WO2005085861A2

WO2005085861A2 - Nucleic acids and encoded polypeptides for use in liver disorders and epithelial cancer

Info

Publication number: WO2005085861A2
Application number: PCT/EP2005/050897
Authority: WO
Inventors: Christian Guelly; Heimo Strohmaier; Charles Buck; Kurt Zatloukal
Original assignee: Oridis Biomed Forschungs- Und Entwicklungs Gmbh
Priority date: 2004-03-03
Filing date: 2005-03-01
Publication date: 2005-09-15
Also published as: WO2005085861A3

Abstract

The invention relates to nucleic acids and to corresponding encoded polypeptides and to their use for the diagnosis, prevention and/or treatment of liver disorders and neoplastic disorders, especially cancer of the liver and other epithelial tissues, benign liver neoplasms such as adenoma and other proliferative liver disorders such as focal nodular hyperplasia (FNH) and cirrhosis. The invention further relates to methods of diagnosing and treating these disorders.

Description

Description NUCLEIC ACIDS AND ENCODED POLYPEPTIDES FOR USE IN LIVER DISORDERS AND EPITHELIAL CANCER. Technical Field

[001] The invention relates to nucleotides and to corresponding encoded proteins and to their use for the diagnosis, prevention and/or treatment of liver disorders and neoplastic disorders, especially cancer of the liver and other epithelial tissues, benign liver neoplasms such as adenoma and other proliferative liver disorders such as focal nodular hypeφlasia (FNH) and cirrhosis. The invention further relates to methods of diagnosing and treating these disorders.

[002] The development of cancer in general is characterized by genetic mutations that alter activity of important cellular pathways including, for example, proliferation, apoptosis (cell death), response to stress and epithelial/stroma interactions. It is increasingly recognized that identification of nucleic acids that are deregulated in cancer can provide important new insight into the mechanisms of neoplastic transformation. Identification of deregulated nucleic acid expression in precancerous stages, such as macro regenerative nodules and the "large" and "small" cell change in liver cancer, provide understanding of early events in malignant transformation. Similarly, identification of deregulated gene expression in disorders characterized by tissue proliferation and remodeling, such as FNH and cirrhosis in the liver may distinguish nucleic acids involved in proliferation and malignant transformation. Together such deregulated nucleic acids and the encoded gene products have potential as new diagnostic markers for cancer. Moreover, the products of these deregulated nucleic acids per se are targets for therapeutic intervention in the prevention and/or treatment of these disorders in human patients.

[003] The liver plays a vital role in the metabolism of proteins, lipids, carbohydrates, nucleic acids and vitamins. There are numerous disorders effecting the liver that cannot be diagnosed, prevented or treated effectively, such as hepatocellular carcinoma (HCC). Examination of HCC is particularly well suited for the identification of deregulated gene expression in cancer. This is because tissue samples of HCC can be obtained from surgically resected tumors and the tumors are well circumscribed solid structures with little stromal tissue. Furthermore, as indicated above, there is the possibility for comparative analyses of benign and malignant tumors as well as cirrhosis, a non-neoplastic condition. If the limitations in the art of identifying differentially expressed genes associated with liver disorders could be overcome, this comparative approach may enable identification of deregulated nucleic acids specifically involved in the processes of cellular proliferation and tissue remodeling in a mature organ (e.g., in cirrhosis) as well as the identification and discrimination of gene expression alterations associated with hypeφlasia (such as FNH) and with benign and malignant neoplasms (e.g., adenoma and HCC). In HCC there is an urgent need for new and better diagnostic and therapeutic capabilities. Deregulated genes in liver cancer may also be highly relevant to other cancers of the gastrointestinal tract and indeed with other carcinomas (epithelial derived cancers) as these tissues share a common embryological origin.

[004] On a global basis, hepatocellular carcinoma (HCC) belongs to the most common malignant tumors accounting for about 1 million deaths/year (Ishak et al., 1999, Atlas of Tumor Pathology. Fascicle 31. Armed Forces Institute of Pathology, Washington, DC).

[005] Definitive diagnosis of neoplastic liver disorders such as HCC and many other tumors relies upon histopathological evaluation of biopsy specimens. This invasive surgical procedure is generally not undertaken until symptoms appear and the disease is then most often in advanced stages, thereby limiting therapeutic intervention options. Thus there is a need to improve diagnostics and methods of diagnosis. In addition, early diagnosis is crucial but hampered by late onset or even a lack of specific clinical symptoms. At diagnosis most HCC tumors are no longer amenable to surgical resection (except encapsulated tumors or the fibrolamellar variants) (Chen and Jeng, 1997, J. Gastroenterol. Hepatol.,, 12:329-34); moreover, they are highly resistant to _;>. cytostatic therapy (Kawata et al., 2001, Br. J. Cancer, 84:886-91). Overall, death usually occurs within 1 year after diagnosis. Thus, markers for early detection, prognostic indicators, and effective prevention and or treatment regimens for HCC are highly desirable in this field.

[006] In contrast, unlike the well-studied situation in colorectal cancer, liver adenoma may not represent a precursor lesion of HCC. Similarly, although cirrhosis and hepatitis viral infections are clearly risk factors for HCC, these conditions are not prerequisite for the development of HCC. Certain liver lesions may represent HCC prestages such as macro regenerative nodular hypeφlasia, but this is not yet confirmed (Shortell and Schwartz, 1991, Surg Gynecol Obstet., 173:426-31; Anthony, P. in MacSween et al, eds. Pathology of the Liver. 2001, Churchill Livingstone, Edinburgh). Although these disorders are diagnosed by histopathological investigation of liver resections and liver biopsies, no efficient method exists for earlier or non-invasive detection of these conditions. Again, there is immediate need for diagnostic and prognostic markers for these neoplasms and for non-invasive detection of these disorders.

[007] Within the past decade, several technologies have made it possible to monitor the expression level of a large number of transcripts within a cell at any one time (see, e.g., Schena et al., 1995, Science, 270:467-470; Blanchard et al., 1996, Nature Biotechnology, 1996, 14:1649). Transcript array technology has been utilized for the identification of genes that are up regulated or down regulated in various disordered states. Several recent studies have utilized this technology to examine changes in gene expression in HCC. These studies have variously revealed deregulation (i.e., over- and underexpression) of genes encoding liver specific proteins in HCC cell lines and HCC tissues relative to controls. Moreover the studies revealed genes essential for cell cycle control, stress response, apoptosis, lipid metabolism, cell-cell-interaction, DNA repair and cytokine and growth factor production (e.g., Graveel et al, 2001, Oncogene, 20:2704-12; Tackels-Horne et al, 2001, Cancer, 92: 395-405; Xu et al, 2001, Cancer Res., 61:3176-81). However, there is little concordance in the gene expression patterns reported in these studies that may be due to differences in experimental design and/or to the heterogeneity of HCC tissue per se. Moreover, the etiologies of these HCCs are an important factor. Chronic hepatitis B and C virus infections are the major causes of HCC but damage from alcohol and chronic liver metabolic disorders are also recognized to result in HCC and the mechanisms responsible for development of a tumor from these different etiologies are likely to differ. Taken together, until now no satisfactory diagnostics and methods of diagnosing have been developed in order to be able to intervene in liver disorders. The same applies to the therapy of liver disorders, and epithelial cancers. For HCC for instance, there is no effective therapeutic option except resection and transplantation but these approaches are only applicable in early stages of HCC, limited by the access to donor livers, and associated with severe risks for the patient. In addition, these approaches are extremely expensive. These cancers respond very poorly to chemotherapeutics, most likely due the normal liver function in detoxification and export of harmful compounds. Several other therapeutic options, such as chemoem- bolization, cryotherapy and ethanol injection are still in an experimental phase and the efficacy of these is not established. Surgical intervention remains the best treatment option but it is not possible to define with precision the extent of the tumor. This invasive procedure therefore, is suboptimal from the perspective of treatment. Furthermore, the lack of early diagnostics for specific liver dysfunctions leads most often to advanced progression of the disease that further confounds therapeutic options and dramatically increases patient mortality from these diseases (Jansen P.L., 1999, Neth. J. Med., 55:287-292). Thus until now no satisfactory therapies have been developed in order to be able to intervene in liver disorders, and other epithelial cancers. Furthermore, in the state of the art, recognition of the different subtypes of liver disorders such as HCC precursor lesions, benign liver neoplasms, and metabolic liver diseases such as alcoholic liver disease and cirrhosis, as revealed by differential gene expression, have not been disclosed. A summary of the key disease features of some of the disorders evaluated in the invention is provided in Table 1. [009] Table 1: Diseases features Table 1

Summary of the Invention

[010] The invention relates to nucleotides and to corresponding encoded proteins and their use for the diagnosis, prevention and/or treatment of liver disorders, especially of hepatocellular carcinoma (HCC), and epithelial cancers, pre-cancerous liver lesions, benign neoplasms such as adenoma, and other proliferative liver disorders such as focal nodular hypeφlasia (FNH) and cirrhosis. The invention also relates to vectors and cells comprising such nucleic acids, and to antibodies or antibody fragments directed against said polypeptides and nucleic acids.

[011] The invention further relates to methods of diagnosing and treating these disorders. The evaluation of multiple disorders with overlapping but distinct moφhological and clinical features provides new information for identification and discrimination and ultimately new therapeutic strategies for these disorders according to invention. Disclosure of Invention

[012] A unique approach employed in this invention utilizes discrete, pathologist- confirmed liver cancer pathologies for production of disease specific cDNA libraries enriched in genes specifically up- and down-regulated in HCC compared with a pool of non-neoplastic human livers. The library is a genome-wide representation of deregulated gene expression in HCC and therefore includes all potential HCC deregulated genes. Repetitive hybridization to these library clones with labeled expressed nucleic acids from many additional discrete, pathologist-confirmed liver cancer samples (HCCs) and non-malignant liver lesions indicated nucleic acids highly deregulated in HCC. The suφrising finding is that this approach provides deregulated nucleic acids that had not previously been identified as well as many deregulated nucleic acids that were not before associated with HCC, the elevated expression of which can also be associated with other neoplasms. These HCC deregulated genes and proteins are the subject of this invention.

[013] The screening and verification strategy is already inventive per se owing to the elaborate and defined choice of parameters. Identification of differentially expressed genes according to the invention relies upon histopathologically distinguished liver disease tissue for comparison of gene expression changes in disorders of the human liver. Non-diseased reference liver samples for the experiments are also diagnostically confirmed.

[014] The object of the invention is a method of diagnosis of a liver disorder, liver cancer and/or epithelial cancer, wherein at least one compound selected from the group consisting of a polypeptide according to the sequence SEQ ID 1 to SEQ ID 93 (Table 2A to 2D), a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, or variants thereof, is identified in the sample of a patient and compared with at least one compound of a reference library or of a reference sample.

[015] Another object of the invention is a method of treating a patient suffering from a liver disorder or an epithelial cancer, wherein at least one compound selected from the group consisting of a polypeptide according, to the SEQ ID 1 to SEQ ID 93, a functional variant of one of the aforementioned polypeptides, a nucleic acid encoding one of the aforementioned polypeptides or a functional variant thereof, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of one of the aforementioned antibodies directed against one of the aforementioned polypeptides or against a functional variant thereof, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, is administered to the patient in need of a the treatment in a therapeutically effective amount.

[016] Another aspect of the invention is a pharmaceutical composition comprising at least one compound selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides or a functional variant thereof, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody directed against one of the aforementioned polypeptides, an antibody directed against a functional variant of one of the aforementioned polypeptides, a fragment of one of the aforementioned antibodies, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments and, optionally, suitable additives or auxiliaries.

[017] The accession numbers of the polypeptides according to the invention and their cDNAs are shown in Table 2A to 2D.

[018]

[019]

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[024]

[025]

[026]

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[028]

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[030]

[031]

[032]

[033]

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[035]

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[037]

[038] [039] [040] [041] [042] [043] [044] [045] [046] [047] Table 2A to 2D: Polypeptides and cDNAs with their respective SEQ ID numbers and accession numbers from the GenBank database.

[048] Table 2A

[049] [050] [051] Table 2B

Table 2C

[053] Table 2D

[054] [(**)c-syn represents three alternative nucleotide transcripts with corresponding three protein products]

[055] [056] A subset of these nucleic acids according to the invention have been shown by RT- PCR analysis to be specifically expressed or deregulated in other cancers of epithelial origin and preferably not in corresponding normal human tissue(s). These nucleic acids include SEQ ID Nos. 94 to 186 (provided in Table 2A to 2D). Deregulated nucleic acids in liver cancer may preferably be highly relevant to other cancers of the gastrointestinal tract as these tissues share a common embryological origin. Consequently, these nucleic acids and the encoded polypeptides may preferably be similarly utilized for diagnostics methods, pharmaceutical compositions and methods of prevention and/ or treatment of these epithelial cancers.

[057] The polypeptides and nucleic acids according to the invention have in common that they are differentially expressed in a sample isolated from a patient suffering from a disorder according to the invention compared to a reference sample. The regulation of the polypeptides and nucleic acids according to the invention is essential for the pathologic process and which are thus in a direct or indirect relationship with diagnosis, prevention and/or treatment of disorders according to the invention. The polypeptides and the nucleic acids according to the invention do not belong to the targets known until now such that suφrising and completely novel approaches for diagnosis and therapy result from this invention.

[058] Generally, the analysis of differentially expressed genes in tissues is less likely to result in errors in the form of artifactual false-positive clones than the analysis of cell culture systems. In addition to the fact that existing cell culture systems cannot adequately simulate the complexity of pathological processes in the tissue, the variations in cell behavior in the culture environment lead to nucleic acid and polypeptide expression patterns with questionable relation to the actual pathologic state. These problems may be less pronounced by an approach that utilizes gene expression in normal and diseased human tissue but again multiple variables confound clear identification of differential gene expression that is directly relevant to disease. For example, differentially expressed nucleic acids may result from inter-individual differences, metabolic state and/or clinical treatment paradigm. Further, large scale gene expression studies using cDNA microarrays do not indicate the cellular source of variation in gene expression. In addition, a differential gene expression study including all or most genes produces a very large volume of data that confounds identification of key disease-associated gene expression changes. Consequently, an approach that includes large scale profiling of gene expression from tissue from liver disorders that are defined only generally (as for example, "liver tumors") is unlikely to illuminate key genes involved in the disease process and it is these key genes that represent best targets for diagnostics and therapeutic intervention.

[059] On account of these difficulties, the success of the screening is significantly dependent on the choice of the experimental parameters. While the methods used are based on established procedures, the screening and verification strategy is already inventive per se owing to the elaborate and defined choice of parameters. A unique approach employed in this invention utilizes discrete, pathologist-confirmed liver cancer pathologies for production of disease specific cDNA libraries enriched in nucleic acids specifically up- and down-regulated in HCC compared with a pool of non-neoplastic human livers. Non-diseased reference liver samples for the experiments are also diagnostically confirmed and pooled from 3 independent samples to reduce detection of false positives resulting from inter-individual variations. Nucleic acids commonly expressed at similar levels in the reference liver pool and in diseased liver (i.e., HCC) are removed by the generation of subtractive suppressive hybridization (SSH) cDNA libraries (Diatchenko et al., 1996, Proc. Natl. Acad. Sci. USA, 93:6025-6030). These cDNAs are highly enriched for nucleic acids both up- and down-regulated in HCC but do not represent those that are not differentially expressed. Each of several thousand SSH clones were amplified by the polymerase chain reaction (PCR) and affixed to glass slides in custom cDNA microarrays. RNA from additional pathologist-confirmed liver disorders is converted to fluorescently-labeled cDNA for competitive hybridization with the pooled non-diseased liver RNA on the microarrays. The resulting ratio of hybridization intensity reveals nucleic acids specifically deregulated in liver disorders. In addition to providing a pool of candidate cDNAs highly enriched for differentially expressed genes, the SSH library represents on a genome-wide scale most if not all differentially expressed genes with far fewer clones than in standard cDNA libraries. This feature thereby focuses on nucleic acids specifically deregulated in disease. The SSH libraries generated in this invention include cDNA clones from nucleic acids that are essentially not expressed in normal liver and thereby not represented in conventional cDNA libraries or on genome-scale cDNA microarrays. Overexpression of the sequences according to the invention in liver disorder tissue compared to normal liver is confirmed by independent analysis of RNA levels with sequence-specific quantitative RT-PCR (Q-PCR). In these verification experiments, PCR product corresponding to the cellular RNA levels of the sequences according to the invention are monitored by fluorescent detection of the specific PCR product. The fluorescent signal is provided either by a sequence specific hydrolysis probe oligonucleotide (primer) in the TaqMan/Assay-on-Demand procedure (Figure 100 to 103) or by a fluorescent double stranded DNA binding dye such as SYBR green (Figure 104). Levels of PCR products corresponding to the sequences according to the invention are normalized for experimental variability by comparison with the levels of 'housekeeping' genes including β-actin, which are considered relatively invariant in disease or following experimental manipulations. The reference gene primers used for TaqMan Q-PCR analyses are GAPDH-pl, (SEQ ID 187); GAPDH-p2, (SEQ ID 188); GAPDH-p3, (SEQ ID 189); 6Actin-pl, (SEQ ID 190); BActin-p2, (SEQ ID 191); and 6Actin-p3, (SEQ ID 192). The reference gene primers used for SYBR Green analyses are βActin-p4, (SEQ ID 193); and 6Actin-p5, (SEQ ID 194). The determination of RNA levels relative to these housekeeping genes in Q-PCR experiments is performed according to the method of Pffafl (Nucleic Acids Research, 2001, 29(9):e45). These techniques are well known to a person skilled in the art.

[061] Furthermore, expression of HCC deregulated genes according to this invention correlates with proliferation of hepatoma cells (Hep3B, HepG2) following for example 8 hours and 12 hours serum stimulation of quiescent cells. This finding supports the suggestion that overexpression of the sequences according to the invention is functionally significant for proliferative liver disorders such as liver cancer.

[062] Compared to the state of the art, these polypeptides and nucleic acids suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or epithelial cancers. The nucleic acids and polypeptides according to the invention can be utilized for the diagnosis, prevention and treatment of liver disorders, and epithelial cancers.

[063] The present invention relates to at least one polypeptide comprising a sequence according to one of the SEQ ID 1 to SEQ ID 93, or a functional variant thereof. The invention also relates to a nucleic acid coding for the polypeptide or a functional variant thereof.

[064] In preferred embodiment the polypeptide consists of the sequence according to the SEQ ID 1. In another preferred embodiment the nucleic acid consists of the sequence according to the SEQ ID 94.

[065] Compared to the state of the art, these polypeptides and nucleic acids suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or epithelial cancers.

[066] In another aspect of the invention the invention relates to the use of at least one polypeptide according to the SEQ ID 1 to SEQ ID 93, a functional variant of the polypeptide, a nucleic acid encoding one of the aforementioned polypeptides, a nucleic acid encoding the functional variant, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody directed against one of the aforementioned polypeptides, an antibody directed against a functional variant of one of the aforementioned polypeptides, a fragment of one of the aforementioned antibodies, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and/or at least one cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, for the diagnosis, prevention and/or treatment of disorders according to the invention. Further embodiments of the invention are described in detail below.

[067] When compared to the state of the art of therapy of liver disorders, and/or epithelial cancers the use of these components suφrisingly provide an improved, sustained and/ or more effective diagnosis, prevention and/or treatment of disorders according to the invention.

[068] The term "polypeptide" refers to the full length of the polypeptide according to the invention. In a preferred embodiment the term "polypeptide" also includes isolated polypeptides and polypeptides that are prepared by recombinant methods, e.g. by isolation and purification from a sample, by screening a library and by protein synthesis by conventional methods, all of these methods being generally known to the person skilled in the art. Preferably, the entire polypeptide or parts thereof can be synthesized, for example, with the aid of the conventional synthesis such as the Merrifield technique. In another preferred embodiment, parts of the polypeptides according to the invention can be utilized to obtain antisera or specific monoclonal antibodies, which may be used to screen suitable gene libraries prepared to express the encoded protein sequences in order to identify further functional variants of the polypeptides according to the invention.

[069] The term "polypeptide according to the invention" refers to the polypeptides according to the SEQ ID 1 to SEQ ID 93 (Table 2A to 2D).

[070] The term "functional variants" of a polypeptide within the meaning of the present invention refers to polypeptides which have a sequence homology, in particular a sequence identity, of about 70%, preferably about 80%, in particular about 90%, especially about 95%, most preferred of 98 % with the polypeptide having the amino acid sequence according to one of the SEQ ID 1 to SEQ ID 93. Such functional variants are, for example, the polypeptides homologous to a polypeptide according to the invention, which originate from organisms other than human, preferably from non- human mammals such as, for example mouse, rats, monkeys and pigs. Other examples of functional variants are polypeptides that are encoded by different alleles of the gene, in different individuals, in different organs of an organism or in different developmental phases.

[071] Functional variants, for example, also include polypeptides that are encoded by a nucleic acid which is isolated from non-liver-tissue, e.g. embryonic tissue, but after expression in a cell involved in liver disorders have the designated functions. Functional variants preferably also include naturally occurring or synthetic mutations, particularly mutations that quantitatively alter the activity of the peptides encoded by these sequences. Further, such variants may preferably arise from differential splicing of the encoding gene.

[072] "Functional variants" refer to polypeptides that have essentially the same biological funtion(s) as the corresponding polypeptide according to the invention. Such biological function can be assayed in a functional assay.

[073] In order to test whether a candidate polypeptide is a functional variant of a polypeptide according the invention, the candidate polypeptide can be analyzed in a functional assay generally known to the person skilled in the art, which assay is suitable to assay the biological function of the corresponding polypeptide according to the invention. Such functional assay comprise for example cell culture systems; enzymatic assays, the generation of mice in which the genes are deleted ("knocked out") or mice that are transgenic for gene encoding the candidate polypeptide, etc. If the candidate polypeptide demonstrates or directly interferes with essentially the same biological function as the corresponding polypeptide according to the invention, the candidate polypeptide is a functional variant of the corresponding polypeptide, provided that the candidate polypeptide fulfills the requirements on the level of % sequence identity mentioned above.

[074] Furthermore, the term "functional variant" encompasses polypeptides that are preferably differentially expressed in patients suffering from liver disorders, or other epithelial cancers relative to a reference sample or a _^reference library, including polypeptides expressed from mutated genes or from genes differentially spliced, provided that the candidate functional variant polypeptide fulfills the criteria of a functional variant on the level of % sequence identity. Such expression analysis can be carried out by methods generally known to the person skilled in the art.

[075] "Functional variants" of the polypeptide can also be parts of the polypeptide according to the invention with a length of at least from about 7 to about 1000 amino acids, preferably of at least 10 amino acids, more preferably at least 20, most preferred at least 50, for example at least 100, for example at least 200, for example at least 300, for example at least 400, for example at least 500, for example at least 600 amino acids provided that they have essentially the same biological function(s) as the corresponding polypeptide according to the invention. Functional variants, such as in fusion proteins, may contain either on one or both ends additional aminoacid stretch(es), preferably 1 to 50 amino acids, more preferably 20 amino acids. Also included are deletions of the polypeptides according to the invention, in the range from about 1-30, preferably from about 1-15, in particular from about 1-5 amino acids provided that they have essentially the same biological function(s) as the corresponding polypeptide according to the invention. For example, the first amino acid methionine can be absent without the function of the polypeptide being significantly altered. Also, post-translational modifications, for example lipid anchors or phosphoryl groups may be present or absent in variants.

[076] "Sequence identity" refers to the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example BLASTP 2.0.1 and in the case of nucleic acids by means of for example BLASTN 2.014, wherein the Filter is set off and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).

[077] "Sequence homology" refers to the similarity (% positives) of two polypeptide sequences determined by means of for example BLASTP 2.0.1 wherein the Filter is set off and BLOSUM is 62 (Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402).

[078] The term "liver disorder" refers to and comprises all kinds of disorders that preferably affect the anatomy, physiology, metabolic, and/or genetic activities of the liver, that preferably affect the generation of new liver cells, and/or the regeneration of the liver, as a whole or parts thereof preferably transiently, temporarily, chronically or permanently in a pathological way. Preferably also included are inherited liver disorders and neoplastic liver disorders. Liver disorder is further understood to preferably comprise liver disorders caused by trauma, intoxication, in particular by alcohol, drugs or food intoxication, radiation, infection, cholestasis, immune reactions, and by inherited metabolic liver diseases. Prefened examples of liver disorders include cirrhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, and haemochromatosis. Preferably further included are autoimmune-disorders wherein the autoimmune response is directed against at least one polypeptide according to the invention. Within the meaning of the present invention the term "liver disorder" preferably also encompasses liver cancer, for example hepatocellular carcinoma (HCC), benign liver neoplasms such as adenoma and/or FNH. Preferably HCC further comprises subtypes of the mentioned disorders, preferably including liver cancers characterized by intracellular proteinaceous inclusion bodies, HCCs characterized by hepatocyte steatosis, and fibrolamellar HCC. For example, precancerous lesions are preferably also included such as those characterized by increased hepatocyte cell size (the "large cell" change), and those characterized by decreased hepatocyte cell size (the "small cell" change) as well as macro regenerative (hypeφlastic) nodules (Anthony, P. in MacSween et al, eds. Pathology of the Liver, 2001, Churchill Livingstone, Edinburgh).

[079] The term "epithelial cancer" within the meaning of the invention includes adeno- carcinomas of any organ other than the liver, preferably of the lung, stomach, kidney, colon, prostate, skin and breast, and refers to disorders of these organs in which epithelial cell components of the tissue are transformed resulting in a malignant tumor identified according to the standard diagnostic procedures as generally known to a person skilled in the art.

[080] Within the meaning of the invention the term "disorder according to the invention" encompasses epithelial cancer and liver disorders as defined above.

[081] In the case of polypeptides, the term "differential expression of a polypeptide" refers to the relative level of expression of the polypeptide in an isolated sample from a patient compared to the expression of the polypeptide in a reference sample or a reference library. The expression can be determined by methods generally known to the person skilled in the art. Examples of such methods include immunohistochemical or immunoblot or ELISA detection of the polypeptide with antibodies specific for the polypeptide. Detection of the polypeptide through genetic manipulation to label the polypeptide and detection in a model system is preferably also included such as by tagging the polypeptide in a transgene for expression in a model system.

[082] The term "sample" refers to a biomaterial comprising liver tissue or liver cells, also tissue from another organ subject to malignant transformation or a cell from this organ, blood, serum, plasma, ascitic fluid, pleural effusions, cerebral spinal fluid, saliva, urine, semen or feces.

[083] The sample can be isolated from a patient or another subject by means of methods including invasive or non-invasive methods. Invasive methods are generally known to the skilled artisan and comprise for example isolation of the sample by means of puncturing, surgical removal of the sample from the opened body or by means of endoscopic instruments. Minimally invasive and non-invasive methods are also known to the person skilled in the art and include for example, collecting body fluids such as blood, serum, plasma, ascitic, pleural and cerebral spinal fluid, saliva, urine, semen, and feces. Preferably the non-invasive methods do not require penetrating or opening the body of a patient or subject through openings other than the body openings naturally present such as the mouth, ear, nose, rectum, urethra, and open wounds.

[084] The term "minimally invasive" procedure refers to methods generally known, especially by persons skilled in the art, for obtaining patient sample material that do preferably not require anesthesia, can be routinely accomplished in a physician office or clinic and are either not painful or only nominally painful. The most common example of a minimally invasive procedure is venupuncture.

[085] The term "reference sample" refers to a sample that serves as an appropriate control to evaluate the differential expression of a nucleic acid and/or a polypeptide according to the invention in a given sample isolated from a patient; the choice of such appropriate reference sample is generally known to the person skilled in the art. Examples of reference samples include samples isolated from a non-diseased organ or tissue or cell(s) of the same patient or from another subject, wherein the non-diseased organ or tissue or cell(s) is selected from the group consisting of liver tissue or liver cells, blood, or the samples described above. For comparison to expression in the sample isolated from a patient with the liver disorder, the reference sample may also include a sample isolated from a non-diseased organ or tissue or cell(s) of a different patient, wherein the liver disordered- tissue or cell(s) is selected from the sample group listed above. Moreover the reference may include samples from healthy donors, preferably matched to the age and sex of the patient.

[086] The term "reference library" refers to a library of clones representing expressed genes, which library is preferably prepared from non-diseased liver tissue or cells. The reference library may also derive from mRNA from non-diseased liver tissue or cells and may also comprise a data base comprising data on non-diseased tissue expression of nucleic acids. For comparison of the expression of the nucleic acids or polypeptides according to the invention in a sample isolated from a patient with the disordered liver, the reference library may comprise an expression library prepared from liver disorder- diseased liver tissue or cells and a data base comprising data on liver disorder-specific expression of nucleic acids.

[087] The term "patient" within the meaning of the invention includes animals, preferably mammals, and humans, dead or alive. The patient is either suffering from a liver disorder, and/or other epithelial cancer, subject to analysis, preventive measures, therapy and/or diagnosis in the context of liver disorder and/or other epithelial cancer.

[088] The term "subject" within the meaning of the invention includes animals, preferably mammals, and humans, dead or alive that is not suffering from a liver disorders and/or other epithelial cancer and thus represent a preferred appropriate control for the determination of differential expression of nucleic acids and/or polypeptides according to the invention in a patient.

[089] The term "effective treatment" within the meaning of the invention refers to a treatment that preferably cures the patient from at least one disorder according to the invention and/or that improves the pathological condition of the patient with respect to at least one symptom associated with the disorder, preferably 3 symptoms, more preferably 5 symptoms, most preferably 10 symptoms associated with the disorder; preferably on a transient, short-term (in the order of hours to days), long-term (in the order of weeks, months or years) or permanent basis, wherein the improvement of the pathological condition may be preferably constant, increasing, decreasing, continuously changing or oscillatory in magnitude as long as the overall effect is a significant improvement of the symptoms compared with a control patient. Therapeutic efficacy and toxicity, e.g. ED and LD may be determined by standard pharmacological procedures in cell cultures or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index and may be expressed by the ratio LD /ED . Pharmaceutical compositions that exhibit large therapeutic indexes are prefened. The dose must be adjusted to the age, weight and condition of the individual patient to be treated, as well as the route of administration, dosage form and regimen, and the result desired, and the exact dosage should of course be determined by the practitioner.

[090] The actual dosage depends on the nature and severity of the disorder being treated, and is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect. However, it is presently contemplated, that pharmaceutical compositions comprising of from about 0.1 to 500 mg of the active ingredient per individual dose, preferably of from about 1 to 100 mg, most prefened from about 1 to 10 mg, are suitable for therapeutic treatments.

[091] The active ingredient may be administered in one or several dosages per day. A satisfactory result can, in certain instances, be obtained at a dosage as low as 0.1 mg/kg intravenously (i.v.) and 1 mg perorally (p.o.). Prefened ranges are from 0.1 mg/kg/day to about 10 mg/kg /day i.v. and from 1 mg/kg/day to about 100 mg/kg/day p.o.

[092] In another aspect the invention relates to a fusion protein comprising a polypeptide according to the SEQ ID 1 to 93, or a functional variant thereof.

[093] A "fusion protein" refers to a polypeptide comprising at least one polypeptide according to the SEQ ID 1 to SEQ ID 93, a functional variant or part thereof and at least one component A selected from polypeptide, peptide and/or peptide analogue that is linked to the polypeptide according to the invention by means of covalent or non- covalent binding such as e.g. hydrogen bonds, generally known to the person skilled in the art. Prefened examples of component A for fusion proteins are polypeptide, peptide and/or peptide analogues that facilitate easier detection of the fusion proteins; these are, for example, "green-fluorescent-protein", or variants thereof. Also included are fusion proteins that facilitate purification of the recombinant protein such as "His- tags", or fusions that increase the immunogenicity of the protein.

[094] Fusion proteins according to the invention can be produced by methods generally known to the person skilled in the art. The fusion proteins according to the invention can be used for the diagnosis, prevention and or treatment of liver disorders and/or epithelial cancer.

[095] Compared to the state of the art, these fusion proteins suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis and/or improved, sustained and/or more effective treatment of the liver disorders and/or epithelial cancers.

[096] Prefened nucleic acids according to the invention comprise a sequence according to one of SEQ ID 94 to SEQ ID 186, or a variant thereof. In particular the invention relates to nucleic acids according to the invention that have been isolated.

[097] Compared to the state of the art, these nucleic acids and polypeptides suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis and/or improved, sustained and/or more effective treatment of the liver disorders and/or epithelial cancers.

[098] The term "nucleic acid according to the invention" refers to the nucleic acids corresponding to the SEQ ID 94 to SEQ ID 186 and/or variants thereof.

[099] The term "encoding nucleic acid" relates to a DNA sequence that codes for an isolatable bioactive polypeptide according to the invention or a precursor thereof. The polypeptide can be encoded by a sequence of full length or any part of the coding sequence as long as the biological function, such as for example receptor-activity, is essentially retained (cf. definition of functional variant).

[100] It is known that small alterations in the sequence of the nucleic acids described above can be present, for example, due to the degeneration of the genetic code, or that untranslated sequences can be attached to the 5' and/or 3' end of the nucleic acid without significantly affecting the activity of the encoded polypeptide. This invention, therefore, also comprises so-called naturally occurring and artificially generated "variants" of the nucleic acids described above.

[101] Preferably, the nucleic acids used according to the invention are DNA or RNA, preferably a DNA, in particular a double-stranded DNA. In particular the nucleic acid according to the invention may be an RNA molecule, preferably single-stranded or a double-stranded RNA molecule. The sequence of the nucleic acids may further comprise at least one intron and or one polyA sequence.

[102] Nucleic acids according to the invention can be produced by methods generally known to the skilled artisan and have also been described in detail below.

[103] "Variant" within the meaning of the invention refers to all DNA sequences that are complementary to a DNA sequence, which hybridize with the reference sequence under stringent conditions and have a similar activity to the conesponding nucleic acid according to the invention. The nucleic acids according to the invention can also be used in the form of their antisense sequence.

[104] "Variant" of the nucleic acids can also be homologues from other species with sequence identity preferably 80%, in particular 90%, most prefened 95%.

[105] "Variant" of the nucleic acids can also be parts of the nucleic acid according to the present invention with at least about 8 nucleotides length, preferably with at least about 16 nucleotides length, in particular with at least about 21 nucleotides length, more preferably with at least about 30 nucleotides length, even more preferably with at least about 40 nucleotides length, most preferably with at least about 50 nucleotides length as long as the parts have a similar activity to the conesponding polypeptide according to the invention. Such a functional activity of an expressed polypeptide encoded by such a nucleic acid can be assayed using the functional assays described further above.

[106] In a prefened embodiment of the invention the nucleic acid comprises a nucleic acid having a sequence complementary to a nucleic acid according to the invention, or a variant thereof. Preferably the nucleic acid comprises a non-functional mutant variant of the nucleic acid according to the invention, or a variant thereof.

[ 107] In particular the invention relates to a nucleic acid having a complementary sequence wherein the nucleic acid is an antisense molecule or an RNA interference molecule.

[108] The term "non-functional mutant variant of a nucleic acid" refers to a nucleic acid derived from a nucleic acid according to the invention, or a variant thereof having been mutated such that the polypeptide encoded by the non-functional mutant variant of the nucleic acid exhibits a biological activity which in comparison the non-mutated polypeptide is significantly decreased or abolished. Such activity of the polypeptide encoded by the non-functional mutant variant nucleic acid can be determined by means of a functional assay as described above for the evaluation of functional variants. The construction and screening of such non-functional mutant variant derived from a nucleic acid according to the invention are generally known to the person skilled in the art. Such "non-functional mutant variant of a nucleic acid" according to the invention can be expressed in a patient and will preferably abolish or diminish the level of expression of the targeted nucleic acid by competing with the native mRNA molecules for translation into polypeptides by the ribosomes.

[109] "Stringent hybridization conditions" refer to those conditions in which hybridization takes place at 60°C in 2.5 xSSC buffer and remains stable following a number of washing steps at 37°C in a buffer of lower salt concentration.

[110] The term "differential expression of a nucleic acid" refers to the relative level of expression of the nucleic acid in an isolated sample from a patient compared to the expression of the nucleic acid in a reference sample or a reference library. Definitions of reference samples and reference libraries have been described in detail above. The expression can be determined by methods generally known to the person skilled in the art. Examples of such methods include RNA blot (northern) analysis, nuclease protection, in situ hybridization, reverse transcriptase PCR (RT-PCR; including quantitative kinetic RT-PCR). cDNA and oligonucleotide microanays are also included as such methods.

[Il l]

[112] Prefened embodiment of the invention relates to the HCC up-regulated phos- phatidylinositol 4-kinase type II (PI4K2) polypeptide (Accession. No. NP_060895, SEQ ID 1) and to the nucleic acid PI4K2 (Accession. No. NM_018425, SEQ ID 94) coding for the polypeptide. The prevalent phosphatidylinositol (Ptdlns) phosphate kinase activity in many mammalian cell types is confened by the widespread type 2 kinase (PI4K2). The human type 2 isoform has been partially purified from plasma membrane rafts of human A431 epidermoid carcinoma cells. (Minogue S. et al., 2001. J Biol Chem., 18; 276(20): 16635-40. Epub 2001 Feb 13). The predicted amino acid sequence revealed two isoforms: 2alpha and 2beta. The type 2alpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes, but the gene encoding this Ptdlns family member, however, has not previously been reported to be expressed at elevated levels in disorders according to the invention, in particular in HCC.

[113] Expression of this mRNA is elevated on average almost 2-fold relative to non- diseased liver in 46% of the HCC cases profiled (see Figure 1 , Table 3A). Elevated expression of the encoding mRNA is also evident in FNH (to even a higher extent than in HCC; Figure 9/Table 4A), but not in cinhotic livers subjected to this cDNA microanay expression profiling procedure (Figure 9 and Table 3A). For this and the other nucleic acids according to the invention, this value for expression includes the expression value ratio data from all of the (28) HCC samples subjected to the cDNA microanay expression profiling experiments, including the values from samples that are not elevated by 2-fold or greater.

[114] These results should confirm that the differential upregulated expression of the PI4K2 cDNA sequence is highly specific for disorders according to the invention. Therefore the PI4K2 polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention

[115] In another prefened embodiment the nucleic according to the invention is the Zinc finger protein 216, ZNF216 cDNA (SEQ ID 95) which includes the open reading frame encoding ZNF216 polypeptide (SEQ ID 2). The ZNF216 polypeptide (GenBank sequence NP_005998) is another embodiment of the invention. The ZNF216 gene is identical to the aheady reported cochlear-expressed gene (Scott DA. et al., 1998, Gene, 215(2): 461- 469) that maps to the DFNB7/11 interval for autosomal recessive non- syndromic hearing loss (ARNSHL) located on human chromosome 9ql3-q21. Although ZNF216 gene is highly conserved between human and mouse, containing two regions that show homology to the putative zinc finger domains of other proteins, the polypeptide sequence has unknown function. Based on homology to bovine cDNA tag A2, ZNF216 may play a role in development of vessel endothelium from precursor cells suggesting a potential regulatory role in neovascularization. In this line it was recently suggested that ZNF216 and its A20-like zinc finger domain (ZnF-A20) have redundant and distinct role in regulating NF-kappaB activation and apoptosis (Huang J, published online ahead of print January 30, 2004, J. Biol. Chem, 10.1074/jbc.M309491200). The gene encoding this zinc finger family member, however, has not previously been reported to be expressed at elevated levels in disorders according to the invention, in particular in HCC.

[116] The expression in HCC of RNA conesponding to assembled sequence SEQ ID 95 is confirmed experimentally. The initial sequence upregulated in HCC relative to non- diseased liver identified as an SSH cDNA clone conesponds to GenBank sequence NM_006007. The expression of sequences of this clone has not previously been reported in liver or in HCC.

[117] In a prefened embodiment the polypeptide according to the invention is the ZNF216 polypeptide (SEQ ID 2) which is siuprisingly identified from an mRNA identified to be upregulated in HCC by an average of 16-fold relative to non-diseased liver (Figure 1) in 54% of the profiled cases (Table 3A). Similarly, elevated expression of the encoding mRNA relative to non-diseased liver is also evident in FNH but not in cinhotic livers (see Figure 10, Tables 4 A/5 A).

[118] cDNA sequences encoding this polypeptide and overlapping with this mRNA might be identified with reverse transcriptase PCR analysis and these nucleic acids can be similarly elevated in HCC. Furthermore, high expression specificity of the ZNF216 cDNA can be confirmed by quantitative assessment (Q-PCR) in HCC, FNH and Cinhosis in comparison to expression pattern in normal tissue(s). The TaqMan procedure utilizing the parallel examination of both GAPDH and β-actin as reference genes should verify a large over expression of ZNF216 cDNA (SEQ ID 95) in HCC .., when compared to FNH and Cinhosis. For TaqMan analyses ZNF 216 expression might be determined with gene specific oligonucleotide primers including ZNF216-pl, 5'-gagaggacaaaataactaccc-3', SEQ ED 195 (from nucleotide 611- 631 of SEQ ID 95 forward strand), ZNF216-p2, 5'-caattcaggagctttttcttca-3', SEQ ID 196 (from nucleotide 726-705 of SEQ ID 95 reverse strand) and the "hydrolysis" probe ZNF216-pr, 5'-tactgggctgagaaactgatggactgggctga-3' SEQ ID 198 (from nucleotide 694-663 of SEQ ID 95 reverse strand).

[119] Furthermore, the expression of this HCC-deregulated gene conelates with proliferation of hepatoma cells, showing 2-fold and 3-fold increase of ZNF216 mRNA in Hep3B cell line upon 8 hours and 12 hours serum stimulation of quiescent cells, respectively (see Figure 106).

[120] These results demonstrate that ZNF216 polypeptide (SEQ ID 2) and the nucleic acid encoding the polypeptide (SEQ ID 95) can be employed in the prevention and therapy of disorders according to the invention, in particular for the treatment of hy- peφlastic (including neoplastic) liver diseases. With regard to the treatment it is prefened to cany out the treatment such that the expression of the ZNF216 polypeptide or of the nucleic acid encoding the polypeptide is reduced and/or inhibited, for example by administering antisense oligonucleotides or RNA interference molecules that specifically interact with the nucleic acid encoding the ZNF216 polypeptide. Alternatively the treatment may be carried out such that the activity of the ZNF216 polypeptide is reduced and/or inhibited, for example by administering an antibody directed against the ZNF216 polypeptide or an antibody fragment thereof which block the activity of the ZNF216 polypeptide to a patient in need of such treatment. Compared to the state of the art, this ZNF216 polypeptide and/or ZNF216 nucleic acid suφrisingly allow improved, more sensitive, earlier, faster, and/or non- invasive diagnosis and/or improved, sustained and/or more effective.

[121] In another prefened embodiment the nucleic acid according to the invention is the AKR1C1 nucleic acid (SEQ ID 96) that represents the sequence of an HCC deregulated cDNA clone. This gene encodes the Aldo-keto reductase family 1 member Cl sharing high sequence identity with three other gene members and is localized at chromosome 10pl5-pl4 (Stolz, A. et al, 1993, J. Biol. Chem., 268: 10448-10457). These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and or NADPH as cofactors. The enzymes display overlapping but distinct substrate specificity and may assist in the rapid intracellular transport of bile acids from the sinusoidal to the canalicular pole of the cell, and thereby having a role in monitoring the intrahepatic bile acid concentration. The AKRICI regulates progesterone action by converting the hormone into its inactive metabolite 20 alpha-hydroxyprogesterone, and toxicologically this enzyme activates polycyclic aromatic hydrocarbon trans-dihydrodiols to redox-cycling o-quinones. However, the significance of its potent induction by Michael acceptors and oxidative stress is unknown (Burczynski ME. et al., J Biol Chem., 2001, 276(4): 2890- 2897). Expression of sequences conesponding to this clone has been aheady reported in several tissues (including liver) and some tumors (including prostate, breast; e.g., Wiebe JP and Lewis ML, 2003, BMC Cancer, 3(1): 9) but the sequence has not previously been described to be upregulated in HCC.

[122] In liver samples from HCC patients expression of the mRNA encoding this polypeptide is suφrisingly elevated relative to non-diseased liver by an average value of 7-fold in 79% cases profiled (Figure 1, Table 3A). Elevated expression of the encoding mRNA relative to non-diseased liver is also evident in FNH but not in cinhotic livers (Figure 11, Table 4A/5A).

[123] Independent RT-PCR analysis of expression levels of AKR 1 C 1 mRNA in HCC relative to normal liver are determined with gene specific oligonucleotide primers including: AKRlCl-pl, 5'- ttggaaaggtcactgaaaaatct-3' (SEQ ID 199) and AKRlCl-ρ2, 5'-gctggctgcggttgaagttgg-3' (SEQ ID 200) verifying the specific expression of this gene (SEQ ID 96) in HCCs when compared to normal liver samples (Figure 104). [124] Furthermore, the expression of this HCC-deregulated mRNA is showing 2-fold and 5 -fold increase by serum stimulation of quiescent hepatoma cells (HepG2) upon 8 hours and 12 hours, respectively (Figure 107).

[125] The target gene encoded polypeptide enzymatic activity clearly shows the conelation between the upregulation of AKRICI gene transcript in HCC with the approximately 2-fold induction of the AKRICI enzymatic activity suggesting that elevated expression of this sequence is conelated with human liver tumor cell proliferation (Table 9).

[126] In yet another prefened embodiment the nucleic acid according to the invention is the dUTP pyrophosphatase, dUT nucleic acid (SEQ ID 97) which has been disclosed before (Accession. No NM_001948) encoding the dUT polypeptide (Accession. No NP_001939, SEQ ID 4). dUTP pyrophosphatase involved in nucleotide metabolism produces dUMP (through hydrolysis of dUTP), the immediate precursor of thymidine nucleotides and decreases the intracellular concentration of dUTP so that uracil cannot be incoφorated into DNA (Mclntosh E.M.et al., 1992; PNAS, 89: 8020-8024). Nuclear DUT- DUT-N (18 kDa) and mitochondrial DUT-M (23 kDa) isoforms of the protein have been identified in humans and arise from the same gene by the alternative use of 5' exons. DUT-N protein and mRNA levels are tightly regulated to coincide with DNA replication. DUT-N is phosphorylated by cyclin-dependent kinases (Ladner R.D., 1996, J. Biol. Chem., 271: 7745-7751). Recently, it has been shown that these isoforms are abenantly expressed in some cancers (Pugacheva E.N. et al., 2002, Oncogene, 21(30): 4595- 4600) but the geneencoding these isoforms has not previously been reported to be expressed at elevated levels in HCC.

[127] Expression of the mRNA encoding the dUT polypeptide is induced by an average of 7-fold relative to non-diseased liver in 47% of the HCC cases profiled (Figure 1, Table 3A). Similarly, elevated expression of the encoding mRNA is also evident in FNH by an average 10.6-fold induction relative to non-diseased liver in 40% of the FNH cases profiled but not in the cinhotic livers (Figure 12, Tables 4A/5A). '

[ 128] Independent RT-PCR analyses of expression levels of dUT mRNA might be determined with gene specific oligonucleotide primers including primers for TaqMan analysis, for example: dUT-pl: 5'-ccgcgggctacgacctg-3', SEQ ED 201 (from nucleotide 153-169 of the SEQ ID 97 forward strand), dUT-p2, 5'-agccactcttccataacacc-3', SEQ ID 202 (from nucleotide 268-249 of the SEQ ID 97 reverse strand) and fluorescently- labeled probe dUT-pr, 5'-tgtccgttttcacaacagctttctccataggt-3', SEQ ID 203 (spanning bases from 227-197 of the SEQ ID 97 reverse strand).

[129] Furthermore, a specific high-affinity inhibitor blocks proliferation of hepatoma cells (Hep3B HepG2); the specific small molecule inhibitor (DMT-dU (5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyuridine; Sigma; No. D7279) (Persson, T. et al., 1996, Bioorg. Med. Chem., 4: 553-556) stimulates a cytostatic and anti-proliferative response (Figures 108 to 109) in these cells.

[130] These results should confirm that the differential upregulated expression of the dUT cDNA sequence is highly specific for disorders according to the invention. Therefore the dUT polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[131] Another prefened embodiment of the invention relates to the HCC up-regulated Paired basic amino acid cleaving enzyme 4, PACE4 polypeptide (Accession. No. NP_002561, SEQ ID 5) and to the nucleic acid PACE4 (Accession. No. NM_002570, SEQ ID 98) coding for the polypeptide. The protein encoded by this gene belongs to the subtilisin kexin-like proprotein convertase family while representing a calcium- dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites [consensus site: RX(K/R)R]. Expression of this gene has been already reported in several tissues (including liver) and suggested to play a role in tumor progression (in colon cancer, e.g. Khatib AM. et al., J Biol Chem., 2001, 276(33):30686-30693), but the sequence has not previously been described to be upregulated in HCC.

[132] Expression of this mRNA is elevated on average by 24-fold relative to non-diseased liver in 57% of the HCC cases profiled (see Figure 1, Table 3 A). Elevated expression of the encoding mRNA is also evident in FNH (to a lesser extent than in HCC; Figure 13/Table 4A), but not in cinhotic livers subjected to this cDNA microanay expression profiling procedure (Figure 13 and Table 5 A).

[133] Taqman RT-PCR analyses of expression levels of PACE4 mRNA (Assay ID Catalogue Number: Hs00159844_ml, Applied Biosystems, USA, see Table 6) verify and confirm the specific elevation of the PACE4 cDNA (Figure 3A) showing up- regulation in 7/17 HCCs, 3/3 FNHs, in 3/3 Cirrhosis and in 0/3 non-neoplastic livers (NNL).

[134] Furthermore, the expression of this HCC-deregulated mRNA is showing 2.4-fold and 6.7-fold increase by serum stimulation of quiescent hepatoma cells (HepG2) upon 8 hours and 12 hours, respectively (Figure 107).

[135] These findings suggest a functionally significant role for PACE4 in disorders according to the invention, especially in HCC. Therefore the PACE4 polypeptide and/ or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[136] In another prefened embodiment invention relates to the HCC up-regulated Transforming growth factor Beta-induced I, BIGH3 polypeptide (Accession number NP_000349; SEQ ID 6) and to the nucleic acid BIGH3 (Accession number NM_000358; SEQ ID 99) coding for the polypeptide. cDNA conesponding to this mRNA has been identified in cDNA libraries expressed in many tissues but at low levels; and highly expressed in the corneal epithelium. This gene known to be induced by TGF-beta binds specifically to collagens and may regulate cell adhesion (Skonier J. et al., 1994, DNA Cell Biol., 6: 571- 584). BIGH3 gene has been shown to be upregulated in oesophageal adenocarcinoma tissue (Hourihan RN. et al., 2003, Anticancer Res., 23(1 A): 161-5), but the sequence has not previously been reported to be up-regulated in disorders according to the invention, in particular in HCC.

[137] Expression of this mRNA is elevated on average by 5-fold relative to non-diseased liver in 79% of the HCC cases profiled (see Figure 1 and Table 3A). Similar analysis reveals elevated expression of this mRNA in 80% of the FNH cases profiled (Figure 14/Table 4A).

[138] The HCC induction of the BIGH3 gene is then verified by amplification of the sequence from the cDNA with primer pairs specific to BIGH3 nucleic acid (Assay ID Catalogue Number: Hs00154671_ml) in the Assay-On-Demand (Applied Biosystems, USA) quantitative PCR method and also confirming that the BIGH3 mRNA is not deregulated in cinhosis (Figure 100).

[139] These findings suggest that the BIGH3 polypeptide and/or a functional variant thereof and/or the encoding nucleic acid and/or a variant thereof can be utilized for the diagnosis, prevention and treatment of disorders according to the invention (in particular for the diagnosis of in HCC and FNH).

[140] In another.prefened embodiment the polypeptide according to the invention is the PRKARIA polypeptide (Accession number NP_002725; SEQ ID 7) which is surprisingly identified from an mRNA identified to be upregulated in HCC (Accession number NM_002734; SEQ ID 100). PRKARIA, a critical component of the cAMP signaling pathway represents a type I regulatory alpha subunit of cAMP-dependent protein kinase, suggested as a dominant negative regulator of transcription in somatic cell hybrids (Sandberg, M. et al., 1987, Biochem. Biophys. Res. Commun., 149:939-945). The inactive form of the enzyme is composed of two regulatory chains and two catalytic chains. Activation by cAMP produces two active catalytic monomers and a regulatory dimer that binds four cAMP molecules (Jones, K.W. et al., 1991, Cell, 66:861-872). Structural information of the protein is not yet obtained. PRKARIA is likely to be expressed in many tissues. However, the sequence has not previously been reported to be up-regulated in disorders according to the invention, in particular in HCC.

[141] The mRNA encoding this polypeptide is elevated an average of 3-fold relative to non-diseased liver in 39% HCCs profiled (see Figure 1 and Table 3A) and similarly in FNH, but not in cinhotic livers (Figure 15 and Tables 4A/5A).

[142] Independent verification analyses of expression levels of PRKARIA mRNA might be determined with gene specific oligonucleotide primers including, for example primer pairs specific to PRKARIA nucleic acid (Assay ID Catalogue Number: Hs0000267597_ml) in the Assay-On-Demand (Applied Biosystems, USA) quantitative PCR method.

[143] These results suggest that the strongly upregulated expression of the PRKARIA cDNA sequence is highly specific for disorders according to the invention, especially in HCC and FNH. Therefore the PRKARIA polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[144] In a further prefened embodiment the invention relates to the s.t. Ocia nucleic acid (Accession number NM_017830; SEQ ID 101) coding for the Ovarian carcinoma im- munoreactive antigen, s.t. Ocia polypeptide (Accession number NP_060300; SEQ ID 8) which may be expressed at low levels in many tissues and known to be elevated in ovarian cancer (Luo LY. et al., 2001,Biochem Biophys Res Commun., 12; 280(1): 401- 406). The gene encoding this putative tumor antigen, however, has not previously been described in liver cancer and not being reported to be expressed at elevated levels in disorders according to the invention, in particular in HCC.

[145] The mRNA encoding this polypeptide is elevated an average of 2.4-fold relative to non-diseased liver (NL) in 32% HCCs profiled (Figure 1 and Table 3A).mRNA levels are marginally elevated in FNH relative to non-diseased liver(Figure 16 and Table 4A). This mRNA is otherwise detected only infrequently in normal and cinhotic livers subjected here to expression profiling.

[146] Independent RT-PCR analyses of expression levels of s.tOcia mRNA are determined with gene specific oligonucleotide primers (Assay ID Catalogue Number: Hs00215197_ml, Applied Biosystems, USA) in the Assay-On-Demand quantitative PCR method confirming that the s.t.Ocia mRNA is not deregulated in cinhosis (Figure 101/ Table 6).

[147] These results suggest that the upregulated expression of the s.t.Ocia cDNA sequence is highly specific for disorders according to the invention, especially HCC. Therefore the s.t.Ocia polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis,prevention and treatment of disorders according to the invention, in particular for the diagnosis of HCC and FNH.

[148] In yet another prefened embodiment the invention relates to the serologically defined colon cancer antigen 28, SDCCAG28 nucleic acid (Accession number NM_006645; SEQ ID 102). The cDNA clones conesponding to the SDCCAG28 mRNA have been identified in many tissues including colon and other cancers (Scanlan, M.J. et al., 1998, Int. J. Cancer, 76:652-658), but neither this mRNA nor the encoded polypeptide have been previously implicated in disorders according to the invention, in particular in liver disorders or in HCC. The invention further relates to the polypeptide encoding for the SDCCAG28, a predicted polypeptide of 40.5 kDa (SDCCAG28, SEQ ID 9; NP_006636 in the GenBank database). The presence of this polypeptide has not been described in any cell or tissue and its function has not been reported, primary sequence suggests similarity to phosphatidylcholine transfer protein 2 (Lai,C.-H., 2000, Genome Res., 10: 703- 713).

[149] mRNA encoding this polypeptide is elevated an average 3-fold in 71% of the HCCs examined and similarly by nearly 7-fold in FNH (40% cases), all relative to non- diseased liver (Figures 1 and 17, Tables 3 A/ 4A).

[ 150] Independent RT-PCR analyses of expression levels of SDCCAG28 mRNA are determined with gene specific oligonucleotide primers (Assay ID Catalogue Number: Hs00246405_ml) as described for the BIGH3 gene, confirming that the SDCCAG28 mRNA is not deregulated in cinhosis (Figure 3B). The Assay-on-Demand Q-PCR shows upregulation in 8/17 HCCs, 2/3 FNHs, 1/3 Cinhosis and 0/3 NNL of profiled cases.

[151] Additionaly, expression of this HCC-deregulated gene conelates with proliferation of hepatomacells, showing almost 2-fold and 4- fold increase of SDCCAG28 mRNA in hepatoma cell line (Hep3B) upon 8 hours and 12 hours serum stimulation of quiescent cells, respectively (see Figure 106).

[152] Furthermore, the protein expression analyses show increase of SDCCAG28 protein signal in HCCs when compared to normal liver (Figure 105). The results support, the functional significance of SDCCAG28 for disorders according to the invention, in particular for HCC.

[153] These data suggest that SDCCAG28 polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[154] In yet another prefened embodiment the nucleic acid according to the invention is the Peroxiredoxin 1 transcript variant 1, PRDXl nucleic acid (SEQ ID 103) which has been disclosed before (Accession. No. NM_002574) encoding the PRDXl polypeptide (Accession. No. NP_002565; SEQ ID 10), a member of the peroxiredoxin family of antioxidant enzymes (Prxs) that also control cytokine-induced peroxide levels which mediate signal transduction in mammalian cells. Prxs can be regulated by changes to phosphorylation, redox and possibly oligomerization states (Wood, Z.A., et al., 2003, Trends Biochem. Sci., 28 (1): 32- 40). Three transcript variants encoding the same protein have been identified for this gene. The PRDXl has been shown to be upregulated in human breast cancer (Noh DY et al., 2001, Anticancer Res., 21 (3B): 2085- 2090). However, neither PRDXl nucleic acid nor the PRDXl polypeptide had been recognized with respect to elevated levels in HCC. [155] Expression of the mRNA encoding this polypeptide is elevated an average of 3.6-fold relative to non-diseased liver in 71% HCC cases profiled (Figure 1, Table 3A). Elevated expression of the encoding mRNA is also evident in other liver disorders (FNH, Cirrhosis) (Figure 18 and Tables 4A/5A).

[156] Independent verification analyses of expression levels of PRDXl mRNA might be determined with gene specific oligonucleotide primers including, for example primer pairs specific to PRDXl nucleic acid (Assay ED Catalogue Number: Hs00602020_ml) in the Assay-On-Demand (Applied Biosystems, USA) quantitative PCR method.

[157] These findings suggest that the PRDXl polypeptide and/or a functional variant thereof and or the encoding nucleic acid and/or a variant thereof can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[158] In yet another prefened embodiment the nucleic acid according to the invention is the Transmembrane trafficking protein, TMP21 nucleic acid (SEQ ID 104) which has been disclosed before (Accession. No NM_006827) encoding the TMP21 polypeptide (Accession No. NP_006818, SEQ ID 11). Tmp21 is involved in biosynthetic transport from the endoplasmic reticulum to the Golgi complex (Blum,R., et al., 1996, J. Biol. Chem. 271, 17183- 17189).There are two known Tmp21 isoforms -I and -II, wherein hum-Tmp21-II is transcribed, but not translated (Horer J et al., 1999, DNA Seq., 10(2): 121-6). Recent data report that phorbol esters translocate beta2-chimaerin (member of "non-protein kinase C" (PKC) phorbol ester/diacylglycerol receptors family) to the perinuclear region and promote its association with Tmp21-I in a PKC-independent manner (Wang H and Kazanietz MG,J Biol Chem, 2002; 277(6): 4541- 4550). Thus, Tmp21-I might be serving as an anchoring protein that determines the intracellular localization of these novel phorbol ester receptors. The gene encoding both isoforms has not previously been reported to be expressed at elevated levels in disorders according to the invention, in particular in HCC.

[159] Expression of the mRNA encoding the TMP21 polypeptide is induced by an average of 8.5-fold relative to non-diseased liver in 26% of the HCC cases profiled (Figure 1, Table 3A). Similarly, elevated expression of the encoding mRNA is also evident in FNH but not in the cinhotic livers (see Figure 19 and Tables 4A 5A).

[160] Furthermore, the expression of this HCC-deregulated mRNA is showing 2.6-fold and 3.5-fold increase by serum stimulation of quiescent hepatoma cells (HepG2) upon 8 hours and 12 hours, respectively (Figure 107).

[161] These results show that the differential upregulated expression of the TMP21 cDNA sequence is highly specific for disorders according to the invention. Therefore theTMP21 polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention

[162] In yet another prefened embodiment the nucleic acid according to the invention is the IQ motif containing GTPase-activating protein 2, IQGAP2 nucleic acid (SEQ ID 105) which has been disclosed before (Accession No. NM_006633) encoding the IQGAP2 polypeptide (Accession No. NP_006624, SEQ ID 12). This liver specific protein has been reported to harbor a potential actin binding domain and to interact with calmodulin and Rho family GTPases (Brill S et al., 1996, Mol Cell Biol.; 16(9): 4869-4878). The recent observations identify a physiologic scaffolding function for IQGAP2 representing a functional genomic unit in humans uniquely evolved to regulate thrombin-induced plateletcytoskeletal actin reorganization (Schmidt VA., 2003, Blood, 101(8): 3021-3028), but the gene encoding these isoforms has not previously been reported to be expressed at elevated levels in HCC.

[163] Expression of the mRNA encoding the IQGAP2 polypeptide is induced by an average of 4-fold relative to non-diseased liver in 71% of the HCC cases profiled (Figure 1, Table 3A). Similarly, elevated expression of the encoding mRNA is also evident in FNH but not in the cirrhotic livers (Figure 20 and Tables 4A/5A).

[164] The HCC induction of the IQGAP2 gene can then be verified by amplification of the sequence from the cDNA with primer pairs specific to IQGAP2 nucleic acid (Assay ID Catalogue Number: Hs00183606_ml) in the Assay-On-Demand (Applied Biosystems, USA) quantitative PCR method. These data suggest that the IQGAP2 polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[165] In yet another prefened embodiment the nucleic acid according to the.invention is the member of RAS oncogene family, Rab2 nucleic acid (SEQ ID 106) which has been disclosed before (Accession No. NM_002865) encoding the Rab2 polypeptide (Accession No. NP_002865, SEQ ID 13). The small GTPase Rab2 is a resident of pre- Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J. et al., 1992, J. Cell Biol., 119: 749- 761). The Rab2 protein, like all small GTPases, contains conserved GTP-binding domains as well as hypervariable carboxyl-terminal and amino-terminal domains. It is suggested that the NH2 terminus of Rab2 is required for its function and for direct interaction with components of the transport machinery involved in the maturation of pre-Golgi intermediates. Rab2 interacts directly with atypical protein kinase C (aPKC) iota/ lambda and inhibits aPKC iota lambda-dependent glyceraldehyde-3-phosphate dehy- drogenase phosphorylation (Tisdale, E.J.2003, J Biol Chem.; 278(52):52524-30). Though overexpression in lymphoid and myeloid malignancies has been reported, neither Rab2 nucleic acid nor the Rab2 polypeptide has been recognized with respect to elevated levels in disorders according to the invention, preferably in HCC.

[166] Expression of the mRNA encoding this polypeptide is elevated an average of 5-fold relative to non-diseased liver in 71% of the HCC cases profiled (Figure 2, Table 3A). Elevated expression of the encoding mRNA is also evident in FNH but not in cinhosis (Figure 21 and Tables 4A/ 5A).

[167] Furthermore, the expression of this HCC-deregulated mRNA is 5.5-fold and almost 8-fold increased by serum stimulation of quiescent hepatoma cells (Hep3B) upon 8 hours and 12 hours, respectively (Figure 106).

[168] These findings suggest that the Rab2 polypeptide and/or the encoding nucleic acid can be utilized for the diagnosis, prevention and treatment of disorders according to the invention.

[169] In another prefened embodiment the nucleic according to the invention is the Clone 6 cDNA (OBCL6, SEQ ID 125), which is assembled by identification of overlapping sequences from the non-redundant GenBank sequence databases. The initial EST sequence upregulated in HCC relative to non-diseased liver identified with cDNA microanay analysis shows the highest similarity (almost 100% identical) to human genomic clone AL035420 øiuman DNA sequence from clone RP4-550H1 on chromosome 2Oql l.l-11.22 containing a high mobility group protein pseudogene). It may be that extending the length of this HCC-deregulated cDNA sequence will reveal that the conesponding RNA encodes a not yet described human protein. Another alternative is that the encoded polypeptide may result from one of the small open reading frames in this sequence. Even further, this RNA may be not translated into polypeptide but may have functional (e.g., regulatory) properties itself.

[170] Suφrisingly the sequence from this mRNA is represented at much higher levels in HCC than in normal human liver. This mRNA is elevated an average of 6-fold or more relative to non-diseased liver in 68% of HCC samples profiled (Table 3B, Figure 3). Clone 6 is also elevated 8-fold or more relative to non-diseased liver in FNHs examined, but not in cinhosis (Figure 40, Tables 4b/5B). Independent RT-PCR analyses of expression levels of might be determined with gene specific oligonucleotide primers. These results show that the strongly upregulated expression of the Clone 6 cDNA sequence is highly specific for disorders according to the invention, especially in HCC and FNH.

[171] Overexpression of the polypeptide and/or the encoding RNA therefore, may be useful for diagnosis of liver disorders. These results clearly demonstrate that the Clone 6 polypeptide and the nucleic acid (SEQ ID 125) encoding the polypeptide (SEQ ID 32) and a functional variant thereof can be utilized for diagnosis, prevention and treatment of disorders according to the invention, in particular for HCC and FNH.

[172] With regard to the treatment it is prefened to cany out the treatment such that the expression of the OBCL6 polypeptide and/or a functional variant thereof; or of the nucleic acid encoding the polypeptide and/or a functional variant thereof is reduced and/or inhibited, for example by administering antisense oligonucleotides or small in- terfering RNA molecules that specifically interact with the nucleic acid defined in SEQ ID 125 potentially encoding the OBCL6 polypeptide and/or a functional variant thereof.

[173] The treatment may be carried out, for example, such that the activity of the Clone 6 polypeptide and/or a functional variant thereof are reduced and/or inhibited, for instance by administering an antibody directed against the OBCL6 polypeptide and/or a functional variant thereof, or an antibody fragment thereof which block the activity of the Clone 6 polypeptide and/or a functional variant thereof to a patient in need of such treatment. Compared to the state of the art, the OBCL6 polypeptide and/or a functional variant thereof; and/or OBCL6 nucleic acid suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis and/or improved, sustained and/or more effective treatment of the liver disorders and/or other epithelial cancer.

[174] Alternatively, the OBCL6 RNA may be not translated into a polypeptide but may have functional (e.g., regulatory) properties itself. The disease relevance of non-coding regulatory RNAs is now becoming apparent as evidenced, for example, by the role of the non-coding RNA "bantam" involved in cellular proliferation in the eukaryote Drosophila (Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM. Cell (2003) Apr4; 113(l):25-36), and by microRNA-23 that interacts with the transcription factor HES-1 to hinder neuronal differentiation (Kawasaki, H. and Tiara, K. Nature, 2003, 423:838-842). «

[175] For example, reduction of the level of Clone 6 RNA (knock-down) in proliferating human hepatoma cells with small interfering RNA (siRNA) oligonucleotides can support a functionally significant role for elevated expression of Clone 6 RNA in liver disorders, especially liver cancer.

[176] Further aspect of the invention represents an isolated polypeptide comprising a sequence according to the SEQ ID 32 or a functional variant thereof. Another prefened embodiment is a fusion protein, wherein the fusion protein contains the polypeptide according to the SEQ ID 32 or a functional variant thereof.

[177] Yet another prefened feature of the invention is an isolated nucleic acid according to the SEQ ID 125 or a variant thereof. Further prefened embodiment represents the nucleic acid according to the SEQ ID 125 or a variant thereof, wherein the nucleic acid is a single-stranded or double-stranded RNA.

[178] Still another aspect of the invention represents a nucleic acid according to the SEQ ID 125 or a variant thereof encoding the polypeptide according to the SEQ ID 32 or a functional variant thereof.

[179] Yet another feature of the invention is a vector, wherein the vector contains a nucleic acid selected from the group consisting of a nucleic acid according to the SEQ ID 125 or a variant thereof encoding the polypeptide according to the SEQ ID 32 or a functional variant thereof. The vector is preferably selected from the group consisting of a knock-out gene construct, a plasmid, a shuttle vector, a phagemid, a cosmid, a viral vector, and an expression vector.

[ 180] Another aspect of the invention represents a cell, wherein the cell contains the nucleic acid according to the SEQ ID 125 or a variant thereof encoding the polypeptide according to the SEQ ID 32 or a functional variant thereof. In another prefened embodiment the cell is transformed with a vector containing a nucleic acid selected from the group consisting of a nucleic acid according to the SEQ ED 125 or a variant thereof encoding the polypeptide according to the SEQ ID 32 or a functional variant thereof. In still further embodiment of the invention the cell is a transgenic embryonic non-human stem cell.

[181] Yet another feature of the invention represents a transgenic non-human mammal, wherein the transgenic non-human mammal contains the nucleic acid according to the SEQ ID 125 or a variant thereof encoding the polypeptide according to the SEQ ID 32 or a functional variant thereof.

[182] Further aspect is an antibody or an antibody fragment thereof, wherein the antibody is directed against the polypeptide according to the SEQ ED 32 or a functional variant thereof, or against a nucleic acid coding for the polypeptide.

[183] The cDNA expression levels relative to a non-diseased liver (NL) reference sample of sequences according to the invention assessed in tissues from human liver disorders, including Hepatocellular Carcinoma (HCC), Focal Nodular Hypeφlasia (FNH) and Cinhosis (Cinh.) samples are shown in Tables 3A to 3D, 4A to 4D and 5A to 5D, respectively (median of log values data between diseased and non-diseased samples obtained from competitive hybridisation to custom-made cDNA microanays). Median represents 50 percentile of values for each sequence (SEQ ID 94 to 186) per group (HCC, FNH and Cinh). Number of the samples profiled and the calculated percentage of valid detectable signals (% detected) are provided. (*) annotates duplicates of the HCCs, FNHs, and Cinh. profiled.

[184]

[185]

[186]

[187]

[188]

[189]

[190]

[191]

[192] [193] [194] [195] [196] Tables 3A to 3D: Summary of c DNA microarray expression level ratios (HCC vs NL). Table 3A

Ki 2.55 5.85 28 68

[197] [198] Table 3B

[199] Table 3C

[200] Table 3D

[201]

[202] [(**) c-syn represents three alternative nucleotide transcripts with conesponding three protein products [203] [204] Tables 4A to 4D: Summary of c DNA microarray expression level ratios (FNH vs NL). Table 4A

Table 4B

Table 4C

[207] Table 4D

[208]

[209] [(**) c-syn represents three alternative nucleotide transcripts with conesponding three protein products [210] [211] Tables 5A to 5D: Summary of c DNA microarray expression level ratios (Cirrh. vs NL). Table 5A

Table 5B Median-fol Cirrh. Gene Median log Detected (%) d microarray

[213] [214] Table 5C

Table 5D

[216] [217] [(**) c-syn represents three alternative nucleotide transcripts with conesponding three protein products]

[218] [219] The quantitative assessment of gene expression (SEQ IDs: 102; 99; 101; 106; 98; 96) by RT-PCR (Q-PCR) in Hepatocellular Carcinoma (HCC), Focal Nodular Hypeφlasia (FNH) and Cinhosis (Cirrh) samples is compared to expression pattern in normal liver (NL), shown inTable 6 (median of log values). Median represents 50 percentile of values for each sequence per group (HCC, FNH and Cinh). Number of the samples profiled (SDCCAG28, BIGH3, s.t.OCIA, Rab2 and PACE4) represent 18 HCC, 3 FNH/Cirrh. NL; and for AKRICI 7 HCC and 4 NL. Percentage of valid/ detectable signals for SEQ IDs 102; 99; 101; 106; 98; 96 (% detected) is equal to 100%, with exception of PACE4 (*) for which 94.45% HCC cases are detected.

[220] [221] Table 6: Summary of differential gene expression levels (SEQ IDs: 102; 99; 101; 106; 98; 96) verified by RT-PCR Table 6

[222] [223] The quantitative assessment of gene expression of TMF4SF4 and DAD-1 in Hepatocellular Carcinoma (HCC), Focal Nodular Hypeφlasia (FNH) and Cinhosis (Cinh) samples is compared to expression pattern in normal liver (NL), shown in Tables 7A/7B respectively (median of log values). Median represents 50 percentile of values for each sequence (SEQ ID 112 and SEQ ID 140) per group (HCC, FNH and Cirrh). Median- fold induction is calculated according to following formula: "2 " ("x" represents median of log values). Number of the samples profiled (TM4SF4 and DAD-1 genes) represent 18 HCC, 3 FNH/Cinh./NL.

[224] [225] Table 7A/7B: Summary of differential gene expression levels (SEQ ID 112 and SEQ ID 140) verified by RT-PCR.

[226] 6 Table 7A

[227] Table 7B

[228] [229] En another prefened embodiment of the invention the nucleic acid according to the invention can be used for the construction of antisense oligonucleotides (Zheng and Kemeny, 1995, Clin. Exp. Immunol., 100: 380-382) and/or ribozymes (Naish et al., 1998, Nucleic Acids Res., 26: 5237-5242; Persidis, 1997, Nat. Biotechnol., 15: 921-922) and/or small interfering double stranded RNAs (Elbashir et al., 2001, Namre, 411: 494-498; Brummelkamp et al., 2002, Science, 296:550-553). In further prefened embodiments of the invention, the stability of the nucleic acid according to the invention can be decreased and/or the translation of the nucleic acid according to the invention inhibited by using RNA interference molecules (oligonucleotides). Thus, for example, the expression of the conesponding genes in cells can be decreased both in vivo and in vitro. Oligonucleotides can therefore be suitable as therapeutics. This strategy is also suitable, for example, for liver cells, in particular if the antisense oligonucleotides are complexed with liposomes. For use as a probe or as an "antisense" oligonucleotide, a single-stranded DNA or RNA is prefened. Small interfering RNA (siRNA) double stranded oligonucleotides can also be suitable as therapeutics. With this approach a short sequence or sequences of 15 to 22 nucleotides including sequence complementary to the sequence to be therapeutically targeted are exposed to the diseased tissue and serve to dramatically reduce or "knock down" the level of expression of the therapeutic target RNA sequence. siRNA therapeutic approaches in other diseases have been recently reported and are also applicable to liver disorders, liver cancers and other epithelial cancers (Filleur S. et al., Cancer Res., 2003; 63(14): 39-22).

[230] In a prefened embodiment a nucleic acid according to the invention has been prepared by recombinant methods, by screening a library or isolation from a sample obtained from a patient or a subject. In another prefened embodiment of the invention the nucleic acid according to the invention has been prepared synthetically. Thus, the nucleic acid according to the invention can be synthesized, for example, chemically with the aid of the DNA sequences described in SEQ ID 94 to SEQ ID 186 and/or with the aid of the protein sequences described in SEQ ID 1 to SEQ ID 93 with reference to the genetic code, e.g. according to the phosphotriester method (see, for example, Uhlmann and Peyman, 1990, Chemical Reviews, 90:543-584).

[231] In another prefened embodiment, the invention relates to a nucleic acid according to the invention or a nucleic acid which is a non-functional mutant variant the nucleic acid or a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, which has been modified by attachment of chemical moieties to the nucleic acid to stabilize it against degradation, so that a high concentration of the nucleic acid is maintained in the cell over a long period (Beigelman et al., 1995, Nucleic Acids Res., 23: 3989-94; Dudycz, 1995, WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116). Typically, such stabilization can be obtained by the introduction of one or more internucleotide phosphorus groups or by the introduction of one or more non-phosphorus internucleotides.

[232] Prefened suitable modified internucleotides are summarized in Uhlmann and Peymann (1990 Chem. Rev. 90, 544; see also Beigelman et al., 1995 Nucleic Acids Res., 23: 3989-94; Dudycz, 1995, WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116).

[233] In a further embodiment the invention relates to a vector comprising a nucleic acid according to the invention and/or a variant thereof, or a nucleic acid which is a nonfunctional mutant variant of the nucleic acid, or a nucleic acid having a sequence complementary to one the aforementioned nucleic acids. Preferably the vector is a knock-out gene construct, a plasmid, a shuttle vector, a phagemid, a cosmid, a viral vector, an expression vector and/or a vector applicable in gene therapy. The preparation of such constructs is generally known to the person skilled in the art.

[234] An "expression vector" within the meaning of the present invention preferably comprises at least one promoter or enhancer, i.e. at least one regulatory element comprising at least one translation initiation signal, at least one of the nucleic acids according to the invention or a nucleic acid which is a non-functional mutant variant the nucleic acid or a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, one translation termination signal, a transcription termination signal, and a polyadenylation signal for the expression in eukaryotes.

[235] For the expression of the gene concerned, in general a double-stranded DNA is prefened, the DNA region coding for the polypeptide being particularly prefened. In the case of eukaryotes this region begins with the first start codon (ATG) lying in a Kozak sequence (Kozak, 1987, Nucleic. Acids Res., 15: 8125-48) up to the next stop codon (TAG, TGA or TAA), which lies in the same reading frame to the ATG. In the case of prokaryotes this region begins with the first AUG (or GUG) after a Shine- Dalgarno sequence and ends with the next stop codon (TAA, TAG or TGA), which lies in the same reading frame to the ATG.

[236] Differentially expressed genes in HCC can contain liver or liver cancer gene- specific regulatory sequences. These non-transcribed sequences, found in the tissue- or disease-specific gene may be used to drive tissue- or disease-specific expression of included therapeutic and/or tumor cell-cytotoxic genes. These regulatory sequences may be used for liver cancer specific expression of a nucleic acid according to the invention or a nucleic acid which is a non-functional mutant variant the nucleic acid or a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids. The screening and construction of such regulatory sequences is generally known to the person skilled in the art.

[237] Suitable expression vectors can be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are, for expression in E. coli, e.g. the vectors pGEM or pUC derivatives, examples of eukaryotic expression vectors are for expression in Saccharomyces cerevisiae, e.g. the vectors p426Met25 or p426GALl (Mumberg et al., 1994, Nucl. Acids Res., 22, 5767-5768), for expression in insect cells, e.g. Baculovirus vectors such as disclosed in EP-B1-0 127 839, and for expression in mammalian cells, e.g. the vectors Rc/CMN and Rc/RSN or SN40 vectors, which are all generally obtainable. Specific vectors for production of.RΝA interference following transfection, such as the pSUPER vector (Brummelkamp et al., 2002, Science, 296:550-553) are also included.

[238] In general, the expression vectors also contain promoters suitable for the respective cell, such as, for example, the tip promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the MET 25, GAL 1 or ADH2 promoter for expression in yeast (Russel et al., 1983, J. Biol. Chem., 258, 2674-2682; Mumberg, supra), the Baculovirus polyhedrin promoter, for expression in insect cells (see, for example, EP- Bl-0 127 839). For expression in mammalian cells, for example, suitable promoters are those which allow a constitutive, regulatable, tissue-specific, cell-cycle-specific or metabohcally specific expression in eukaryotic cells. Regulatory elements according to the present invention preferably are promoters, activator sequences, enhancers, silencers and or repressor sequences.

[239] Examples of suitable regulatory elements which make possible constitutive expression in eukaryotes preferably are promoters which are recognized by the RΝA polymerase EEE or viral promoters, CMN enhancer, CMN promoter, SN40 promoter or LTR promoters, e.g. from MMTN (mouse mammary tumor virus; Lee et al., 1981, Nature, 214, 228-232) and further viral promoter and activator sequences, derived from, for example, adeno- and adeno-like viruses, HBN, HCN, HSN, HPN, EBN, HTLN or HIN.

[240] Examples of regulatory elements which make possible regulated expression in eukaryotes are the tetracychne operator in combination with a conesponding repressor (Gossen et al., 1994, Curr. Opin. Biotechnol., 5:516-20).

[241] Translation initiation signals, translation termination signals, transcription termination signals, and polyadenylation signals are generally known to the person skilled in the art and can be readily obtained from commercial laboratory suppliers.

[242] Preferably, the expression of the genes relevant for liver disorders and/or epithelial cancer takes place under the control of tissue-specific promoters, for example, under the control of liver-specific promoters such as albumin, alpha fetoprotein, apolipoprotein AI, alpha- 1 antitrypsin, and the complement C5 and C8A genes (Schrem et al., 2002, Pharmacol. Rev., 54 129-58; Pontoglio et al., 2001, J. Expt. Med., 194:1683-1689). The regulatory sequences associated with genes highly deregulated in HCC as described herein also provide a preferable method for specific gene expression in these disorders.

[243] Further examples of regulatory elements which make tissue-specific expression in eukaryotes possible are promoters or activator sequences from promoters or enhancers of those genes which code for proteins which are only expressed in certain cell types.

[244] Examples of regulatory elements which make possible metabohcally specific expression in eukaryotes are promoters which are regulated by hypoxia, by oxidative stress, by glucose deficiency, by phosphate concentration or by heat shock.

[245] Examples of regulatory elements which make cell cycle-specific expression in eukaryotes possible are promoters of the following genes: cdc25A, cdc25B, cdc25C, cyclin A, cyclin E, cdc2, E2F-1 to E2F-5, B-myb or DHFR (Zwicker J. and MuUer R., 1997, Trends Genet., 13:3-6). The use of cell cycle regulated promoters is particularly prefened in cases, in which expression of the polypeptides or nucleic acids according to the invention is to be restricted to proliferating cells.

[246] In order to make possible the introduction of nucleic acids as described above, or a nucleic acid which is a non-functional mutant variant of the nucleic acid and thus the expression of the polypeptide in a eukaryotic or prokaryotic cell by transfection, transformation or infection, the nucleic acid can be present as a plasmid, as part of a viral or non-viral vector. Suitable viral vectors here are particularly: baculoviruses, vaccinia viruses, adenoviruses, adeno-associated vimses, retroviruses and heφesviruses. Suitable non-viral vectors here are particularly: virosomes, liposomes, cationic lipids, or poly ly sine-conjugated DΝA or naked DΝA.

[247] Plasmids, shuttle vectors, phagemids, and cosmids suitable for use according to the invention are also known to the person skilled in the art and are generally obtainable from commercial laboratory suppliers.

[248] Examples of vectors applicable in gene therapy are virus vectors, for example adenovirus vectors, retroviral vectors or vectors based on replicons of RNA viruses (Lindemann et al., 1997, Mol. Med. 3: 466-476; Springer et al., 1998, Mol. Cell. 2:549-558). Eukaryotic expression vectors are suitable in isolated form for gene therapy use, as naked DNA can penetrate, for example, into liver cells upon local application or via the blood supply.

[249] Compared to the state of the art, this fusion construct suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive diagnosis and/or improved, sustained and/or more effective treatment of the liver disorders, and/or other epithelial cancers.

[250] In another aspect the invention furthermore relates to a cell comprising a nucleic acid according to the invention and/or a variant thereof. Preferably the cell is transformed with a vector according to the invention. The cell preferably contains a nucleic acid wherein the nucleic acid is either a non-functional mutant variant of a nucleic acid according to the invention. In particular the cell contains a vector comprising a nucleic acid wherein the nucleic acid is a non-functional mutant variant of a nucleic acid according to the invention. Preferably the cell contains a nucleic acid having a sequence complementary to a nucleic acid according to the invention, or a variant thereof. Moreover the cell preferably contains a vector comprising a nucleic acid coding for an antibody according to the invention or a fragment of the antibody. The cell according to the invention may for example be a liver cell, comprising at least one of the aforementioned nucleic acids or a cell which is transformed using one of the above described vectors. Cells can be either prokaryotic or eukaryotic cells, heterologous or autologous cells. Examples of prokaryotic cells are E. coli and examples of eukaryotic cells include primary hepatocytes cells, hepatocytes cell lines such as HepG2 and Hep3B cells, yeast cells, for example Saccharomyces cerevisiae or insect cells.

[251] Compared to the state of the art, the cell according to the invention suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive diagnosis and/or improved, sustained and/or more effective treatment of the liver disorders and or other epithelial cancers.

[252] In a prefened embodiment of the invention the cell is a transgenic embryonic non-human stem cell which comprises at least one nucleic acid according to the invention, at least one vector, at least one knock-out gene construct and/or at least one expression vector as described above.

[253] Processes for the transformation of cells and/or stem cells are well known to a person skilled in the art and include, for example, electroporation or microinjection. [254] In another aspect the invention relates to the provision of a transgenic non-human mammal comprising a compound selected from the group consisting of a nucleic acid according to the invention and/or a variant thereof, a nucleic acid which is a nonfunctional mutant variant the nucleic acid, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, one of the aforementioned nucleic acids in the form of a vector, of a knock-down or knock-out gene construct, and of an expression vector.

[255] Transgenic animals in general show a tissue-specifically increased expression of the nucleic acids and or polypeptides and can be used for the analysis of liver disorders and/or epithelial cancers, such as for example HCC, and for development and evaluation of therapeutic strategies for such disorders. Transgenic animals may further be employed in the production of polypeptides according to the invention. The polypeptide produced by the animal may for example be enriched in a body fluid of the animal. The polypeptides according to the invention may for example be isolatable from a body fluid such as the milk.

[256] Compared to the state of the art, this transgenic non-human mammal suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive analysis and/or diagnosis of liver disorders and/or other epithelial cancers.

[257] Processes for the preparation of transgenic animals, in particular of transgenic mice, are likewise known to the person skilled in the art from e.g., US 5,625,122; US 5,698 765; US 5,583,278 and US 5,750,825 and include transgenic animals which can be produced, for example, by means of direct injection of expression vectors according to the invention into embryos or spermatocytes or by injection of the expression vectors into the pronucleus of the fertilized ovum or by means of the transfection of expression vectors into embryonic stem cells or by nuclear transfer into appropriate recipient cells (Polites and Pinkert, DNA Microinjection and Transgenic Animal Production, page 15 to 68 in Pinkert, 1994, Transgenic animal technology: a laboratory handbook, Academic Press, London, UK; Houdebine, 1997, Harwood Academic Publishers, Amsterdam, The Netherlands; Doetschman, Gene Transfer in Embryonic Stem Cells, page 115 to 146 in Pinkert, 1994, supra; Wood, Retrovirus- Mediated Gene Transfer, page 147 to 176 in Pinkert, 1994, supra; Monastersky, Gene Transfer Technology; Alternative Techniques and Applications, page 177 to 220 in Pinkert, 1994, supra).

[258] If the above described nucleic acids are integrated into so-called "targeting vectors" or "knock-out" gene constructs (Pinkert, 1994, supra), it is possible after transfection of embryonic stem cells and homologous recombination, for example, to generate knockout mice which, in general, as heterozygous mice, show decreased expression of the nucleic acid, while homozygous mice no longer exhibit expression of the nucleic acid. The animals thus produced can also be used for the analysis of liver disorders, such as for example HCC, and/or epithelial cancers.

[259] Knock-out gene constructs are known to the person skilled in the art, for example, from the US patents 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825.

[260] In a further aspect the invention relates to an antibody or a fragment, wherein the antibody or antibody fragment is directed against a polypeptide according to the invention, a functional variant thereof or against a nucleic acid coding for the polypeptide, or a variant thereof.

[261] Compared to the state of the art, these antibody or a fragment thereof suφrisingly allow improved, more sensitive, earlier, faster, and or non-invasive diagnosis and/or improved, sustained and or more effective treatment of the liver disorders and/or other epithelial cancers.

[262] The term "antibody" or "antibody fragment" is understood according to the present invention as also meaning antibodies or antigen-binding parts thereof prepared by genetic engineering and optionally modified, such as, for example, chimeric antibodies, humanized antibodies, multifunctional antibodies, bi- or oligospecific antibodies, single-stranded antibodies, F(ab) or F(ab) fragments (see, for example, EP- Bl-0 368 684, US 4,816,567; WO 98/24884). The antibodies according to the invention can for example be used for diagnosis, prevention and/or treatment of disorders according to the invention such as liver disorders, for example HCC, and/or epithelial cancers.

[263] The invention further relates to a method for producing an antibody or antibody fragment, preferably a polyclonal or monoclonal antibody, specific for the polypeptides or functional variants thereof encoded by the nucleic acids according to the invention, or variants thereof for example for the diagnosis and/or prevention and/ or treatment of disorders according to the invention. The process is carried out according to methods generally known to the person skilled in the art by immunizing a mammal, for example a rabbit, with a nucleic acid according to the invention or their variants thereof, or with a polypeptide according to the invention or parts thereof or functional variants thereof, having at least 6 amino acid length, preferably having at least 8 amino acid length, in particular having at least 12 amino acid length, if appropriate in the presence of, for example, Freund' s adjuvant and/or aluminum hydroxide gels (see, for example, Harlow and Lane, 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, USA, Chapter 5, pp. 53-135). The polyclonal antibodies formed in the animal as a result of an immunological reaction can then be easily isolated from the blood according to generally known methods and purified, for example, by means of column chromatography. Monoclonal antibodies can be produced, for example, according to the known method of Winter & Milstein (Winter and Milstein, 1991, Nature, 349:293-299).

[264] The present invention further relates to an antibody or antibody fragments directed against a polypeptide described above and reacts specifically with the polypeptides described above, where the above-mentioned parts of the polypeptide are either im- munogenic themselves or can be rendered immunogenic by coupling to suitable carriers, such as, for example, bovine serum albumin or keyhole limpet hemocyanin to increase in their immunogenicity. This antibody is either polyclonal or monoclonal; preferably it is a monoclonal antibody.

[265] Still further, the present invention relates to the generation and/or production of an antibody or antibody fragment specific for the polypeptide according to the invention from a recombinant antibody expression library, such as for example described by Knappik et al. (2000, J. Molec. Biol., 296:57-86).

[266] In another embodiment of the invention, it is provided an array, wherein the anay contains at least two compounds selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding the polypeptide, a non-functional mutant variant nucleic acid and an antibody or an antibody fragment directed against the polypeptide. Alternatively, the anay may contain at least one component according to the invention in combination with previously described components implicated in neoplastic or metabolic liver disorders or epithelial cancers.

[267] Within the meaning of the invention the term "anay" refers to a solid-phase or gellike carrier upon which at least two compounds are attached or bound in one-, two- or three-dimensional anangement. Such anays are generally known to the person skilled in the art and are typically generated on glass microscope slides, specially coated glass slides such as polycation-, nitrocellulose- or biotin- coated slides, cover slips, and membranes such as for example membranes based on nitrocellulose or nylon.

[268] The aforementioned anays include bound polypeptides according to the invention or functional variants thereof or nucleic acids coding for the polypeptides, or variants thereof, fusion proteins according to the invention or antibodies or antibody fragments directed against polypeptides according to the invention or functional variants thereof or cells expressing polypeptides according to the invention or functional variants thereof or at least two cells expressing at least one nucleic acid according to the invention, or variants thereof. Nucleic acids coding for these, or variants thereof can also be part of an anay. Such anays can be employed for analysis and/or diagnosis of liver disorders, preferably of HCC, and/or epithelial cancer.

[269] The invention further relates to a method of producing anays according to the invention, wherein at least two compounds according to the invention are bound to a carrier material. [270] Methods of producing such arrays, for example based on solid-phase chemistry and photo-labile protective groups are generally known (US 5,744,305). Such anays can also brought into contact with substances or a substance libraries and tested for interaction, for example for binding or change of conformation.

[271] The invention further relates to a process for preparing an array immobilized on a support material for analysis and/or diagnosis of disorders according to the invention such as a liver disorder, preferably of HCC, in which at least two nucleic acids, at least two polypeptides or at least two antibodies or antibody fragments, and/or at least two cells, or at least one of the aforementioned components in combination with other components relevant to neoplastic and metabolic liver disorders or epithelial cancers, as described above, is used for preparation. The arrays produced by such process can be employed for the diagnosis of disorders according to the invention.

[272] In another aspect the invention relates to a diagnostic comprising at least one compound selected from the group consisting of a polypeptide according to the SEQ ID 1 to SEQ ID 93 or functional variants thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, and an antibody or an antibody fragment directed against one of the aforementioned polypeptides, combined or together with suitable additives or auxiliaries.

[273] In a prefened embodiment the invention relates to a diagnostic comprising a polypeptide according to the SEQ ID 1 or a functional variant thereof, a nucleic acid encoding the aforementioned polypeptide, a variant of the aforementioned nucleic acid, and an antibody or an antibody fragment directed against the aforementioned polypeptide, combined or together with suitable additives or auxiliaries.

[274] In a further aspect the invention relates to a diagnostic comprising at least one compound selected from the group consisting of a nucleic acid according to the SEQ ID 94 to SEQ ID 186 or variants thereof, combined with suitable additives or auxiliaries.

[275] In a prefened embodiment the invention relates to a diagnostic comprising a nucleic acid according to the SEQ ID 94 or a variant thereof, combined with suitable additives or auxiliaries

[276] Compared to the state of the art, this diagnostic suφrisingly allows improved, more sensitive, earlier, faster, and or non-invasive diagnosis of liver disorders and/or other epithelial cancers.

[277] Within the meaning of the invention "suitable additives" or "auxiliaries" are generally known to the person skilled in the art and comprise, for example, physiological saline solution, demineralized water, gelatin or glycerol-based protein stabilizing reagents. Alternatively, the nucleic acid or polypeptide according to the invention may be lyophilized for stabilization. [278] In another examplea diagnostic kit based on the nucleic acid sequences according to the invention could be generated. Such a kit may be designed specifically to detect cells altered as a result of the described disorders resident in the circulatory system and thereby detectable in serum from test patients. Additional examples of diagnostic kits includes enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and detection of an immune reaction or specific antibodies to the polypeptides according to the invention including detection of specific responding immune cells.

[279] In a prefened embodiment the diagnostic according to the invention contains a probe, preferentially a DNA probe.

[280] For example, it is possible according to the present invention to prepare a diagnostic based on the polymerase chain reaction (PCR). Under defined conditions, preferably using primers specific for a nucleic acid according to the invention as a DNA probe PCRs specific for the nucleic acid sequences of the invention will be utilized to monitor both the presence, and especially the amount, of specific nucleic acids according to the invention in a sample isolated from a patient obtained for diagnostic or therapeutic puφoses. This opens up a further possibility of obtaining the described nucleic acids, for example by isolation from a suitable gene or cDNA library, for example from a liver disorder-specific or liver specific gene bank, with the aid of a suitable probe (see, for example, J. Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Chapter 8 pages 8.1 to 8.81,. Chapter 9 pages 9.47 to 9.58 and Chapter 10 pages 10.1 to 10.67).

[281] Suitable probes are, for example, DNA or RNA fragments having a length of about 50-1000 nucleotides, preferably having a length of about 10 to about 100 nucleotides, preferably about 100 to about 200 nucleotides, in particular having a length of about 200-500 nucleotides, whose sequence can be derived from the polypeptides according to SEQ ID 1 to SEQ ID 93, and functional variants thereof, and nucleic acids coding for the polypeptides, preferably according to SEQ ID 94 to SEQ ID 186, and variants thereof.

[282] Alternatively, it is preferably possible with the aid of the derived nucleic acid sequences to synthesize oligonucleotides that are suitable as primers for a polymerase chain reaction. Using this, the nucleic acid described above or parts of this can be amplified and isolated from cDNA, for example HCC-specific cDNA. Suitable primers are, for example, DNA fragments having a length of about 10 to 100 nucleotides, preferably having a length of about 15 to 50 nucleotides, in particular having a length of 17 to 30 nucleotides, whose sequence can be derived from the polypeptides according to SEQ ED 1 to SEQ ED 93 from the nucleic acids according to SEQ ID 94 to SEQ ID 186. The design and synthesis of such primers is generally known to the person skilled in the art. The primers may additionally contain restriction sites, e.g. suitable for integration of the amplified sequence into vectors, or other adapters or overhang sequences, e.g. having a marker molecule such as a fluorescent marker attached, generally known to the skilled worker.

[283] In another aspect of the invention it is provided a method of diagnosis of a disorder according to the invention, wherein at least one compound selected from the group consisting of a polypeptide according to the sequence of SEQ ID 1 to SEQ ID 93, functional variants thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, and an antibody directed against one of the aforementioned polypeptides or antibody fragment thereof, is identified in the sample of a patient and compared with at least one compound of a reference library or of a reference sample.

[284] In a prefened embodiment of the method the disorder of the liver is a disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia.

[285] In a prefened embodiment of the method the epithelial cancer is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[286] Compared to the state of the art, this diagnostic suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or other epithelial cancers.

[287] Preferably the sample is isolated from a patient by non-invasive methods as described above.

[288] For example, serum detection of specific deregulated gene proteins via ELISA assay is one application, alternatively one or a panel of antibodies to deregulated gene products may be used, from which a diagnostic score is deduced based on the combinations of, and the expression levels of gene products expressed in the diseased tissue or in serum from diseased individuals.

[289] A prefened diagnostic according to the invention contains the described polypeptide or the immunogenic parts thereof described in greater detail above. The polypeptide or the parts thereof, which are preferably bound to a solid phase, e.g. of nitrocellulose or nylon, can be brought into contact in vitro, for example, with the body fluid to be investigated, e.g. blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, in order thus to be able to react, for example, with autoimmune antibodies present in e.g. the blood of the patient. The antibody- peptide complex can then be detected, for example, with the aid of labeled antihuman IgG antibodies. The labeling involves, for example, an enzyme, such as peroxidase, which catalyses a color or chemiluminescent reaction. The presence and the amount of autoimmune antibody present can thus be detected easily and rapidly by means of the color.

[290] En addition the diagnostic may be directed to detecting an endogenous antibody or fragment thereof present in the sample isolated from a patient which antibody or fragment thereof is directed against a polypeptide according to the invention. Detection of such autoimmune antibodies may be accomplished by methods generally known to the skilled artisan, e.g. by immunoaffinity assays using polypeptides according to the invention or functional variants thereof or parts thereof as a probe. Preferably the presence of such autoimmune antibodies is indicative of the patient suffering from a disorder according to the invention.

[291] A further diagnostic, that is subject matter of the present invention, contains the antibodies according to the invention themselves. With the aid of these antibodies, it is possible, for example, to easily and rapidly investigate a tissue sample as to whether the concerned polypeptide according to the invention is present in an increased amount in order to thereby obtain an indication of possible disease including liver disorders, for example HCC. In this case, the antibodies according to the invention are preferably labeled directly, or more commonly for example these are detected with a specific secondary antibody indirectly, such as with an enzyme or fluorescent molecule, as already described above. The specific antibody-peptide complex can thereby be detected easily,and rapidly, e.g., by means of an enzymatic color reaction. ,-,.,.

[292] In still another aspect of the invention it is provided a method for identifying at least one nucleic acid according to the SEQ ED 94 to SEQ ID 186, or a variant thereof differentially expressed in a sample isolated from a patient relative to a reference library or a reference sample comprising the following steps: (a) detecting the expression of at least one nucleic acid according to the SEQ ED 94 to SEQ ED 186, or a variant thereof in a sample isolated from a patient, (b) comparing the expression of said nucleic acid(s) detected in step (a) with the expression of the same nucleic acid(s) in a reference library or in a reference sample, (c) identifying said nucleic acid(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample.

[293] Compared to the state of the art the method suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive identification of differentially expressed nucleic acids according to the invention that provides a useful basis for diagnosing a disorder according to the invention.

[294] Preferably at least 2, at least 3, at least 4 at least 5, at least 6, or at least 7 nucleic acids are identified.

[295] In another prefened embodiment of the method said nucleic acid(s) is (are) detected by PCR based detection or by a hybridization assay.

[296] In another prefened embodiment of the method the expression of said nucleic acid is compared by a method selected from the group consisting of solid-phase based screening methods, hybridization, subtractive hybridization, differential display, and RNase protection assay.

[297] In a further prefened embodiment of the method the sample isolated from the patient is selected from the group consisting of liver tissue, a liver cell, tissue from another organ subject to cancerous transformation, a cell from this organ, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[298] Preferably the reference sample is isolated from a source selected from a non-diseased sample of the same patient or a non-diseased sample from another subject. The selection of appropriate reference samples is generally known to the person skilled in the art. In particular the reference sample may be selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[299] In another prefened embodiment of the method, the reference library is an expression library or a data base comprising clones or data on non-diseased expression of at least one nucleic acid according to the invention in samples that preferably may be selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[300] In another aspect of the invention it is provided a method of diagnosing a liver disorder, or an epithelial cancer comprising the following steps: (a) detecting the expression of at least one nucleic acid according to the SEQ ID 94 to SEQ ED 186, or a variant thereof in a sample isolated from a patient, (b) comparing the expression of said nucleic acid(s) detected in step (a) with the expression of the same nucleic acid(s) in a reference library or in a reference sample, (c) identifying said nucleic acid which is differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample, and (d) matching said nucleic acid(s) identified in step (c) with said nucleic acid(s) differentially expressed in a pathologic reference sample or pathologic reference library, wherein the matched nucleic acid(s) is (are) indicative of the patient suffering from a liver disorder or an epithelial cancer.

[301] Compared to the state of the art, this method of diagnosing suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or other epithelial cancers.

[302] In another prefened embodiment of the method of diagnosis, the pathologic reference sample is isolated from a diseased sample from another patient. The latter patient having been diagnosed as suffering from the disorder according to the invention which is to be diagnosed. The selection of appropriate pathologic reference samples is generally known to the person skilled in the art. In particular the pathologic reference sample may be selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[303] In another prefened embodiment of the method of diagnosis, the pathologic reference library is a data base comprising data on differential expression of the at least one nucleic acid according to the invention in samples isolated from at least one patient, excluding the patient under diagnosis, suffering from the disorder according to the invention to be diagnosed in the inventive method relative to control expression in a reference sample or reference library. The pathologic reference library preferably also relates to a differential expression library comprising nucleic acids according to the invention which are differentially expressed in samples isolated from at least one patient, excluding the patient under diagnosis, suffering from the disorder according to the invention to be diagnosed in the inventive method relative to control expression in a reference sample or reference library. The selection of an appropriate pathologic reference library is generally known to the person skilled in the art.

[304] Preferably the liver disorder is a disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia. In particular the epithelial cancer is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[305] Within the meaning of the invention the term "detecting a nucleic acid" refers to a method that preferably uncovers, visualizes, separates or allows recognition of the nucleic acid according to the invention from the background of the other components present in the sample. Such methods are generally known to the person skilled in the art and include in situ hybridization, PCR amplification, gel electrophoresis, northern blots, solid phase anay (gene chips) based methods, nuclease protection methods (as described and referenced in Alberts, et al. 2002, The Molecular Biology of the Cell, 4 ed. Garland, New York, USA).

[306] Within the meaning of the invention the term "comparing the expression of said nucleic acid(s) detected in step (a) with the expression of the same nucleic acid(s) in a reference library or in a reference sample" refers to a comparison of the expression of the two groups of said nucleic acid(s) on a quantitative or qualitative level by means of an experimental procedure such as differential display, subtractive hybridization, RNAse protection assay, or especially DNA chip hybridization. Moreover a comparison of experimental data on said nucleic acid(s) detected in step (a) with the expression of the same nucleic acid(s) in a reference library as defined above is also included herein.

[307] The term "identifying said nucleic acid(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample" within the meaning of the present invention is understood to mean selecting said nucleic acid(s) which is (are) differentially expressed compared to the reference library or the reference samples which fulfills the following criteria: the level of differential expression of the detected said nucleic acid(s) compared to the reference library or the reference samples is greater than about 2 fold, preferably greater than about 5 fold, more prefened greater than about 10 fold upregulated.

[308] The term "matching said nucleic acid(s) identified in step (c) with said nucleic acid(s) differentially expressed in a pathologic reference sample or pathologic reference library " within the meaning of the invention is understood to mean that said nucleic acid(s) identified in step (c) is (are) compared with said nucleic acid(s) differentially expressed in a pathologic reference sample or pathologic reference library. Then said nucleic acid(s) identified in step (c) that is (are) also differentially expressed in the pathologic reference sample or pathologic reference library is (are) matched, i.e. said identical pair is identified and allocated. Since the differential expression of said nucleic acid(s) in the pathologic reference sample or pathologic reference library is (are) indicative of a disorder according to the invention, such conespondence with the ..^differential expression in the sample then indicates that the patienkguffers from that disorder.

[309] Preferably the sample is isolated from a patient by non-invasive or preferably minimally invasive methods such as described above, including venupuncture.

[310] The methods of diagnosing according to the invention allows early detection of a liver disorder and/or an epithelial cancer, and/or non-invasive diagnosis of the disorder, based on an essentially concordant expression pattern of the nucleic acids according to the invention detected in the samples isolated from an animal and/or a human patient suffering from a liver disorder and or an epithelial cancer relative to a reference sample or relative to a reference library. The method has the additional advantage that it also provides additional and novel diagnostic parameters to characterize different subtypes of liver disorders, such as for example subtypes of HCC.

[311] The term "essentially concordant expression pattern" of the nucleic acids according to the invention refers to a pattern of expression that is essentially reproducible from patient to patient or subject to subject, provided that the patients or subjects compared are in the same or comparable pathological condition or healthy condition, respectively. [312] In still another aspect of the invention it is provided a method for identifying at least one polypeptide according to the SEQ ID 1 to SEQ ID 93, or a functional variant thereof differentially expressed in a sample isolated from a patient relative to a reference library or a reference sample comprising the following steps: (a) detecting the expression of at least one polypeptide according to the SEQ ID 1 to SEQ ID 93, or a functional variant thereof in a sample isolated from a patient, (b) comparing the expression of said polypeptide(s) detected in step (a) with the expression of said polypeptide(s) in a reference library or in a reference sample, (c) identifying said polypeptide(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample.

[313] Compared to the state of the art, this method suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive identification of differentially expressed polypeptides according to the invention that provides a useful basis for diagnosing a disorder according to the invention.

[314] Preferably at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 polypeptides are identified.

[315] Preferably the sample is isolated from a patient by non-invasive or minimally invasive methods such as described above, including venupuncture.

[316] In another embodiment of the method the sample is a sample as defined further above. Preferably the reference sample is a reference sample as defined above.

[317] In another prefened embodiment of the method, the reference library is an expression library or a data base comprising clones or data on non-diseased expression of the at least one polypeptide according to the invention in samples that preferably may be selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, or feces. Such databases are generated as a result of the cDNA microanay expression analysis according to the invention and are known to persons skilled in the art. Further reference libraries useable according to the invention have been described above.

[318] In another aspect of the invention it is provided a method of diagnosing a liver disorder or an epithelial cancer comprising the following steps: (a) detecting the expression of at least one polypeptide according to the SEQ ID 1 to SEQ ED 93, or a functional variant thereof in a sample isolated from a patient, (b) comparing the expression of said polypeptide(s) detected in step (a) with the expression of said polypeptide(s) in a reference library or in a reference sample, (c) identifying said polypeptide(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample, and (d) matching said polypeptide(s) identified in step (c) with said polypeptide(s) differentially expressed in a pathologic reference sample oφathologic reference library, wherein the matched polypeptide(s) is (are) indicative of the patient suffering from a liver disorder or an epithelial cancer.

[319] Compared to the state of the art, this method of diagnosing suφrisingly allows improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or other epithelial cancers.

[320] Preferably at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 polypeptides are identified.

[321] Within the meaning of the invention the term "detecting a polypeptide" refers to a method that preferably uncovers, visualizes, separates and/or allows recognition of the polypeptide according to the invention from the background of the other components present in the sample. Such methods are generally known to the person skilled in the art and includes gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, enzyme based immunoassay, mass spectroscopy, high pressure liquid chromatography, surface plasmon resonance, and/or antibody and protein anays as described above (Ausubel, F.A. et al., eds., 1990, Cunent Protocols in Molecular Biology. Greene Publishing and Wiley-lnterscience, New York, USA, Chapter 10; Myszka and Rich 2000, Pharm. Sci. Technol. Today 3:310-317). Preferably proteins and polypeptides are prepared from the sample by disruption of the cells with physical sheering or ultrasonic means, for example. Protein is denatured and stabilized with reducing agent treatment and heating and the protein is size fractionated on electrophoretic polyacrylamide gels.

[322] Within the meaning of the invention the term "comparing the expression of said polypeptide(s) detected in step (a) with the expression of the same polypeptide(s) in a reference library or in a reference sample " refers to a comparison of the expression of the two groups of polypeptide(s) on a quantitative and/or qualitative level by means of an experimental procedure such as two dimensional gel electrophoresis, chromatographic separation techniques, immunoblot analysis, surface plasmon resonance, immunohistochemistry, and enzyme based immunoassay. En two dimensional gel electrophoresis, all peptides are first resolved according to isoelectric point in the first electrophoretic dimension and then by size according to methods well known to persons experienced in the art. Moreover a comparison of experimental data on the at least one polypeptide detected in step 1 with the expression of the polypeptide in a reference library as defined above is also included herein.

[323] The term "Identifying said polypeptide(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample" within the meaning of the present invention is understood to mean selecting said polypeptide(s) which is (are) differentially expressed compared to the reference library or the reference samples which fulfills the following criteria: the level of dif- ferential expression of the detected polypeptide(s) compared to the reference library or the reference samples is greater than about 2 fold, preferably greater than about 5 fold, more prefened greater than about 10 fold upregulated.

[324] The term "matching said polypeptide(s) identified in step (c) with said polypeptide(s) differentially expressed in a pathologic reference sample or pathologic reference library " within the meaning of the invention is understood to mean that said polypeptide(s) identified in step (c) is compared with said polypeptide(s) differentially expressed in a pathologic reference sample or pathologic reference library. Then said polypeptide(s) identified in step (c) that is (are) also differentially expressed in the pathologic reference sample or pathologic reference library is (are) matched, i.e. said identical pair(s) is (are) identified and allocated. Since the differential expression of said polypeptide(s) in the pathologic reference sample or pathologic reference library is (are) indicative of a disorder according to the invention, such conespondence with the differential expression in the sample then indicates that the patient suffers from that disorder.

[325] Preferably the sample is isolated from a patient by non-invasive or minimally invasive methods such as described above, including venupuncture.

[326] En another embodiment of the method the sample is a sample as defined further above. Preferably the reference sample is a reference sample as defined above.

[327] En another prefened embodiment of the method of diagnosis, the reference library is an expression library or a dataset comprising clones or data on non-diseased expression of the at least one polypeptide according to the invention in samples that preferably may be selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[328] An example of a data base according to the invention and further experimental reference libraries useable according to the invention have been described above.

[329] En another prefened embodiment of the method of diagnosis, the pathologic reference sample is a pathologic reference sample as has been defined above.

[330] En another prefened embodiment of the method of diagnosis, the pathologic reference library is a data base comprising data on differential expression of said polypeptide(s) according to the invention in samples isolated from at least one patient, excluding the patient under diagnosis, suffering from the disorder according to the invention to be diagnosed in the inventive method relative to control expression in a reference sample or reference library. The pathologic reference library also relates to a differential expression library comprising polypeptides according to the invention which are differentially expressed in samples isolated from at least one patient, excluding the patient under diagnosis, suffering from the disorder according to the invention to be diagnosed in the inventive method relative to control expression in a reference sample or reference library. The selection of an appropriate pathologic reference library is generally known to the person skilled in the art.

[331] Preferably the liver disorder is a disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia. In particular the epithelial cancer is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[332] The methods of diagnosing according to the invention allows early detection of a liver disorder and/or epithelial cancer, and/or non-invasive diagnosis of the disorder, based on an essentially concordant expression pattern of the polypeptides according to the invention detected in the samples isolated from an animal and/or a human patient suffering from a liver disorder and/or epithelial cancer relative to a reference sample or relative to a reference library. The method has the additional advantage that it also provides additional and novel diagnostic parameters to characterize different subtypes of liver disorders, such as for example subtypes of HCC.

[333] The term "essentially concordant expression pattern" of the polypeptides according to the invention refers to a pattern of expression that is essentially reproducible from patient to patient or subject to subject, provided that the patients or subjects compared are in the same or comparable pathological condition or healthy condition, respectively.

[334] En another aspect of the invention it is provided a pharmaceutical composition comprising at least one compound selected from the group consisting of a polypeptide according to SEQ ID 1 to 93, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, a vector comprising a nucleic acid coding for the aforementioned antibody, a cell comprising the vector comprising a nucleic acid coding for the aforementioned antibody, and a cell comprising the vector comprising a nucleic acid coding for the aforementioned antibody fragment, combined or together with suitable additives or auxiliaries. En a prefened embodiment the pharmaceutical composition contains at least one cell according to the invention, combined or mixed together with suitable additives or auxiliaries. [335] When compared to the state of the art of therapy of liver disorders, and/or other epithelial cancers the pharmaceutical composition according to the invention surprisingly provide an improved, sustained and/or more effective treatment.

[336] A pharmaceutical composition in the sense of the invention encompasses medicam ents which can be used for preventing and/or treating liver disorders and/or epithelial cancer. The pharmaceutical composition includes, for instance, a stabilized recombinant antibody that has been produced by expression of specific antibody gene fragments in a cellular system, preferably a eukaryotic system. A recombinant antibody therapeutic for instance, is delivered by injection into the diseased liver region or into the venous or arterial vascularsystems or into the hepatic portal system. The injections can be repeated at regular intervals to achieve therapeutic efficacy. Therapeutics according this invention may also be employed in combinations with other chemical, antibody, or any other therapeutic application to improve efficacy.

[337] An antibody or other specific-binding partner can be conjugated to a second molecule, such as a cytotoxic agent, and used for targeting the second molecule to a tissue-antigen positive cell (Niteta E.S. et al, 1993, Immunotoxin therapy, in DeNita Jr. N.T. et al., eds, Cancer: Principles and Practice of Oncology, 4 ed., J.B. Lippincott Co., Philadelphia, 2624-2636). Examples of cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes and chenotherapeutic agents. Techniques for conjugating ,.» therapeutic agents to antibodies are well known in prior art.

[338] In addition to immunotherapy, polynucleotides and polypeptides can be used as targets for non-immunotherapeutic applications, e.g. using compounds which interfere with function, expression, assembly of the genes according to the invention, including but not limited to modulation(s) of the enzymatic active site(s) of the polypeptide(s), change of the protein(s) structure(s), interaction(s) via small molecules, etc.

[339] The present invention also relates to a process producing a pharmaceutical ' composition for the treatment and or prevention of disorders according to the invention, for example, HCC, in which at least one component selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, is combined or mixed together with suitable additives.

[340] The present invention furthermore relates to a pharmaceutical composition produced by this process for the treatment and/or prevention of liver disorders and/or epithelial cancers, for example, HCC, which contains at least one component selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a nonfunctional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, if appropriate together with suitable additives and auxiliaries.

[341] The invention furthermore relates to the use of this pharmaceutical composition for the prevention and/or treatment of liver disorders, for example, HCC and/or epithelial cancer.

[342] Preferably the pharmaceutical composition is employed for the treatment of a liver disorder selected from the group consisting of cirrhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia. In particular the phramaceutical composition is employed for the treatment of an epithelial cancer that is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[343] Therapy can also be carried out in a conventional manner generally known to the person skilled in the art, e.g. by means of oral application or via intravenous injection of the pharmaceutical compositions according to the invention. It is thus possible to administer the pharmaceutical composition comprising the suitable additives or auxiliaries, such as, for example, physiological saline solution, demineralized water, stabilizers, proteinase inhibitors.

[344] A therapy based on the use of cells, which express at least one polypeptide according to the invention, functional variants thereof or nucleic acids coding for the polypeptide, or variants thereof can be achieved by using autologous or heterologous cells. Prefened cells comprise liver cells, for example primary cultures of liver cells, liver populating stem or progenitor cells, or blood cells. The cells can be applied to the tissue, preferably to the blood or injected into the liver, with suitable carrier material. Such therapy is preferably based on the notion that upon expression and/or release of a polypeptide according to the invention the polypeptide stimulates an immune response in the patient in need of the treatment.

[345] Preferably the therapeutical approach is directed toward inhibiting the function and/ or expression of at least one polypeptide according to the invention and/or the function and/or expression of at least one nucleic acid according to the invention. Such inhibition of the expression and/or function preferably reduces the expression and/or function of the targeted nucleic acid/polypeptide significantly, for example by 50%, in particular by 80% and most preferably by 95%. The inhibition of the expression and/or function preferably abolishes the expression and/or functioning of the targeted nucleic acid/polypeptide. The inhibition can occur at any level, including transcription, translation, and or perdurance of the nucleic acid (e.g. degradation, stability) in the cell. For inhibiting the biological activity of polypeptides according to the invention e.g. antibodies and small molecules can be targeted to cell-surface, exposed, extracellular, ligand binding, functional, etc. domains of the polypeptide. The term "antagonist/inhibitor" in the sense of the present invention can be directed to, or targeted to any part of the nucleotide and polypeptide according to the invention.

[346] Such reduction or abolished expression and/or functioning of the targeted nucleic acid/polypeptide can be determined using conventional assays for determining the expression and or functioning of a nucleic acid/polypeptide generally known to the person skilled in the art. In particular such assays for determining the function comprise methods for comparing the biological activity of the targeted nucleic acid/ polypeptide before and after administration of the pharmaceutical composition. Preferably such assays for determining the expression comprise methods for comparing the level of expression of the targeted nucleic acid/polypeptide before and after administration of the pharmaceutical composition.

[347] Such therapy is preferably accomplished by the use of a nucleic acid having a sequence complementary to one of nucleic acids according to the invention, i.e. an antisense molecule or a RNA interference molecule which reduces or abolishes the translation of transcribed nucleic acids according to the invention and thereby inhibits the function and/or expression of the targeted nucleic acid/polypeptide.

[348] In a prefened embodiment, the pharmaceutical composition comprises a nucleic acid having a complementary sequence which is an antisense molecule or an RNA interference molecule. [349] Preferably such nucleic acid having a complementary sequence may be employed in the form of a vector or a cell comprising such nucleic acid. On the polypeptide level the therapy may in particular be carried out by the use of an antibody or an antibody fragment directed against a polypeptide according to the invention. The antibody or antibody fragment may be administered directly to the patient or preferably the nucleic acid encoding the antibody is contained in a vector which is preferably contained in a cell. The cell or vector may then be administered to the patient in need of such treatment.

[350] When compared to the state of the art of therapy of liver disorders, and/or other epithelial cancers the method of treating according to the invention suφrisingly provide an improved, sustained and/or more effective treatment.

[351] The invention further relates to a method of treating a patient suffering from of a liver disorder, wherein at least one component selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding the polypeptide, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the vector, an antibody directed against the polypeptide, a fragment of the antibody, a vector comprising a nucleic acid coding for ihe antibody, a cell comprising the vector comprising a nucleic acid coding for the antibody, and a cell comprising the vector comprising a nucleic acid coding for the antibody fragment, optionally combined or together with suitable additives and or auxilaries, is administered to the patient in need of a treatment in a therapeutically effective amount.

[352] Preferably the method of treatment is directed to a liver disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia. En particular the method of treatment is directed to an epithelial cancer that is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[353] Methods of administering such compounds or cells have been described in detail above.

[354] The term "therapeutically effective amount" refers to the administration of an amount of the compound to the patient that results in an "effective treatment" as defined above. Determination of the therapeutically effective amount of the compound(s) is generally known to the person skilled in the art. [355] Such methods of treating allow effective treatment of a liver disorder and/or epithelial cancers as described above.

[356] In another aspect of the invention it is provided a method of stimulating an immune response in a patient suffering from a liver disorder and/or an epithelial cancer to a polypeptide according to the invention, or a functional variant thereof, wherein at least one component selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, and a cell comprising the aforementioned vector, is administered to the patient in need of such treatment in an amount effective to stimulate the immune response in the patient.

[357] When compared to the state of the art of therapy of liver disorders and/or other epithelial cancers, the method of stimulating an immune response according to the invention suφrisingly provides an improved, sustained and/or more effective immunization.

[358] In another aspect of the invention it is provided a method of preventing a patient from developing a liver disorder and/or an epithelial cancer, wherein at least one component selected from the group consisting of a polypeptide according to the invention, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, and a cell comprising the aforementioned vector, is administered to the patient in need of such preventive treatment in a therapeutically effective amount.

[359] When compared to the state of the art of therapy of liver disorders, and/or other epithelial cancers the method of preventing according to the invention suφrisingly provides an improved, sustained and/or more effective preventive measure.

[360] Preferably the method of preventing and/or method of stimulating an immune response is directed to a liver disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia. In particular, preferably the method of preventing and/or method of stimulating an immune response is directed to an epithelial cancer which is an adenocarcinoma of any organ other than liver, preferably of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast. [361] In another aspect of the invention it is provided a method of identifying a pharmacologically active compound comprising the following steps (a) providing at least one nucleotide according to the SEQ ID 94 to SEQ ID 186, or a variant thereof, (b) contacting said nucleotide(s) with suspected to be pharmacologically active compound(s), (c) assaying the interaction of said nucleotide(s) of step (a) with said compound(s) suspected to be pharmacologically active, (d) identifying said compound(s) suspected to be pharmacologically active which directly or indirectly interact with said nucleotide(s) of step (a).

[362] In a further aspect the invention relates to a method of identifying at least one pharmacologically active compound comprising the following steps: (a) providing at least one polypeptide according to the SEQ ID 1 to SEQ ED 93, or a functional variant thereof, (b) contacting said polypeptide(s), with suspected to be pharmacologically active compound(s), (c) assaying the interaction of said polypeptide(s) of step (a) with saidcompound(s) suspected to be pharmacologically active, (d) identifying said compound (s) suspected to be pharmacologically active which directly or indirectly interact with said polypeptide(s) of step (a).

[363] Preferably said nucleotide(s) or said polypeptide(s) is (are) provided in a form selected from the group of said nucleotide(s) or said polypeptide(s) is (are) attached to a column, said nucleotide(s) or said polypeptide(s) is (are) attached to an anay, said nucleotide(s) or said polypeptide(s) is (are) contained in an electrophoresis gel, said ,«. nucleotide(s) or said polypeptide(s) is (are) attached to a membrane, and said nucleotide(s) or said polypeptide(s) is (are) expressed by a cell.

[364] It is prefened but not intended to be limited to assay the interaction by a method selected from the group of enzyme and fluorescence based cellular reporter assays in which interaction of the compound suspected to be pharmacological active with a recombinant fusion protein including said polypeptide(s) of step (a) is detected. The interaction may preferably also be assayed by displacement of specific nucleic acid binding aptamer molecule(s) on the recombinant fusion protein, surface plasmon resonance, HPLC and mass spectroscopy.

[365] Preferably the direct or indirect interaction is selected from the group consisting of induction of the expression of said nucleotide(s) or said polypeptide(s), inhibition of the expression of said nucleotide(s) or said polypeptide(s), activation of the function of said nucleotide(s) or said polypeptide(s), inhibition of the function of said nucleotide(s) or said polypeptide(s).

[366] In a prefened embodiment a method for identifying an antagonist/inhibitor against the nucleotide according to the SEQ ED 94 to SEQ ED 186, or a variant thereof, comprising (a) contacting at least one nucleotide according to the SEQ ED 94 to SEQ ED 186 with a putative antagonist/inhibitor, and (b) determining whether the putative antagonist inhibitor prevents the activity of the nucleotide.

[367] In a further aspect of the invention, a method for identifying a putative antagonist/ inhibitor against the polypeptide according to the SEQ ED 1 to SEQ ID 93, or a functional variant thereof, comprising (a) contacting at least one polypeptide according to the SEQ ID 1 to SEQ ID 93 with the putative antagonist/inhibitor, and (b) determining whether the putative antagonist/ inhibitor prevents the activity of the polypeptide.

[368] The term "pharmacologically active substance" in the sense of the present invention is understood as meaning all those molecules, compounds and/or compositions and substance mixtures which can interact under suitable conditions with a nucleotide according to the SEQ ID 94 to 186 or variants thereof, if appropriate together with suitable additives and/or auxiliaries.

[369] The term "pharmacologically active substance" in the sense of the present invention is also understood as meaning all those molecules, compounds and/or compositions and substance mixtures which can interact under suitable conditions with polypeptide according to the SEQ ED 1 to 93 or functional variants thereof, if appropriate together with suitable additives and/or auxiliaries.

[370] Possible pharmacologically active substances are simple chemical (organic or inorganic) molecules or compounds, but can also include peptides, proteins or complexes thereof. Examples of pharmacologically active substances are organic molecules that are derived from libraries-of compounds that have been analyzed for their pharmacological activity. On account of their interaction, the pharmacologically active substances can influence the expression and/or function(s) of the nucleotide or polypeptide in vivo or in vitro or alternatively only bind to the nucleotides or polypeptides described above or enter into other interactions of covalent or non- covalent manner with them.

[371] A suitable test system, for example, that can be used in accordance with the invention is based on identifying interactions with the two hybrid system (Fields and Stemglanz, 1994, Trends in Genetics, 10, 286-292; Colas and Brent, 1998 TIBTECH, 16, 355-363). In this test system, cells are transformed with expression vectors that express fusion proteins that consist of at least one polypeptide according to the invention and a DNA-binding domain of a transcription factor such as Gal4 or E xA. The transformed cells also contain a reporter gene whose promoter contains binding sites for the conesponding DNA-binding domain. By means of transforming a further expression vector, which expresses a second fusion protein consisting of a known or unknown polypeptide and an activation domain, for example from Gal4 or heφes simplex virus NP16, the expression of the reporter gene can be greatly increased if the second fusion protein interacts with the investigated polypeptide according to the invention. This increase in expression can be used for identifying new interacting partners, for example by preparing a cDNA library from e.g., liver tissue, or diseased liver tissue for the puφose of constructing the second fusion protein. In a prefened embodiment, the interaction partner is an inhibitor of at least one of the polypeptides according to the SEQ ID 1 to 93 (encoded by the SEQ ED 94 to 186) or functional variants thereof. This test system can also be used for screening substances that inhibit an interaction between the polypeptide according to the invention and an interacting partner. Such substances decrease the expression of the reporter gene in cells that are expressing fusion proteins of the polypeptide according to the invention and the interacting partner (Nidal and Endoh, 1999, Trends in Biotechnology, 17: 374-81). En this way, it is possible to rapidly identify novel active compounds that can be employed for the therapy of and/or prevention of liver disorders and/or epithelial cancer.

[372] Assays for identifying pharmacologically active substances that exert an influence on the expression of proteins are well known to the skilled person (see, for example, Sivaraja et al., 2001, US 6,183,956). Thus, cells that express a polypeptide according to the SEQ ED 1 for example, or functional variants thereof can be cultured as a test system for analyzing gene expression in vitro, with preference being given to liver cells. Gene expression is analyzed, for example, at the level of the mRΝA or of the proteins using methods generally known to the person skilled in the art. In this connection, the quantity of a polypeptide according to the SEQ ID 1 to 93.(encoded by the SEQ ID 94 to 186) or mRΝA present after adding one or more putative pharmacologically active substances to the cell culture is measured and compared with the corresponding quantity in a control culture. This is done, for example, with the aid of an antibody specifically directed against the polypeptide according to the SEQ ED 1 to 93 (encoded by the SEQ ED 94 to 186), or a functional variant thereof, which can be used to detect the polypeptide present in the lysate of the cells. The amount of expressed polypeptide can be quantified by methods generally known to the person skilled in the art using, for example, an ELISA or a Western blot. En this connection, it is possible to carry out the analysis as a high-throughput method and to analyze a very large number of substances for their suitability as modulators of the expression of at least one of the polypeptides according to the SEQ ID 1 to 93 (encoded by the SEQ ED 13 to 24) (Sivaraja et al., 2001, US 6,183,956). In this connection, the substances to be analyzed can be taken from substance libraries (see, e.g. DE19816414) that can contain many thousands of substances, which are frequently very heterogeneous.

[373] The invention will now be further illustrated below with the aid of the figures and examples, representing prefened embodiments and features of the invention without the invention being restricted hereto. [374]

[375] Figure 1 to 8 RNA expression levels in hepatocellular carcinoma (HCC) samples

[376] Summary boxplot of expression values inHCC versus non-diseased liver cDNA microanay experiments is provided. The box plot is a graphical representation of log expression value ratios with the median value indicated by a horizontal line in each box. The extent of each box indicates the iqr = interquartile range (+/- 25 percentile of median value); whiskers indicate of 1.5 times the iqr. Ratios that do not fall within this range are indicated with small circles. For each nucleic acid according to the invention (SEQ ED 95 to 186) elevated expression is apparent in HCC in comparison to non- diseased liver samples. For gene abbreviations see Tables 2A to 2D (**) c-syn represents three alternative nucleotide transcripts with conesponding three protein products.

[377]

[378] Figure 9 to 99: RNA expression levels in various diseased liver samples and normal tissue(s)

[379] Summary boxplots of expression values (SEQ ED 94 to 186) inHepatocellular Carcinoma (HCC), Focal Nodular Hypeφlasia (FNH) and Cinhosis samples (Cinh.) versus non-neoplastic liver cDNA microarray experiments are provided. The box plot analogs are used as described in Figure 1. For each nucleic acid according to the invention, elevated expression is apparent in HCCs and most of the FNHs samples, legend: A= HCC; B= FNH; C= Cinh. For gene abbreviations see Table 2A to 2D (**) c-syn represents three alternative nucleotide transcripts with conesponding three protein products.

[380]

[381] Figure 100 to 104 : Verification of differential gene expression when compared to normal tissue(s) and other types of cancer

[382] The Assay-On-Demand (Applied Biosystems, USA) quantitative PCR (Q-PCR) method is utilized for verification of disease deregulated expression of nucleic acids PACE4; BIGH3; s.t.OCIA; SDCCAG28; Rab2; TM4SF4; DAD-1. In Figures 100 to 103, for example, the following commercially available Assay-On-Demand primers are employed: Hs00159844_ml for PACE4 (SEQ ID 98); Hs00154671_ml for BIGH3 (SEQ ED 99); Hs00215197_ml for s.t.OCIA (SEQ ID 101); Hs00246405_ml for SDCCAG28 (SEQ ID 102); Hs00234094_ml for Rab2 target (SEQ ID 106), Hs00270335_ml for TM4SF4 (SEQ ED 112); Hs00154671_ml for DAD-1 (SEQ ED 140). En another example (Figure 104), the AKRICI PCR product is monitored accordingly by incoφoration of fluorescent double-stranded DNA intercalating molecules such as SYBR green. The increased expression of AKRICI (SEQ ID 96) in HCC when compared to normal liver (NNL) is verified by using the SEQ ID 199 and SEQ ID 200 primers; data for B and C are not available. Overall, Mann-Whitney-U Test (non-parametric test applied for non-normally distributed data) is performed as Wilcoxon Rank Sum Test (Hollander & Wolfe, 1973, Nonparametric statistical inference. New York: John Wiley & Sons, pgs. 27-33, 68-75; Bauer, D.F., 1972, J. Amer. Statistical Assoc. 67, pgs 687-690). The expression values typically do not fit to a normal distribution so averaging the values may be misleading. However, analysis of the median values demonstrates significant differences in most of the cases between experimental and reference values, particularly in the large data sets.

[383] E_^egend: A= Hepatocellular Carcinoma (HCC); B= Focal Nodular Hypeφlasia (FNH); C= Cinhosis (Cinh.); D= Non-Neoplastic liver (NNL). For gene abbreviations see Table 2A to 2D.

[384] Figure 105: SDCCAG28 protein expression in tissues

[385] Protein extracts are subjected to immunoblot analysis with HuCAL™ antibodies (Moφhosys AG, Germany) specific to recombinant SDCCAG28 protein (e.g., MOR3491 anti OREOIO), in order to determine the level of expression of the protein in human tissues [a= OREOIO (human SDCCAG28 recombinant protein); b= Hepatocellular Carcinoma (HCC); c= Normal Liver (NL); d= hepatoma HepG2 cell line. Annotated 33kDa position reflects a size of the predicted SDCCAG28 protein. Following incubation with an anti-HIS mouse antibody to specifically detect the HuCAL™ antibody and a horse-radish peroxidase (HRP) conjugated anti-mouse antibody the immune complexes are detected with a chemiluminescent HRP substrate. It is evident that the native SDCCAG28 protein migrates slightly faster than the recombinant SDCCAG28 protein (approximately 44 kDa band in lane a compared with 40.5 kDa bands prominent in lanes b and d). The intensities of the SDCCAG28 protein band are clearly stronger in the HCC tissue and in the HepG2 hepatoma cell line lysate (lanes b and d, respectively) than in the normal liver tissue (lane c). These analyses indicate that SDCCAG28 protein, the functional product of the SDCCAG28 mRNA specifically upregulated in HCC, is also highly overexpressed in HCC when compared to NNL.

[386]

[387] Figure 106 to 107: Expression of HCC deregulated genes correlates with proliferation of hepatoma cells

[388] Proliferation-dependent expression of target gene sequences according to the invention in hepatoma cells Hep3B (Figure 106) and HepG2 (Figure 107) following serum stimulation for 8 hours (black columns) and for 12 hours (white columns) of quiescent cells. The log -transformed ratios of serum-stimulated vs. quiescent expression values from cDNA microanay experiment readout are provided. The substantial increase in the level of expression of these sequences (for example, (ZNF216) SEQ ID 95; (AKRICI) SEQ ID 96; (PACE4) SEQ ID 98; (SDCCAG28) SEQ ID 102; (TMP21) SEQ ED 104 and (RAB2) SEQ ED 106) in proliferating compared to quiescent hepatoma cells suggests that elevated expression of these sequences is functionally significant for liver cancer cell growth. For gene abbreviations see Table 2 A to 2D.

[389]

[390] Figure 108 to 109: Effect of dUT specific inhibitor on growth of proliferating liver cancer (hepatoma) cell lines

[391] Specific dUT enzyme inhibitor (DMT-dU (5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyuridine) (Sigma; No. D7279) is added to the hepatoma cells (Hep3B in Figure 108 and HepG2 in Figure 109) at the 10, 25, 50, 100, 250 and 500 μM final concentrations in a maximum of 3 μl of the appropriate solvent. Following incubation of the cells for 24 (black columns) and 48 hours (white columns) respectively, cell viability is assessed via an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay known in the prior art (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega), and plotted relative to the number of cells in wells not treated with the inhibitor (control = 0; no inhibitor added). The relationship between the increased concentration of the inhibitor and absorbance (A= 495nm) reflects the Hep3B/ HepG2 cytostatic/ anti-proliferative response, suggesting.that dUT gene conelates with human liver tumor cell proliferation. Examples

[392]

[393] Example 1: Preparation of HCC subtracted cDNA libraries

[394] RNA is isolated from three pathologist-confirmed HCC tumor samples and from three pathologist-confirmed non-diseased human liver samples using the TRIZOL reagent (Invitrogen) according to standard methods (Chomczynski & Sacchi, 1987, Anal. Biochem. 162:156-159). The tissues used for the generation of cDNA libraries is from patients that provided specific informed consent for utilization of this material for research puφoses, including commercial research. mRNA is converted to double stranded cDNA with reverse transcriptase and DNA polymerase as described in the instructions provided in the "PCR select cDNA subtraction kit" from Clontech Laboratories. To enrich for cDNAs specifically increased and decreased in HCC, cDNAs expressed in common and at similar levels in the reference liver pool and in HCC are removed by subtractive suppressive hybridization (SSH) according to the instructions provided in this kit and as described by Diatchenko et al. (1996, Proc. Natl. Acad. Sci. USA 93:6025-6030). The SSH steps are performed in both directions (subtracting non- diseased liver cDNAs from HCC cDNAs and subtracting HCC cDNAs from non- diseased liver cDNAs) so the resulting cDNA molecules represent nucleic acid sequences both up- and down-regulated in HCC but do not represent those that are not differentially expressed. In addition a normalized but not subtracted HCC cDNA library is generated to better represent rare mRNA transcripts in HCC tissues. These cDNAs are separately cloned into the pCREl vector (Invitrogen) by ligation into this plasmid followed by electrophoretic transformation into E. coli XL- 1 -Blue elec- troporation-competent cells (Stratagene). The cloning is canied out as described by the supplier of the vector and competent cells. Cloned differentially expressed cDNAs are plated onto selective (ampicillin) media to isolate individual clones. 960 clones are isolated from each SSH library and 384 clones isolated from the normalized HCC library and cultures established in 96- well microtiter plates. Together these cDNA clones provide a unique representation of mRNA expression specific for human HCC tissue. [395]

[396] Example 2: Preparation and hybridization of HCC cDNA microarrays

[397] 1 ml cultures of the SSH cDNA library clones described above are established and the cDNA inserts amplified by PCR with primers specific to the vector sequence flanking the cDNA inserts. The M13 forward (5'- gtaaaacgacggccag-3'; SEQ ED 42) and M13 reverse primers (5I-caggaaacagctatgac-3'; SEQ ED 43) are employed for the PCR amplification of clone inserts. Fifty microliters of the bacterial cultures are heat denatured at 95°C for 10 minutes, debris removed by centrifugation, and 2 μl of the supernatant included in a standard PCR [IX Amplitaq PCR buffer, 2.5 mM MgCl , 37.5 nM each primer, 0.5 mM each of dATP, dCTP, dGTP and dTTP and 1.5 units Amplitaq DNA polymerase (Applied Biosystems)]. Reaction conditions are 95°C for 5 minutes followed by 35 cycles of: 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds; then followed by 72°C for 7 minutes and then cooled to 4°C. Amplification of cDNA inserts is confirmed by electrophoresis of a 5% of the PCR on a 1% agarose gel comprising 0.4 mg/ml ethidium bromide and run in IX Tris Acetate EDTA (TAE; 40mM Tris-acetate, lmM EDTA, pH 7.5) buffer. Each of the SSH clone amplified insert sequences is affixed to sialinized glass microscope slides (GAPS Coming) using a GeneticMicrosystems 417 cDNA anayer robot to generate custom HCC cDNA microanay s. The protocol for spotting the cDNA inserts to the slides is according to that published by Hedge et al. (2000, Biotechniques 29:548-560) except that PCR products are spotted directly from the PCR microtiter plates without purification or adjustment of the cDNA buffer. In addition to the SSH cDNA clone inserts, numerous control DNAs are spotted onto the microarrays as controls for hybridization reactions. Further, approximately 2000 publicly available cDNA clones conesponding to genes previously reported to be involved in cancer are purchased from the German Genome Research Center (RZPD), expanded, amplified and spotted onto these microaπays as described above. For preparation of hybridization probes, 20 micrograms of RNA from additional pathology-confirmed liver disorders and from the same quantity of pooled non-diseased liver RNA is converted to cy5-fluorescence-labeled and cy3-fluorescence-labeled cDNA, respectively (cy5-CTP and cy3-CTP, Pharmacia) using reverse transcriptase according to the standard methods (Hedge et al., 2000, Biotechniques 29: 548-560). Using this protocol, these labeled cDNAs are competitively hybridized to the HCC microarrays. Following prehybridization at 42°C for 45 minutes in 5X SCC (0.75 M sodium citrate, 75 mM sodium citrate, pH 7.0); 0.1% SDS (sodium dodecyl sulfate) and 1% BSA (bovine serum albumin), the hybridization is carried out overnight at 42°C in buffer comprising 50% formamide, 5XSSC, and 0.1% SDS. Hybridized slides are washed in stringent conditions (twice at42°C in IX SSC, 0.1% SDS for 2 minutes each; twice at room temperature in 0.1X SSC, 0.1% SDS for 4 minutes each; and twice at room temperature in 0.05X SSC for 2 minutes each), dried and analyzed with the GeneticMicrosystems 418 cDNA microarray scanner and associated Imagene 4.1 image analysis software according to the manufacture's recommendations. [398] _*. [399] Example 3: Independent verification of differential expression of the nucleic acids and polypeptides according to the invention [400] RNA is isolated from human patient samples as described in detail above. HCC samples for this analysis are not from the same patients as employed for production of the HCC SSH library or for cDNA microanay chip hybridization. In addition to HCC samples, RNA is prepared from independent non-diseased liver samples to assess expression of the nucleic acids according to the invention in non-diseased liver tissue. Further, RNA is prepared from additional non-diseased and cancer tissues to assess expression of the nucleic acids according to the invention in other normal human tissues and other human cancers.One mg of RNA is converted to single-strand cDNA with the aid of Superscript reverse transcriptase (Invitrogen) in dATP, dCTP, dGTP, and dTTP (0.4 mM each), 7.5 nM random 6-nucleotide primer (hexamers), 10 mM dithiothreitol and 1 unit RNAse inhibitor using standard procedures known in the art (Sambrook et al., Molecular Cloning, 2^nd ed., 1989, Cold Spring Harbor Press, NY, USA, pp. 5.52-5.55). The presence or absence and the relative concentration of the nucleic acids according to the invention is then confirmed and verified by amplification of these sequences from the cDNA with primer pairs specific to each nucleic acid according to the invention in quantitative kinetic PCR experiments. The Assay-On-Demand (Applied Biosystems, USA) quantitative PCR method well known for the person skilled in the art might be utilized for verification of disease deregulated expression of nucleic acids according to the invention (Figure 3A/3B). For example, the Assay-On-Demand ID primer numbers for PACE 4, BIGH3, s.LOCIA, SDCCAG28, Rab2, PRKARIA, PRDXl, 1QGAP2, TM4SF4, DAD-1 target genes are given in the following Table 8.

[401] [402] Table 8: Target clones and their Assay-On-Demand ID Table 8

[403] [404] In further example, AKRICI PCR product is monitored accordingly by incoφoration of fluorescent double-stranded DNA intercalating molecules such as SYBR green. The AKRICI cDNA is validated by using following primers: AKRlCl-pl, 5'- ttggaaaggtcactgaaaaatct-3" (SEQ ED 199) and AKRlCl-p2, 5'-gctggctgcggttgaagttgg-3' (SEQ ED 200) verifying the specific expression of this gene (SEQ ID 96) in HCCs when compared to normal liver samples (Figure 104).

[405] Usally PCR is performed according to the manufacturer's instructions using TaqMan Universal PCR Mastermix (CatNr. 4304437; Applied Biosystems, Branchburg, New Jersey USA). Kinetic quantitative PCR analyses are performed by using the 7000 Sequence Detection System (Applera). The PCR Setup included two reference genes [GAPDH and Beta- Actin (GAPDH primers used = GAPDH-pl, SEQ ID 187; GAPDH-P2, SEQ ED 188; GAPDH-p3, SEQ ED 189) (B- Actin primers used = BActin-pl, SEQ ID 190; BActin-p2, SEQ ED 191; BActin-p3, SEQ ID 192)] which are used for independent normalisation of the investigated target genes. A standard curve (125ng, 25 ng, 5ng and Ing) is used for proper calculation of the expression data. The PCR sample contained 12.5 ng of cDNA, 12.5 μl Universal PCR Mastermix and 1.25μl Assay-On-Demand reagent to give a final volume of 25μl. PCR conditions are used according to the manufacture's instructions (2 min 50°C, 10 min 95°C followed by 40 cycles of 15 sec 95°C and 1 min at 60°C). Amplification of cDNA inserts is additionally confirmed by electrophoresis of a 10% of the PCR on a 2.5% agarose gel comprising 0.5 mg/ml ethidium bromide and run in IX Tris Acetate EDTA (TAE) buffer. Standard controls for RT-PCR including RNAse treatment of samples prior to cDNA synthesis and omission of reverse transcriptase routinely demonstrate the specificity of these reactions. The kinetic quantitative RT-PCR (Q-PCR) verifies the over expression of sequences according to the invention in liver cancer and other liver disorder relative to non-diseased liver (Figures 100 to 104). Furthermore, the protein expression analyses indicate that for example SDCCAG28 protein, the functional product of SDCCAG28 mRNA specifically upregulated in HCC, is also significantly overexpressed in HCC (Figure 105). To detect SDCCAG28 protein expression in HCC samples standard western blot analysis known in the prior art is performed using protein extracts derived from frozen tissues (stored in liquid nitrogen). The 50 μm sections are obtained (HCC, normal liver) using a refrigerated microtome (cyrocut, Leica CM3050), wherein the identity and homogeneity of the tissues under scrutiny is verified by H&E-staining of sections taken before, in between and after each cutting process. Tissues sections (HCC, normal liver), SDCCAG28 antigen (Moφhosys AG, Germany) and HepG2 cells are resuspended in ice-cold RIPA-buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 0.1% SDS, 1% deoxycholate, 1% NP-40) supplemented with 2 μg/ml leupeptin, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride (PMSF), and 2 mM dithiothreitol followed by homogenization through sonication (2 bursts of 5 seconds) on ice. After incubation for 20 minutes on ice, the lysates are cleared by two centrifugational steps in a microcentrifuge at 13 000 rpm for 15 minutes at 4°C and the supematants are collected. Protein concentrations are determined by the Bradford assay(Biorad) using bovine serum albumin as a standard. Equal amounts of protein (typically 10-30 μg) are separated on a 12% SDS-PAGE gel and transfened electrophoretically to a polyvinylidene diflouride (PVDF) membrane (Hybond-P, Amersham Biosciences) through Semidry-blotting (TE 70, Amersham). The membrane is blocked for 1 hour (or overnight) at room temperature in blocking solution [5 to 10% milkpowder (Micrbiology/Lactan: 1.15363.0500) in TBS-T (25 mM Tris-HCl pH 7.4, 137 mM NaCl, 3 mM KC1, comprising 0.1% Tween-20 (Merck: 822184) and 2% BSA (Sigma:A-7906)] and incubated with the primary antibody specific for the SDCCAG28 recombinant protein (Moφhosys AG, Germany), usually in the concentration between 30ng to 50ng/ml in TBS-T/1% milk solution at 4°C overnight with agitation. After removal of the primary antibody solution and several washes in TBS-T, the membrane is incubated with a mouse anti-HIS antibody to specifically detect the primary antibody (Dianova, 1 :25000) followed by a rabbit anti-mouse HRP (horse-radish peroxidase)-conjugated antibody (Dako, 1: 1000) for one hour at room temperature. Following several washes in TBS-T, detection is performed through chemilu- miniscence (ECL, Amersham) detection of HRP activity and exposing the membrane to x-ray film.

[407] These data provide independent verification of deregulated expression of the nucleic acids and polypeptides according to the invention in HCC. Expression of the nucleic acids and polypeptides according to the invention is either absent or observed only at very low levels in non-diseased liver, thereby validating the differential expression of these nucleic acids identified by hybridization to the cDNA microarray. The results provide suφrising evidence that the nucleic acids and polypeptides according to the invention can be used to diagnose, prevent and/or treat disorders according to the invention.

[408]

[409] Example 4: Sequences according to the invention are increased in proliferating liver cancer (hepatoma) cell lines

[410] Human hepatoma cell lines (Hep3B, HepG2) are cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO at 37°C. The cells are split to about 20% confluency and subsequently rendered quiescent by culturing in the absence of serum for 3 days. After the starvation period, the cells are stimulated to proliferate by the addition of 10 % FBS to the media. Samples are taken before and following the induction of cell growth (0, 8 and 12 hours) for the preparation of RNA and for determination of the position of the cells in the cell cycle by FACS (fluorescence activated cell sorting) analysis. Accordingly, to determine the cell cycle distribution by propidium iodide (PI) staining, the cells are harvested by trypsinization, washed twice with phosphate buffered saline (PBS) and finally resuspended in 500 μl PBS. Subsequently, 5 ml prechilled methanol is added. After 10 minutes incubation at -20°C the cell suspension is directly used for FACS analysis following 3 times washing in PBS, resuspended in 500 μl propidium iodide (PI) staining buffer (DNA-Prep Stain, Part No. 6604452; Beckman Coulter) and incubated for 15 minutes at 37°C. Finally, 70 μl of IM NaCl is added and the samples are kept on ice protected from light prior to analysis on an EPICS XL-MCL flow cytometer (Beckman Coulter). Cells prepared from an asynchronous cell population are used as reference.

[411] The isolated RNA is used to monitor the expression of genes in quiescent vs. pro- liferating hepatoma cells by cDNA microanay analysis. Following labeling with fluorescent dyes as described in example 2, the RNAs are hybridized on a specifically developed HCC- specific cDNA microanay chip that also contained control genes which are known to be expressed in a cell cycle dependent manner. Finally, the data are analysed using ImaGene 4.1 and GeneSight software packages. The signals obtained for 0 hours samples isolated before the addition of serum are used as reference. The log -transformed ratios of serum-stimulated vs. quiescent expression values from the cDNA experiment readout is provided in Figure 106 to 107.

[412] These data indicate that the sequences according to the invention are conelated with human liver tumor cell proliferation. Compared to the state of the art, these nucleic acids and polypeptides therefore suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or epithelial cancers.

[413]

[414] Example 5: Effect of dUT specific small molecule inhibitor on growth of proliferating liver cancer (hepatoma) cell lines

[415] To determine the effects of small molecule inhibitors of activity of enzyme polypeptides according to the invention on the growth of human hepatoma cells, for example a specific dUT inhibitor (DMT-dU (5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyuridine) (Sigma; No. D7279) is employed. Hep3B or HepG2 cells are seeded into 96-well plates at 10,000 and 7,500 cells, respectively, in a total volume of 150 μl of growth DMEM media supplemented with 10% fetal calf serum. The next day of incubation at 37 °C, the dUT enzyme inhibitor is added to the cells at the 10, 25, 50, 100, 250 and 500 μM final concentrations in a maximum of 3 μl of the appropriate solvent. Following incubation of the cells for 24 and 48 hours, cell viability is assessed via an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay known in the prior art (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega) according to the manufacturer's instructions. Thirty μl of the assay reagent are added directly to the culture wells, incubated for 1-2 hours and then absorbance at 495 nm is recorded using a microtiter plate reader (Anthos 2010; Anthos Labtec Instruments). Each value represents the mean of at least 4 replicates. Control cells (= 0) receive solvent only (Figures 108 to 109)

[416] The relationship between the increased concentration of the inhibitor and absorbance indicates that application of the aforementioned specific dUT inhibitor to hepatoma cells elicits a cytostatic/ anti-proliferative response, suggesting a specific role of the dUT gene in human liver tumor cell proliferation.

[417]

[418] Example 6: Elevation of enzymatic activity in hepatoma cells correlates with AKRICI target gene overexpression in liver disorders

[419] A comparison of the enzymatic activity of a target gene encoded polypeptide gives insight whether a deregulation of mRNA transcript is also reflected by a significant increase in activity that indicates its functional role in tumor biology. In a substrate- specific reaction, the activity of AKRICI (SEQ ID 96) is determined (see below Table 9).

[420] Enzymatic assays are performed by using lysates prepared from frozen tissues (stored in liquid nitrogen) or from cell pellets derived from asynchronously growing human hepatoma cell lines (Hep3B, HepG2). 50 μm sections obtained from pieces of frozen tissues using a freezing microtome (Cryocut, Leica CM3050) and the identity and homogeneity of the tissues under scrutiny is verified by a pathologist following H &E-staining of sections taken before, in between, and after each cutting process. Tissues sections as well as frozen cell pellets are resuspended in ice-cold lysis buffer (50 mM KPO 4 pH 7.0, 10 mM KOAc, 2 mM MgCl 2 ) supplemented with 2 μg/ml leupeptin, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, and 2 mM dithiothreitol followed by homogenization through sonication (2 bursts of 3 seconds) on ice. After incubation for 15 minutes on ice, the lysates are cleared by two centrifugation steps in a microcentrifuge at 13,000 rpm for 15 minutes at 4°C and the supematants are collected. Protein concentrations are determined by the Bradford assay (Biorad) using bovine serum albumin as a standard.

[421] The AKRICI enzymatic activity is measured spectrophotometrically based on the oxidation of 1-acenaphthenol in 1.0 ml systems containing 1 mM 1-acenaphthenol (in 4% methanol), 2.3 mM NAD⁺, and various amounts of whole cell lysate in 100 mM potassium phosphate buffer (pH 7.0). Reactions runs at 25°C wherein the change in absorbance of pyridine nucleotide over time is monitored at 340 nm on a Beckman DU640 spectrophotometer. Absorbance values are plotted versus time, and slope- values versus time (min^* ) are calculated from initial reaction velocities using linear least-squares regression analysis, see Table 9 (HCC = Hepatocellular Carcinoma; NNL = Non-Neoplastic (Normal) Liver).

[422]

[423]

[424]

[425]

[426] Table 9: Enzymatic assay for AKRICI (SEQ ID 96)

[427] Table 9 Tissue Protein con- Slope Weighted Mean 'of

[428] [429] The HCC samples (HCC11, HCC28, HCC30 and HCC2) are characterized by a weighted mean of the slope approximately 2-3-fold higher than the NNL samples. These data clearly show the conelation between the upregulation of AKRICI gene transcript in HCC with the induction of the AKRICI enzymatic activity in hepatoma cell lines, suggesting that the sequences according to the invention are conelated with human liver tumor cell proliferation. Compared to the state of the art, these nucleic acids and polypeptides therefore suφrisingly allow improved, more sensitive, earlier, faster, and/or non-invasive diagnosis of the liver disorders and/or epithelial cancers.

[430] [431] [432] Example 7: A method of diagnosing using HCC specific probes [433] A diagnostic method for disorders according to the invention preferably based on the polymerase chain reaction (PCR) can be established. A standard PCR detection of nucleic acid sequences of the invention can be sufficient to identify, for example, circulating HCC tumor cells in the blood stream of the patient. Detection of expression of nucleic acid sequences of the invention in tumor biopsy material however, such as from a fine needle biopsy, would also be a prefened indication for this diagnostic procedure. Nucleic acid sequences of the invention, ZNF216 (SEQ ID 95) for example, are not detected in most non-diseased tissues and relatively specifically expressed in e.g. HCC. Elevated expression of this nucleic acid in FNH and HCC is also demonstrated indicating the potential discriminatory power of such an approach for differential diagnosis of liver diseases (Figures 1 and 9; Tables 3 A/4 A).

[434] The PCR diagnostic would preferably require approximately 1 pg, preferably at least 100 ng, more preferably at least 1 μg of RNA isolated from patient material. In the prefened utilization the RNA would be isolated according to standard procedures from, e.g., the white blood cell fraction preferably from circulating blood obtained by the minimally invasive venupuncture procedure. In this prefened case, the procedure would detect the presence of HCC tumor cells in the blood circulatory system. RNA could similarly be isolated from liver or other tissue biopsy material.

[435] For specific detection of ZNF216, the PCR diagnostic would include several primers specific for ZNF216 nucleic acid sequence, including a specific primer set for cDNA synthesis from the RNA generated from the patient sample, such as for example (ZNF216-pl, 5'-ttctttctgcacatgaaacatctg-3' (SEQ ID 195). Also included would be forward and reverse PCR primers specific for ZNF 216 nucleic acid sequence such as for example, ZNF216-p2, 5'-gagaggacaaaataactaccc-3' (SEQ ID 196) and ZNF216-p3, 5'-caattcaggagctttttcttca-3' (SEQ ED 197), and for increased specificity and heightened sensitivity a fluorescently-labeled hydrolysis probe would be included such as, for example, ZNF216-pr, 5'-tactgggctgagaaactgatggactgggctga-3', SEQ ED 198 (from nucleotide 694 to 663 of the SEQ ID 95 reverse strand). The specificity of this detection assay may be further heightened with alternative primers specific for the ZNF216 sequence including an independent pair of specific PCR forward and reverse primers ("nested" primers) located on the amplicon of the outer forward and reverse PCR primers. En this case the probe primer would be specific for the amplicon the nested PCR primer pair.

[436] Quantitative assessment of AKRICI mRNA levels, for example, may also be achieved in such detection strategies as illustrated in Figure 3C using kinetic quantitative PCR with, for example:

[437] cDNA may be prepared from the patient RNA sample following digestion of the RNA with RNAse-free DNAse-1 (Roche) to eliminate potential contamination by genomic DNA. This contamination possibility is further controlled by including primers for PCR amplification from sequences of different exons of the gene such that PCR products resulting from a genomic DNA template (and thereby not reflective of expression of the mRNA conesponding to for example ZNF216) would be larger than the RNA specific PCR products. cDNA synthesis can e.g. be primed by the ZNF216-specifιc ZNF216-pl (SEQ ID 195;at about 1 μM) with the aid of reverse tran- scriptase [such as Maloney murine leukemia virus reverse transcriptase (Roche) at about 2 unit/reaction] in an appropriate buffer such as 50 mM Tris-HCl, 6 mM MgC12, 40 mM KC1, and 10 mM dithiotreitol, pH 8.5. Also required in the cDNA synthesis reaction is dATP, dCTP, dGTP and dTTP, each at about 1 mM, RNAse inhibitor, such as placental RNAse inhibitor (Roche) at about 1-10 units/reaction. cDNA synthesis would be preferably carried out at 42°C for 30 to 60 minutes followed by heating at 95° C for 10 minutes to denature the RNA template. The resulting cDNA can be employed as the template for a PCR to detect ZNF 216 in the blood (or liver or tissue biopsy sample). The additional reagents required for PCR detection of ZNF216 would preferably also be provided including: 10X Taq DNA polymerase buffer (500 mM Tris-Cl pH 8.3, 25 mM MgCl₂, 0.1% Triton X-100); a mixture of dATP, dCTP, dGTP and dTTP for a final concentration of 0.2 mM each; Taq DNA polymerase (2.5U/reaction), and ZNF216 specific primers such as ZNF216-pl (SEQ ID 195), ZNF216-p2 (SEQ YD 196) and ZNF216-p3, (SEQ ID 197), and for increased specificity and heightened sensitivity a fluorescently-labelled hydrolysis probe ZNF216-pr, SEQ YD 198 (0.1 - 1 μM final concentration). A positive control for PCR amplification such DNA from a plasmid clone with the ZNF216 sequence insert would preferably also be included (1-10 ng/reaction). The PCR can e.g. be carried out over 22-40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds. As indicated above, prefened additional sensitivity and specificity may be achieved in this diagnostic procedure by utilization of the additional ZNF216primer set located within the sequence amplified with the original PCR primer set. En this case a subsequent PCR under conditions similar to those utilized in the first PCR reaction except that would be employed to amplify the nested sequence in a reaction that included 1-10 μl of the first PCR as the template DNA. Alternatively, the reaction may preferably be carried with the first primer set for 10-15 cycles after which and 1-10 μl of this reaction then included as template in a new PCR reaction with nested primers (and including all the necessary PCR components). Detection of ZNF216 specific PCR product(s) should preferably utilize agarose gel electrophoresis as is known in the art and described in previous examples. Included in the diagnostic should preferably be a comparable fluid or tissue extract as a control for such PCR-based diagnostic test. This may include serum or plasma from non-diseased individuals and/or semm, plasma or tissue extracts from an appropriate animal model. If the PCR-determined expression of the nucleic acid according to the invention such as the product of the reaction with primers ZNF216-pl (SEQ ID 195), ZNF216-p2 (SEQ ED 196) , ZNF216-p3 (SEQ ID 197) and ZNF 216-pr (SEQ ED 198) is upregulated in the sample isolated from the patient relative to the control and if in particular the upregulated expression essentially matches the disorder specific (mean) expression ratios then such matching is indicative of the patient suffering from the disorder. Variations on this approach can also be appreciated. The cDNA synthesis and PCR amplifications can be carried our sequentially or simultaneously in a single reaction vessel utilizing heat stabile DNA polymerases with reverse transcriptase activities, such as provided by the Titan one-tube or Car- boxydothermus DNA polymerase one-set RT-PCR systems from Roche. Alternatively the PCR product can be monitored by incoφoration of fluorescently labeled primers or various fluorescence-based indicators of PCR product including the Taqman probe hydrolysis systems, as described above and with fluorescent double-stranded DNA intercalating molecules such as SYBR green. The fluorescent-based approaches provide advantage as the accumulation of PCR product can be continuously monitored to achieve sensitive quantitative assessment of expression of the nucleic acid according to the invention. This should be particularly advantageous for nucleic acids increased in blood or tissues of disorders according to the invention but also present at lower levels in non-diseased patients and tissues such that quantitative information about the level of expression of the nucleic acid is acquired. Further, as with this example, accurate quantitation of nucleic acid expression levels contributes to differential diagnosis, between cirrhosis and HCC for example. Comparison of this data with supplied standards indicative of disease and absence of disease provides an important advantage for such a diagnostic procedure.

[438] Additional variations on this diagnostic strategy include simultaneous detection of multiple nucleic acids according .to the invention and/or of nucleic acids according to the invention together with other nucleic acids implicated in the disorder. Further hybridization-based diagnostic detection of nucleic acids according to the invention is also envisioned. In this case mRNA detection preferably utilizing detection of RNA transfened to a membrane by capillary or electrophoretic blotting, RNAse protection or in situ hybridization on patient cells or tissue biopsy samples is also effective.

[439] By similar methods and variants thereof the nucleic acids according to the invention and/or of nucleic acids according to the invention together with other nucleic acids can be utilized for diagnosis of the disorders according to the invention.

[440]

[441] Example 8: A method of diagnosing via antibody detection of polypeptides according to the invention

[442] A prefened diagnostic method for disorders according to the invention is based on antibodies directed against a polypeptide according to the invention. For example, a diagnostic procedure may preferably employ serum detection of specific upregulated gene proteins via enzyme-linked immunosorbent assay (ELISA) assay. In a simple form the diagnostic assay preferably includes a microtiter plate or strip of microtiter wells, e.g., thoroughly coated with an isolated and purified antibody specific to a polypeptide according to the invention such as, ZNF216 (SEQ ID 2), AKRICI (SEQ ID 3). The antibody may for example be an affinity purified polyclonal antibody, such as is commonly raised in rabbits, for example, or a purified monoclonal antibody such as is commonly produced in mice according to procedures well established in the art (Cooper, H.M. & Paterson, Y., (2000), In Current Protocols in Molecular Biology (Ansubel, F.A. et al., eds.) pp. 11.12.1 - 11.12.9, Greene Publ. & Wiley Intersci., NY); (Fuller S.A. et al., (1992), In Cunent Protocols in Molecular Biology (Ansubel, F.A. et al., eds.) pp. 11.4.1 - 11.9.3, Greene Publ. & Wiley Intersci., NY). Preferably, the antibody may a recombinant antibody obtained from phage display library panning and purification as has been described by Knappik et al. (2000, J. Molec. Biol. 296:57-86) or by Chadd and Chamow (2001 Cun. Opin. Biotechnol.12: 188-94), or a fragment thereof. The antibody coating is preferably achieved by dilution of the anti-ZNF216.pr antibody or anti-AKRICl.pr antibody to 1-100 μg/ml in a standard coating solution such as phosphate buffered saline (PBS). The antibody is preferably bound to the absoφtive surface of the microtiter well (such as a Nunc Maxisoφ immunoplate) for 60 minutes at 37°C, or overnight at room temperature or 4°C. Prior to binding sample to the coated wells, the wells are preferably thoroughly blocked from non-specific binding by incubation for 15-60 minutes at room temperature in a concentrated protein solution such as 5% bovine serum albumin in phosphate buffered saline or 5% non-fat dry milk powder resuspended in the same buffer. Preferably, the patient sample material is then applied to the microtiter wells, diluted into the blocking solution to increase specificity of detection. The sample may be for example plasma or serum or protein extract from tissue biopsy or surgical resection prepared according to methods well known in the art (Smith, J.A. (2001) In, Cunent Protocols in Molecular Biology, Ausubel, FA. et al., eds) pp. 10.0.1- 10.0.23, Greene Publ. & Wiley Intersci., NY). In particular, the patient sample is brought into contact with the antibody-coated well for 30-120 minutes (or longer) at room temperature or at 4°C. Non-specifically interacting proteins are preferably removed by extensive washing with a standard wash buffer such as 0.1 M Tris-buffered saline with 0.02-0.1% Tween 20, for example. Washes are preferably carried out for 3-10 minutes and repeated 3-5 times. Detection of ZNF216 polypeptide in the patient sample is for example achieved by subsequent binding reaction with a second, independent anti-ZNF216 antibody, generated as described above, recognizing a distinct epitope on the ZNF216 polypeptide in the standard two- site 'sandwich' type ELISA. Binding of the second anti-ZNF216 antibody or AKRICI antibody is for example achieved by incubating the wells in the antibody (at a concentration of 1-100 μg/ml in blocking solution, for example) at room temperature for 30-60 minutes followed by extensive washing as in the previous step. The second antibody may preferably be directly coupled to an enzyme capable of producing a colorigenic or fluorgenic reaction product in the presence of an appropriate substrate, such as alkaline phosphatase. Alternatively, for example an anti-species and anti- isotype specific third antibody, so coupled to an enzyme, is employed to generate a reaction product that preferably can be detected in a standard spectrophotometric plate reader instrument. For the reaction product development, the washed (as above) antibody-antigen-enzyme complex is preferably exposed to the colorigenic substrate, such as AttoPhos from Roche for about 10 minutes at room temperature, the reaction may be stopped with a low pH buffer such as 50 mM Tris-HCl pH 5.5, or can instead be directly assayed. The amount of specifically bound ZNF216 polypeptide or AKRICI polypeptide is for example determined by measurement of the amount of the enzymatic reaction product in each well following excitation at the appropriate wavelength in the spectrophotometer (420 nm in this case). Measurement is preferably made in the plate reader at the emission wavelength (560 nm in this case). Preferably included in the diagnostic is a ZNF216 protein standard or an AKRICI protein standard, such as purified recombinant ZNF216 polypeptide or AKRICI polypeptide, for example. A dilution series of this protein standard is preferably included in parallel in the ELISA as a control for the reactions and to deduce a protein standard curve for comparison of polypeptide expression levels as is well known in the art. A concentration range conesponding indicative of the particular liver disorder(s) should preferably be provided in the diagnostic. In addition, a comparable fluid or tissue extract should preferably also be included as a control for such ELISA test. This may preferably include semm or plasma from non-diseased individuals and/or serum, plasma or tissue extracts from an appropriate animal model. Such ELISA detection diagnostics are common in the art (see for example, Hauschild et al., 2001, Cancer Res. 158:169-77). The sample: control protein levels determined by ELISA are compared with ELISA-determined disorder specific protein expression ratio values preferably determined in pathologist-confirmed tissues of patients suffering from a disorder according to the invention in relation to control samples. En case the protein level of the sample: control essentially matches the disorder specific protein expression ratio values such matching is preferably indicative of the patient suffering from the disorder. Preferably such diagnosis is carried out for more than 1 polypeptide according to the invention. In addition the diagnostic may be directed to detecting an endogenous antibody directed against a polypeptide according. to the invention, or a functional variant thereof or fragment thereof present in the sample isolated from a patient which antibody or fragment thereof is directed against a polypeptide according to the invention. Detection of such autoimmune antibodies may be accomplished by methods generally known to the skilled artisan, e.g. by immunoaffinity assays such the ELISA described in detail above using polypeptides according to the invention or functional variants thereof or parts thereof as a probe. The presence of such autoimmune antibodies is indicative of the patient suffering from a disorder according to the invention. [444] In addition or alternatively, a relevant diagnostic kit based upon immunohis- tochemical detection of at least one polypeptide according to the invention can be formulated. In such a kit, for example a purified antibody or antibodies specific for the polypeptide(s) according to the invention can be included as well as preferably the reagents necessary to detect the binding of the antibody (ies) to patient cells or tissue sections. These reagents include, for example a specific anti-species and subtype specific secondary antibody -directed against a polypeptide according to the invention of a functional variant thereof- preferably coupled to an enzyme capable of catalysis of e.g. a colorigenic substrate or coupled to a fluorophore (such as Texas Red, for example). Preferably the enzymatic substrate would also be included as well as washing and incubation buffers. An additional optional component of such a kit may be a section of positive control tissue, e.g. liver, or tissues or a section from a packed pellet of cells specifically expressing the polypeptide(s) as a positive tissue control. Instructions provided would include prefened and/or alternative methods of antigen retrieval for detection of the polypeptide(s) according to the invention or e.g., indication that frozen, rather than formalin fixed and paraffin-embedded tissue material should be employed. In this case, recommendations would preferably be included for fixation of frozen tissue sample sections, such as immersion in ice-cold acetone for 10 minutes. Further instructions would preferably provide recommendations for the concentration of antibodies to use in the detection of the gene product(s) as well as e.g., recommended and suggested incubation times and temperatures for exposure of the tissue to the immunological reagents provided. Prefened reaction buffers for the antibody incubations, such as 0.01% - 0.1% tween-20 comprising phosphate buffered saline including 3% normal sheep serum, could also be included. Further, specific conditions for washing of the tissue sections prior to and following incubation in the specific antibody would be preferably included, such as for example, 4 washes with 0.1% tween-20 comprising phosphate buffered saline for 5 minutes each. Such im- munohistochemical detection protocols are known to a person skilled in the art. In general the kit would preferably include a panel of images of specific immunohis- tochemical staining results from positive and negative tissue examples and in particular tables indicating which result is indicative of the patient suffering from the disorder to be diagnosed as a user guide. Utilization of such a kit would preferably rule out, support or confirm diagnoses of the aforementioned liver disorders, liver cancer, or epithelial cancers according to the invention. [445] As specified above for nucleic acid-based diagnostic approaches, diagnostics based on detection and/or quantitation of polypeptides according to the invention may include 1 or more of such polypeptides. Moreover, simultaneous detection of such polypeptides together with other peptides implicated in the disorders according to the invention may be employed in such diagnostics.

[446] It will be apparent to those skilled in the art that various modifications can be made to the compositions and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents. All publications cited herein are incoφorated in their entireties by reference.

[447]

Claims

[001] A diagnostic comprising at least one compound selected from the group consisting of the polypeptide according to SEQ ID 1 to 93, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, and an antibody or an antibody fragment directed against one of the aforementioned polypeptides, combined or together with suitable additives or auxiliaries.

[002] The diagnostic according to claim 1, wherein the nucleic acid is a probe.

[003] The diagnostic according to claim 2, wherein the probe is a DNA probe.

[004] A pharmaceutical composition comprising at least one component selected from the group consisting of the polypeptide according to claim 1, a polypeptide according to SEQ ED 1 to 93, a functional variant of one of the aforementioned polypeptides, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a nonfunctional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, an antibody or a fragment of the antibody directed against a functional variant of one of the aforementioned polypeptides, a vector comprising a nucleic acid coding for one of the aforementioned antibodies, a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibodies, and a cell comprising the vector comprising a nucleic acid coding for one of the aforementioned antibody fragments, combined or together with suitable additives or auxiliaries.

[005] The pharmaceutical composition according to claim 4, wherein the nucleic acid having a complementary sequence is an antisense molecule or an RNA interference molecule.

[006] A method of diagnosis of a liver disorder or an epithelial cancer, wherein at least one compound selected from the group consisting of a polypeptide according to the sequence of SEQ ED 1 to SEQ ID 93, a functional variant of one of the aforementioned polypeptides, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, and an antibody or a fragment of the antibody directed against a functional variant of one of the aforementioned polypeptides, is identified in the sample of a patient and compared with at least one compound of a reference library or of a reference sample.

[007] The method according to claim 6, wherein the liver disorder, is a disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia.

[008] The method according to claim 6, wherein the epithelial cancer is an adenocarcinoma of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[009] A method of treating a patient suffering from a liver disorder or an epithelial cancer, wherein at least one component selected from the group consisting of a polypeptide according SEQ ED 1 to 93, a functional variant of one of the aforementioned polypeptides, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, a cell comprising the aforementioned vector, an antibody or a fragment of the antibody directed against one of the aforementioned polypeptides, an antibody or a fragment of the antibody directed against a functional variant of one of the aforementioned polypeptides, a vector comprising a nucleic acid coding for the antibody, a cell comprising the vector comprising a nucleic acid coding for the antibody, and a cell comprising the vector comprising a nucleic acid coding for the antibody fragment, combined or together with suitable additives or auxiliaries, is administered to the patient in need of a the treatment in a therapeutically effective amount.

[010] The method of treating according to claim 9, wherein the nucleic acid having a complementary sequence is an antisense molecule or an RNA interference molecule.

[011] The method of treating according to claim 10, wherein the RNA interference molecule is administered in the form of a double stranded RNA or a vector expressing the double stranded RNA.

[012] The method according to claim 10, wherein the RNA interference molecule has a size range selected from the group consisting of from 15 to 30 nucleotides.

[013] The method according to one of claims 9 to 12, wherein the liver disorder, is a disorder selected from the group consisting of cinhosis, alcoholic liver disease, chronic hepatitis, Wilson's Disease, haemochromatosis, hepatocellular carcinoma, benign liver neoplasms, and focal nodular hypeφlasia.

[014] The method according to one of claims 9 to 13, wherein the epithelial cancer is an adenocarcinoma of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[015] A method of stimulating an immune response to a polypeptide according to the sequence of SEQ ID 1 to SEQ ID 93, or a functional variant thereof in a patient suffering from a liver disorder or an epithelial cancer, wherein at least one component selected from the group consisting of a polypeptide according to the sequence of SEQ ID 1 to SEQ ID 93 , a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, a cell comprising one of the aforementioned nucleic acids, and a cell comprising the aforementioned vector, is administered to the patient in need of such treatment in an amount effective to stimulate the immune response in the patient.

[016] A method for identifying at least one nucleic acid according to SEQ ED 94 to SEQ ED 186, or a variant thereof differentially expressed in a sample isolated from a patient relative to a reference library or a reference sample comprising the following steps: (a) detecting the expression of at least one nucleic acid . according to SEQ ID 94 to SEQ ID 186, or a variant thereof in a sample isolated from a patient, (b) comparing the expression of said nucleic acid(s) detected in step (a) with the expression of the said nucleic acid(s) in a reference library or in a reference sample, (c) identifying said nucleic acid(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample.

[017] A method of diagnosing a liver disorder or an epithelial cancer comprising the following steps: (a) detecting the expression of at least one nucleic acid according to SEQ ED 94 to SEQ ED 186, or a variant thereof in a sample isolated from a patient, (b) comparing the expression of said nucleic acid(s) detected in step (a) with the expression of said nucleic acid(s) in a reference library or in a reference sample, (c) identifying said(s) nucleic acid which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample, and (d) matching said nucleic acid(s) identified in step (c) to said nucleic acid(s) differentially expressed in a pathologic reference sample or pathologic reference library, wherein the matched nucleic acid(s) is (are) indicative of the patient suffering from a liver disorder or an epithelial cancer.

[018] The method according to claim 17, wherein in step (a) at least 2 nucleic acids are identified.

[019] The method according to claim 17, wherein in step (a) the detection of said nucleic acid(s) is (are) by PCR based detection or by a hybridization assay.

[020] The method according to one of claims 17 to 19, wherein in step (b) the expression of said nucleic acid(s) is compared by a method selected from the group consisting of solid-phase based screening methods, hybridization, subtractive hybridization, differential display, and RNase protection assay.

[021] The method according to one of claims 17 to 20, wherein the sample isolated from the patient is selected from the group consisting of liver tissue, a liver cell, tissue from another organ subject to cancerous transformation, a cell from this organ, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[022] The method according to one of claims 17 to 21, wherein the reference sampleis isolated from a source selected from a non-diseased sample of the same patient and a non-diseased sample from another subject.

[023] The method according to one of claims 17 to 22, wherein the reference sampleis selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[024] The method according to one of claims 17 to 23, wherein the reference libraryis an expression library or a data base comprising clones or data on liver disorder-specific expression of said nucleic acid(s) of step (a).

[025] The method according to one of claims 17 to 24, wherein the pathologic reference sample is isolated from a source selected from a diseased sample from another patient suffering from a liver disorder or epithelial cancer.

[026] The method according to claim 17 to 25, wherein the pathologic reference library is a data base comprising data on differential expression of said nucleic acid(s) in step (a) in samples isolated from another patient suffering from a liver disorder or epithelial cancer relative to control expression in a reference sample or reference library.

[027] The method according to claim 17 to 26, wherein the liver disorder, is a disorder selected from the group consisting of hepatocellular carcinoma, benign liver neoplasms, and cinhosis.

[028] The method according to claim 17 to 26, wherein the epithelial cancer is an adenocarcinoma of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin and the breast. [029] A method for identifying at least one polypeptide according to SEQ ID 1 to SEQ ED 93, or a functional variant thereof differentially expressed in a sample isolated from a patient relative to a reference library or a reference sample comprising the following steps: (a) detecting the expression of at least one polypeptide accord ing to SEQ ID 1 to SEQ ID 93, or a functional variant thereof in a sample isolated from a patient, (b) comparing the expression of said polypeptide(s) detected in step (a) with the expression of said polypeptide(s) in a reference library or in a reference sample, (c) identifying said polypeptide(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample.

[030] A method of diagnosing a liver disorder or epithelial cancers comprising the following steps: (a) detecting the expression of at least one polypeptide according to SEQ ID 1 to SEQ ID 93, or functional variants thereof in a sample isolated from a patient, (b) comparing the expression of said polypeptide(s) detected in step (a) with the expression of said polypeptide(s) in a reference library or in a reference sample, (c) identifying said polypeptide(s) which is (are) differentially expressed in the sample isolated from the patient compared to the reference library or the reference sample, and (d) matching said polypeptide(s) identified in step (c) with said polypeptide(s) differentially expressed in a pathologic reference sample or pathologic reference library, wherein the matched polypeptide(s) are indicative of the patient suffering from a liver disorder, or an epithelial cancer.

[031] The method according to claim 30, wherein at least 2 polypeptides are identified.

[032] The method according to claim 30 or 31, wherein the polypeptides are detected by a method selected from the group consisting of gel electrophoresis, chromatographic techniques, immunoblot analysis, immunohistochemistry, enzyme based immunoassay, surface plasmon resonance, HPLC, mass spectroscopy, immunohistochemistry, and enzyme based immunoassay.

[033] The method according to one of claims 30 to 32, wherein the polypeptides are compared by a method selected from the group consisting of two dimensional gel electrophoresis, chromatographic separation techniques, immunoblot analysis, surface plasmon resonance, immunohistochemistry, and enzyme based immunoassay.

[034] The method according to one of claims 30 to 33, wherein the sample isolated from a patient is selected from the group consisting of liver tissue, a liver cell, tissue from another organ subject to cancerous transformation, a cell from this organ, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces. [035] The method according to one of claims 30 to 34, wherein the reference sampleis isolated is from a source selected from a non-diseased sample of the same patient and a non-diseased sample from another subject.

[036] The method according to one of claims 30 to 35 wherein the reference sample is selected from the group consisting of liver tissue, a liver cell, blood, serum, plasma, ascitic fluid, pleural effusion, cerebral spinal fluid, saliva, urine, semen, and feces.

[037] The method according to one of claims 30 to 36, wherein the reference libraryis an expression library or a data base comprising clones or data on liver disorder-specific expression of said polypeptide(s) of step (a).

[038] The method according to claim 30 to 37, wherein the pathologic reference sample is isolated from a source selected from a diseased sample from another patient suffering from a liver disorder and epithelial cancer.

[039] The method according to claim 30 to 38, wherein the pathologic reference library is a data base comprising data on differential expression of said polypeptide(s) of step (a) in samples isolated from another patient, suffering from a liver disorder or epithelial cancer, relative to control expression in a reference sample or reference library.

[040] The method according to claim 30 to 39, wherein the liver disorders is a disorder selected from the group consisting of hepatocellular carcinoma, benign liver neoplasms, and cinhosis.

[041] The method according to one of claims 30 to 40, wherein the epithelial cancer is an adenocarcinoma of an organ selected from the group consisting of the lung, the stomach, the kidney, the colon, the prostate, the skin, and the breast.

[042] A method of preventing a patient from developing a liver disorder or an epithelial cancer, wherein at least one component selected from the group consisting of a polypeptide according to the sequence of SEQ ED 1 to SEQ ED 93, a functional variant thereof, a nucleic acid encoding one of the aforementioned polypeptides, a variant of one of the aforementioned nucleic acids, a nucleic acid having a sequence complementary to one of the aforementioned nucleic acids, a nucleic acid which is a non-functional mutant variant of one of the aforementioned nucleic acids, a vector comprising one of the aforementioned nucleic acids, or a variant thereof, a cell comprising one of the aforementioned nucleic acids, or a variant thereof, and a cell comprising the aforementioned vector, is administered to the patient in need of such preventive treatment in a therapeutically effective amount.

[043] A method of identifying a pharmacologically active compound comprising the following steps: (a) providing at least one polypeptide according to the SEQ ID 1 to 93, or a functional variant thereof, (b) contacting said polypeptide(s) with (a) compound(s) suspected to be pharmacologically active , (c) assaying the interaction of said polypeptide(s) of step (a) with said compound(s) suspected to be pharmacologically active, (d) identifying said compound(s) suspected to be pharmacologically active which directly or indirectly interact with said polypeptide(s) of step (a).

[044] The method according to claim 43, wherein said polypeptide(s) of step (a) is (are) attached to a column, said polypeptide(s) is (are) attached to an anay, contained in an electrophoresis gel, attached to a membrane, or is (are) expressed by a cell.

[045] The method according to claim 43 or 44, wherein the interaction is assayed enzyme or fluorescence based cellular reporter methods.

[046] The method according to claim 43 or 44, wherein the interaction is assayed by surface plasmon resonance, HPLC, or mass spectroscopy.

[047] The method according to claim 43, wherein the direct or indirect functional interaction of step (d) is selected from the group consisting of induction of the expression of said polypeptide(s) of step (a), inhibition of said polypeptide(s), activation of the function of said polypeptide(s), and inhibition of the function of said polypeptide(s).

[048] An isolated polypeptide comprising a sequence according to SEQ ID 32, or a functional variant thereof.

[049] A fusion protein comprising a polypeptide according to claim 48.

[050] An isolated nucleic acid, or a variant thereof encoding the polypeptide according to claim 48.

[051] The nucleic acid according to claim 50, wherein the nucleic acid is a single- stranded or double-stranded RNA.

[052] The nucleic acid according to claim 50, wherein the nucleic acid comprises a nucleic acid according to SEQ ED 125.

[053] A vector comprising a nucleic acid according to claim 50.

[054] The vector according to claim 53, wherein the vector is selected from the group consisting of a knock-out gene construct, a plasmid, a shuttle vector, a phagemid, a cosmid, a viral vector, and an expression vector.

[055] A cell comprising the nucleic acid according to claim 50.

[056] A cell comprising the vector according to claim 53.

[057] The cell according to claim 56, wherein the cell is a transgenic embryonic non-human stem cell.

[058] A transgenic non-human mammal comprising the nucleic acid according to claim 50. [059] An antibody or an antibody fragment thereof, wherein the antibody is directed against the polypeptide according to claim 48 or against the nucleic acid according to claim 50.

[060] A nucleic acid which comprises a nucleic acid having a sequence complementary to the nucleic acid according to claim 50 or a non-functional mutant variant of the nucleic acid according to claim 50.

[061] The nucleic acid according to claim 60, wherein the nucleic acid having a complementary sequence is an antisense molecule or an RNA interference molecule.

[062] A vector comprising the nucleic acid according to claim 60.

[063] The vector according to claim 62, wherein the vector is selected from the group consisting of a plasmid, a shuttle vector, a phagemid, a cosmid, a viral vector, and an expression vector.

[064] A cell comprising the nucleic acid according to claim 62.

[065] A cell comprising the vector according to claim 64.