNA, cDNA) produced by or derived from the expression of the marker gene or a fragment of the polynucleotide (e. g by contacting polynucleotides obtained or derived from the sample with a substrate having affixed thereto a nucleic acid comprising the marker gene sequence or a portion of such sequence).

Any marker gene or combination of marker genes listed within Table 3 or Table 2, 6 and 7, as well as any known adenocarcinoma marker genes in combination with the marker genes set forth within Table 3 or Table 2, 6 and 7, may be used in the compositions, kits, and methods of the present invention. In general, it is preferable to use marker genes for which the difference between the level of expression of the marker gene in adenocarcinoma cells or adenocarcinoma associated body fluids and the level of expression of the same marker gene in a sample from a control subject. Although this difference can be as small as the limit of detection of the method for assessing expression of the marker gene, it may be the case that the difference be at least greater than the standard error of the assessment method, and that the difference is at least 1.5, 2-, 3 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-, 500-, 1000-fold or greater if overexpressed and 0.75, 0.5 or 0.25 if underexpressed.

The methods of the present invention may be performed by assessing the expression of a plurality (e.g 2, 3, 5, or 10 or more) of marker genes. A significantly altered, preferably increased, level of expression in the patient sample of one or more of the marker genes, or some combination thereof, relative to those marker genes' expression levels in samples from subjects serving as a control, is an indication that the patient has a higher risk of progression of adenocarcinoma, preferably colorectal cancer.

Preferred in vivo techniques for detection of a protein encoded by marker gene of the invention include introducing into a subject an antibody that specifically binds the protein, or a polypeptide or protein fragment comprising the protein. In certain embodiments, the antibody can be labeled with a radioactive molecule whose presence and location in a subject can be detected by standard imaging techniques. Expression of a marker gene of the invention may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. Such method may also include physical methods such as liquid and gas chromatography, mass spectroscopy, and nuclear magnetic resonance.

In a preferred embodiment, expression of a marker gene is assessed using an antibody (e.g a radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled antibody), an antibody derivative (e.g. an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g biotin- skeptavidin}), or an antibody fragment (e.g a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a protein encoded by the marker gene or a polypeptide or a protein fragment comprising the protein, wherein the protein may have undergone none, all or a portion of its normal post-translational modification and/or proteolysis during the course of its secretion or release from preferably colorectal cells, cancerous or otherwise.

In another preferred embodiment, expression of a marker gene is assessed by preparing mRNA/cDNA (i. e. a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide which comprises the marker gene sequence or its complement, or a fragment of said sequence or complement. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more marker genes can likewise be detected using quantitative PCR to assess the level of RNA transcripts encoded by the marker gene(s).

In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a RNA transcript encoded by a marker gene of the invention. If polynucleotides complementary to or homologous with a RNA transcript encoded by the marker gene of the invention are differentially detectable on the substrate (e.g detectable using radioactivity, different chromophores or fluorophores), are fixed to different selected positions, then the levels of expression of a plurality of marker genes can be assessed simultaneously using a single substrate (e.g a "gene chip" microarray of polynucleotides fixed at selected positions). When a method of assessing marker gene expression is used which involves hybridization of one nucleic acid with another, it is preferred that the hybridization be performed under stringent hybridization conditions.

Because the compositions, kits, and methods of the invention rely on detection of a difference in expression levels of one or more marker genes of the invention, it is preferable that the level of expression of the marker gene is greater, preferably significantly greater, than the minimum detection limit of the method used.

When the compositions, kits, and methods of the invention are used for characterizing the progression of adenocarcinoma in a patient, it is preferred that the marker gene or panel of marker genes of the invention, whose expression level is assessed, is selected such that a positive result is obtained in at least about 20%, and preferably at least about 40%, 60%, or 80%, and more preferably in substantially all patients afflicted with adenocarcinoma. Preferably, the marker gene or panel of marker genes of the invention is selected such that a positive predictive value (PPN) of greater than about 10% is obtained for the general population.

When a plurality of marker genes of the invention are used in the methods of the invention, the level of expression of each marker gene in a patient sample can be compared with the normal level of expression of each of the plurality of marker genes in non-cancerous samples of the same type, either in a single reaction mixture (i. e. using reagents, such as different fluorescent probes, for each marker gene or a mixture of similiarly labeled probes to access expression level of a plurality of marker genes whose probes are fixed to a single substrate at different positions) or in individual reaction mixtures corresponding to one or more of the marker genes. In one embodiment, a significantly enhanced level of expression of more than one of the plurality of marker genes in the sample, relative to the corresponding normal levels, is an indication that the patient is at risk that adenocarcinoma progresses. When the expression level of a plurality of marker genes is assessed, it is preferred that the expression level of 2, 3, 4, 5, 8, 10, 12, 20 15, 20, 30, or 40 or more individual marker genes is assessed. In order to maximize the sensitivity of the compositions, kits, and methods of the invention (i e. by interference attributable to cells of non-cancer origin in a patient sample), it is preferable that the marker gene of the invention whose expression level is examined therein be a marker gene which is tissue specific. These marker genes are not, of course, included among the marker genes of the invention, although they may be used together with one or more marker genes of the invention in a panel of marker genes, for example. It is well known that certain types of genes, such as oncogenes, tumor suppressor genes, growth factor-like genes, protease- like genes, and protein kinase-like genes are often involved with development of cancers of various types. Thus, among the marker genes of the invention, use of those which encode proteins which resemble known secreted proteins such as growth factors, proteases and protease inhibitors are preferred. Known oncogenes and tumor suppressor genes include, for example, abl, abr, akt2, ape, bcl2a, bcl2,C, bcl3, bcr, brcal, brca2, cbl, ccudl, cdc42, cdk4, crk- II, csprlfins, dbl. ccc, dpc41smad4, e-cad, e2fllrbap, e lerbb-1, elkl, elk3, eph, erg, etsl, ets2, fer, fgrlsrc2, flillergb2, fos, fpslfes,fi al, fia2, fyn, kick, trek, her21erkb- 21nen, 5 her31erbb- 3, her41erbb-4, hrasl, hst2, hstil, igf p2, ink4a, ink4b, int21f f3, jun, junb,; jund, kip2, kit, kras2a, kras2b, Ick, Iyn, mas, mclx, mcc, mdm2, met, mlkl, mmpl O. mos, msh2, msh3, msh6, myb, myba, mybb, n yc, mycll, mycn, nfl, nf2, nme2, eras, pS3, pdgfb, phb, piml, pmsl, pms2, ptc, pten, rafl, rapla, rbl, rel, ret, rosl, ski, srcl, tall, t fbr2, tgfb3, tf br3, thral, thrb, tiaml, timp3, tjpl, tpS3, trk, vav, vhf, vil2, wafl, wntl, 10 wnt2, wtl, and yesl (Hesketh, In: The Oncogene and Tumour Suppressor Gene Facts Book, 2nd ed., Academic Press, 1997; Fishel, R., et al., Science 266 (1994) 1403-1405). Known growth factors include platelet-derived growth factor alpha, platelet derived growth factor beta (simian sarcoma viral {v- sis) oncogene homolog), thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte 5 growth and development factor), erythropoietin, B cell growth factor, macrophage stimulating factor 1 (hepatocyte growth factor-like protein), hepatocyte growth factor (hepapoietin A), insulin-like growth factor 1 (somatomedia C), hepatoma- derived growth factor, amphiregulin (schwannoma-derived growth factor), bone morphogenetic; proteins 1, 2, 3, 3 beta, and 4, bone morphogenetic protein 7 (osteogenic protein 1), bone morphogenetic protein 8 (osteogenic protein 2), connective tissue growth factor, connective tissue activation peptide 3, epidermal growth factor (EGF), teratocarcinoma derived growth factor 1, endothelin, endothelin 2, endothelin 3, stromal cell-derived factor 1, vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, placental growth factor (vascular endothelial growth factor-related protein), transforming growth 25 factor alpha, transforming growth factor beta 1 and its precursors, transforming growth factor beta 2 and its precursors, fibroblast growth factor 1 (acidic), fibroblast growth factor 2 (basic), fibroblast growth factor 5 and its precursors, fibroblast growth factor 6 and its precursors, fibroblast growth factor 7 (keratinocyte growth factor), fibroblast growth factor 8 (androgen- induced), fibroblast growth factor 9 (glia- activating factor), pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1), brain-derived neurotrophic factor, and recombinant glial growth factor 2. Known proteases include interleukin- 1 beta convertase and its precursors, Mch6 and its precursors, Mch2 isoform alpha, Mch4, Cpp32 isoform alpha, Lice2 gamma cysteine protease, Ich-1 S. Ich- 1 L, Ich-2 and its precursors, TY protease, matrix 35 metalloproteinase 1 (interstitial collagenase), matrix metalloproteinase 2 (gelatinase A, 72kD gelatinase, 72kD type IV collagenase), matrix metalloproteinase 7 (matrilysin), matrix metalloproteinase 8 (neutrophil collagenase), matrix metalloproteinase 12 (macrophage elastase), matrix metalloproteinase 13 (collagenase 3), metallopeptidase 1, cysteine-rich metalloprotease (disintegrin) and its precursors, subtilisin-like protease Pc8 and its precursors, chymotrypsin, snake venom-like protease, cathepsin 1, cathepsin D (lysosomal aspartyl protease), skomelysin, aminopeptidase N. plasminogen, tissue 5 plasminogen activator, plasminogen activator inhibitor type II, and urokinase-type plasminogen activator.

It is recognized that the compositions, kits, and methods of the invention will be of particular utility to patients having an enhanced risk of progression of colorectal cancer and their medical advisors.

The methods of the present invention may be practiced using one or more marker genes of the invention in combination with one or more known marker genes. It will be appreciated that the methods and kits of the present invention may also include known cancer marker genes including known marker genes. It will further be appreciated that the methods and kits may be used to identify cancers other than adenocarcinoma or colorectal cancer.

Various specific aspects of the invention are described in further detail in the following subsections. I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules that correspond to a marker gene of the invention. Such nucleic acid molecules comprise sequences of RNA transcripts encoded by the marker gene or portions of such transcripts. Isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify of RNA transcripts encoded by the marker gene or portions of such transcripts, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single- stranded or double-stranded, but preferably is double- stranded DNA.

The invention also encompasses polynucleotides which differ from that of the polynucleotides described herein, but which produce the same phenotypic effect, such as an allelic variant. These altered, but phenotypically equivalent polynucleotides are referred to as "equivalent nucleic acids." This invention also encompasses polynucleotides characterized by changes in non-coding regions that do not alter the polypeptide produced therefrom when compared to the polynucleotide herein. This invention further encompasses polynucleotides, which hybridize to the polynucleotides of the subject invention under conditions of moderate or high stringency. Alternatively, the polynucleotides are at least 85%, or at least 90%, or more preferably, greater or equal to 95% identical as determined by a sequence alignment program when run under default parameters.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule comprises a protein-coding sequence and is free of sequences which naturally flank the coding sequence in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleotide transcript encoded by a marker gene listed in Table 3 or Table 2, 6 and 7, can be isolated using standard molecular biology techniques. Nucleic acid molecule of the present invention also encompass the marker genes of the invention, which can be isolated using standard hybridization and cloning techniques (e.g. as described in Sambrook et al, ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

A process for identifying a larger fragment or the full-length coding sequence of a marker gene of the present invention is thus also provided. Any conventional recombinant DNA techniques applicable for isolating polynucleotides may be employed. One such method involves the 5'-RACE-PCR technique, in which the poly A mRNA that contains the coding sequence of particular interest is first reverse transcribed with a 3'-primer comprising a sequence disclosed herein. The newly synthesized cDNA strand is then tagged with an anchor primer with a known sequence, which preferably contains a convenient cloning restriction site attached at the end.

The tagged cDNA is then amplified with the 3'-primer (or a nested primer sharing sequence homology to the internal sequences of the coding region) and the 5'- anchor primer. The amplification may be conducted under conditions of various levels, of stringency to optimize the amplification specificity. 5'-RACE-PCR can be readily performed using commercial kits (available from, e.g. BRL Life Technologies Inc., Clontech) according to the manufacturer's instructions.

Isolating the complete coding sequence of a gene can also be carried out in a hybridization assay using a suitable probe. The probe preferably comprises at least nucleotides, and more preferably exhibits sequence homology to the polynucleotides of the marker genes of the present invention. Other high throughput screens for cDNAs, such as those involving gene chip technology, can also be employed in obtaining the complete cDNA sequence. In addition, databases exist that reduce the complexity of ESTs by assembling contiguous EST sequences into tentative genes. For example, TIGR has assembled human ESTs into a database called THC for tentative human consensus sequences. The THC database allows for a more definitive assignment compared to ESTs alone.

Software programs exist (TIGR assembler and TIGEM EST assembly machine and contig assembly program (see Huang, X., Genomes 33 (1996) 21-23) that allow for assembling ESTs into contiguous sequences from any organism.

Alternatively, mRNA from a sample preparation is used to construct cDNA library in the ZAP Express vector following the procedure described in Velculescu, V.E., et al, Science 270 (1995) 484-487. The ZAP Express cDNA synthesis kit (Stratagene) is used accordingly to the manufacturer's protocol. Plates containing 250 to 2000 plaques are hybridized as described in Ruppert, J.M., et al., Mol. Cell. Bio. 8 (1988) 3104-3113, to oligonucleotide probes with the same conditions previously described for standard probes except that the hybridization temperature is reduced to a room temperature. Washes are performed in 6X standard-saline-citrate 0. 1% SDS for 30 minutes at room temperature. The probes are labeled with 32P-ATP trough use of T4 polynucleotide kinase.

A partial cDNA (3' fragment) can be isolated by 3' directed PCR reaction. This procedure is a modification of the protocol described in Polyak, K., et al., Nature 389 (1997) 300-305. Briefly, the procedure uses SAGE tags in PCR reaction such that the resultant PCR product contains the SAGE tag of interest as well as additional cDNA, the length of which is defined by the position of the tag with respect to the 3' end of the cDNA.

The cDNA product derived from such a transcript driven PCR reaction can be used for many applications.

RNA from a source to express the cDNA corresponding to a given tag is first converted to double-stranded cDNA using any standard cDNA protocol. Similar conditions used to generate cDNA for SAGE library construction can be employed except that a modified oligo-dT primer is used to derive the first strand synthesis. For example, the oligonucleotide of composition 5'- TCC GGC GCG CCG TTT TCC CAG TCA CGA(30)-3', contains a poly-T stretch at the 3' end for hybridization and priming from poly-A tails, an M13 priming site for use in subsequent PCR steps, a 5' Biotin label (B) for capture to strepavidin-coated magnetic beads, and an Ascl restriction endonuclease site for releasing the cDNA from the strepavidin-coated magnetic beads.

Theoretically, any sufficiently-sized DNA region capable of hybridizing to a PCR primer can be used as well as any other 8 base pair recognizing endonuclease. cDNA construction utilizing this or similar modified oligo-dT primer is then processed as described in U.S. Patent No. 5,695,937 up until adapter ligation where only one adapter is ligated to the cDNA pool. After adapter ligation, the cDNA is released from the streptavidin-coated magnetic beads and is then used as a template for cDNA amplification.

Various PCR protocols can be employed using PCR priming sites within the 3' modified oligo-dT primer and the SAGE tag. The SAGE tag-derived PCR primer employed can be of varying length dictated by 5' extension of the tag into the adaptor sequence. cDNA products are now available for a variety of applications. This technique can be further modified by: (1) altering the length and/or content of the modified oligo-dT primer; (2) ligating adaptors other than that previously employed within the SAGE protocol; (3) performing PCR from template retained on the streptavidin-coated magnetic beads; and (4) priming first strand cDNA synthesis with non-oligo-dT based primers.

Gene trapper technology can also be used. The reagents and manufacturer's instructions for this technology are commercially available from Life Technologies, Inc., Gaithsburg, Maryland. Briefly, a complex population of single-stranded phagemid DNA containing directional cDNA inserts is enriched for the target sequence by hybridization in solution to a biotinylated oligonucleotide probe complementary to the target sequence. The hybrids are captured on streptavidin- coated paramagnetic beads.

A magnet retrieves the paramagnetic beads from the solution, leaving nonhybridized single-stranded DNAs behind. Subsequently, the captured single- stranded DNA target is released from the biotinylated oligonucleotide. After release, the cDNA clone is further enriched by using a nonbiotinylated target oligonucleotide to specifically prime conversion of the single-stranded DNA. Following transformation and plating, typically 20% to 100% of the clones represent the cDNA clone of interest. To identify the desired cDNA clone, the clones may be screened by colony hybridization using the 32P-labeled oligonucleotide, or alternatively by DNA sequencing and alignment of all sequences obtained from numerous clones to determine a consensus sequence.

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence of a RNA transcript encoded by a marker gene of the invention or a complement of said sequence. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only a portion of the nucleotide sequence (RNA or cDNA) of a RNA transcript encoded by a marker gene of the invention or a complement of said sequence. Such nucleic acids can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences of one or more marker genes of the invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mix-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. The invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein which corresponds to a marker gene of the invention, and thus encode the same protein.

In addition to the nucleotide sequences described in the GenBank and IMAGE consortium database records described herein, and in Table 3 or Table 2, 6 and 7, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level ofthat gene (e.g. by affecting regulation or degradation). As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide by a marker gene of the invention. Such natural allelic variations can typically result in 0. 1-0.5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

In another embodiment, an isolated nucleic acid molecule of the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a RNA transcript of a marker gene of the invention or a portion of said transcript or a cDNA corresponding to said transcript or portion thereof. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 75% (80%, 85%, preferably 90%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1 -6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989. A preferred, non-limiting example of stringent hybridization conditions for annealing two single-stranded DNA each of which is at least about 100 bases in length and/or for annealing a single-stranded DNA and a single-stranded RNA each of which is at least about 100 bases in length, are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C. Further preferred hybridization conditions are taught in Lockhart, D.J., et al, Nat. Biotechnol. 14 (1996) 1675-1680; Breslauer, K.J., et al., Proc. Natl. Acad. Sci. USA 83 (1986) 3746-3750; van Ness, J., and Chen, L., Nucleic Acids Res. 19 (1991) 5143- 5151; McGraw, R.A., et al., BioTechniques 8 (1990) 674-678; and Milner, N., et al, Nat. Biotechnol. 15 (1997) 537-541, all expressly incorporated by reference. In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention that can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g. murine and human) may be essential for activity and thus would not be likely targets for alteration. Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from the; naturally- occurring proteins encoded by the marker genes of the invention, yet retain biological activity. In one embodiment, such a protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of one of the proteins encoded by the marker genes of the invention. An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein.

Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been deemed in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

The present invention encompasses antisense nucleic acid molecules, i. e., molecules which are complementary to a sense nucleic acid of the invention, e.g. complementary to the coding strand of a double-stranded cDNA molecule corresponding to a marker gene of the invention or complementary to an mRNA sequence corresponding to a marker gene of the invention. Accordingly, an antisense nucleic acid of the invention can hydrogen bond to (i. e. anneal with) a sense nucleic acid of the invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g. all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g. an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5- bromouracil, 5 chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5 (carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl- 2-thiouridine, 5 carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2- dimethylguanine, 2 methyladenine, 2-methylguanine, 3- methylcytosine, 5- methylcytosine, N6-adenine, 7 methylguanine, 5- methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta D- mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4- thiouracil, 5 methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl 2-thiouracil, 3-(3- amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub- cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide corresponding to a selected marker gene of the invention to thereby inhibit expression of the marker gene, e.g. by inhibiting transcription and/or translation.

The hybridization can be by conventional nucleotide complementarily to forth a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site or infusion of the antisense nucleic acid into a colon- associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g. by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.

To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an a-anomeric nucleic acid molecule. An anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a- units, the strands run parallel to each other (Gautier, C, et al., Nucleic Acids Res. 15 (1987) 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue, H., et al., Nucleic Acids Res. 15 (1987) 6131-6148) or a chimeric RNA-DNA analogue (Inoue, H., et al., FEBS Lett. 215 (1987) 327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haseloff, J., and Gerlach, W.L., Nature 334 (1988) 585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding by a marker gene of the invention can be designed based upon the nucleotide sequence of a cDNA corresponding to the marker gene. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see U.S. Patent No. 4,987,071; and U.S. Patent No. 5,116,742). Alternatively, an mRNA encoding a polypeptide ofthe invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g. Bartel, D.P., and Szostak, J.W., Science 261 (1993) 1411-1418). The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g. the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene, C, Anticancer Drug Des. 6 (1991) 569-584; Helene, C, et al, Ann. N. Y. Acad. Sci. 660 (1992) 27-36; and Maher, L.J. 3^rd, Bioassays 14 (1992) 807-815.

In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup, B., and Nielsen, P.E., Bioorg. Med. Chem. 4

(1996) 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural bases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe, H., et al., Proc. Natl. Acad. Sci. USA 93 (1996) 14670-14675.

PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or anti-gene agents for sequence-specific modulation of gene expression by, e.g. inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g. PNA directed PCR clamping, as artificial restriction enzymes when used in combination with other enzymes, e.g. SI nucleases (Hyrup, 1996, supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra, Perry O'Keefe et al., 1996, supra).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g. RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.

PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the bases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, 1996, supra, and Finn, P.J., et al, Nucleic Acids Res. 24 (1996) 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4 methoxytrityl)amino-5'- deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag, M., and Engels, J.W., Nucleic Acids Res. 17 (1989) 5973-5988).

PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al., 1996, supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al., Bioorganic Med. Chem. Lett. 5 (1975) 1119- 11124).

In other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g. Letsinger, R.L., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 6553-6556; Lemaitre, M., et al., Proc. Natl. Acad. Sci. USA 84 (1987) 648-652; WO 88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). In addition, oligonucleotides can be modified with hybridization- triggered cleavage agents (see, e.g. van der Krol, A.R., et al, Bio/Techniques 6 (1988) 958-976) or intercalating agents (see, e.g., Zon, G., Pharm. Res. 5 (1988) 539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acids having at least one region which is complementary to a nucleic acid of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid of the invention in a sample. A "molecular beacon" nucleic acid is a nucleic acid comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher. When the complementary regions of the nucleic acid are not annealed with one another, fluorescence of the fluorophore is quenched to a lesser degree. Molecular beacon nucleic acids are described, for example, in U.S. Patent 5,876,930.

II. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins encoded by individual marker genes of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide encoded by a marker gene of the invention. In one embodiment, the native polypeptide encoded by a marker gene can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides encoded by a marker gene of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide encoded by a marker gene of the invention can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").

When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i. e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide encoded by a marker gene of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein encoded by the marker gene (e.g., the amino acid sequence listed in the GenBank and IMAGE Consortium database records described herein), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25,50, 100 or more amino acids in length.

Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention. Preferred polypeptides have the amino acid sequence listed in the NCBI Protein Database records described herein. Other useful proteins are substantially identical (e.g. at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = of identical positions/total of positions (e.g. overlapping positions) xlOO).

In one embodiment the two sequences are the same length. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S., and Altschul, S.F., Proc. Natl. Acad. Sci. USA 87 (1990) 2264-2268, modified as in Karlin, S., and Altschul, S.F., Proc. Natl. Acad. Sci. USA 90 (1993) 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, S.F., et al., J. Mol. Biol. 215 (1990) 403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul, S.F., et al., Nucleic Acids Res. 25 (1997) 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS or 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson, W.R., and Lipman, D.J., Proc. Natl. Acad. Sci. USA 85 (1988) 2444-2448. When using the PASTA algorithm for comparing nucleotide or amino acid sequences, a PAM120 weight residue table can, for example, be used with a k- tuple value of 2.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. The invention also provides chimeric or fusion proteins corresponding to a marker gene of the invention. As used herein, a "chimeric protein" or "fusion protein" comprises all or part (preferably a biologically active part) of a polypeptide encoded by a marker gene of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide encoded by the marker gene). Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the amino-terminus or the carboxyl-terminus of the polypeptide of the invention.

One useful fusion protein is a GST fusion protein in which a polypeptide encoded by a marker gene of the invention is fused to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signal sequence at its amino terminus. For example, the native signal sequence of a polypeptide encoded by a marker gene of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence

(Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal

(Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide encoded by a marker gene of the invention is fused to sequences derived from a member of the immunoglobulin protein family.

The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction can be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.

Chimeric and fusion proteins ofthe invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.

Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g. Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g. a GST polypeptide).

A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteolytically cleaved (i. e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods.

Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

The present invention also pertains to variants of the polypeptides encoded by individual marker genes of the invention. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e. g. discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological actiλdties of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g. Narang, Tetrahedron 39 (1983) 3; Itakura, K., et al, Annu. Rev. Biochem. 53 (1984) 323-356; Itakura, K., et al, Science 198 (1977) 1056-1063; Ike, Y., et al, Nucleic Acid Res. 11 (1983) 477-488). In addition, libraries of fragments of the coding sequence of a polypeptide encoded by a marker gene of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin, A.P., and Youvan, D.C., Proc. Natl. Acad. Sci. USA 89 (1992) 7811-7815; Delagrave, S., et al, Prot. Eng. 6 (1993) 327-331).

An isolated polypeptide encoded by a marker gene of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acid residues of the amino acid sequence of one of the polypeptides of the invention, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with a protein encoded by a marker gene of the invention. Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g. hydrophilic regions. Hydrophobicity sequence analysis, hydrophilicity sequence analysis, or similar analyses can be used to identify hydrophilic regions. An immunogen typically is used to prepare antibodies by immunizing a suitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The terms "antibody" and "antibody substance"; as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g. a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention. Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be harvested or isolated from the subject (e.g., from the blood or serum of the subject) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g. affinity chromatography.

For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i. e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention.

At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler, G., and Milstein, C, Nature 256 (1975) 495-497, the human B cell hybridoma technique (see Kozbor et al., Immunol. Today 4 (1983) 72), the EBV hybridoma technique (see Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al., ed., Current Protocols in Immunology, John Wiley 8. Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g. the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; end the Stratagene Su' 4P Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs, P., et al. Bio/Technology 9 ((1991)) 1369-1372; Hay, B.N., et al., Hum. Antibod. Hybridomas 3 (1992) 81-85; Huse, W.D., et al., Science 246 (1989) 1275-1281; Griffiths, A.D., et al., EMBO 12 (1993) 725-734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g. U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g. U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP-A 0 184 187; EP-A 0 171 496; EP-A 0 173 494; WO 86/01533; U.S. Patent No. 4,816,567; EP-A 0 125 023; Better, M., et al., Science 240 (1988) 1041-1043; Liu, AN., et al, Proc. Νatl. Acad. Sci. USA 84 (1987) 3439-3443; Liu, AN., et al, J. Immunol. 139 (1987) 3521-3526; Sun, L.K., et al., Proc. Νatl. Acad. Sci. USA 84 ((1987)) 214-218; Nishimura, Y., et al., Cancer Res. 47 (1987) 999- 1005; Wood, C.R., et al., Nature 314 (1985) 446-449; and Shaw, D.R., et al., Natl. Cancer Inst. 80 (1988) 1553-1559); Morrison, S.L., Science 229 (1985) 1202-1207; Oi et al., Bio/Techniques 4 (1986) 214; U.S. Patent 5,225,539; Jones, P.T., et al., Nature 321 (1986) 522-525; Verhoeyen, M., et al., Science 239 (1988) 1534-1536; and Beidler, C.B., et al., J. Immunol. 141 (1988) 4053-4060.

Antibodies of the invention may be used as therapeutic agents in treating cancers. In a preferred embodiment, completely human antibodies of the invention are used for therapeutic treatment of human cancer patients, particularly those having adenocarcinoma, preferably colorectal cancer. Such antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g. all or a portion of a polypeptide encoded by a marker gene of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.

Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg, N., and Huszar, D., Int. Rev. Immunol. 13 (1995) 65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers, L.S., et al., Bio/Technology 12 (1994) 899-903). An antibody directed against a polypeptide encoded by a marker gene of the invention (e.g. a monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.

Moreover, such an antibody can be used to detect the polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the marker gene. The antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in a colon-associated body fluid) as part of a clinical testing procedure, e.g. to, for example, determine the efficacy of a given treatment regimen.

Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, 6-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, P, S or H. Further, an antibody (or fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B. gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dThydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-fhioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclmes (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In

Cancer Therapy", In: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.

(eds.), Alan R. Liss, Inc., 1985, pp. 243-256; Hellstrom et al, "Antibodies For Drug

Delivery", In: Controlled Drug Delivery, 2nd ed., Robinson et al. (eds.), Marcel

Dekker, Inc., 1987, pp. 623-653; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", In: Monoclonal Antibodies '84: Biological And Clinical

Applications, Pinchera et al. (eds.), 1985, pp. 475-506; Baldwin et al. (eds.),

"Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled

Antibody In Cancer Therapy", In: Monoclonal Antibodies For Cancer Detection

And Therapy, Academic Press, 1985, pp. 303-316; and Thorpe, P.E., and Ross, W.C., Immunol. Rev. 62 (1982) 119-158.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. Patent No. 4,676,980.

Accordingly, in one aspect, the invention provides substantially purif ed antibodies or fragments thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. In various embodiments, the substantially purified antibodies of the invention, or fragments thereof, can be human, non-human, chimeric and/or; humanized antibodies.

In another aspect, the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. Such non- human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the non human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of the present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to an amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.

The substantially purified antibodies or fragments thereof may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a kansmembrane or a cytoplasmic domain or cytoplasmic membrane of a polypeptide of the invention. In a particularly preferred embodiment, the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal; antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of the present invention.

Any of the antibodies of the invention can be conjugated to a therapeutic moiety; or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.

The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.

Still another aspect of the invention is a method of making an antibody that specifically recognizes a polypeptide of the present invention, the method comprising immunizing a mammal with a polypeptide. The polypeptide used as an immunogen comprises an amino acid sequence selected from the group consisting of the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA of the nucleic acid molecules of the present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4) and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0. 1% SDS at 65°C.

After immunization, a sample is collected from the mammal that contains an antibody that specifically recognizes the polypeptide. Preferably, the polypeptide is recombinantly produced using a non-human host cell. Optionally, the antibodies can be further purified from the sample using techniques well known to those of skill in the art.

The method can further comprise producing a monoclonal antibody- producing cell from the cells of the mammal. Optionally, antibodies are collected from the antibody-producing cell.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide encoded by a marker gene of the invention (or a portion of such a polypeptide). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g. replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner which allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology, Vol. 185, Academic Press, San Diego, CA, 1991. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g. tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as; described herein.

The recombinant expression vectors of the invention can be designed for is expression of a polypeptide encoded by a marker gene of the invention in prokaryotic (e.g. E. coli) or eukaryotic cells (end insect cells {using baculovirus expression vectors}, yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed; and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B., and Johnson, K.S., Gene 67 (1988) 31-40), pMAL (New England Biolabs,

Beverly, MA) and pRIT5 (Phannacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann, E., et al., Gene 69 (1988) 301-315) and pET 1 Id (Studier et al., In: Gene Expression Technology: Methods in Enzymology,

Vol. 85, Academic Press, San Diego, CA, 1991, pp. 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 Id vector relies on transcription from a T7 gulO-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gal). This viral polymerase is supplied by host strains

BL21(DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gal gene under the transcriptional control of the lacUV promoter.

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, In: Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Pres, San Diego, CA, 1990, pp. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada, K., et al, Nucleic Acids Res. 20 (1992) 2111- 2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. In another embodiment, the expression vector is a yeast expression vector.

Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, C, et al., EMBO J. 6 (1987) 229-234), pMFa (Kurjan, J., and Herskowitz, L, Cell 30 (1982) 933-943), pJRY88 (Schultz, L.D., et al., Gene 54 (1987) 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego,

CA).

Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g. Sf 9 cells) include the pAc series (Smith, G.E., et al, Mol. Cell Biol. 3 (1983) 2156-2165) and the pVL series (Luckow, V.A., and Summers, M.D., Virology 170 (1989) 31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B., Nature 329 (1987) 840-842) and pMT2NOPC (Kaufman, R.J., et al., EMBO J. 6 (1987) 187-193). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.

For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both; prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, C.A., et al., Genes Dev. 1 (1987) 268-277), lymphoid-specific promoters (Calame, K., and Eaton, S., Adv. Immunol. 43 (1988) 235-275), in particular promoters of T cell receptors (Winoto, A., and Baltimore, D., EMBO J. 8 (1989) 729-733) and immunoglobulins (Banerji, J., et al, Cell 33 (1983) 729-740; Queen, C, and Baltimore, D., Cell 33 (1983) 741-748), neuron-specific promoters (e.g. the neurofilament promoter; Byrne, G.W., and Ruddle, F.H., Proc. Natl. Acad. Sci. USA 86 (1989) 5473-5477), pancreas-specific promoters (Edlund, T., et al, Science 230 (1985) 912-916), and mammary gland-specific promoters (e.g. milk whey promoter, U.S. Patent No. 4,873,316 and EP-A 0 264 166). Developmentally regulated promoters are also encompassed, for example the murine box promoters (Kessel, M., and Gruss, P., Science 249 (1990) 374-379) and the a-fetoprotein promoter (Camper, S.A., and Tilghman, S.M., Genes Dev. 3 (1989) 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention.

Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue- specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., 1986, Trends in Genetics, Vol. 1(1).

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic (e.g. E. coli) or eukaryotic cell (e.g. insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art- recognized; techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran- mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a "selectable marker" (SM) gene (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.

Preferred SM genes include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the SM gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide encoded by a marker gene of the invention. Accordingly, the invention further provides methods for producing a polypeptide encoded by a marker gene of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide encoded by the marker gene is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell. The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide of a marker gene of the invention have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a marker gene of the invention have been introduced into their genome or homologous recombinant animals in which endogenous gene(s) encoding a polypeptide corresponding to a marker gene of the invention have been altered. Such animals are useful for studying the function and/ or activity of the polypeptide corresponding to the marker gene and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the s animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal; develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g. an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing a nucleic acid encoding a polypeptide encoded by a marker gene of the invention into the male pronuclei of a fertilized oocyte, e.g. by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; 4,873,191, and in Hogan, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a marker gene of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g. functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i. e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas, K.R., and Capecchi, M.R., Cell 51 (1987) 503-512 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g. by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g. Li, E., et al., Cell 69 (1992) 915-926). The selected cells are then injected into a blastocyst of an animal (e.g. a mouse) to form aggregation chimeras (see, e.g. Bradley, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, Robertson, ed., IRL, Oxford, 1987, pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A., Curr. Opin. Biotechnol. 2 (1991) 823-829 and in WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage Pi. For a description ofthe cre/loxP recombinase system, see, e.g. Lakso, M., et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman, S., et al., Science 251 (1991) 1351-1355). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g. by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I., et al., Nature 385 (1997) 810-813 and WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") encoded by or corresponding to a marker gene of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid encoded by a marker gene of the invention. Such methods comprise formulating a pharmaceutically; acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid encoded by a marker gene of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid encoded by a marker gene of the invention and one or more additional active compounds. The invention also provides methods (also referred to herein as "screening assays") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the marker gene or its gene products, or (b) have a modulatory (e.g. stimulatory or inhibitory) effect on the activity of the marker gene or, more specifically, (c) have a modulatory effect on the interactions of a protein encoded by the marker gene (hereinafter "marker protein") with one or more of its natural substrates (e.g. peptide, protein, hormone, co-factor, or nucleic acid), or (d) have a modulatory effect on the expression of the marker gene. Such assays typically comprise a reaction between the marker gene or the marker protein and one or more assay components. The other components may be either the test compound itself, or a combination of test compound and a natural binding partner of the marker protein.

Therefore, it is an object of the invention, to provide a method of deriving a candidate compound, said method comprising:

(c) observing the effect of the candidate compound by comparing the level of expression of one or several of said marker genes in the sample contacted with the candidate agent and the level of one or several of said marker genes in the sample not contacted with the candidate agent,

(d) deriving said compound from said observed effect,

wherein an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of one of said marker genes in the sample contacted with the candidate compound and the level of expression of the same marker gene in the sample not contacted with the candidate compound is an indication of an effect of the candidate compound. In the method according to the invention, the candidate compound is a candidate inhibitory compound or a candidate enhancing compound.

The test compounds of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Test compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g. Zuckermann, R.N., et al, J. Med. Chem. 37 (1994) 2678-2685); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S., Anticancer Drug Des. 12 (1997)145-167).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, S.H., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 6909- 6913; Erb, E., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 11422-11426; Zuckermann, R.N., et al., J. Med. Chem. 37 (1994) 2678-2685; Cho, C.Y., et al., Science 261 (1993) 1303-1305; Carrell et al, Angew. Chem. Int. Ed. Erg. 33 (1994) 2059; Carell et al, Angew. Chem. Int. Ed. Engl. 33 (1994) 2061; and in Gallop, M.A., et al., J. Med. Chem. 37 (1994) 1233-1251.

Libraries of compounds may be presented in solution (e.g. Houghten, R.A., et al., Biotechniques 13 (1992) 412-421), or on beads (Lam, K.S., et al, Nature 354 (1991) 82-84), chips (Fodor, S.P., et al., Nature 364 (1993) 555-556), bacteria and/or spores, (US 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci US 1 89:1865- 1869) or on phage (Scott, J.K., and Smith, G.P., Science 249 (1990) 386-390; Devlin, J.J., et al, Science 249 (1990) 404-406; Cwirla, S.E., et al, Proc. Natl. Acad. Sci. 87 (1990) 6378-6382; Felici, F., et al, Mol. Biol. 222 (1991) 301-310; US 5,223,409). In one embodiment, the invention provides assays for screening candidate or test; compounds which are substrates of the marker protein or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to a marker protein or biologically active portion thereof.

Determining the ability of the test compound to directly bind to a marker protein can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to the marker protein can be determined by detecting the marker protein compound in a labeled complex. For example, compounds (e.g. substrates of the marker protein) can be labeled with 25I,35S, 4C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate s substrate to product.

In another embodiment, the invention provides assays for screening candidate or test compounds which modulate the activity of a marker protein or a biologically active portion thereof. In all likelihood, the marker protein can, in vivo, interact with one or more molecules, such as but not limited to, peptides, proteins, hormones, cofactors and nucleic acids. For the purposes of this discussion, such cellular and extracellular molecules are referred to herein as "binding partners" or marker protein "substrate".

One necessary embodiment of the invention in order to facilitate such screening is the use of the marker protein to identify its natural in vivo binding partners. There are many ways to accomplish this which are known to one skilled in the art. One example is the use of the marker protein as "bait protein" in a two-hybrid assay or three-hybrid assay; (see, e.g. U.S. Patent No. 5,283,317; Zervos, A.S., et al, Cell 72 (1993) 223-232; Madura, K., et al., J. Biol. Chem. 268 (1993) 12046-12054; Bartel, P., et al, Biotechniques 14 (1993) 920-924; Iwabuchi, K., et al, Oncogene 8 (1993) 1693-1696; WO 94/10300) in order to identify other proteins which bind to or interact with the marker protein (binding partners) and, therefore, are possibly involved in the natural function of the marker protein. Such marker protein binding partners are also likely to be involved in the propagation of signals by the marker protein or downstream elements of a marker gene-mediated signaling pathway. Alternatively, such marker protein binding partners may also be found to be inhibitors of the marker protein. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that encodes a marker protein fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a marker gene dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be readily detected and cell clones containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the marker protein.

In a further embodiment, assays may be devised through the use of the invention for the purpose of identifying compounds which modulate (e.g. affect either positively or negatively) interactions between a marker protein and its substrates and/or binding partners. Such compounds can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof.

Such compounds may also be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. The preferred assay components for use in this embodiment is a marker protein identified herein (see Table 3 or Table 2, 6 and 7), the known binding partner and/or substrate of same, and the test compound. Test compounds can be supplied from any source.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between a marker protein and its binding partner involves preparing a reaction mixture containing the protein and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the protein and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the protein and its binding partner is then detected. The formation of a complex in the control reaction, but less or no such formation in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the marker protein and its binding partner. Conversely, the formation of more complex in the presence of compound than in the control reaction indicates that the compound may enhance interaction of the marker protein and its binding partner.

The assay for compounds that interfere with the interaction of a marker protein with its binding partner may be conducted in a heterogeneous or homogeneous format.

Heterogeneous assays involve anchoring either the marker protein or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the marker protein and the binding partners (e.g. by competition) can be identified by conducting the reaction in the presence of the test substance, i. e., by adding the test substance to the reaction mixture prior to or simultaneously with the marker protein and its interactive binding partner.

Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either a marker protein or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non- anchored component may be labeled, either directly or indirectly. In practice, microtitre plates are often utilized for this approach. The anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the marker protein or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix. For example, glutathione-S-transferase/marker protein fusion proteins or glutathione-S transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed marker protein or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g:, physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound assay components; the immobilized complex assessed either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of marker protein binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a marker protein or its binding partner can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the protein-immobilized surfaces can be prepared in as advance and stored. In order to conduct the assay, the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted assay components are removed (e.g. by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways.

Where the non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non- immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g. using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled). Depending upon the order of addition of reaction components, test compounds which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may be used. This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test compound. The formed complexes are then separated from unreacted components, and the amount of complex formed is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test compounds modulate (inhibit or enhance) complex formation and which disrupt preformed complexes. In such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, complexes of molecules may be separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sci. 18 (1993 ) 284-287). Standard chromatographic techniques may also be utilized to separate; complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components.

Similarly, the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion- exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g. Heegaard, N.H., J. Mol. Recognit. 11 (1998) 141-148; Hage, D.S., and Tweed, S.A., J. Chromatogr. B. Biomed. Sci. Appl. 699 (1997) 499-525). Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, nondenaturing gels in the absence of reducing agent are typically preferred, but conditions appropriate to the particular interactants will be well known to one skilled in the art. Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g. Ausubel et al., supra). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation. The bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in no complex formation in both the presence and the absence of a test compound can be compared, thus offering information about the ability of the compound to modulate interactions between the marker protein and its binding partner.

Also within the scope of the present invention are methods for direct detection of interactions between a marker protein and its natural binding partner and/or a test compound in a homogeneous or heterogeneous assay system without further sample manipulation. For example, the technique of fluorescence energy transfer may be utilized (see, e.g. U.S. Patent No. 5,631,169; U.S. Patent No. 4,868,103). Generally, this technique involves the addition of a fluorophore label on a first 'donor' molecule (edge, test compound) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule (e.g. test compound), which in turn is able to fluoresce due to the absorbed energy.

Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g. using a fluorimeter). A test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background. In this way, test substances that modulate interactions between a marker protein and its binding partner can be identified in controlled assays.

In another embodiment, modulators of marker gene expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of of mRNA or protein encoded by a marker gene is determined. The level of expression of mRNA or protein in the presence of the candidate compound is compared to the level of expression of mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of marker gene expression based on this comparison. For example, when expression of marker gene or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of marker gene expression. Conversely, when expression of marker gene mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of marker gene expression. The level of marker gene expression in the cells can be determined by methods described herein for detecting marker gene mRNA or protein.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of a marker protein can be further confirmed in vivo, e.g., in a whole animal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by the above- described screening assays. Therefore, the invention provides a candidate agent derived by the method according to the invention and a pharmaceutical preparation comprising an agent according to the invention. The agent according to the invention can be used for the preparation of a composition for the inhibition of progression of colorectal cancer. Further, the invention is related to a method of producing a drug comprising the steps of the method according to the invention; and

(i) synthesizing the candidate compound identified in step (d) or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or (ii) combining the drug candidate the candidate compound identified in step (d) or an analog or derivative thereof with a pharmaceutically acceptable carrier.

Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g. a marker gene or marker protein modulating agent, an antisense marker gene nucleic acid molecule, an marker protein specific antibody, or an marker protein binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.

Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Exemplary doses of a small molecule include milligram or microgram amounts per Idlogram of subject or sample weight (e.g about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per Idlogram, or about 1 microgram per kilogram to about 50 micrograms per Idlogram). Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g about 1 microgram per Idlogram to about 5 grams per Idlogram, about 100 micrograms per kilogram to about 500 milligrams per Idlogram, or about 1 milligram per kilogram to about 50 milligrams per Idlogram). It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g. a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include; parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid,, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g. a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch, a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g. a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transder nal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g. with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the colon epithelium). A method for lipidation of antibodies is described by Cruikshank, W.W., et al., J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14 (1997) 193-203.

The nucleic acid molecules corresponding to a marker gene of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328, 470), or by stereotactic injection (see, e.g., Chen, S.H., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. V. Arrays

The invention also includes an array comprising the nucleotide sequence of a marker gene of the 'present invention. The array can be used to assay expression of one or more genes, including the marker gene, in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, more than 12.000 genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

Therefore, the invention provides an array of nucleic acids wherein a nucleic acid corresponding to a marker gene of the invention is attached at addressable locations wherein all of said marker genes attached are selected from Table 2, 6 or 7 or all of said marker genes attached are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. Most preferred, all marker genes attached are selected from the marker genes analysed in Fig.4 to 14.

In addition to such qualitative determination, the invention allows the quantisation of marker gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, marker genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on marker gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell- cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time course of expression of one or more marker genes in the array. This can occur in various biological contexts, as disclosed herein, for example in development and differentiation of adenocarcionoma, particularly colorectal cancer, tumor progression, progression of other diseases, in vitro processes, such a cellular transformation and senescence, autonomic neural and neurological processes, such as, for example, pain and appetite, and cognitive functions, such as learning or memory.

The array is also useful for ascertaining the effect of the expression of a marker gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated. The array is also useful for ascertaining differential expression patterns of one or more marker genes in normal and abnormal cells. This provides a battery of marker genes that could serve as a molecular target for diagnosis or therapeutic intervention.

VI. Predictive Medicine

The present invention pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the level of expression of polypeptides or nucleic acids encoded by one or more marker genes of the invention, in order to determine whether an individual is at risk of relapse adenocarcinoma, preferably colorectal cancer. Such assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the relapse of the cancer.

Yet another aspect of the invention pertains to monitoring the influence of agents (e.g. drugs or other compounds administered either to inhibit the relapse of colorectal cancer or to treat or prevent any other disorder) on the expression or activity of a marker gene of the invention in clinical trials. These and other agents are described in further detail in the following sections.

A. Diagnostic Assays

The diagnostic assays provided by the invention can make use of the detection of the protein encoded by a marker gene. Therefore, in an embodiment of the invention, in the method according to the invention, the level of expression of said marker genes in the samples is assessed by detecting the presence in the samples of a protein encoded by each of said marker gene or a polypeptide or protein fragment comprising said protein. Preferably, the presence of said protein, polypeptide or protein fragment is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment. More preferably, the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.

The diagnostic assays provided by the invention can also make use of the detection of the polynucleotide encoded by the marker gene, Therefore, in an embodiment of the invention, the level of expression of said marker genes in the sample is assessed by detecting the presence or amount in the sample of a transcribed polynucleotide encoded by each of said marker genes or a portion of said marker genes. Preferably, the transcribed polynucleotide is a cDNA, mRNA or hnRNA. Preferably, the step of detecting further comprises amplifying the transcribed polynucleotide. Preferably, the presence of a transcribed polynucleotide is detected by a probe which anneals with each of said marker genes under stringent hybridization conditions and wherein the probe corresponds to the transcribed polynucleotide or a portion thereof. Preferably, a multitude of probes are used whereby the probes are attached to a solid phase in the form of an array. Preferably, the probe is labeled by the attachment of a label.

An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid encoded by a marker gene of the invention in a biological sample involves obtaining a biological sample (e.g. a biopsy of colon tissue or a lump) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g. mRNA, genomic DNA, or cDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide encoded by a marker gene of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunohistochemistry and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide encoded by a marker gene of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a protein or nucleotide encoded by a marker gene, and a probe, under appropriate conditions and for a time sufficient to allow the protein or nucleotide and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoring the protein or nucleotide on the one hand or probe on the other onto a solid phase supports also referred to as a substrate, and detecting complexes comprising the target marker gene or protein and the probe anchored on the solid phase at the end of the reaction. In; one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of the proteins or nucleotides encoded by the marker genes, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to a solid phase. These include, without limitation, the protein or nucleotide encoded by the marker gene or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g. biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin- coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker gene protein or nucleotide or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, the non immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of complexes comprising the marker protein or nucleotide sequence and the probe anchored to the solid phase can be accomplished in a number of methods outlined herein. In a preferred embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect complexes comprising a marker protein or nucleotide sequence and the probe without further manipulation or labeling of either component (the marker protein or nucleotide or the probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, U.S. Patent No. 5,631,169; U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent; label on a second 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal.

An PET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g. using a fluorimeter). In another embodiment, determination of the ability of a probe to recognize a protein or nucleotide encoded by a marker gene can be accomplished without labeling either assay component (probe or marker gene) by utilizing a technology such as real time Biomolecular Interaction Analysis (BIA) (see, e.g. Sjolander, S., and Urbaniczky, C, Anal. Chem. 63 (1991) 2338-2345 and Szabo, A., et al., Curr. Opin. Struct. Biol. 5 (1995) 699-705). As used herein, "BIA" or "surface plasmon resonance" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable which can be used as an indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with the marker protein or nucleotide and the probe as solutes in a liquid phase. In such an assay, complexes comprising the marker protein or nucleotide and the probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, such complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sci. 18 (1993 ) 284- 287). Standard chromatographic techniques may also be utilized to separate such complexes from uncomplexed components. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complexes may be separated from the relatively smaller uncomplexed components. Similarly, the different charge properties of such complexes as compared to the uncomplexed components may be exploited to; differentiate the complexes from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g. Heegaard, N.H., J. Mol. Recognit. 11 (1998) 141-148; Hage, D.S., and Tweed, S.A., J. Chromatogr. B. Biomed. Sci. Appl. 699 (1997) 499-525). Gel electrophoresis may also be employed to separate such complexes from unbound components (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non- denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

In a particular embodiment, the level of mRNA encoded by a marker gene can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA.

For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from colorectal cells (see, e.g., Ausubel et al., supra). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynsld (U.S. Patent No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA encoded by a marker gene of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of a mRNA with the probe indicates that the marker gene in question is expressed.

In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the a marker gene of the present invention.

An alternative method for determining the level of mRNA encoded by a marker gene of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193), self sustained sequence replication (Guatelli, J.C., et al, Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878), transcriptional amplification system (Kwoh, D.Y., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177), Q-Beta Replicase (Lizard et al., Bio/Technology 6 (1988) 1197), rolling circle replication (U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the colorectal cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA encoded by the marker gene.

As an alternative to maldng determinations based on the absolute expression level of the marker gene, determinations may be based on the normalized expression level of the marker gene. Expression levels are normalized by correcting the absolute expression level of a marker gene by comparing its expression to the expression of a gene that is not a marker gene, e.g. a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g. a non adenocarcinoma, preferably colorectal cancer, sample, or between samples from different sources.

Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker gene, the level of expression of the marker gene is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker gene. The expression level of the marker gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker gene. This provides a relative expression level.

Preferably, the samples used in the baseline determination will be from colorectal cancer or from non-colorectal cancer cells of colon tissue. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker gene assayed is colon specific (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from colorectal cells provides a means for grading the severity of the colorectal cancer state.

In another embodiment of the present invention, a polypeptide encoded by a marker gene is detected. A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide encoded by a marker gene of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab') 2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i. e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. Proteins from colorectal cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane, Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988. A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, immunohistochemistry and enzyme linked immunoabsorbant assay (ELISA).

A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether colorectal cells express a marker gene of the present invention.

In one format, antibodies, or antibody fragments, can be used in methods such as Western blots, immunohistochemistry or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody, proteins, or cells containing proteins, on a solid support. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from colorectal cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

In another embodiment of the invention, a format for the use in the LightCycler^® instrument is provided as described in US 6,174,670. These formats apply the fluorescent resonance energy transfer technology (see, for example, US Patent Nos. 4,996,143; 5,565,322; 5,849,489; and 6,162,603) and are based on the fact that when a donor and a corresponding acceptor fluorescent label are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent labels that can be visualized or otherwise detected and/or quantitated. As used herein, two probes, each containing a fluorescent label, whereby the probes hybridize to the a marker gene according to the invention being the target nucleic acid, can hybridize to an amplification product at particular positions determined by the complementarity of the probes to the target nucleic acid. Upon hybridization of the probes to the amplification product at the appropriate positions, a FRET signal is generated. Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range. As used herein with respect to donor and corresponding acceptor fluorescent labels, "corresponding" refers to an acceptor fluorescent label having an excitation spectrum that overlaps the emission spectrum of the donor fluorescent label. Accordingly, efficient non- radiative energy transfer can be produced there between. The preferred fluorescent label is fluorescein as the donor fluorescent label, whereby the acceptor fluorescent label is rhodamine, however, preferred is a cyanine dye, preferably Cy5 as described in US 6,174,670.

Therefore, in an embodiment of the invention, a method for detecting the presence or absence or amount of a target nucleic acid, i.e. a marker polynucleotide according to the invention, in a sample is provided, comprising the steps of:

performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with primers to produce an amplification product if the target nucleic acid is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of probes, wherein the members of said pair of probes hybridize to said amplification product within no more than five nucleotides of each other, wherein a first probe of said pair of probes is labeled with a donor fluorescent label and wherein a second probe of said pair of probes is labeled with a corresponding acceptor fluorescent label,and detecting the presence or absence of fluorescence resonance energy transfer between said donor fluorescent label of said first probe and said acceptor fluorescent label of said second probe, wherein the presence of FRET is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of FRET is indicative of the absence of the the target nucleic acid in the sample. Then the amount of the marker polynucleotide in the sample containing cells from a patient should be compared to the amount of the marker polynucleotide in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma. The marker polynucleotide is encoded by a marker gene selected from the marker genes listed in Table 3 or Table 2, 6 and 7.

In another preferred embodiment of the invention, a method for detecting a target nucleic acid, i.e. a marker polynucleotide, according to the invention, in a sample is provided, comprising the steps of amplifying the target nucleic acid by polymerase chain reaction in the presence of two nucleic acid probes, , that hybridize to adjacent regions of the target nucleic acid, one of said probes being labeled with an acceptor fluorescent label and the other probe labeled with a donor fluorescent label of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target nucleic acid, the donor and acceptor fluorescent labels are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase, nucleotides and primers for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the biological sample with light at a wavelength absorbed by the donor fluorescent label and detecting fluorescent emission from the fluorescence energy transfer pair. Then the amount of target nucleic acid in the sample containing cells from a patient should be compared to the amount of target nucleic acid in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma. The marker polynucleotide is encoded by a marker gene selected from the marker genes listed in Table 3 or Table 2, 6 and 7.

In another preferred embodiment of the invention, a method for the detection of a target nucleic acid, i.e. a marker polynucleotide according to the invention, in a sample is provided comprising the steps of amplifying the target nucleic acid by polymerase chain reaction in the presence of two nucleic acid probes, that hybridize to adjacent regions of the nucleic acid, one of said probes being labeled with an acceptor fluorescent label and the other probe labeled with a donor fluorescent label of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target nucleic acid, the donor and acceptor fluorescent labels are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase, nucleotides and primers for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the sample with light at a wavelength absorbed by the donor label and monitoring temperature dependent fluorescence from the fluorescence energy transfer pair. Then the amount of target nucleic acid in the sample containing cells from a patient should be compared to the amount of target nucleic acid in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma. The marker polynucleotide is encoded by a marker gene selected from the marker genes listed in Table 3 or Table 2, 6 and 7.

The detection of DNA amplification products can also be done in other "homogeneous" assay system. A "homogeneous" assay system comprises reporter molecules or labels which generate a signal while the target sequence is amplified. Another example for a "homogeneous" assay system is the TaqMan® system that has been detailed in US 5,210,015, US 5,804,375 and US 5,487,972. Briefly, the method is based on a double-labelled probe and the 5'-3' exonuclease activity of Taq DNA polymerase. The probe is complementary to the target sequence to be amplified by the PCR process and is located between the two PCR primers during each polymerisation cycle step. The probe has two fluorescent labels attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched by energy transfer due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of each amplification cycle, the Taq DNA polymerase in the process of elongating a primed DNA strand displaces and degrades the annealed probe, the latter due to the intrinsic 5'-3' exonuclease activity of the polymerase. The mechanism also frees the reporter dye from the quenching activity of TAMRA. As a consequence, the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. Accordingly, amplified target sequence is measured detecting the intensity of released fluorescence label.

Therefore, in another embodiment of the invention a method for the detection of a target nucleic acid, which is a marker polynucleotide according to the invention, in a sample is provided comprising the steps of

(a) providing a sample suspected to contain the target nucleic acid (b) providing an oligonucleotide, which is essentially complementary to a part or all of the target nucleic acid,

(c) optionally amplifying the target nucleic acid with a template-dependent DNA polymerase and primers (d) contacting the sample with the olignucleotide under conditions for binding the oligomeric compound to the target nucleic acid, (e) determining the binding product or the degree of hybridization between the target nucleic acid and the olignucleotide as a measure of the presence, absence or amount of the target nucleic acid.

Preferably in step (d) of the method, the degree of hybridization is determined by the quantity of the first or second fluorescent label that is released from the oligonucleotide hybridized to the target nucleic acid by exonuclease hydrolysis by the template-dependent DNA polymerase. Therefor, preferably, the olignucleotide comprises two labels, preferably two fluorescent labels. Then the amount of target nucleic acid in the sample containing cells from a patient should be compared to the amount of target nucleic acid in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma. The marker polynucleotide is encoded by a marker gene selected from the marker genes listed in Table 3 or Table 2, 6 and 7.

In a very preferred embodiment of the invention related in more detail to the TaqMan^® assay format, a method for the detection of a marker polynucleotide according to the invention, being the target nucleic acid, in a sample is provided comprising the steps of

(a) contacting a sample comprising single-stranded nucleic acids with a first oligonucleotide containing a sequence complementary to a region of the target nucleic acid and a secong oligonucleotide containing a first and a second fluorescent label, and whereby said first oligonucleotide contains a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the second oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first and second oligonucleotide such that the 3' end of the first oligonucleotide is upstream of the 5' end of the oligomeric compound, (b) maintaining the mixture of step (a) having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity of the polymerase to cleave the annealed, oligomeric compound and release labelled fragments; and (c) detecting and/or measuring the release of labelled fragments.

Then the amount of target nucleic acid in the sample containing cells from a patient should be compared to the amount of target nucleic acid in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal of the adenocarcinoma. The marker polynucleotide is encoded by a marker gene selected from the marker genes listed in Table 3 or Table 2, 6 and 7.

The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid encoded by a marker gene of the invention in a biological sample (e.g a colon-associated body fluid). Such kits can be used to determine if a subject is is at increased risk of progression of adenocarcinoma, preferably colorectal cancer. For example, the kit can comprise a labeled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide encoded by a marker gene of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g. an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for interpreting the results obtained using the kit.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g. attached to a solid support) which binds to a polypeptide corresponding to a marker gene of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label. For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide encoded by a marker gene of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoded by a marker gene of the invention. The kit can also comprise e.g, a buffering agent, a preservative, or a protein stabilizing agent. The ldt can further comprise components necessary for detecting the detectable label (e.g. an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

Therefore, the invention is related to a kit for assessing whether a patient carries a risk of progression of adenocarcinoma, particularly colorectal cancer, the kit comprising reagents for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting of the marker genes listed in Table 3. Preferably, all of said marker genes are selected from Table 2, 3, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c

Further, the invention is related to a kit for assessing the suitability of each of a plurality of compounds for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the kit comprising:

Preferably, all of said marker genes are selected from Table 2, 3, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c

B. Pharmacogenomics

Agents or modulators which have a stimulatory or inhibitory effect on expression of a marker gene of the invention can be administered to individuals to treat (prophylactically or therapeutically) adenocarcinoma, preferably colorectal cancer, in the patient. In conjunction with such treatment, the pharmacogenomics (i. e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g. drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the level of expression of a marker gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

In an embodiment of the invention, a method is provided of selecting a composition for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the method comprising:

a) providing a sample comprising cancer cells from the patient; b) separately exposing aliquots of the sample in the presence of a s plurality of test compositions; c) comparing expression of one or several marker genes in each of the aliquots; and d) selecting one of the test compositions which alters the level of expression of one or several of the marker genes in the aliquot containing that test composition relative to other test compositions; wherein at least one of said marker gene is selected from the group consisting of the marker genes listed in Table 3.

Pharmacogenomics deals with clinically significant variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Linder, M.W., et al, Clin. Chem. 43 (1997) 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action." Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti- malarials, sulfonamides, analgesics, nitrofurans) and consumption of Lava beans. As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g. N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug.

These polymorphisms are expressed in two phenotypes in the population, the extensive metabolized (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6 formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the level of expression of a marker gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of expression of a marker gene of the invention.

This invention also provides a process for preparing a database comprising at least one of the marker genes set forth in Table 3 or Table 2, 6 and 7, preferably all of said marker genes are selected from Table 2, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c. Most preferred, all marker genes are selected from the marker genes analysed in Fig. 4 to 14. For example, the polynucleotide sequences are stored in a digital storage medium such that a data processing system for standardized representation of the genes that identify a adenocarcinoma, preferably colorectal cancer, cell is compiled.

The data processing system is useful to analyze gene expression between two cells by first selecting a cell suspected of being of a neoplastic phenotype or genotype and then isolating polynucleotides from the cell. The isolated polynucleotides are sequenced.

The sequences from the sample are compared with the sequence(s) present in the database using homology search techniques. Greater than 90%, more preferably greater than 95% and more preferably, greater than or equal to 97% sequence identity between the test sequence and the polyoucleotides of the present invention is a positive indication that the polynucleotide has been isolated from a adenocarcinoma, preferably colorectal cancer, cell as defined above.

C. Monitoring Clinical Trials

Monitoring the influence of agents (e.g. drug compounds) on the level of expression of a marker gene of the invention can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent to affect marker gene expression can be monitored in clinical trials of subjects receiving treatment for adenocarcinoma, preferably colorectal cancer. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of (i) obtaining a pre- administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of one or more selected marker genes of the invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression of the marker gene(s) in the post- administration samples; (v) comparing the level of expression of the marker gene(s) in the pre-administration sample with the level of expression of the marker gene(s) in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent can be desirable to increase expression of the marker gene(s) to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent can be desirable to decrease expression of the marker gene(s) to lower levels than detected, i. e., to decrease the effectiveness of the agent.

D. Surrogate Marker genes

The marker genes of the invention may serve as surrogate marker genes for one or more disorders or disease states or for conditions leading up to disease states, and in particular, colorectal cancer. As used herein, a "surrogate marker gene" is an objective biochemical marker gene which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such marker genes is independent of the disease. Therefore, these marker genes may serve to indicate whether a particular course oftreatment is effective in lessening a disease state or disorder. Surrogate marker genes are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker gene, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker gene, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS).

Examples of the use of surrogate marker genes in the art include: Koomen, J.M., et al, J. Mass. Spectrom. 35 (2000) 258-264 and James, AIDS Treatment News Archive 209 (1994).

The marker genes of the invention are also useful as pharmacodynamic marker genes. As used herein, a "pharmacodynamic marker gene" is an objective biochemical marker gene whose expression correlates specifically with drug effects. The presence or quantity of expression of a pharmacodynamic marker gene is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker gene expresson is indicative ofthe presence or activity of the drug in a subject. For example, expression of a pharmacodynamic marker gene may be indicative of the concentration of the drug in a biological tissue, in that the marker gene is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level ofthe drug. In this fashion, the distribution or uptake of the drug may be monitored by assessing expression ofthe pharmacodynamic marker gene.

Similarly, the presence or quantity of expression of the pharmacodynamic marker gene maybe related to the presence or quantity ofthe metabolic product of a drug, such that the presence or quantity ofthe marker gene expression is indicative ofthe relative breakdown rate of the drug in vivo. Pharmacodynamic marker genes are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to s activate multiple rounds of marker gene transcription or expression, the amplified marker gene may be in a quantity which is more readily detectable than the drug itself.

Also, expression of the marker gene may be more easily detected due to the nature of the marker gene itself; for example, using the methods described herein, antibodies may be employed in an immune-based detection system for a protein encoded by a marker gene, or marker gene-specific radiolabeled probes may be used to detect a mRNA encoded by a marker gene. Furthermore, the use of a pharmacodynamic marker gene may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples ofthe use of pharmacodynamic marker genes in the art include: US 6,033,862; Hattis, D., Environ. Health Perspect. 90 (1991) 229-238; Schentag, J.J., Am. J. Health Syst. Pharm. 56 Suppl. 3 ((1999) S21-S24; and Nicolau, D.P., Am. J. Health Syst. Pharm. 56 Suppl. 3 (1999) S16-S20.

The marker genes of the invention are also useful as pharmacogenomic marker genes. As used herein, a "pharmacogenomic marker gene" is an objective biochemical marker gene whose expression correlates with a specific clinical drug response or susceptibility in a subject (see, e. g., McLeod, H.L., et al., Eur. J. Cancer 35 (1999) 1650-1652). The presence or quantity of expression of the pharmacogenomic marker gene is related to the predicted response ofthe subject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of expression of one or more pharmacogenomic marker genes in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA or protein encoded by specific tumor marker genes in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in marker gene DNA may correlate with drug response. The use of pharmacogenomic marker genes therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit ofthe invention.

Description ofthe Figures

Figure 1: Dendrogramm of recurrence-positive and recurrence-negative samples (suffix L, local recurrence; suffix D, distant metastasis) of the broad sample set (Example I) resulting from hierarchical cluster analysis. 8663 genes (A) and 163 genes (B) were considered in clustering.

Figure 2: Phylogenetic tree of recurrence-positive and recurrence-negative samples (suffix L, local recurrence; suffix D, distant metastasis) of the limitted sample set (Example II) resulting from hierarchical cluster analysis. 8575 genes (A) and 159 genes (B) were enlisted in clustering.

Figure 3: Phylogenetic tree of recurrence-positive and recurrence-negative samples (suffix D, distant metastasis) of the limitted sample set (Example III) resulting from hierarchical cluster analysis. 8057 genes (A) and 85 genes (B) were enlisted in clustering.

Figure 4-14: Box-whisker plots of expression values of differentially regulated genes, which were selected in Example II (A) as well as in Example III (B). Higher expression of KIAA0769, RIL, PLOD2, DPYSL3, SATB1, FXYD3, IGFBP4, EEF1A1 and BHLHB2 (Fig. 4-12) are associated with recurrence, whereas, MRPL12 and TIMELESS are downregulated in patients with early recurrence (Fig. 13-14). Examples

General remarks concerning

Primary Tumor Samples. A set of overall 37 primary colorectal adenocarcinomas, including 26 metastase-free samples and 11 recurrence positive tumor samples, were analyzed by gene expression analysis (Lockhart et al., 1996; Nature Genetics Supplement, 1999 and 2002) of approx. 12600 genes. The selected node-negative primary colorectal tumors of early TNM stages Tl-4, NO, M0 (Table 1), which were completely resected (R0), were obtained from patients who underwent colectomy at Klinikum rechts der Isar (Munich, Germany). The patients were not additionally treated by chemotherapy or radiation therapy either before or during surgical resection. The follow-up data of 60 months of all 26 recurrence-negative cases was mandatory. Patients having a relapse positive tumor developed distant metastases and local recurrence, respectively, within 60 months.

RNA Preparation, Probe Synthesis and Microarray Analysis. Sections of snap- frozen tumor tissues were transferred in Lysing Matrix D (Bio 101 System, Qbiogene GmbH, Heidelberg, Germany) and homogenized in 1 ml TriPure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) using FastPrep FP120 (BIO101 Savant, Qbiogene GmbH). The total RNA was extracted according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany) and finally purified using RNeasy^® RNA isolation system (Qiagen, Hilden, Germany). Except for minor modifications, biotinylated antisense RNA targets for GeneChip Expression Analysis (Affymetrix Incorporated, Santa Clara, CA) were generated according to the current technical manual (Affymetrix, Inc.). Briefly, cDNA synthesis was started from 20 μg total RNA using cDNA Synthesis System kit and oligo-dT T7 promoter primer (cDNA Synthesis System, Roche Diagnostics GmbH). In-vitro transcription of cDNA product was performed by MEGAscript™ (Ambion, Huntingdon, UK) using biotinylated UTP and CTP, followed by clean-up on a RNeasy" spin column (Qiagen). 10 μg fragmentated tumor probes were hybridized to HG95Av2 Affymetrix GeneChips Array (Affymetrix, Inc.) following the manufacturer's protocol. Raw data were analyzed using Microarray Suite Software MAS5.0 (Affymetrix, Inc.). However, only profiles with proper hybrization signals of internal standards were considered, i.e., the 375' ratio of either human GAPDH or beta-actin should be below 5 and signal intensities of spiked bacterial transcripts of bioB (1.5 pM), bioC (5 pM), bioD (15 pM) and ere (100 pM) should be within the scope. For reliable comparisons of multiple arrays the data were normalized by the ,global scaling' procedure of median values using the software program Roche Affymetrix Chip Experiment Analysis (RACE-A) which performs the publicly available and cited procedures as mentioned but is only a user-friendly collection of the methods and programs described.

Hierarchical Cluster and Statistical Analysis. Unsupervised two-way cluster analysis was performed using the software program cluster 2.11 and treeview 1.5 (Life Sciences Division, Lawrence Berkeley National Laboratory, Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, CA (Eisen, Spellman et al. 1998)). Genes, which were absent or marginal in all 37 samples (detection p-values between 0.06 - 0.1, MAS5.0), were eliminated from the data set. Prior to average linkage clustering, the prefiltered genes were log transformed, median centered on genes and, finally, normalized for genes. As similarity measures, the uncentered Pearson's correlation was used to calculate the resemblance of genes and profiles. Hierarchical cluster analysis were also performed with gene sets which have been reduced by certain filter criteria such as significance of differential expression and fold changes, followed by the procedure described above.

Selection of Genes Associated with Tumor Progression. To identify genes with statistically significant changes in expression, two approaches were carried out. First, the conventional method for analysis was applied. Transcripts displaying more than 1 1/2-fold (-0.5>CHGF>0.5) differential expression with a statistical significance P < 0.05 and P < 0.01, respectively, were selected. The statistical significance P of differently expressed genes between both groups of samples was calculated by Mann-Whitney's Utest (PU). The software program RACE-A (Roche Diagnostics GmbH) was used for the calculation of PU values and fold changes, whereby the expression levels of genes below 4 were set to 4. Second, the statistical significance was computed on the basis of permutation profiles for both conditions using the software program RACE-A (Roche Diagnostics GmbH). This statistical method was adapted from Significance Analysis of Microarrays (SAM) which was published by Tusher et al., 2001. The false discovery rate (FDR) indicating the percentage of genes which were identified by chance were held at FDR < 10 %. The software programs NetAffx (Liu et al., 2003) and Gene Index (Roche Diagnostics GmbH) which are linked to public databases such as UniGene, LocusLink, GeneBank, OMIM, etc. were used for descriptive, functional and sequence annotation.

Example I

Identification of genes which are associated with the tumor progression using the broad sample set

1. Selected Tumor Samples. Expression profiles of 37 primary colorectal adenocarcinomas, including 26 metastases-free samples and 11 recurrence positive tumor samples, were analyzed (Table 1). 70.2 % of all samples, 42 % recurrence negative samples and 100 % recurrence positive samples were male donors. The age of the patients range between 40 - 87 years (recurrence negative samples: 53 - 87 years; recurrence positive samples: 40 - 84 years), the median was 62 years (recurrence negative samples: 64 years; recurrence positive samples: 59 years).

2. Present Calls and R-squared Values. The human GeneChip U95Av2 covers 12625 gene transcripts. 35 - 55 % of the measured transcripts were reliably detected and the median value of these so-called Present Calls is 47. The R- squared value of the median of recurrence-positive and recurrence-negative samples is 0.9765.

3. Cluster Analysis. Unsupervised hierarchical clustering of all samples did not form distinct groups of samples with different risk indicating that only a small set of transcripts are indeed deregulated (Fig.lA). Clustering of 163 differentially expressed genes with a confidence level of 99.99 % shows that recurrence-negative gather within one branch except for 7 samples (Fig. IB).

4. Potential Gene Markers Associated with Tumor Progression. For the identification of gene markers which are differently expressed in relapse- negative and relapse-positive samples, 7 analyses considering all profiles and subsets of profiles, respectively, were performed. Genes which showed 1 1/2 fold change (-0.5>CHGF>0.5) in expression with a confidence level of 99.99 % (PU<0.01) were filtered and the union of all selected genes is listed in Table 2a, b and c. These criteria were met by a total of 249 genes, 105 were up-regulated in recurrence-positive samples and 144 genes were down-regulated. The SAM- based algorithm was used as an alternative method to identify 85 transcripts showing the most significant expression changes (Table 2a,b,c). 72 genes were up-regulated in recurrence-positive samples and 13 genes were downregulated. Both lists of genes generated by conventional filtering and SAM- based algorithm share 58 genes (Table 2c) including 48 up-regulated and 10 down-regulated genes in recurrence-positive samples. The reference ID's ofthe selected genes are given in Table 3 and the complete sequences are listed in the sequence listing.

Example II

Identification of genes which are associated with the tumor progression using a limited sample set

1. Selected Tumor Samples. In the following study only tumor specimens with more than 60 % tumor cells were considered. All samples were evaluated by a pathologist and the sample set was reduced to 18 primary colorectal adenocarcinomas, including 9 metastases-free samples and 9 recurrence positive tumors (Table 4). Furthermore the scaling factor for normalization of the profiles should be in the range of 0.5 to 2. 78 % of all samples, 55 % recurrence negative samples and 100 % recurrence positive samples were male donors. The age of the patients range between 40 - 85 years (recurrence negative samples: 61 - 85 years; recurrence positive samples: 40 - 62 years), the median was 62 years (recurrence negative samples: 67 years; recurrence positive samples: 59 years).

2. Present Calls and R-squared Values. On average 47.9 % ± 3.1 SD of the transcripts were detected. The R-squared value of median values of metastases- free and recurrence-positive samples was determined in a scatter blot as 0.962.

3. Cluster Analysis. Unsupervised hierarchical clustering of all samples did not form distinct groups of samples with different risk (Fig. 2A). But clustering of differentially expressed genes with a confidence level of 99.99 % shows that recurrence-negative and metastases-free samples group separately on two different branches ofthe dendrogramm (Fig. 2B). '

4. Identification of Significantly Expressed Genes. First the genes which were below detection limit in all 18 samples were excluded. The gene set was therefore reduced from 12625 to 8575 genes. The genes exhibiting expression changes of more than 1.5-fold (-0.5 > CHGF > 0.5) were selected. 9.6 % of measured transcripts are differentially expressed. 54 % were up-regulated in recurrence-positive samples and 46 % genes were down-regulated. Primarily those genes are of interest, which show significant changes in expression levels. 2.9 % of the transcripts are 1.5-fold differentially expressed with a confidence level of 95 % (PU<0.05). 0.5 % of the transcripts are 1.5-fold differentially expressed with a confidence level of 99 % (PU<0.01). The latter criteria were met by 103 genes, which are listed in Table 6a and c. 54 genes were up- regulated in recurrence-positive samples and 49 genes were down-regulated. The SAM-based algorithm identified 18 transcripts with a FDR < 10 % (Table 6b and c). 14 were up-regulated in recurrence-positive samples and 4 genes were down-regulated. The union of genes which were identified by conventional filtering and SAM are given in Table 6c. 16 genes were selected independently by both filtering methods. 3 genes seem to be associated with tumorgenesis (RIL (LIM domain protein; accession: X93510; AFFY_ID: 32610_at); FXYD3 (FXYD3 domain-containing ion transport regulator 3; accession: U28249; AFFYJD: 39087_at); IGFBP4 (insulin-like growth factor binding protein 4; accession: U20982; AFFY_ID: 3978 l_at)) and 8 genes belong to different functional categories (SEPP1 (selenoprotein P, plasma 1; accession: Z11793; AFFYJD: 34363_at); PLOD2 (procollagen-lysine, oxoglutarate 5-dioxygenase 2 ; accession: U84573; AFFY_ID: 34795_at); SATB1 (special AT-rich sequence binding protein 1; accession: M97287; AFFY_ID 36899_at); MRPL12 (mitochondrial ribosomal protein L12; accession: X79865 AFFY_ID: 39812_at); EFNA1 (ephrin Al; accession: M577730; AFFYJD 40425_at); IL4R (interleukin 4 receptor; accession: X52425; AFFYJD: 404_at) EEF1A1 (eukaryotic translation elongation factor 1 alpha 1; accession: W28170: AFFYJD: 40888_at); TIMELESS (timeless homolog (Drosophila); accession AF098162; AFFYJD: 41626_at)). The molecular function of the remaining 5 genes is not described yet. Of particular interest are the 2 genes which are not or low expressed in recurrence-free samples, such as RIL (LIM domain protein; accession: X93510; AFFYJD: 32610_at) and FXYD3 (FXYD3 domain- containing ion transport regulator 3; accession: U28249; AFFYJD: 39087_at).

5. Tumor Progression Markers which are not expressed in Colorectal Normal Tissue. For diagnostic and therapeutic application, the search for tumor progression markers is e.g. focused on transcripts which show no or low expression levels in colorectal adjacent normal tissue (colorectal tissue collection, Roche Diagnostic GmbH). 3 of those 105 (Table 6) genes which have been identified as tumor progression associated are not or low expressed in colorectal normal tissue. All 3 genes are down-regulated in recurrence positive samples. Candidate genes are the following: TIMELESS (timeless homolog (Drosophila); accession: AF098162; AFFYJD: 41626_at), RFC4 (replication factor C (activator 1) 4 (37kD); accession: M87339; AFFYJD: 1054_at) and CKS2 (CDC28 protein ldnase 2; accession: X54942; AFFYJD: 40690_at).

6. Identification of Tumor-Cell Associated Progression Markers. The list of potential tumor progression markers were compared with a list of genes which have been identified to be over-expressed in tumor-cell enriched samples compared to colorectal tumor homogenates (Poster at the 87^th meeting of the Deutsche Gesellschaft fur Pathologen in Bamberg 2003: Gene expression profiling of microdissected colorectal carcinoma cells). Among the 105 genes (Table 6) associated with tumor progression are 2 which were predominantly expressed in tumor cells. These represent potential candidate genes for therapeutic targets. GOSR1 (golgi SNAP receptor complex member 1; accession: AF047438; AFFYJD: 40725_at) is up-regulated in recurrence positive samples. CKS2 (CDC28 protein kinase 2; accession: X54942; AFFYJD: 40690_at) is down-regulated in recurrence positive samples.

Example III

Identification of genes which are associated with the development of distant metastasis

1. Selected Tumor Samples. The following study is analogous to that one of example II but cases of local recurrence were excluded. The sample set was reduced to 15 primary colorectal adenocarcinomas, including 9 metastases-free samples and 6 tumors which developed distant metastases within 5 years (Table 4). 73 % of all samples, 55 % recurrence negative samples and 100 % recurrence positive samples were male donors. The age of the patients range between 40 - 85 years (recurrence negative samples: 61 - 85 years; recurrence positive samples: 40 - 62 years), the median was 63 years (recurrence negative samples: 67 years; recurrence positive samples: 59 years). 2. Present Calls and R-squared Values. On average 47.9 % ± 2.5 SD of the transcripts were detected. The R-squared value of median values of metastases- free and recurrence-positive samples was determined in a scatter blot as 0.9629.

3. Cluster Analysis. Unsupervised hierarchical clustering of all samples did not form distinct groups of samples with different risk (Fig. 3A). But clustering of differentially expressed genes with a confidence level of 99.99 % shows that recurrence-negative and metastases-free samples group separately on two different branches ofthe dendrogramm (Fig. 3B).

4. Identification of Significantly Expressed Genes. First the genes which were below detection limit in all 15 samples were excluded. The gene set was therefore reduced from 12625 to 8057 genes. The genes exhibiting expression changes of more than 1.5-fold (-0.5 > CHGF > 0.5) were selected. 8.9 % of measured transcripts are differentially expressed. 52 % were up-regulated in recurrence-positive samples and 48 % genes were down-regulated. Primarily those genes are of interest, which show significant changes in expression levels. 2.1 % of the transcripts are 1.5-fold differentially expressed with a confidence level of 95 % (PU<0.05). 0.5 % of the transcripts are 1.5-fold differentially expressed with a confidence level of 99 % (PU<0.01). The latter criterion was met by 64 genes, which are listed in Table 7a and c. 36 genes were up-regulated in recurrence-positive samples and 28 genes were down-regulated. The SAM- based algorithm identified 72 transcripts with a FDR < 10 % (Table 7b and c). 68 were up-regulated in recurrence-positive samples and 4 genes were downregulated. Both sets share 34 genes (Table 7c). The 34 genes fall into the following main categories: signal transduction (18 %), oncogeneses (12 %), translation (3 %), apoptose (3 %) and others. Of particular interest are 8 genes which are not or low expressed in recurrence-free samples, such as ATM (ataxia telangiectasia mutated; accession: U26455; AFFYJD: 2000_at), RIL (LIM domain protein; accession: X93510; AFFYJD: 32610_at), KIAA0356 (KIAA0356 gene product; accession: AB002354; AFFYJD: 35650_at), ZNF288 (zinc finger protein 288; accession: AL050276; AFFYJD: 3821 l_at), KIAA0563 (KIAA0563 gene product; accession: AB011135; AFFYJD: 38915_at), FXYD3 (FXYD3 domain-containing ion transport regulator 3; accession: U28249; AFFYJD: 39087_at), UGCG (UDP-glucose ceramide glucosyltransferase; accession: D50840; AFFYJD: 40215_at) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1; accession: W28170; AFFYJD: 40888_at). 5. Intersection of Gene Lists from Example II and Example III. 11 of these 34 genes were also identified in Example II. The expression changes of these 11 genes in recurrence-free and recurrence-positive samples were visualized in Box-Whiskerplots (Fig. 4-15). Of particular interest are the 2 genes which are not or low expressed in recurrence-free samples, such as RIL (LIM domain protein; accession: X93510; AFFYJD: 32610_at) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1; accession: W28170; AFFYJD: 40888_at).

References

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Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, Valmeekam V, Sun S, Kulp D and Siani-Rose MA (2003). " NetAffx: Affymetrix probesets and annotations." Nucleic Acids Res. 2003 31(l):82-6.

Lockhart, D. J., Dong, H., Byrne, M. C, Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C, Kobayashi, M., Horton, H., Brown, E. L. (1996). "Expression monitoring by hybridization to high-density oligonucleotide arrays." Nat Biotechnol 14 (13): 1675-80.

Nature Genetics Supplement (1999) The Chipping Forecast Vol 21.

Nature Genetics Supplement (2002) The Chipping Forecast Vol 32.

Tusher, V. G., Tibshirani, R. and Chu, G. (2001). "Significance analysis of microarrays applied to the ionizing radiation response". Proc Natl Acad Sci U S A 98 (9): 5116-21. Legend of tables:

Table 1: Clinical data of all samples.

Table 2a,b,c: List of differentially expressed genes (Example I). 276 genes were identified by conventional filtering (1.5 fold change, PU < 0.01, present call in at least one sample) (table 2a), by SAM algorithm (FDR < 10 %) (table 2b) or by both methods (table 2c). The function ofthe genes is indicated with a one letter code (O, oncogene(sis), tumor; A, apoptosis, cell death; S, signal transduction; R, receptor, G-protein, P, kinase, protein (tyrosine) phosphatase; T, transcription; t, translation; n, nucle(ar/us); m, membrane; o, mitochondri(on/al); r, ribosomal; c, cytosol; i, integral; s, secreted; p, transport).

Table 3: Table of genes and reference ID.

Table 4: Clinical data of samples analyzed in example II and III.

Table 5: Table absent. All sequences are disclosed in the sequence listing

Table 6: Differentially expressed genes (Example II). 105 genes were identified by conventional filtering (1.5 fold change, PU < 0.01, present call in at least one sample) (table 6a), by SAM algorithm (FDR < 10 %) (table 6b) or by both methods (table 6c). The function ofthe genes is indicated with a one letter code (O, oncogene(sis), tumor; A, apoptosis, cell death; S, signal transduction; R, receptor, G-protein, P, kinase, protein (tyrosine) phosphatase; T, transcription; t, translation; n, nucle(ar/us); m, membrane; o, mitochondri(on/al); r, ribosomal; c, cytosol; i, integral; s, secreted; p, transport). The gene expression value of the house keeping gene GAPDH is specified for the determination of relative gene expression values. The median value of probe set AFFX-HUMGAPDH/M33197_M_at is 14612.10.

Table 7: Differentially expressed genes (Example III). 102 genes were identified by conventional filtering (1.5 fold change, PU < 0.01, present call in at least one sample) (table 7a), by SAM algorithm (FDR < 10 %) (table 7b) or by both methods (table 7c). The function ofthe genes is indicated with a one letter code (O, oncogene(sis), tumor; A, apoptosis, cell death; S, signal transduction; R, receptor, G-protein, P, ldnase, protein (tyrosine) phosphatase; T, transcription; t, translation; n, nucle(ar/us); m, membrane; o, mitochondri(on/al); r, ribosomal; c, cytosol; i, integral; s, secreted; p, transport). The 11 genes which were highlighted are also differentially expressed in Examples II. For the calculation of relative gene expression values, the median value of the house-keeping gene GAPDH (probe set AFFX-HUMGAPDH/M33197_M_at) was determined as 15263.09.

Table 1:

Table 2a: CONVENTIONAL FILTERING

Table 2b: SAM-ALGORITHM

Table 2c GENES IDENTIFIED BY BOTH METHODS

Table 3:

Table 4:

Table 6a: Conventional Filtering

Table 6b: SAM algorithm

Table 6c: Both methods

Table 7a: CONVENTIONAL FILTERING

-134

Table 7b: SAM

Table 7c: Both methods

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Claims

Patent Claims

1. A method of evaluating the progression of cancer of a patient who is afflicted with an adenocarcinoma, the method comprising comparing: a) the level of expression of one or several marker genes in a patient sample, and b) the level of expression of one or several of said marker genes in a sample from a control subject afflicted with an adenocarcinoma which did not recur within 5 years after surgical removal ofthe adenocarcinoma, wherein at least one of said marker genes is selected from the group consisting ofthe marker genes listed in Table 3, a significant difference between the level of expression of one or several of said marker genes in the patient sample and the level of one or several of said marker genes in a sample from a control subject is an indication that the patient carries the risk of progression of cancer.

2. The method according to claim 1, wherein the adenocarcinoma is a colorectal cancer.

3. The method according to claim 1 or 2, wherein several of said marker genes are selected from the group consisting ofthe marker genes listed in Table 3 or Table 2, 6 and 7.

4. The method according to any of the claims 1 to 3, wherein all of said marker genes are selected from Table 2, 3, 6 or 7 or all of said marker genes are selected from Table 2a and c, Table 2 b and c, Table 6a and c, Table 6b and c, Table 7a and c, or Table 7b and c.

5. The method according to any ofthe claims 1 to 4, wherein the patient sample comprises cells obtained from the patient.

6. The method according to claim 5, wherein the cells are in stool, in urine, in a blood fluid or in a lymph fluid.

7. The method according to any of the claims 1 to 5, wherein the sample is a colon tissue sample.

8. The method according to any of the claims 1 to 7, wherein the level of expression of said marker genes in the samples is assessed by detecting the presence in the samples of a protein encoded by each of said marker gene or a polypeptide or protein fragment comprising said protein.

9. The method of claim 8, wherein the presence of said protein, polypeptide or protein fragment is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment.

10. The method of claim 9, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.

11. The method of claim according to any of the claims 1 to 7, wherein the level of expression of said marker genes in the sample is assessed by detecting the presence or amount in the sample of a transcribed polynucleotide encoded by each of said marker genes or a portion of said marker genes.

12. The method of claim 11, wherein the transcribed polynucleotide is a cDNA, mRNA or hnRNA.

13. The method according to any of the claims 11 to 12, wherein the step of detecting further comprises amplifying the transcribed polynucleotide.

14. The method according to any of the claims 11 to 13, wherein the presence of a transcribed polynucleotide is detected by a probe which anneals with each of said marker genes under stringent hybridization conditions and wherein the probe corresponds to the transcribed polynucleotide or a portion thereof.

15. The method according to claim 14 wherein a multitude of probes are used whereby the probes are attached to a solid phase in the form of an array.

16. The method according to claim 14 wherein the probe is labeled by the attachment of a label.

17. The method according to any of the claims 1 to 16, wherein said significant difference comprises an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of one of said marker genes in the patient sample and the level of expression of the same marker gene in the sample from the control subject.

18. The method of the claims 1 to 16, wherein said significant difference comprises an at least 2 fold difference or a less than 0.5 fold difference between the level of expression of one of said marker genes in the patient sample and the level of expression of the same marker gene in the sample from the control subject.

19. A method of selecting a composition for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the method comprising: a) providing a sample comprising cancer cells from the patient; b) separately exposing aliquots of the sample in the presence of a s plurality of test compositions; c) comparing expression of one or several marker genes in each of the aliquots; and d) selecting one of the test compositions which alters the level of expression of one or several of the marker genes in the aliquot containing that test composition relative to other test compositions; wherein at least one of said marker gene is selected from the group consisting ofthe marker genes listed in Table 3.

20. A kit for assessing whether a patient carries a risk of progression of adenocarcinoma, particularly colorectal cancer, the kit comprising reagents for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting ofthe marker genes listed in Table 3.

21. A kit for assessing the suitability of each of a plurality of compounds for inhibiting the progression of adenocarcinoma, particularly colorectal cancer, in a patient, the kit comprising: a) the plurality of compounds; and b) a reagent for assessing expression of one or several marker genes, wherein at least one of said marker genes is selected from the group consisting ofthe marker genes listed in Table 3.

22. A method of deriving a candidate agent, said method comprising:

(a) contacting a sample containing adenocarcinoma cells, preferably colorectal cancer cells, with said candidate agent; (b) determining the level of expression of one or several marker genes in the sample contacted with the candidate agent and determining the level of expression of one or several of said marker genes in a sample not contacted with the candidate agent; (c) observing the effect of the candidate agent by comparing the level^' of expression of one or several of said marker genes in the sample contacted with the candidate agent and the level of one or several of said marker genes in the sample not contacted with the candidate agent, (d) deriving said agent from said observed effect, wherein at least one of said marker genes is selected from the group consisting ofthe marker genes listed in Table 3 and wherein an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of one of said marker genes in the sample contacted with the candidate agent and the level of expression of the same marker gene in the sample not contacted with the candidate agent is an indication of an effect ofthe candidate agent.

23. The method according to Claim 22, wherein said candidate agent is a candidate inhibitory agent.

24. The method according to Claim 22, wherein said candidate agent is a candidate enhancing agent.

25. A candidate agent derived by the method according to any ofthe claims 22 to 24.

26. A pharmaceutical preparation comprising an agent according to claim 25.

27. Use of an agent according to claim 25 for the preparation of a composition for the inhibition of progression of colorectal cancer.

28. A method of producing a drug comprising the steps ofthe method of any one of Claims 22 to 24; and

(i) synthesizing the candidate agent identified in step (d) or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or (ii) combining the drug candidate the candidate agent identified in step (d) or an analog or derivative thereof with a pharmaceutically acceptable carrier.