AU2007247497A1 - Biomarkers for endometrial proliferation - Google Patents

Description

WO 2007/128429 PCT/EP2007/003694 BIOMARKERS FOR ENDOMETRIAL PROLIFERATION FIELD OF THE INVENTION [01] The present invention relates to a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample. The invention also relates to compositions or kits and arrays for qualitatively and/or quantitatively determining endometrial proliferation. Further, the invention relates to a method for the diagnosis of a disorder that is associated with a dysfunction of endometrial proliferation or a predisposition therefore in a subject. BACKGROUND OF THE INVENTION [02] The endometrium is the mucous membrane of the cavity that lines the uterus. Under the influence of ovarian hormones, essential anatomic and functional changes in the endometrium take place. [03] Thus, under physiological conditions, the endometrium thickens under hormonal control and, if pregnancy does not occur, is shed in menstruation and regenerates each menstrual cycle throughout the reproductive life of a female. By the fifth day of the menstrual cycle, the endometrium shows proliferation of its stroma and glands, the latter elongating. The cells lining the glands are cuboidal with definite limiting membranes and the stroma cells are thin and spindly, indicating the early proliferative phase. One week later, at the twelfth day, the glands are very large and are now dilated, indicating the late proliferative phase. At this stage, the blood vessels are also prominent and capillaries are dilated. These proliferative changes are due to the influence of estrogen, in particular of 17-fl-estradiol secreted by the ovary at this time. Following ovulation the corpus luteum produces large quantities of progesterone which induce secretory changes in the glands and swelling of the stromal cells. There is a rich blood supply and the capillaries become sinusoidal. Towards the end of the twenty eight day cycle, the stroma becomes even more vascular and oedematous, small haemorrhages and thrombi appear and the endometrium ultimately breaks down due to the withdrawal of the hormonal support. The superficial layers of endometrium, together with blood and leucocytes, are shed and discharged, indicating menstruation. Within a day or two the raw surface is healed over by epithelium proliferating from the basal portions of the glands.

WO 2007/128429 PCT/EP2007/003694 -2 [041 However, disordered endometrial proliferation may be associated with several gynecological diseases including endometriosis, adenomyosis, endometrial hyperplasia and gynecological cancer. [05] So far little is known about the physiological and pathophysiological mechanisms involved in endometrial proliferation. Moreover, no efficient therapy and/or prevention of diseases associated with endometrial proliferation has been established until now. [06] Insight into these physiological and pathophysiological mechanisms and into the function of genes and proteins involved therein might provide tools for the diagnosis and prognosis of endometrial proliferation and therefore tools for the prevention and/or therapy of diseases associated with endometrial proliferation. SUMMARY OF THE INVENTION [07] The invention provides a method for identifying the stage of endometrial proliferation in a subject. This object is achieved according to the invention by providing a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising determining the level of expression and/or activity of at least one gene which is modulated by estrogens and/or Selective Estrogen Receptor Modulators (SERMs) in said sample. [08] Surprisingly, several genes were identified to be modulated by estrogens and SERMs, wherein said modulation of said genes correlates with the different potential of estrogens and/or SERMs to induce endometrial proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [09] Estrogens are steroid hormones essential for normal sexual development and functioning of female reproductive organs such as the endometrium. Estrogens also have important non-reproductive effects on bones and the heart. Estrogens comprise a group of natural and synthetic substances. Natural estrogens include estradiol (i.e. 17fl-estradiol), estrone and estriol. Particularly under the influence of 1706 estradiol, the proliferation of the endometrium takes place. 17fi-estradiol induces the synthesis of its own receptors as well as the progesterone receptors. Therefore, the endometrium proliferates in the first half of the cycle, i.e. in the proliferation phase, resulting in a certain thickness of the endometrium which reaches a maximum of 8 to WO 2007/128429 PCT/EP2007/003694 -3 10 mm. Besides the natural estrogens, estrogens are sometimes given therapeutically in the form of a conjugate, such as ethinyl estradiol for example, conjugated estrogens or diethylstilbestrol. [10] Tissues in the body that are responsive to estrogens are called ,estrogen sensitive" or ,,estrogen-responsive" tissues and include cells of the urogenital tract, cardiovascular system and skeletal system. The cells that comprise estrogen sensitive tissues contain estrogen receptors (ER). ER can be of the alpha type or beta type. Estrogens enter cells and bind to ER in the cytoplasm of such cells and an estrogen-ER complex is formed. A molecule such as estrogen that binds to a receptor is termed a ,,ligand". [11] Once the estrogen ligand binds to ER, the estrogen-ER complex migrates to the nucleus of the cell and binds to specific sequences of DNA within the cellular genome called ,,estrogen response elements". Such estrogen response elements are located in the promoters of the specific genes in the cell nucleus. [12] Binding of the estrogen-ER complex to estrogen-responsive elements causes activation or suppression of the transcription of the specific genes. The activation or suppression of specific gene transcription is one type of molecular and/or cellular response that can result from the formation of a ligand-receptor complex. When such a response occurs, the receptor is said to have been ,,activated". [13] Estrogen-ER complexes, therefore, act as transcription factors to regulate the expression of these genes. When a ligand binds to a receptor and a molecular and/or cellular response (e.g. transcriptional regulation of genes) occurs, such ligands are referred to as ,agonists" and the response produced is called ,,agonism". [14] In addition to the role estrogens and ER play in the normal development and functioning of cellular tissues, estrogens and ER play significant roles in certain human disease states, such as enhanced endometrial proliferation, endometriosis, gynecological cancers such as breast cancer, etc. [15] Substances that bind to the ER and prevent the molecular and/or cellular responses caused by estrogens are given the general name ,selective estrogen receptor modulators" or SERMs. SERMs can also be called ,,anti-estrogens". SERMs encompass ER ligands that produce different responses. For example, one particular SERM may be an antagonist. Another particular SERM may be a partial agonist. Still another particular SERM may bind to ER and produce a molecular and/or cellular WO 2007/128429 PCT/EP2007/003694 -4 response that is only slightly less in magnitude than the response produced by estrogens. Such a SERM would result in a molecular and/or cellular response of a greater magnitude than the response to produced by a partial agonist, but would not be referred to as an agonist because the molecular and/or cellular response is less than that produced by an agonist-like estrogen. [16] Chemically, SERMs can be classified into three groups. The first group comprises triphenylethylene derivatives, of which tamoxifen, an anti-cancer agent, is one. Other substances that are triphenylethylene derivatives are toremifene, droloxifene, (3-hydroxytamoxifen), idoxifene, TAT-59 (a phosphorylated derivative of 4 hydroxytamoxifen) and GW5638 (a carboxylic acid derivative of tamoxifen). The second group of SERMs comprises other non-steroidal compounds. This group comprises EM 800, EM652 (benzopyranes), raloxifene, LY353381 (SERM3) and LY357489. The third group of SERMs comprises steroidal compounds that have a better ability to inhibit the response produced by estrogens. ICE-182,780 (ICE) is a member of this third group. The listings of sub-sensors that comprise each group is not complete and others may exist. [171 In accordance with the present invention, it has been discovered that the level of expression and/or activity of certain genes is modulated by estrogens and/or SERMs that correlated with the different potential of said estrogens and SERMs to induce endometrial proliferation. Therefore, measuring the levels of expression and/or activity of said certain genes provides a method for qualitatively and/or quantitatively determining endometrial proliferation in a sample. [18] Preferably, the estrogen is estradiol and the SERMs are tamoxifene and/or raloxifene. [19] In a preferred embodiment, the method of the invention, the genes which are modulated by estrogens and/or SERMs are mammalian genes, preferably human genes. [20] The method of the present invention is contemplated for application in human, veterinary and/or diagnostic medicine. [21] Preferably, the at least one gene which is modulated by estrogens and/or SERMs is selected from the group consisting of the genes anterior gradient 2 homolog (Xenopus laevis) (Gene ID 10551); chondroitin sulfate proteoglycan 2 (versican) (Gene ID 13003); collagen, type I, alpha I (Gene ID 1277); collagen, type I, alpha 2 (Gene ID WO 2007/128429 PCT/EP2007/003694 -5 1278); collagen, type I1, alpha 1, (Ehlers-Danlos syndrome type IV autosomal dominant) (Gene ID 1281); collagen, type IV, alpha 1 (Gene ID 1282); collagen, type VI, alpha 3 (Gene ID 1293); creatine kinase, brain (Gene ID 24264); dynein, cytoplasmic, light polypeptide 1 (Gene ID 8655); glyceraldehyde-3-phosphate dehydrogenase (Gene ID 2597); heat shock 27kDa protein 1 (Gene ID 3315); likely ortholog of mouse limb bud and heart gene (Gene ID 81606); lysyl oxidase-like I (Gene ID 4016); melanoma antigen, family D,1 (Gene ID 9500); membrane interacting protein of RGS16 (Gene ID 51573); procollagen C-endopeptidase enhancer (Gene ID 5118); prostaglandin D2 synthase 21kDa (brain) (Gene ID 5730); secreted frizzled-related protein 4 (Gene ID 6424); serine (or cysteine) proteinase inhibitor, clade H (heat shock protein 47), member 1, (collagen binding protein 1) (Gene ID 871); tropomyosin 1 (alpha) (Gene ID 7168) and/or insulin-like growth factor binding protein 5 (Gene ID 3488) and/or combinations thereof. [22] More preferably, the at least one gene is collagen, type 1, alpha 1 (Gene ID 1277); collagen, type I, alpha 2 (Gene ID 1278); collagen, type Ill, alpha 1, (Ehlers Danlos syndrome type IV autosomal dominant) (Gene ID 1281); creatine kinase, brain (Gene ID 24264) and/or prostaglandin D2 synthase 21 kDa (brain) (Gene ID 5730). [23] Most preferably, the at least one gene is prostaglandin D2 synthase 21 kDa (brain) (Gene ID 5730). [24] The accession numbers of the above-mentioned genes are indicated by reference in the public gene database NCBI Entrez Gene database (http//www.ncbi.nlm.nih.gov/entrezlquery.fcgi?db=gene). [251 In a preferred embodiment of the invention, the level of gene expression and/or activity of the at least one gene which is modulated by estrogens and/or SERMs is determined by measuring the transcript level of said genes. [26] Surprisingly, it was found that the transcript level of the transcripts (see example 1) is associated with the different estrogenic potency of estrogens and SERMs to induce endometrial proliferation. [271 Preferably, the determination of significance may be carried out as described in Example 1. The determination of significance may be carried out by a gene expression analysis of a sufficient number, e.g. of at least 500, preferably 500 to 700 probe sets (genes). The tissue from which the transcripts are obtained is preferably pituitary and uterus tissue.

WO 2007/128429 PCT/EP2007/003694 -6 [28] Methods for measuring the level of gene transcription comprise measuring the level of mRNA. RNA can be isolated from the samples by methods well known to those skilled in the art, for example as described in Molecular Cloning, A Laboratory Manual, Volume 1, Chapter 7, pp 7.4 to 7.12, Cold Spring Harbor Laboratory Press, New York, 3 rd Edition (2001). [291 In a preferred embodiment of the invention, the determination of the transcript level is carried out using a hybridization reaction. Usually, the hybridization reaction involves the hybridization of at least one oligonucleotide or polynucleotide to a transcript product, an mRNA. Preferably, the determination further comprises an elongation and/or amplification reaction. [301 In a preferred embodiment of the invention, the amplification comprises at least one technique selected from the group consisting of reverse transcriptase PCR and/or real time PCR. [31] The oligonucleotide or polynucleotide are preferably of sufficient length to specifically hybridize to complementary transcripts of the above genes, according to the invention. As used herein, the terms "olignucleotide" or "polynucleotide" refer to a single-stranded nucleic acid. Generally, the olignucleotide or polynucleotide will be at least 16 to 20 nucleotides in length, although in some cases longer probes of at least 20 to 25 nucleotides will be desirable. The olignucleotide or polynucleotide can be labelled with one or more labelling moieties to permit detection of the hybridized probe/target polynucleotide complexes. Labelling moieties can include compositions that can be detected by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, electrical, optical or chemical means. Examples of labelling moieties include, but are not limited to, radioisotopes, e.g. 3P, 1P, 31S, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, linked enzymes, mass spectrometry tags and magnetic labels. [321 According to another preferred embodiment of the invention, the level of gene expression is determined by measuring the level of a protein product of the at least one gene being modulated by estrogens and/or SERMs. Preferably, the protein product is selected from the group consisting of the protein products with the corresponding accession numbers anterior gradient 2 homolog (NP _006399), chondroitin sulfate proteoglycan 2 (versican) (NP_004376), alpha 1 type I collagen preproprotein WO 2007/128429 PCT/EP2007/003694 -7 (NP_000079), alpha 2 type I collagen (NP_000080), alpha 1 type Ill collagen (NP_000081), alpha 3 type IV collagen isoform 1, precursor (NP_000082), alpha 4 type IV collagen precursor (NP_000083), alpha 1 type IV collagen preproprotein (NP_001836), alpha 3 type IV collagen isoform 1 precursor (NP_004360), brain creatine kinase (NP_001814), cytoplasmatic dynein light polypeptide (NP_003737), glyceraldehyde-3-phosphate dehydrogenase (NP 002037), glycyl-tRNA synthetase (NP_002038), growth arrest-specific 1 (NP_002039), heat shock 27kDa protein 1 (NP_001531), hypothetical protein DKFZp 566J091 (NP_112177), lysyl oxidase-like 1 (NP_005567), melanoma antigen family D, 1 isoform b (NP 008917), membrane interacting protein of RGS16 (NP_057725), procollagen C-endopeptidase enhancer (NP 002584), prostaglandin D2 synthase 21 kDa (NP_000945), secreted frizzled related protein 4, (NP_003005), serine (or cysteine) proteinase inhibitor, clade H1, member 1 precursor (NP_001226), tropomyosin 1 (alpha) (NP_000357) and/or insulin like growth factor binding protein 5 (NP_000590). 1331 More preferably, the protein product is alpha 1 type I collagen preproprotein (NP_000079), alpha 2 type I collagen (NP_000080), alpha 1 type Ill collagen (NP_000081), brain creatine kinase (NP_001814) and/or prostaglandin D2 synthase 21kDa (NP_000945). [34] Most preferably, the protein product is prostaglandin D2 synthase 21 kDa (NP 000945). [35] All of the above-mentioned proteins were selected from the publicly available database for protein sequences NCBI Entrez Protein database (http://www.ncbi.nlm.nih.gov/entry/query.fcgi?db=protein). [36] Preferably, the protein product level is determined by enzymatic binding, immunological and/or physical methods, which are suitable for protein determination such as for example immunoassays or mass spectrometry. [37] Expression of the at least one protein or a fragment thereof, e.g. catalytic domain, can be detected by a probe which is detectably labelled, or which can be subsequently labelled. Generally, the probe can be an antibody, an antibody derivative or an antibody fragment which is able to recognise the expressed protein. As used herein, the term ,antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional WO 2007/128429 PCT/EP2007/003694 -8 antibody fragments, which are those fragments sufficient for binding of the antibody fragment to the protein or a fragment thereof. [38] For the production of antibodies to a protein encoded by one of the disclosed genes or to a fragment of the protein, various host animals may be immunized by injection with the polypeptide or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice and rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (bacille Calnett-Guerin) and Corinebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a target product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals, such as those described above, may be immunized by injection with the encoded protein, or a portion thereof, supplemented with adjuvants as is also described above. [39] Monoclonal antibodies (mABs), which are homogenous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (Nature, Vol. 256, pp. 495-497 (1975); and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, Vol. 4, p. 72 (1983); Cole at al. Proc. NatI. Acad. Sci. USA, Vol. 80, pp. 2026-2030 (1983), and the EBV-hybdridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the production of ,,chimeric antibodies" (Morrison et al., Proc. NatI. Acad. Sci. USA, Vol. 81, pp. 6851-6855 (1984); Neuberger et al., Nature, Vol. 312, pp. 604-608 (1984); Takeda et al., Nature, Vol. 314, pp. 452-454 (1985) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity, together with genes from a human WO 2007/128429 PCT/EP2007/003694 -9 antibody molecule of appropriate biological activity, can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single-chain antibodies (U.S. Patent No. 4,946,778; Bird, Science, Vol. 242, pp. 423-426 (1988); Huston et al., Proc. Nati. Acad. Sci. USA, Vol. 85, pp. 5879 5883 (1988); and Ward et al., Nature, Vol. 334, pp. 544-546 (1989)) can be adapted to produce differentially expressed gene-single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Most preferably, techniques useful for the production of ,,humanized antibodies" can be adapted to produce antibodies to the proteins, fragments or derivatives thereof. Such techniques are disclosed in U.S. Patent Nos. 5,932,448; 5,693,762; 5,693,761; 5.585,089; 5,530,101; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661,016 and 5,770,429. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule, and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., Science, Vol. 246, pp. 1275-1281 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. [401 The level of protein (fragment) expressed in a biological sample may then be determined by immunoassay methods which utilize the antibodies, antibody derivatives, or antibody fragments described above. Such immunoassay methods include, but are not limited to, Western blotting, fluorescence-activated cell sorting faces) , immunohistochemistry, enzyme-linked immunosorbant assays (ELISA), enzyme linked immuno-spot assay (ELISPOT), dot blotting, competitive and noncompetitive protein binding assays, and other methods commonly used and widely described in scientific and patent literature, and many employed commercially. [41] Particularly preferred, for ease of detection, is the sandwich ELISA, of which a number of variations exist, all of which are intended to be encompassed by the present invention. For example, in a typical forward assay, unlabelled antibody, antibody derivative or antibody fragment is immobilized on a solid substrate and the WO 2007/128429 PCT/EP2007/003694 - 10 sample to be tested is brought into contact with the bound molecule and incubated for a period of time sufficient to allow formation of an antibody-antigen binary complex. At this point, a second antibody, antibody derivative, or antibody fragment labelled with a molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal or may be quantified by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include the simultaneous assay in which both sample and antibody are added simultaneously to the bound antibody or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and added to the unlabelled surface-bound antibody. These techniques are well known to those skilled in the art and the possibility of minor variations will be readily apparent. As used herein, ,,sandwich assay" is intended to encompass all variations of the basic two-site technique. [42} The most commonly used reporter molecules for labelling an antibody, antibody fragment or derivative in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules. In the case of an enzyme immunoassay (EIA), an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different ligation techniques exist which are well-known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production of a detectable color change upon hydrolysis by the corresponding enzyme. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates which yield a fluorescent product, rather than the chromogenic substrates noted above. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate reacts with the enzyme linked to the second antibody giving a qualitative visual signal which may be further quantified, usually spectrophotometrically, to give an evaluation of the amount of protein or fragment thereof. Alternately, fluorescent compounds such as fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with WO 2007/128429 PCT/EP2007/003694 - 11 light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use. [431 Preferably, the sample which is subjected to the method according to the invention is a body fluid or tissue sample. Preferably, the body fluid is blood, plasma, urine and/or serum. [441 A further aspect of the invention relates to a composition or kit for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising reagents for determining the level of gene expression and/or activity of at least one gene being modulated by estrogens and/or SERMs. Preferably, the gene expression and/or activity of the at least one gene being modulated by estrogens and/or SERMs is selected from the group consisting of the genes as defined above. [45] In a preferred embodiment of the invention, the composition or kit comprises reagents for determining the transcript level of at least one gene being modulated by estrogens and/or SERMs. Preferably, said reagents for determining the transcript level comprise a hybridization probe and, optionally, primers for elongation and/or amplification. Preferably, the hybdrization probe and primers are able to bind to a transcript of the above-mentioned genes. [46] Another preferred embodiment of the invention comprises a composition or kit for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising reagents for determining the level of a protein product encoded by at least one gene as defined above. [47] Preferably, the composition or kit comprises reagents for enzymatic, binding, physiological and/or immunological determination in order to determine the level of a protein product encoded by at least one gene as defined above. More preferably, the composition or kit comprises at least one antibody, antibody derivative or antibody fragment able to bind at least one protein product encoded by at least one gene as WO 2007/128429 PCT/EP2007/003694 -12 defined above. Preferably, said composition or kit comprises a monoclonal antibody as described in detail above. [48] In another preferred embodiment, a composition or kit is provided, further comprising a device for collecting a biological sample. More preferably, the composition or kit of the invention further comprises a device for collecting a biological sample of a subject and, in addition, may also comprise instructions for use of the kit and interpretation of the determined level of gene expression and/or activity and/or level of protein product. [49] Still a further aspect of the invention relates to an array for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising a carrier and a probe for determining the level of expression and/or activity and/or the transcript level of at least one gene being modulated by estrogens and/or SERMs in said sample. [501 An array is a particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of genes which involves hybridization of labelled mRNA to an ordered array of olignucleotides or polynucleotides. Typically, the olignucleotides or polynucleotides utilized in this hybridization method are bound to a solid support. Examples of solid supports include, but are not limited to, membranes, filters, slides, paper, nylon, wafers, fibres, magnetic or non-magnetic beds, gels, tubing, polymers, polyvinyl chloride dishes, etc. Any solid surface to which the olignucleotides or polynucleotides can be bound, either directly or indirectly, either covalently or non covalently, can be used. Such probe arrays for expression monitoring can be prepared and used and techniques which are well known to those skilled in the art as described in the manuals of the Affymetrix company, for example. Such a method allows the level of transcription of a plurality of genes to be measured simultaneously to generate gene expression profiles or patterns. [51] Further, an array is a preferred method for determining the presence of at least one polymorphism in the genes as defined above. Such an array preferably comprises a carrier having immobilized thereto at least one probe for determining the presence of at least one polymorphism of a gene being modulated by estrogens and/or SERMs. Preferably, the array carrier, e.g. a planar carrier or a microchannel device, has immobilized thereto a plurality of different probes located at different areas of the carrier which are designed such that they can bind nucleic acid molecules, e.g. RNA molecules or DNA molecules, amplification products, primer elongation products, etc, WO 2007/128429 PCT/EP2007/003694 -13 containing the sequence in which the polymorphism to be tested is located. Thus, an identification of the polymorphism to be analysed by detection of a site-specific binding event of the nucleic acid sample molecule to the probe immobilized on the carrier may be accomplished. [52] A further aspect of the invention relates to a method for the diagnosis of a disorder and/or condition that is associated with endometrial proliferation or a predisposition therefor in a subject, comprising determining the level of expression and/or activity of at least one gene as defined above in said sample, wherein a level of expression and/or activity substantially deviating from the reference value for a healthy subject is indicative of a dysfunction of endometrial proliferation or a predisposition therefor. [53] The term "disorder" is defined as a pathological state of a mammal, preferably a human, and the term "condition" is defined as a non-pathological state of a mammal, preferably a human. In the context of the invention, a condition which is associated with endometrial proliferation is, for example, pregnancy or ovulation, whereas a disorder that is associated with endometrial proliferation is, for example, endometriosis or endometritis. Preferably, a method for the diagnosis of a disorder according to the invention is associated with a dysfunction of endometrial proliferation. [54] The present invention discloses for the first time genes which are differentially expressed in subjects having a dysfunction of endometrial proliferation or a predisposition therefor. The gene expression profile derived from the biological sample obtained from the subject can be compared with a gene expression profile derived from the sample obtained from a subject or subject population not affected by the dysfunction of endometrial proliferation or a predisposition therefor. It can thereby be determined whether the subject is affected or is at risk of being affected by a dysfunction of endometrial proliferation. [55] Preferably, the subject is a mammal, more preferably a human. [56] In a preferred embodiment, the level of gene expression and/or activity is determined by measuring the transcript level of the at least one gene and/or the level of a protein product. [57] The proteins codified by the above-mentioned genes can be used as biomarkers for endometrial proliferation, if measurable in body fluids.

WO 2007/128429 PCT/EP2007/003694 - 14 [58] Preferably, the disorder and/or condition associated with endometrial proliferation or a predisposition therefor is selected from the group consisting of endometrial atrophy, underdeveloped endometrium amenorrhea, endometritis, enhanced endometrial proliferation, endometriosis, disordered menstrual cycle, pregnancy, ovulation, infertility, endometrial hyperplasia, endometrial polyps and/or gynecological tumours. [59] Endometrial atrophy is a disorder wherein the endometrium resists proper development. A typical cause for endometrial atrophy is long-term treatment with gestagens, causing a strong depletion of estrogen in the organism. [60] The endometrium may be underdeveloped as a consequence of a failure of uterine development. [61] Further, menstrual abnormalities such as amenorrhea are associated with a dysfunction of endometrial proliferation. Amenorrhoea is defined as the absence of menstruation under a wide variety of circumstances. Under physiological conditions, amenorrhea occurs at four distinct phases of life: the early stage of the menarche, during pregnancy, lactation and following the menopause. Under pathological conditions, amenorrhea occurs as a cause of endometrial atrophy or an underdeveloped endometrium, for example. Then, conditions relating to the menstrual cycle such as pregnancy and ovulation are associated with endometrial proliferation. [62] A further disorder associated with a dysfunction of endometrial proliferation is infertility, e.g. as a result of endometriosis or endometritis, which are further explained below. [63] Another important disease associated with a dysfunction of endometrial proliferation according to the invention is endometriosis. [64] Endometriosis is the second most common disease in women and is defined as the occurrence of endometrial cells outside the uterus. Endometriosis affects about 1 in 5 women of reproductive age and as many as I in 2 women with fertility problems. [65] Under normal circumstances, the endometrium is only found in the uterus. In endometriosis, tissue with a histological appearance resembling the endometrium is found outside the uterus, for example externally on the uterus, on the intestine or even in the pancreas or the lung. Although these endometriotic foci are located outside the uterus, they also bleed during menstruation, thus they are influenced by hormones of the female cycle. Since endometriotic foci, like the endometrium, go through volume WO 2007/128429 PCT/EP2007/003694 -15 changes during the cycle, these changes may cause pain depending on location. Moreover, the body reacts to endometriotic cells and inflammatory response which again causes pain. Furthermore, inflammation leads to adhesions in the area of the ovaries and Fallopian tubes and, as a result, is responsible for the so-called mechanical sterility of affected women. Apparently, however, in endometriosis, messengers are also released (e.g. cytokines, prostaglandins) which can reduce the fertility of affected women even in the absence of adhesions. [66] In view of their pathobiological properties, endometriotic cells could be classified as being between normal cells and tumour cells: on the one hand they show no neoplastic behaviour, on the other hand, however, like metastasising tumour cells, they are capable of moving across organ boundaries in the organism and of growing into other organs, i.e. they show invasive behaviour. For this reason, endometriotic cells are defined as ,benign tumour cells" in the literature, although up until now no tumour specific mutations in proto-oncogenes have been found in cells of this type. [67] A further disorder associated with a dysfunction of endometrial proliferation or a predisposition therefor is endometritis. This disorder is defined as the acute inflammation of the endometrium, which may develop in response to infection following childbirth or abortion or in response to the insertion of a contraceptive device or as part of a gonorrheal infection. [68] A further associated disorder according to the invention is the endometrial hyperplasia which is defined as an enhanced endometrial proliferation, particularly of the glandular epithelium. The disorder is sub-divided into grades I to Ill (atypical hyperplasia). Firstly, grade IlIl is a precancerous condition of the endometrial carcinoma. Endometrial hyperplasia develops under the protracted influence of estrogens in the absence of gestagens (,,unopposed estrogens"), mostly in the climacterium and the post-menopause. [69] Further, endometrial polyps occur mainly in climacterium in 10% of all women. Most polyps grow as circumscribed hyperplasia in the basalis of the endometrium. In patients with endometrial polyps the incidence of myoma is greater than that of the normal population. This is an indication of general proliferative activity in the uterus. Only in less than 1 % of cases is a carcinoma found in an endometrial polyp. In endometrial polyps irregular or ongoing bleeding from the uterus can occur.

WO 2007/128429 PCT/EP2007/003694 - 16 [70] Further, several gynecological tumours are associated with a dysfunction of endometrial proliferation selected from the group consisting of endometrial cancer, breast cancer, ovarian cancer, vulva cancer and/or vaginal cancer. [711 Endometrial cancer is one of the most common gynecological cancers. It is usually found in post-menopausal women (75%), most frequently between the ages of 55 and 65 years and the majority, 60%, in the latter part of that span. The majority of tumours (60%) are pure adenocarcinomas. They can be divided into three groups according to the degree of glandular differentiation. In general, this cancer is slow to spread from the uterine cavity, probably because the endometrium lacks lymphatics. Distant metastases do occur in advanced cases. Pulmonary deposits are the most common. The actual cause of this cancer is unknown. Argument has centred around the undoubted association between this tumour and estrogen. Increased incidences have been reported in women given estrogen alone as post-menopausal hormone replacement therapy and in patients who develop estrogen secreting tumours of the ovary. The central position of estrogen in the aetiology of endometrial cancer is strengthened by the fact that the addition of progestogen to the estrogen in hormone replacement therapy appears to abolish the increased incidence of endometrial cancer. [72] A further aspect of the invention relates to a method for screening for a modulator for a disorder that is associated with a dysfunction of endometrial proliferation or a predisposition therefor, comprising determining the level of expression and/or activity of at least one gene as defined above in a sample of cells in the presence of a candidate agent, the effect of which on endometrial proliferation is to be examined, compared to said level of expression and/or activity in the absence of said candidate agent. Such methods include in vivo or cell-based and/or cell-free assays to identify compounds which are capable of interfering with the expression and/or activity of at least one gene as defined above which is modulated by estrogens and/or SERMs. [73] The invention also relates to the use of at least one gene and/or protein product as defined above as target for the development of a drug which is active against a disorder and/or condition that is associated with endometrial proliferation. Drug targeting is a strategy aiming at the delivery of a compound to a particular tissue of the body. The drugability of a given target is defined either by how well a compound, such as small molecule drugs or antibodies, can excess the target, or by the efficiency with which a compound can actually achieve the target. A long list of parameter WO 2007/128429 PCT/EP2007/003694 - 17 influences drugability of a given target; these include cellular location, development of resistance, transport mechanisms such as export pumps, side effects, toxicity and others. As targets for therapeutic intervention, receptors, proteins, enzymes, DNA, RNA and ribosomal targets are to be considered. In accordance with the present invention, genes which are modulated by estrogens and/or selective estrogen receptor modulators (SERMs), the transcripts of said genes as well as the protein products thereof are particularly useful as targets. By using said genes, transcripts and/or protein products as markers as drugable targets, it is possible to discover new and better therapies for disorders and/or conditions associated with endometrial proliferation. [74] According to the invention, a pharmaceutical composition preferably contains the active ingredient in combination with one or more pharmaceutically acceptable carriers, excipients and/or additives. Such a composition may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to, saline, buffered saline, dextrose and water. The composition may be administered alone or in combination with other agents, drugs or hormones to a patient, wherein the level of the at least one gene and/or the level of the at least one protein product is substantially deviating from the reference for a healthy subject which is indicative for a dysfunction of endometrial proliferation or a predisposition therefor. [751 The pharmaceutical compositions may be administered by any number of routes. The pharmaceutical compositions may be administered by any number of routes, including, but not limited to, oral, sublingual, intravenous, intramuscular, intraarticular, intraarterial, intramedullary, intrathecal, intraventricular, intraoccular, intrathecal, intracereberal, intracranial, respiratoral, intratracheal, nasopharyngeal, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, or via rectal means, infusion or implant. Preferably, said route of administration is oral. [76] When administered, the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a mammals including humans. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

WO 2007/128429 PCT/EP2007/003694 - 18 [771 The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents. [78] When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. [79] The pharmaceutical compositions may contain suitable buffering agents, including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. [80] The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as benzalkonium chloride; chlorobutanol; parabens and thiomerosal. [811 The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. [82] Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion. [83] Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of a polypeptide or nucleic acid encoding the polypeptide, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable WO 2007/128429 PCT/EP2007/003694 - 19 diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. [84] Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. [85] A further aspect of the invention encompasses the use of a modulator of at least one gene as defined above for the manufacture of a medicament for the prevention and/or treatment of a disorder and/or condition that is associated with endometrial proliferation. [86] Preferably, the modulator of the pharmaceutical composition is an estrogen and/or a Selective Estrogen Receptor Modulator (SERM), an antagonist and/or agonist thereof such as an antibody or an antibody fragment, a siRNA (small interfering RNA), a miRNA (microRNA), an antisense molecule and/or a ribozyme. [87] Agonists of proteins or hormones, e.g. estrogen or SERM, are substances such as enzymes, coenzymes, membrane receptors etc., which increase the activity of the respective protein or hormone. [88] In one aspect, antibodies, which are specific for the protein products of the invention and homologous protein products may be used directly as a modulator, e.g. an antagonist or an agonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein. The antibodies may be generated using methods that are well-known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies (i.e. those which inhibit dimer formation) are especially preferred for therapeutic use. [89] For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the protein or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. It is WO 2007/128429 PCT/EP2007/003694 - 20 preferred that the peptides, fragments or oligopeptides used to induce antibodies to the protein products have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids. [901 Monoclonal antibodies to the protein products may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. Proc. NatI. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). [91] In addition, techniques developed for the production of 'chimeric antibodies', the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S. L. et al. (1984) Proc. NatI. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce single chain antibodies specific for the protein products of the invention and homologous protein products. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Nati. Acad. Sci. 88:11120-11123). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. NatI. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299). [92] Antibody fragments, which contain specific binding sites for the protein products may also be generated. For example, such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by Pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

WO 2007/128429 PCT/EP2007/003694 -21 [931 Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding and immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox, supra). [94] In another embodiment of the invention, polynucleotides or fragments thereof or nucleic acid modulator molecules such as antisense molecules, aptamers, siRNA molecules, miRNA molecules or ribozymes may be used for therapeutic purposes. in one aspect, aptamers, i.e. nucleic acid molecules, which are capable of binding to a protein product of the invention and modulating its activity, may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries. [95] In a further aspect, antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding the protein products as defined above and homologous protein products. Thus, antisense molecules may be used to modulate protein product activity or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligomers or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding the protein products. Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding the proteins of the invention and homologous protein products. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra). Genes encoding the protein products of the invention and homologous protein products can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode the protein products of the invention and homologous WO 2007/128429 PCT/EP2007/003694 - 22 protein products or fragments thereof. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system. [96] As mentioned above, modifications of gene expression can be obtained by designing antisense molecules, e.g. DNA, RNA or nucleic acid analogues such as PNA, to the control regions of the genes encoding the above defined protein products and homologous protein products, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In; Huber, B. E. and B. 1. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). The antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. [97] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples, which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding the protein products of the invention and homologous protein products. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated WO 2007/128429 PCT/EP2007/003694 -23 by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. [98] Nucleic acid modulator molecules, e.g. antisense molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g. the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. [991 Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

WO 2007/128429 PCT/EP2007/003694 - 24 EXAMPLE GENE EXPRESSION ANALYSIS MATERIALS AND METHODS ANIMALS [100] The OVX cynomolgus monkey (Macaca fascicularis), which has been validated as a useful model predictive of outcomes in human clinical trials of estrogen and SERMs, was used as a model of estrogen deficiency. The study was conducted in compliance with the Animal Health regulation, Council Directive No. 86/609/EEC pertaining to the laws, regulations or administrative provisions relating to the protection of animals used for experimental or other scientific purposes. The animals were acclimated to the treatment facility for at least 14 days before beginning treatment. Animals received water ad libitum and approximately 180 g of OWM pelleted diet (Dietex France, SDS, Saint Gratien, France) daily at least 1 hour after dosing, except on the last day of the treatment where the animals were fasted. In addition, each animal received 2 fruits or vegetables daily. Sexually mature female cynomolgus monkeys (Macaca fascicularis obtained from R.C. Hartelust BV, Tilburg, the Netherlands) which were approximately 48 months old were subjected to surgery (OVX or sham) 10 weeks prior to drug treatments. SURGICAL PROCEDURES [1011 Ovariectomy was performed by standard surgical protocols at CIT (Evreux, France) 10 weeks prior to the first day of treatment. Ovaries were removed following anesthesia with a combined intramuscular injection of xylazine (Rompun@: 0.4 mUanimal, Bayer Pharma Division Sant6 Animale, Puteaux, France) and ketamine hydrochloride (Imalgene@: 0.6 mL/kg, Merial, Lyon, France). Sham-operated animals were subjected to the same surgical procedure, except for the removal of ovaries. ESTRADIOL ASSAY [102] Estradiol serum levels were determined in each animal approximately 2 weeks after surgery to verify the effect of ovariectomy. Venous blood samples (approximately 1.5 mL) of non-fasted animals were collected in tubes without anticoagulant and analyzed using a radioimmunoassay (Sorin, Ecole Nationale WO 2007/128429 PCT/EP2007/003694 -25 Veterinaire de Lyon, France). In addition, an estradiol assay was performed at approproximately 2 and 4 weeks after initiating drug treatments to determine if any animals had developed ectopic ovarian tissue growth; any such animals were removed from the study and subsequent analysis. TREATMENT PROTOCOL [103] Ovariectomized and sham-operated animals were divided into groups of 4. Sham-operated and one group of OVX animals received only vehicle, while the remaining groups of OVX monkeys received estradiol (Sigma No. E8875), or tamoxifen (Tamofene@, Aventis Pharma), or raloxifene (Evista@, Lilly France SA). The test items were prepared as suspensions in vehicle and administered daily by oral gavage. On the final day of treatment, animals were sacrificed by deep anesthesia induced by intramuscular ketamine hydrochloride and intravenous sodium pentothal followed by exsanguination. PITUITARY AND UTERINE GENE EXPRESSION Tissue Preparation [104] Gene expression was determined in the pituitary and uterus at the end of drug therapy. Tissues to be analyzed (pituitary glands and uterus) were excised, snap frozen in liquid nitrogen, and stored at -80 0 C until RNA extraction was performed. DNA Microarray Analysis [1051 Total RNA was obtained by acid guanidinium isothiocyanate-phenol chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, CA, USA) and purified on an affinity resin column (RNeasy; Quiagen, Hilden, Germany) according to manufacturer instructions. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002). Prior microarray studies have proven the validity of cross-species DNA chip analysis, therefore the human gene expression probe arrays HGU133A (Affymetrix Inc., Santa Clara, CA, USA) containing 22,283 probe sets interrogating primarily annotated human genes were used. One GeneChip was used per tissue, per animal. The resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab-delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).

WO 2007/128429 PCT/EP2007/003694 - 26 [106] The transcripts which were found to be differentially expressed are indicated by reference by the Affymetrix Probe Set IDs 209173_at, 221731_x_at, 202310_s_at, 202403_s_at, 201852_x_at, 215076_s_at, 211161_s_at, 211980_at, 201438_at, 200884_at, 200703_at, 217398_x_at, 213453_x_at, 212581_x_at, 201841_sat, 221011_s_at, 203570_at, 209014_at, 202593_s_at, 202465_at, 211663_x_at, 204051_s_at, 207714_s-at, 206116_s_at and/or 211959_at. QUANTIFICATION OF ESTROGENIC POTENCY Estrogenic Score Axis [107] To define a reference axis to represent the estrogenic potency of drug treatments, two conditions were chosen to represent the extremes - the ovariectomized animals (OVX group; estrogen deficient) and the estradiol-treated OVX (OVX-EE) group (maximal estrogen). These two groups were used to quantitatively scale the axis by setting their relative estrogenic potency scores to 0% and 100%, respectively. The gene expression from sham-operated animals and from animals treated with tamoxifen or raloxifene were then positioned on this axis to rank their estrogenic potency. [108] The selection of genes (probe sets) used for this analysis were chosen on the basis of mathematical and statistical criteria to meet an operational definition for regulation by estrogen; in addition genes were categorized into groups based on a priori knowledge of biological function. Probe set A (n = 544) was defined by applying the following filtering criteria to the gene expression data; (1) the mean signal intensity for a given probe must have been greater than 50 in at least one of the test conditions, (2) there must have been a 1.5-fold or greater change (up or down) in expression level when comparing the OVX condition to sham and (3) the altered expression pattern must have been reversed with estradiol treatment (OVX-EE). Probe set B (n = 74) was identified as a subset of set A based on using a priori knowledge of gene functions and grouping genes into categories (signal transduction, transport, extracellular matrix, and growth factors). Probe set C (n = 225) was defined using stringent, unbiased mathematical and statistical parameters to filter the data, including only those probes that (1) had a median signal intensity of greater than 50 in either one or both of the OVX and OVX-EE conditions, (2) exhibited a 1.5-fold or greater change in expression between these conditions, and (3) exhibited a P .05 (using a t-test for statistical significance on the log10 transformed intensity values).

WO 2007/128429 PCT/EP2007/003694 -27 [109] The covariance matrix of the log10-transformed gene expression (GEP) data of the OVX and OVX-EE animals were subjected to a principal component analysis (PCA), a statistical dimensional reduction method that has been used previously for describing microarray gene expression data. The PCA algorithm calculates the so called "loadings" and "scores". The loadings are the weights in the linear combinations of the variables (genes), which define the axes (Principal Components, PCs) of the coordinate system. The scores represent the coordinates of the individual animals based on their GEP data in the same coordinate system. [110] All other log-transformed GEP data were then used as input of this PCA model and their scores calculated. Only one PC was calculated and the scores of all samples on this PC were used in the subsequent calculations. The median of the scores of the OVX samples was subtracted from all scores. All scores were then scaled by dividing them by the median of the scores of the OVX-EE animals and multiplied by 100. The calculations described in this section were done using the SIMCA software for multivariate analysis (Version 10, Umetrics., Umea, Sweden) and Excel 2002 (Microsoft Corp., Redman WA, USA). [111] This expression profiling study confirms the particular influence of the corresponding genes of the analysed transcripts, which are modulated by estrogens and/or or SERMs as regulators of endometrial proliferation. Thus, the proteins codified by these genes can be used as biomarkers of enodmetrial proliferation. [112] The selection of candidates included in Table I below was based on scientific criteria, and on a good level of expression of the gene (at least 180 for the average control) and a regulation of at least average 2 fold by estrogen ethinyl etradiol. The values in table 1 denote arbitrary units that reflect the average expression level of a gene of the measured individual gene expression level indicated in Table 2, i.e. the calculated average number of transcripts present. The fold increase given in the headline of the grey columns of table 1 indicates the changes in gene expression of the respective genes of the analysed transcripts which are modulated due to the influence of ethinyl estradiol and the SERMs Tamoxifen and Raloxifene.

WO 2007/128429 PCT/EP2007/003694 - 28 TABLE 1 SELECTED MEAN VALUES Selected biomarkers (candidates) uterus SERMs a) E 0 a. a .C a - poeolya 2(vricn - -a colaen typ lt, alh0 C)(1 2 E U c OLME E 6 0 -a ) C9C ccc -~ - yp , utsoa -2 .2)a 2190at. olgn yeIapa1 113 16 9 2'56 37 1'86 2.7 85 12 E 2 (13 0 0 0 - 0.0 ~ . 00 ZC 0' 0. 0 1~ w Ca) LL LL -CLL)L 209173_at anterior gradient 2 homolog 835 1.78 976 3790 3.89 '093 3.17. 1'021 1.05 - (Xenepuslaevis) ______ 22173 x atchondroitin sulfate 30 13 4 9 .9 76 28- 32 14 22173_X~at proteoglycan 2 (versican) 34 1.7 28 91 .5 76 289 52 .2 202310_s_at collagen, type 1, alpha 1 8502 2.78 3056 20114 6.58 10714 3.51 5576 1.82 202403_sat collagen, type , alpha 2 1758 2.81 2757 14'144 5.13 10180 '3.69 6124 2.22 collagen, type 11l, alpha 1 201852 x at (hlers-Danlos syndrome 2,668 2.88 1'8 4'5 2.39 3'0 1.61 1'561.82 213453 x typeraueo-psht 324 1.0 2'984 7'28 6.7 3 '7 3.42 24 1.8 -- ehydominant)as collagen, type Ill, alpha 1 215076_s_ at (Ehlers-Danlos syndrome 1 '317 13 5'83 12'173 2.0 9 '882 1.8 3'91 0 _ type IV, autosomal 734 21 45 110 41 90 -28 91 20 ___________dominant)________ ____ ____ ___ 211980at collagen, type IV, alpha 1 1'143 1.66 690 2'565 3.72 1'864 2.70 851 1.23 201438_at collagen, type VI, alpha 3 6'008 1.75 3'428 12'617 3.68 6'770 1.97 4*191 1.22 200884_at creatine kinase, brain 487 1.13 430 2'302 5.36 831 1.94 381 0.89 200703_at dynein, cytoplasmic, light 17396 1.51 927 2735 2.95 1'615 1.74 1132 1.22 _____ _ am p lypeptide 1 1 217398_x at glyceraidehyde-3-phosphate 1 '668 0.88 1 '899 4'533 2.39 3*055 -1.61 1'561 0.82 _ dehydrogenaseI 213453_x at glyceraidehyde-3-phosphate 3'124 1105 2*984 7'248 2.43 4'177 1.40 2'934 0.98 -- dehydrogenase 212581__x_at glyceraldehyde-3-phosphate 2733 0.94 2*913 6*646 2.28 3*857 1.32 2'575 0.88 dehydrogenase____ 201841_sat heat shock 27kDa protein 1 6'017 1.03 5'831 12'173 2.09 69882 1.18 3'919 0.67 221011_s at likely ortholog of mouse 842 1.10 763 2'272 2.98 1'013 1.33 688 0.90 _ limb-bud and heart gene ______________ 203570_at lysyl oxidase-like 1 293 1.60 183 451 2.46 329 1.80 226 1.23 291_t melanoma antigen. 879 1.14 771 11737 2.25 1*310 1.70 724 0.94 _ family D, 1 ___ _____________________

___

WO 2007/128429 PCT/EP2007/003694 - 29 00 E ox Uo u cc .) NC 0D E C 0 x a)) COE - - m N sq e c - -_21k~ (brain Ei E C3 04051sa)ertdfrize-rltd '1 2.7 3 3'8 4.1 2'3 .9 9 0.8 s e i n ( c s t protinas iniioold proei 1).2?9 E 2o 0 x~ C: 0 , 0 0 6U 5 CU CL IU ICU-21 LLL 206116_s at tropomyosint1e(acpha) 3'14 1.33 2357 4'63 2.7 11 6 2'48 19 5 8 1'1 1.81 Unkn(protein fr 1 211959_sat PMAGE:18332 [Homo 1',978 1.56 17 6'12 4.83 21'67 .31 109 1.8 spsmRNA sequence procolage - C 202593_s_at merotgandinte2acting 520 1.85 281 7626 2.35 3616 26.19 385 1.24 _ 1~ prtenbfri 211161_s at 2'011[oo ains, 98 2.2 417 93 13883 4.43 2739 2.94 808 1.94 202465_at protein __________ enoeiase enhtoace 44.9 25 66 32 0 .3 35 17 207166_xat (etokprostgt ei2snthase 397112.74 227 162 9.35 165 16.08 285 1.25 mema bran ,_ (_oaeninin protein 4) 206116_sat tropomyosin 1 (alpha) T'145 1.33 2'357 4'963 2:11 2'948 1.25 11919 0.81 Unknown (protein for 211959_at IMAGE:4183312) (H-omo V978 1.56 V270 6'126 4.83 2'677 2.11 1'095 0.86 .sapiens],_mRNA sequence _________________________________ WO 2007/128429 PCT/EP2007/003694 - 30 TABLE 2 MEASURED INDIVIDUAL VALUES Selected biomarkers (candidates) uterus SERMs ooo o 4) 0 ME 2 Q E E E E U) (n M CO j__ J_ CU 0 0 0 0 Cu C.) 0U) U) U) U) C4 C(00 to to toLi U LO an LAOL E E 0A W) V) to v2 1:2 LA LA 2 ZC7 CIL G D G 0 t CO CO (0 cc CO CO I n U C n anterior gradient 209173_at 2 homolog 1479 5'326 518 15 2'406 911 2'667 246 79 1'183 (Xenepus laevis) chondroitin 221731_x_at sulfate 369 293 499 199 127 337 271 133 252 85 proteoglycan 2 39 23 49 19 17 37 21 13 22 8 (versican) 202310_s_at collagen, type 1, 16'425 6'445 6'871 4'269 5'403 3'992 2'663 3'159 2'410 697 _ apha 11 202403_s_at collagen, type l' 12'122 7'125 6'867 4'918 3072 4'016 2'781 2'420 1'813 929 _ alpha 2 collagen, type ll, alpha 1 201852 x at (Ehlers-Danlos 2'354 2'593 4'257 2765 860 1'317 706 1'636 678 471 - - syndrome type IV, autosomal dominant) collagen, type liI, alpha 1 215076 s at (Ehlers-Danlos 6'649 6'898 9'510 6360 1'454 3'831 2'610 4'465 2'874 859 - - syndrome type IV, autosomal dominant) 211980 at collagen, type 963 936 1'136 1'535 276 812 397 842 708 203 - IV, alpha 1 201438 at collagen, type 6'347 4'025 6'888 6772 1'342 3'639 2'841 3758 3'474 408 - VI, alpha 3 200884 at creatine kinase, 680 232 423 612 202 689 464 385 180 211 - brain dynein, 200703 at cytoplasmic, 1'331 1565 1'430 1'259 133 930 759 978 1'042 121 light polypeptide 1 glyceraldehyde 217398_x at 3-phosphate 1'941 2'021 1'306 1'402 366 3'531 1'669 1'654 742 1171 dehydrogenase glyceraldehyde 213453_x at 3-phosphate 3'470 3'901 2'515 2'611 673 3'904 3'014 3'184 1'834 858 dehydrogenase glyceraldehyde 212581_x at 3-phosphate 3'280 3'157 2'284 2'213 563 4'335 2763 2'388 2'167 979 dehydrogenase 1 201841_s at heat shock 8'345 3'953 5713 6'055 1'805 8'028 4'851 5'571 4'874 1'502 _ 27k~a protein 1 likely ortholog of 221011_s at mouse limb-bud 887 989 1'089 402 305 651 837 743 823 86 and heart gene I I II WO 2007/128429 PCT/EP2007/003694 - 31 o oc 0 Q0Da o0 E 0 0 6- 0 E E E E 2e 30 0 0 0 u 2004aUnien)5 '4 4 678 20 07 62 104 6409 C1 M 't to ( (D I-- (U 0) ) W) W) WA LA LA LAOL LrA LO LA LA LA LA L memane 202593_s at interacting 508 640 529 402 98 336 326 266 196 65 protein of RGS16I PRO3121 211161_s at [oosapiens], ,'332 663 1'040 758 302 425 353 528 363 80 sequence procollagen C 202465at endopeptidase 572 448 443 335 97 305 280 182 93 97 enhancer prostaglandin 211663x_at D2 synthase 844 428 155 160 324 317 267 158 168 77 21kDa (brain) secreted 204051s at frizzled-related 2'715 1616 2'938 803 995 983 787 1261 694 251 protein 4 serine (or cysteine) proteinase inhibitor, clade H 207714_sat (heatshock 943 615 1'319 168 306 500 499 484 521 15 protein 47), member 1, (collagen bdg. protein 1) 206116 s at tropomyosin 1 3'163 2'151 3194 4'073 786 3'187 2'300 1'991 1950 575 - - (alpha) Unknown (protein for 211959_at I MAGE:4183312 ,'678 1'867 1'598 2'770 540 1 '669 1'277 1'131 1'001 289 ) [Homo sapiens], mRNA sequence WO 2007/128429 PCT/EP2007/003694 - 32 TABLE 2 MEASURED INDIVIDUAL VALUES Selected biomarkers (candidates) uterus SERMs _ V0 0 5, CD x x x C o oO o 0 0 0 0 Ci) C C~ 72 E E CE , E 7 -2 W 0 C) ) W0) '0C a) C 4 C F 0) 0) 0) a) n' CO 0 (D 0 CM~C C C C (0- Cu E E EE ;E G E (7;E __CO__ ___ (0 Co M COl Ml~ anterior gradient 209173_at 2 homolog 2'489 143 10'456 2'073 4'560 5'032 2'657 2973 1'709 1'400 (Xenepus Iaevis) chondroitin 221731_x_at sulfate 1'120 887 385 1'171 359 895 534 333 1'102 347 proteoglycan 2 (versican) 202310_s_at collagen, type 1, 24'989 16'234 12'804 26'429 6'636 10'002 7787 9'008 16'058 3'676 _ alpha 1 202403_s_at collagen, type 1, 14'895 12'607 13'648 15'427 1'267 9575 9'076 1 V533 10'535 1'086 _ alpha 2 collagen, type Ill, alpha 1 201852_x_at (Ehlers-Danlos 9'395 4'411 4'436 11'189 3'466 3'379 3'138 3'979 4'330 546 _ syndrome type IV, autosomal dominant) collagen, type Ill, alpha 1 215076_s_at (Ehers-Danlos 15'992 10'345 11'585 18'678 3'871 8'926 8'534 9'909 12'511 1'790 - syndrome type IV, autosomal dominant) 211980 at collagen, type 3'377 1'205 1'772 3'905 1'282 1'849 914 976 3'717 1'307 - IV, alpha 1 201438_at collagen, type 14'143 9'992 11'686 14'647 2'177 7'541 4'366 5'419 9'753 2'387 - VI, alpha 3 200884_at creatine kinase, 1'605 3'322 2'243 2'036 730 1'095 617 568 1'046 277 - brain dynein, 200703_at cytoplasmic, 2'138 4'650 2'502 1'651 1'324 1'481 1'658 1314 2'007 297 - light polypeptide 1 glyceraldehyde 217398_x_at 3-phosphate 3'951 5'596 3'925 4'661 786 3'184 3'865 3'329 1'840 861 dehydrogenase glyceraldehyde 213453_x_at 3-phosphate 7'042 8'310 7'017 6'623 733 3741 5'479 4'261 3227 965 dehydrogenase glyceraldehyde 212581_x_at 3-phosphate 5'793 8'402 5'973 6'417 1'199 3709 4'913 3'763 3'045 776 dehydrogenase 201841_s_at heat shock 15'197 16753 6716 10'026 4'637 6'430 8'855 5'121 7122 1'555 _ 27kDa protein 1 likely ortholog of 221011 s at mouse limb-bud 2'996 362 3'171 2'559 1'299 1'238 846 795 1'173 225 and heart gene I I I I I I II__I _ WO 2007/128429 PCT/EP2007/003694 - 33 o o o ~ o~ o C 205at ke1xdae4 54 354 455 78 23 41 28 35 8 in(V a) C C C ( a) a) a) > a 4?- > E C -4 0 0 0 0. 20 t 830 23 14 62 1 0 0 0 am a D, 1. 2059_ at rot ng 480 663 1'8 539 418 756 618 538 55 09 0) pro e 0) C C.I 0> AC 0j NCMCO C W) to m6 (0 c0) (Do C 0- 9 Et E EtoE ~ E ?E v E !P CME 0 tE E (a~ C.'t q: C 't CD~ E o (Do E M Mn 6 0 1n M (DC MC O C> MC) C 203570 at lysyl oxidase- 448 545 354 455 78 233 421 288 375 85 - like 1 melanoma 209014 at antigen. 1'939 830 2230 1'949 620 985 798 804 1'650 603 family D, 1 ___ ___ membrane 202593_s_at interacting 480 663 1'383 539 418 756 618 538 554 99 protein of RGS16___ PR03121 211161_s_at (Hom sapiens] 1'817 1'194 1'439 2'947 775 1'571 667 880 2'502 827 mRNA sequence _______ procollagen C 202465_at endopeptidase 732 712 346 994 266 482 536 558 424 60 enhancer____ prostaglandin 211663_xat D2 synthase 826 948 4'841 6'888 871 T846 1'626 941 4'204 3128 2lkl~a (brain) secreted 204051_sat frizzled-related 4'81 0 3'625 T358 3737 638 3'534 2'221 1 '986 3213 751 protein 4o serine (or cysteine) proteinase inhibitor, cdade H 207714_sat (heat shock 2'027 1'410 1'159 2'052 448 V'227 471 752 2'012 674 protein 47), member 1, (collagen bdg. protein 1) 206116_s at tropomyosin 1 6'313 2'995 4'281 6'262 V'617 2'407 2'140 2'188 5'057 1'411 _ (alpha) Unknown (protein for 211959_at IMAGE:4 183312 2'916 8!645 8'442 4'502 2'866 T237 1'919 1767 3787 991 [Homo sapiens), mRNA __________sequence ____ ____ ____________ ____________ WO 2007/128429 PCT/EP2007/003694 -34 TABLE 2 MEASURED INDIVIDUAL VALUES Selected biomarkers (candidates) uterus SERMs z .2 U) a) C M) E _ , x 2 _ax (e c o>. 0 0>1 321 2 _O CUa CcU w F 1z Ca). 0) C -) - lh (0- CO 5'6 '207 5 E E vn 0) ~ vD U CD CO CO - ap 2' 11 30 7 3 anterior gradient 209173_at 2 homotog Xenepuslaevis ) 400 1-103 1'377 1'203 429 chondroitin 221731_x at sulfate _ proteoglycan 2 (versican) 225 650 214 321 204 202310_s at collagen, type 1, alpha 1 1143 13888 5067 2207 5784 202403_s at collagen, type 1, d i alpha 2 n5192 11320 7605 3380 4'171 collagen, type IVl, alpha 1 (Ehlers 201852_x at Danlos _ syndrome type IV, autosomal dominant) 1 '565 3'547 1*255 1 *726 1'034 collagen, type II, alpha 1 (Ehlers 215076_ sat Danlos3 syndrome type IV, autosomal dominant) 5-565 12'120 5-670 4'608 3-453 211980_at collagen, type IV, alpha 1 802 1249 610 744 277 201438_ at collagen, type VI. alpha 3 3'246 5585 4267 3667 1019 200884_at creatine kinase, brain 256 376 487 404 95 dynein, 200703_at cytoplasmic, light polypeptide 1 972 1*498 1'044 1'015 245 glyceraldehyde 217398_x at 3-phosphate dehydrogenase 1'067 1983 1'413 1782 405 glyceraldehyde 213453_xat 3-phosphate dehydrogenase 2-191 3-454 2791 3-298 570 glyceraldehyde 212581_xat 3-phosphate dehydrogenase 2'050 3T074 2'425 2*749 439 201841 s at heat shock - - 27kDa protein 1 2'652 3'605 4'484 4'936 1'009 likely ortholog of 221011_s at mouse limb-bud and heart gene 628 725 711 686 43 203570_at lysyl oxidase like 1 192 304 229 179 56 WO 2007/128429 PCT/EP2007/003694 -35 E CD 20 antigen, fa C ml D, 10 8 7 57 142 M W)2 M02 : r 0 00) 0) 0) 0D CD 0)0 D C - CD- I--- E E Wn) W) 0 e 0 ) t)o CD ) ~E ~E E ~E melanoma 209014_at antigen, family D, 1 660 899 768 570 142. membrane 202593 s at interacting 20 _ - protein of RGS16 250 497 290 353 108 PR03121 [Homo 211161_s_at sapiens], mRNA sequence 504 1'682 498 549 583 procollagen C 202465_at endopeptidase enhancer 126 716 362 294 248 prostaglandin D2 211663_x_at synthase 21kDa (brain) 132 403 432 172 155 204051s at secreted frizzled - related protein 4 718 927 799 722 98 serine (or cysteine) proteinase inhibitor, clade H 207714_s_at (heat shock protein 47), member 1, (collagen binding protein 1) 522 1'053 813 349 311 206116 s at tropomyosin 1 - - (alpha) 1'477 2'620 2'013 1'567 522 Unknown (protein for 211959_at IMAGE:4183312 ) [Homo sapiens], mRNA sequence 891 1'112 1'569 807 341

Claims (19)

1. 2. The method of claim 1, wherein the gene is collagen, type 1, alpha 1 (Gene ID 1277); collagen, type I, alpha 2 (Gene ID 1278); collagen, type l1l, alpha 1, (Ehlers-Danlos syndrome type IV autosomal dominant) (Gene ID 1281); creatine kinase, brain (Gene ID 24264) and/or prostaglandin D2 synthase 21 kDa (brain) (Gene ID 5730).
2. 3. The method of claim 2, wherein the gene is prostaglandin D2 synthase 21 kDa (brain) (Gene ID 5730). WO 2007/128429 PCT/EP2007/003694 - 37 4. The method of claims 1 to 3, wherein the level of gene expression and/or activity is determined by measuring the transcript level.
3. 5. The method of any one of claims 1 to 4, wherein the determination comprises a hybridization reaction.
4. 6. The method of any one of claims 4 to 5, wherein the determination further comprises an amplification and/or elongation reaction.
5. 7. The method of claim 6, wherein the amplification comprises at least one technique selected from the group consisting of reverse transcriptase PCR and/or real-time PCR.
6. 8. The method of any one of claims 1 to 3, wherein the level of gene expression is determined by measuring the level of a protein product selected from the group consisting of the protein products anterior gradient 2 homolog (NP_006399), chondroitin sulfate proteoglycan 2 (versican) (NP_004376), alpha 1 type I collagen preproprotein (NP 000079), alpha 2 type I collagen (NP_000080), alpha 1 type Ill collagen (NP 000081), alpha 3 type IV collagen isoform 1, precursor (NP_000082), alpha 4 type IV collagen precursor (NP_000083), alpha 1 type IV collagen preproprotein (NP_001836), alpha 3 type IV collagen isoform 1 precursor (NP 004360), brain creatine kinase (NP_001814), cytoplasmatic dynein light polypeptide (N P003737), glyceraldehyde-3-phosphate dehydrogenase (NP_002037), glycyl-tRNA synthetase (NP_002038), growth arrest-specific 1 (NP_002039), heat shock 27kDa protein I (NP_001 531), hypothetical protein DKFZp 566J091 (NP_112177), lysyl oxidase-like 1 (NP_005567), melanoma antigen family D, 1 isoform b (NP_008917), membrane interacting protein of RGS16 (NP 057725), procollagen C-endopeptidase enhancer (NP_002584), prostaglandin D2 synthase 21kDa (NP_000945), secreted frizzled-related protein 4, (NP_003005), serine (or cysteine) proteinase inhibitor, clade H1, member 1 precursor (NP_001226), tropomyosin 1 (alpha) (NP_000357) and/or insulin-like growth factor binding protein 5 (NP_000590). WO 2007/128429 PCT/EP2007/003694 - 38 9. The method of claim 8, wherein the protein product is alpha 1 type I collagen preproprotein (NP_000079), alpha 2 type I collagen (NP_000080), alpha 1 type IlIl collagen (NP_000081), brain creatine kinase (NP_001814) and/or prostaglandin D2 synthase 21kDA (NP_000945).
7. 10. The method of claim 9, wherein the protein product is prostaglandin D2 synthase 21kDa (NP_000945).
8. 11. The method of any one of claims 8 to 10, wherein the protein product level is determined by enzymatic, binding, physical and/or immunological methods.
9. 12. The method of any one of claims 1 to 11, wherein the sample is a body fluid or tissue sample.
10. 13. The method of claim 12, wherein the body fluid is blood, plasma, urine and/or serum.
11. 14. An array for qualitatively and/or quantitatively determining endometrial proliferation in a sample, comprising a carrier and a probe for determining the level of expression and/or activity and/or the transcript level of at least one gene being modulated by estrogens and/or SERMs in said sample, wherein the at least one gene is selected from the group consisting of the genes as defined in any one of claims 1 to 3.
12. 15. A method for the diagnosis of a disorder and/or condition that is associated with endometrial proliferation or a predisposition therefor in a subject, comprising determining the level of expression and/or activity of at least one gene as defined in any one of claims 1 to 4 in said sample, wherein a level of expression and/or activity substantially deviating from the reference value for a healthy subject is indicative for a dysfunction of endometrial proliferation or a predisposition therefor. WO 2007/128429 PCT/EP2007/003694 - 39 16. The method of claim 15, wherein the subject is a mammal.
13. 17. The method of claim 16, wherein the mammal is a human.
14. 18. The method of any one of claims 15 to 17, wherein the level of gene expression and/or activity is determined by measuring the transcript level of the at least one gene and/or the level of a protein product.
15. 19. The method of any one of claims 15 to 18, wherein the disorder and/or condition is selected from the group consisting of endometrial atrophy, underdeveloped endometrium, amenorrhea, endometritis, enhanced endometrial proliferation, endometriosis, disordered menstrual cycle, pregnancy, infertility, endometrial hyperplasia, endometrial polyps and/or gynecological tumors.
16. 20. The method of claim 19, wherein the gynecological tumors are selected from the group consisting of endometrial cancer, breast cancer, ovarian cancer, vulva cancer and/or vaginal cancer.
17. 21. A method for screening for a modulator of a disorder and/or condition that is associated with endometrial proliferation or a predisposition therefor, comprising determining the level of expression and/or activity of at least one gene as defined in any one of claims 1 to 3 in a sample of cells in the presence of a candidate agent, whose effect on endometrial proliferation is to be examined, compared to said level of expression and/or activity in the absence of said candidate agent.
18. 22. Use of at least one gene as defined in any one of claims 1 to 3 and/or at least one protein product as defined in any one of claims 8 to 10 as target and/or modulator of endometrial proliferation.
19. 23. The use of claim 22 for the manufacture of an agent which is active against a disorder and/or condition that is associated with endometrial proliferation. WO 2007/128429 PCT/EP2007/003694 -40 24. Use of a modulator of at least one gene as defined in any one of claims 1 to 3 for the manufacture of a medicament for the prevention and/or treatment of a disorder and/or condition that is associated with endometrial proliferation.