Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/36—Gynecology or obstetrics
  • G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity

Definitions

• the present invention relates to means and methods for determining the likelihood of endometriosis in a female subject. Furthermore, kits for performing the inventive methods are provided. The invention also provides for novel uses of binding molecules interacting with and/or specifically binding to ezrin and/or of binding molecules interacting with and/or specifically binding to insulin receptor substrate- 1 (IRS-I) in the preparation of diagnostic compositions for endometriosis.
• IRS-I insulin receptor substrate- 1
• Endometriosis is a common, benign, estrogen dependent, chronic gynaecological disorder associated with pelvic pain and infertility. It is characterised by the presence of uterine endometrial tissue outside of the normal location (i.e. ectopic sites) — mainly on the pelvic peritoneum, but also on the ovaries and in the rectovaginal septum, and more rarely in the pericardium, pleura, and even in the brain. The prevalence of pelvic endometriosis approaches 6-10% in the general female population in the reproductive age (Giudice and Kao, 2004).
• Endometriosis is linked to dysmenorrhoea (60-80%), pelvic pain (30-50%), infertility (30- 40%), dyspareunia (25-40%), and menstrual irregularities (10-20%) (Shaw, 1995).
• the term "endometriosis” includes lesions of the peritoneum, of the ovaries and of the rectovaginal septum that recently have been described as three separate entities with different pathogeneses (Nisolle and Donnez, 1997) as follows.
• Peritoneal endometriosis consists of red, flame-like lesions that probably reflect the first stage of early implantation of endometrial glands and stroma, regurgitated through the fallopian tubes during menstruation. Their significantly higher stromal vascularization and epithelial mitosis are responsible for the invasion of cells into the serous layer of the peritoneal cavity, referred as ectopic site.
• MMPs matrix-metalloproteinases
• Ovarian endometriosis (endometrioma) is considered an invagination of the metaplastic celomic epithelium into the ovarian cortex (Donnez et al., 1996).
• Adenomyotic nodules and endometriosis located in the rectovaginal septum are equal entities, evolving from Muellerian rests by metaplasia (Nisolle et al., 1993; Donnez et al., 1995; Donnez et al., 1996).
• the new endometriotic foci are associated with striking proliferation of surrounding smooth muscle, creating an adenomyomatous appearance.
• hyperplasia of smooth muscle provokes perivisceritis due to the inflammatory process and secondary retraction of the rectal serosa.
• Intrinsic molecular aberrations in pelvic endometriotic implants were proposed to contribute significantly to the development of endometriosis. These include aberrant expression of cytokines (Khorram et al., 1993) and matrix-metalloproteinases (Osteen et al., 1996), resistance to the protective action of progesterone (Bruner et al., 1995), deficiency of 17 ⁇ - hydroxysteroid dehydrogenase (17 ⁇ -HSD) type 2 (Zeitoun et al., 1998), and aberrant expression of aromatase (Noble et al., 1996; Zeitoun et al., 1999; Kitawaki et al., 1999). Because endometriosis is an estrogen-dependent disorder, aromatase expression appeared to be of paramount importance in the pathophysiology.
• the prior art provides for diagnostic methods that are inconvenient (for the patient as well as for the attending physician), time-consuming and cost-intensive.
• WO 01/62959 relates to markers of endometriosis that are differently expressed in the endometrial cells of females with endometriosis compared to endometriosis-free females.
• WO 00/43789 Al is directed to a method and a kit for the diagnosis of endometriosis using blood and endometrial leukocyte markers.
• the marker is a surface antigen from endometrial or blood leukocytes.
• WO 00/47739 A2 describes a technique to discover auto-antigens found in patients having endometriosis.
• EP-Al 1 321 768 proposes a method for diagnosing endometriosis by measuring gene products in basal cells whereby said gene product measured is estrogen receptor, progesterone receptor or aromatase. Furthermore, this EP application proposes the measurement of a protein of the cytoskeleton or 17 ⁇ HSD-type II when the functionalities of the endometrium are measured as a method for the determination of functionalities of the endometrium.
• JP 2001 124776-A describes a method of endometriosis detection linked to the detection of aromatase cytochrome P450 in biopsies.
• estrogen-dependent diseases of the uterus such as endometrial carcinoma (Bulun et al., 1994), endometriosis (Noble et al., 1996; Kitawaki et al., 1997), adenomyosis (Yamamoto et al., 1993), and leiomyomas (Bulun et al., 1994), show the up-regulation of aromatase both at transcriptional and translational level, suggesting that these tissues produce locally estrogens, which may interfere with the normal cell proliferation through the interaction with its receptors.
• PGs proliferative and inflammatory features of endometriosis.
• Steril 78, 825-829 taught that aromatase P450 mRNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis.
• Bedaiwy 2004; loc. cit.
• the use of aromatase P450 is limited by observation that large numbers of women with endometriosis do not express aromatase P450 in their eutopic endometrium.
• the present inventions relates to a method for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, said method comprising the steps of
• the inventive method as characterized above may further comprise a step in which an additional marker is measured.
• Said additional marker may, e.g. be aromatase (also known as aromatase P450 or C19 aromatase; see, e.g. W ⁇ lfler (2005; loc. cit.) or Bedaiwy (2004; loc. cit.)).
• aromatase also known as aromatase P450 or C19 aromatase
• W ⁇ lfler 2005; loc. cit.
• Bedaiwy 2004; loc. cit.
• the present methods and means also provide for possibility to verify positive or negative results obtained by measuring the expression level(s) of other (also previously described) endometriosis markers provided in the art, in particular aromatase.
• This additional verification of previously obtained data is necessary since as pointed out above, the currently used markers or marker systems are not very reliable.
• the present invention now provides for diagnostic tools with high sensitivity and specificity.
• the present invention also provides for method for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, said method comprising the steps of (a) assaying in a biological sample to be analysed the expression level of aromatase and assaying, sequentially or concomitantly in a biological sample to be analysed the expression level of ezrin and/or of insulin receptor substrate-1 (IRS-I); and
• an elevated level of ezrin/phospho-ezrin and/or of IRS-I in comparison to the corresponding expression levels of said markers in (a) non- endometriotic control sample(s) or in (a) non- endometriotic standard(s) is diagnostic for an endometriotic event in said sample to be analyzed/tested. Accordingly, the methods and means provided herein are useful to verify whether a (human) patient suspected to suffer from or being prone to suffer from endometriosis has said disease or whether there is a certain (high) likelihood (i.e. a potential risk of) that this individual will suffer from said disease.
• the present means and methods of this invention will also be employed to determine the susceptibility (or vulnerability) of a patient to suffer from endometriosis. This also includes the possibility to determine whether said patient has a given predisposition (tendency) and a certain risk to develop endometriosis.
• ezrin in particular phospho- ezrin
• IRS-I also in phosphorylated form
• This higher expression level of either of these two specific markers disclosed herein in a (patient) sample is the indication that said patient suffers from endometriosis or is at least likely to suffer from endometriosis.
• the test provided herein is particularly useful to diagnose the presence of endometriosis and also to verify diagnostic results obtained with other markers for endometriosis, in particular to verify results obtained with the marker "aromatase”.
• the inventive method also relates to a method for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, as disclosed above which further comprises in step (a) a step (a') of assaying in said biological sample to be analyzed the expression level of aromatase and in step (b) a step (b') of comparing said expression level of said aromatase to a baseline expression level established and/or obtainable by assaying the expression level of aromatase in (an) endometriosis-free reference sample(s), i.e. (a) non-endometriotic sample(s).
• the present invention provides for a particular sensitive and specific method for the detection of endometriosis when the expression level of e.g. aromatase and ezrin (or phospho-ezrin) in combination, of aromatase and IRS-I (or phosphorylated versions thereof), of ezrin (or phospho-ezrin) and IRS-I (or phosphorylated versions thereof), it is also envisaged that all three herein discussed markers for endometriosis are measured in combination in a sample to be tested in accordance with this invention. Said combinational measurement of these markers may be sequentially or concomitantly.
• markers ezrin in particular phospho- ezrin
• insulin receptor substrate-1 insulin receptor substrate-1
• additional markers may be in particular aromatase (C 19 aromatase/aromatase P450), but also further markers (as, inter alia, described in Bedaiwy (2004, loc. cit.)) are envisaged to be determined in addition to and/or in combination with the herein described novel and inventive markers ezrin and/or IRS-I.
• Such additional markers may be tumor markers, like CA-115, CA 19-9, SICAM-I or glycodelin-A (PP14), immunological markers like cytokines EL-6 or TNF, autoantibodies, genetic markers, like early growth response (EGR)-I gene, placenta protein (PP14) or cytokeratines.
• EGR early growth response
• PP14 placenta protein
• the present invention provides ezrin (phospho-ezrin) and/or IRS-I as a reliable marker system for the determination of the susceptibility, the predisposition, the presence and/or even the potential risk of developing endometriosis in a female subject.
• ezrin phospho-ezrin
• IRS-I IRS-I
• the teachings of the present invention can also be employed on other biological samples than menstrual blood and these biological samples comprise, but are not limited to serum, cells, like cells derived from the endometrium, the rectovaginal septum and the like. Also it is envisaged that as biological samples tissue and/or cells obtained during biopsies can be employed and scrutinized for the expression pattern of ezrin and/or IRS-I (either alone or in combination or even in combination with the detection of the known marker aromatase). Further examples of biological samples, which are not limiting are provided herein below.
• the methods provided herein are preferably carried out on a biological sample derived from a patient suspected to suffer from endometriosis or showing a prevalence or disposition for suffering from endometriosis and/or an endometriosis-free reference sample(s) which is/are derived from human patients and/or healthy human volunteers.
• the samples provided by or obtained from healthy volunteers may be employed as "endometriosis-free reference sample".
• the person skilled in the art is readily in a position to also generate control samples or control values by the determination of the ezrin (phospho-ezrin) expression level or of the IRS-I (also phosphorylated forms thereof) levels in healthy control samples.
• control values either of these two markers disclosed herein in control samples (healthy, non-endometriotic samples) and to thoroughly establish “norm values”, “reference interavals” or “reference ranges” to which the corresponding values from a given (patient) sample can be compared.
• Such "reference intervals” etc. can easily be established by the person skilled in the art, for example by approved recommendations on the theory of reference values/norm values of the International Federation of Clinical Chemistry, Expert panel on Theory of Reference values.
• Quantitative and qualitative methods in diagnosis comprise, but are not limited to the herein described immunological methods, like Western blots (and their quantitative analysis) or assays like ELISA-, RIA-tests, etc. Also envisaged are expression tests based on the quantitative methods like PCR. However, as documented in the appended examples, of particular interest in the methods and means of the present invention is protein-expression diagnostic.
• the term higher "expression level" of ezrin (phospho-ezrin) or of IRS-I means that said expression level in quantitative or qualitative measurements is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% higher than in a corresponding non-endometriotic sample.
• the "eutopic endometrium” is uterine mucosa, which is localized within the normal, physiologic site, the uterine cavity.
• “ectopic endometrium” defines mucosa that appears with the histologic criteria of uterine mucosa located within sites outside of the uterine cavity, i.e. localized within the peritoneal cavity, the ovaries, or embodied within the myometrium (smooth muscle cells of the uterus) and/ or the rectovaginal septum, or rarely located within extraperitoneal sites, i.e. lungs, heart, and/ or brain.
• ezrin in particular phospho- ezrin
• Endometriosis is a painful, chronic disease that affects 5 1/2 million women and girls in the USA and Canada, and millions more worldwide. It occurs when tissue like that which lines the uterus (tissue called the endometrium) is found outside the uterus — usually in the abdomen on the ovaries, fallopian tubes, and ligaments that support the uterus; the area between the vagina and rectum; the outer surface of the uterus; and the lining of the pelvic cavity. Other sites for these endometrial growths may include the bladder, bowel, vagina, cervix, vulva, and in abdominal surgical scars.
• ..endometriosis as employed herein, in particular relates to perineal endometriosis, ovarian endometriosis (endometrioma) and also adenomyotic nodules as also defined herein above.
• the methods, means and kits of the present invention are particularly useful in the diagnosis of perineal endometriosis.
• the ezrin to be assessed/measured is in one embodiment phospho-ezrin.
• the detection of phosphorylated ezrin e.g.. ezrin as shown in SEQ ID.
• No.2 provided herein below and comprising a phosphorylation on the Tyr354 and/or on the Thr- 567 position is of particular usefulness in the diagnostic of endometriosis as provided by the methods and means of this invention.
• the person skilled in the art is readily in a position to deduce the corresponding phosphorylation sited of other (human) ezrin isoforms or variants which correspond to the phosphorylations sites Tyr354 and/or on the Thr-567 of the specific, exemplified ezrin form provided in SEQ ID No.2 and encoded by the DNA as provided in SEQ ID. NO. 1.
• the ezrin to be assessed/measured is preferably human ezrin and the insulin receptor substrate- 1 (IRS-I) is preferably human IRS-I.
• the other marker molecule for endometriosis namely human insulin receptor substrate- 1/IRS-l.
• activating" phosphorylations of this molecule are particularly useful as positive markers for endometriosis in a human subject.
• SEQ ID NO: 4 Such “activating phosphorylations” of IRS-I are known in the art and comprise potential phosphorylations on Tyr612, Tyr632, Tyr662, Tyr732, Tyr896 and Tyr 941 of the exemplified sequence for IRS-I as shown in herein shown SEQ ID NO: 4.
• Ezrin is described in more detail here below and is well known in the art.
• SEQ ID NO: 1 may encode one human ezrin: (also accession number NM_003379).
• ezrin isoforms/variants are known in the art and are, inter alia, available under accession numbers: NM_003379.3,BC068458.1, AL162086.1, J05021.1 , BC013903.2 or X51521.1. Accordingly, the present invention is not limited to the ezrin isoform as shown in SEQ ID NO. 2 (or as encoded by the nucleic acid sequence provided herein as SEQ ID NO.l).
• IRS-1/insulin receptor substrate-1 is well known in the art and also in detail described herein below.
• One human IRS-I is encoded by the sequence as shown here under SEQ ID NO: 3 (also accession numberNM_005544).
• IRS-I also further naturally occurring isoforms/variants of IRS-I are known in the art and are, inter alia, available under accession numbers: BC053895.1 and S62539.1. Accordingly, the present invention is not limited to the ERS-I isoform as shown in SEQ ID NO. 4 (or as encoded by the nucleic acid sequence provided herein as SEQ ID NO. 3).
• markers/additional markers may be scrutinized/tested/analyzed in the biological samples obtained from patients suspected to be susceptible, to having a predisposition for, or suspected of suffering from endometriosis.
• an additional marker may, inter alia, be aromatase (aromatase P450/aromatase C 19).
• Such an aromatase is accessible under accession number NM_031226, Homo sapiens cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), transcript variant 2, mRNA and is also depicted herein in form of its coding sequence (see SEQ ID NO: 5 herein below). A furtherisoform is accessible under Ace. No. NM_00103.
• the present invention is not limited to the specific detection of the specific ezrin amino acid sequence (or translation products derived from the above shown nucleotide sequences) nor is it limited to the specific sequences provided herein for IRS-I or for aromatase.
• derivatives and/or variants of the above-identified specific ezrin/IRS-1/aromatase sequences are envisaged for the methods provided herein.
• Such derivatives or variant may, e.g. be mutants or isoforms.
• the nucleotide and amino acid sequences of "ezrin", “IRS-I” or “aromatase” given herein below are not limiting. Accordingly, the terms “ezrin”, “IRS-I” or “aromatase” also encompasses corresponding proteins/genes having amino acid or nucleotide sequences being derivatives of those given sequences.
• the term “derivatives” or “derivatives thereof refers to amino acid or nucleotide sequences being homologous to the amino acid or nucleotide sequences shown herein, e. g. those of human ezrin, IRS-I or aromatase, and/or amino acid or nucleotide sequences as shown herein, e. g. those of human ezrin, IRS-I or aromatase, having (a) particular conservative amino acid(s) exchanged.
• “homologous” means that amino acid or nucleotide sequences have identities of at least 80%, 90%, 95%, 98% or 99% to the sequences shown herein, e. g. those of human ezrin, IRS-I or aromatase, wherein the higer identity values are preferred upon the lower ones.
• the inventive methods for the determination of either a predisposition or the presence of an endometriosis in a human patient are related to the detection of the expression levels of ezrin (in particular phospho-ezrin) as well as or in combination with insulin receptor substrate- 1 (IRS-I, also in its phosphorylated form). It is of note that the corresponding expression level cannot only be determined by the expression level of ezrin or IRS-I protein but also by the measurement of the corresponding translation products like, for example, the measurements of the corresponding RNA. Furthermore, it is envisaged that the in vitro diagnostic as provided herein can also be or can also be related to a DNA diagnostic where the presence/absence of ezrin and/or insulin receptor substrate- 1 genetic variant and/or mutation are determined.
• the measurement of the expression level of the herein identified and characterized independent endometriosis markers ezrin (in particular phospho-ezrin) and IRS-I (also in form of phosphorylated IRS- 1), which both can be used either alone or in combination, comprise, for example, immunological methods and/or corresponding protein detecting methods.
• the corresponding measurement for these markers may comprise quantitative as well as qualitative measuring methods which are known in the art; see, for example, Cell Biology: Laboratory Manual 3 rd Edition, J. Celis (Ed.), Academic Press (NY).
• These immunological methods comprise, inter alia, but are not limited to Western Blot analysis, ELISA-tests, RIA-tests and the like.
• the corresponding tests may, for example, be densitrometric, spectrophotometric, luminescent, autoradiographic or fluorescent methods. All these methods are very well known in the art and can easily be used. Further corresponding methods are disclosed herein below in context of the specific uses of binding molecules against the herein identified novel and inventive markers and the above identified methods to establish and/or deduce the expression levels of these markers are in no way limiting to the present invention.
• RNA translation products of the ezrin gene
• IRS-I gene the IRS-I gene
• aromatase gene the above-identified translation products (in particular RNA) of the ezrin gene, the IRS-I gene or, optionally, the aromatase gene.
• Corresponding methods comprise, but are not limited to, the polymerase chain reaction technology as described, inter alia, in "PCR Primer - a laboratory Manual” Dissenbach (1995), Cold Spring Harbor Press or as described in US 4,383,195; US 4,683,202 and the like.
• PCR technology like for example RC-PCR may be employed in order to measure/deduce and/or assess the amount or quality of ezrin translation products and/or IRS-I translation products.
• the invention also relates to a method for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, said method comprising the steps of:
• step (c) comparing the expression level of said at least two endometriosis-related markers in said sample of step (a) to a baseline expression level established and/or obtained by assaying the expression level of said at least two endometriosis-related markers in (an) endometriosis-free reference sample or in a negative reference group of endometriosis-free women, wherein said at least two endometriosis-related markers are selected from the group consisting of:
• insulin receptor substrate-1 insulin receptor substrate-1 (IRS-I);
• the expression level of aromatase and ezrin (phospho-ezrin) and/or IRS-I (also in phosphorylated from) is to bed deduced in the diagnostic means and methods for endometriosis provided herein. It is also part of the invention that expression levels of aromatase and ezrin/phospho-ezrin are to be determined in a given biological sample. In another embodiment of this invention, expression levels of aromatase and IRS-l/IRS-1 in phosphorylated form are to be determined in a given biological sample. In a further embodiment expression levels of IRS-l/IRS-1 in phosphorylated form and ezrin/phospho-ezrin is to be determined in a given biological sample.
• the inventive method for the detection of endometriosis may also comprise (besides the assessment of ezrin/phospho-ezrin either above or in combination with IRS-I) the determination of other markers like aromatase.
• the method as described above, wherein both ezrin as well as IRS-I (also phosphorylated forms are envisaged) are measured may further comprise a step wherein the expression level of aromatase is measured in said biological sample and wherein the expression level of aromatase in the sample to be tested is compared to a baseline expression level established and/or obtained by assaying the expression level of aromatase in (an) endometriosis-free reference sample(s).
• the methods as provided herein relate to the assessment of the specific markers ezrin (or phospho-ezrin) and IRS-I (also in its phosphorylated form), either alone or in combination, in biological samples. These samples may be selected from the group consisting of cells, tissues or body fluids.
• the samples to be assessed are compared to control (endometriosis-free) samples obtained/obtainable from healthy (endometriosis-free) individuals.
• Cell samples may be derived, for example, from endometrial glands, endometrial stroma, celomic epithelium, Muellerian duct, endometrial vasculature, endometrial lymphatic system, and endometrial immune system.
• Tissue samples may, inter alia, be derived from the endometrium, the rectovaginal septum, the peritonaeum parietale, from peritonaeum viscerale of intraabdominal organs, from extraabdominal sites.
• the body fluids to be assessed as samples may be blood, urine, menstrual discharge or vaginal mucus.
• biopsy material may be employed as samples to be assessed in accordance with the present invention such biopsy material may be, inter alia, derived from endometrial biopsies currettage, hysterectomy, uterine biopsy, Fallopian tube biopsies, ovarian biopsies, biopsies of tissue of the rectovaginal septum.
• the inventive method can also be carried out on any other biopsy material or full material from organs and/ or tissue, which could be affected by endometriosis and which are substrates of access via surgery.
• the samples to be analyzed for the expression level of ezrin (phospho-ezrin), IRS-I (also in its phosphorylated form) (or optionally aromatase) is blood, in particular menstrual blood.
• said blood or said menstrual discharge may, e.g. be obtained from extra- or intravaginal plugs.
• a method for determining the likelihood of endometriosis in a female subject comprising the steps of: obtaining a biological sample (for example and in a particular embodiment of menstrual blood) from said female subject; assaying said sample for the expression level of at least one, preferably two endometriosis-related markers or even three endometriosis-related markers as defined herein; comparing the expression level of said endometriosis-related marker(s) to a baseline expression level, established by assaying the expression level of said endometriosis- related marker(s) in a negative reference group of endometriosis-free women, wherein said endometriosis-related markers are
• insulin receptor substrate-1 insulin receptor substrate-1 (IRS-I); and, optionally,
• aromatase may be measured in the diagnostic in vitro methods provided herein.
• a preferred embodiment is characterized in that ezrin is selected, wherein ezrin is present as phospho-ezrin.
• the phosphorylated form(s) may be detected/analyzed in context of this invention.
• Corresponding phosphorylations have been identified herein above and below in form of non-limiting examples, i.e. "activating phosphorylations" of IRS-I may comprise potential phosphorylations on Tyr612, Tyr632, Tyr662, Tyr732, Tyr896, and Tyr 941 of the exemplified sequence for IRS-I as shown in herein shown SEQ ID NO: 4 and phosphorylation sites of ezrin comprise Tyr354 and Thr567 in exemplified SEQ ID NO: 2.
• kits useful for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject comprising: at least are binding molecule specifically binding to and/or interacting with
• insulin receptor substrate- 1 (ii) insulin receptor substrate- 1 (IRS-I) gene.
• a “fragment” of ezrin that is characteristic for said protein is e.g. the amino acid stretch from 2 to 206, 200 to 292 and 210 to 586 in the sequence as shown in SEQ ID No. 2.
• a “fragment” of IRS-I that is characteristic for said protein is e.g. the amino acid stretch from 12 to 113 or 160 to 263 in the sequence as shown in SEQ ID No. 4.
• the "fragments” provided herein or epitopes comprised in said fragments are particullrly useful in the generation od "specific binding molecules" directed against the endometriosis markers of this invention.
• fragments or epitopes comprised in said fragments it is within the normal skills of the artisan to generate antibodies, e.g. polyclonal or monoclonal antibodies which can then be used in the assays, methods, means and kits provided herin.
• the generation of corresponding antibody molecules or binding molecules is described herein and known in the art.
• the kit as provided in the present invention may also comprise at least one binding molecule specifically binding to (i) ezrin protein product or a fragment thereof and at least one binding molecule specifically binding to (ii) insulin receptor substrate-1 (IRS-I) protein product or a fragment thereof or comprising at least one binding molecule capable of specifically amplifying or detecting the translation product of the (i) ezrin gene and (ii) the insulin receptor substrate-1 (IRS-I).
• IRS-I insulin receptor substrate-1
• kits comprising binding molecules for ezrin/phospho-ezrin and also binding molecules for IRS-I are particularly useful when both markers are to be assessed and/or measured.
• the invention also relates to a kit that comprises as additional part binding molecules capable of detecting said additional marker, like aromatase. Therefore, the invention also provides for a kit as described above which further comprises at least a binding molecule specifically binding to (iii) aromatase protein product or fragment thereof or further comprises at least one binding molecule capable of specifically amplifying or detecting the translation product of the aromatase gene.
• binding molecules described above to be comprised in the inventive kits are tool for the detection of ezrin (phospho-ezrin) and/or IRS-I, either alone or in combination.
• Said binding molecules may, for example, be detectably labelled or said kit may, optionally, also comprise further substances for the detection of said binding molecules.
• the inventive kits may also comprise at least one substance for the detection of said specifically binding molecules to
• insulin receptor substrate- 1 insulin receptor substrate- 1 (IRS-I) protein product or fragment thereof;
• binding molecules to be employed in context of this invention should be specific for ezrin (or protein fragments thereof which are characteristic for ezrin) and the binding molecules directed against IRS-I should be specific for said ERS-I.
• binding molecules bind to or interact specifically and individually with the herein identified markers ezrin (phospho-ezrin), IRS-I and, optionally, aromatase.
• binding molecules react with ezrin (or phospho- ezrin), with IRS-I or with the further (optionally) to be determined marker, like aromatase and does not react/detect other, non-related proteins or translation products. It is, however, also envisaged that the binding molecules to be employed in this invention also comprise binding molecules which react with, bind to and/or detect variants or mutant forms of ezrin and/or of ERS-I (and, optionally, of aromatase).
• the binding molecule to be employed in context of the present invention and also comprised in the kits of the present invention may be selected from the group consisting of antibodies, affybodies, trinectins, anticalins, aptamers, RNAs, PNAs and the like.
• binding molecules useful in the methods, kits, assays provided herein.
• These molecules are directed and bind specifically to or specifically label ezrin (phospho-ezrin)/IRS-l, and/or aromatase and described herein.
• Non-limiting examples of these binding molecules may be selected from aptamers (Gold, Ann. Rev. Biochem. 64 (1995), 763-797), aptazymes, antisense RNA, si RNA, antibodies (Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988), affibodies (Hansson, Immunotechnology 4 (1999), 237-252; Henning, Hum Gene Ther.
• aptamer means nucleic acid molecules that can bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995), Ann. Rev. Biochem 64, 763-797).
• a preferred binding molecule in context of the present invention is an antibody specific for ezrin (phospho-ezrin)/IRS-l (also phosphorylated form(s) thereof), and/or aromatase.
• the appended examples also provide for specific antibodies directed against ezrin, IRS-I or aromatase.
• Such antibodies e. g., may bind to the amino acid stretches or amino acid peptides of ezrin (phospho-ezrin)/IRS-l, and/or aromatase.
• Said presence, absence, identity or amount of ezrin (phospho-ezrin) and/or IRS-I (also phosphorylated form(s)) in a given sample to be tested may then be compared to the corresponding molecule of a healthy control or an internal or normal standard.
• the methods, kits and uses provided herein are particularly useful in the in vitro diagnoses of endometriosis.
• Particularly useful are in this context antibodies, antibody molecules, antibody derivatives, which specifically interact with ezrin (phospho-ezrin), ERS- 1 or with aromatase as described herein.
• the term "antibody/antibodies” as employed herein also comprise antibody derivatives, antibody fragments and the like.
• the antibody useful in context of the present invention can be, for example, polyclonal or monoclonal.
• the term “antibody” also comprises derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used for the monitoring of the presence, absence, amount, identiy and/or ratio of ezrin (phospho-ezrin), IRS-I or with aromatase, in particular in diagnosis.
• phage antibodies which bind to an epitope of the polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Accordingly, also phage antibodies can be used in context of this invention.
• the present invention furthermore includes the use of chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments.
• Antibody fragments or derivatives further comprise F(ab')2, Fv or scFv fragments; see, for example, Harlow and Lane, loc. cit.
• F(ab')2, Fv or scFv fragments see, for example, Harlow and Lane, loc. cit.
• the (antibody) derivatives can be produced by peptidomimetics.
• techniques described for the production of single chain antibodies see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies to polypeptide(s) as defined in context of this invention.
• transgenic animals may be used to express humanized antibodies to ezrin (phospho-ezrin), IRS-I and, optionally, aromatase.
• the antibody to be employed in context of this invention is a monoclonal antibody.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (K ⁇ hler and Milstein Nature 256 (1975), 495- 497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
• antibody molecule relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules.
• the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies.
• the term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies.
• antibody molecule also comprises bifunctional antibodies, trifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
• binding molecules provided herein are to be used in the methods, kits, uses as well as assays provided in the context of the evaluation of ezrin (phospho-ezrin), IRS-I (also phosphorylated form(s)) and, optionally, aromatase in a given sample to be tested or analyzed.
• ezrin phospho-ezrin
• IRS-I also phosphorylated form(s)
• aromatase in a given sample to be tested or analyzed.
• sample any biological sample obtained from an individual, or other source containing polynucleotides or polypeptides or portions thereof.
• biological samples include body fluids (such as blood, sera, plasma, urine, sputum) and tissue sources found to express the polynucleotides coding for ezrin (phospho-ezrin), IRS-I or aromatase.
• body fluids such as blood, sera, plasma, urine, sputum
• tissue sources found to express the polynucleotides coding for ezrin (phospho-ezrin), IRS-I or aromatase.
• Methods for obtaining tissue biopsies and body fluids from mammals, in particular human are well known in the art.
• a biological sample which includes menstrual blood/menstrual discharge is preferred as a source.
• the diagnostic composition and/or kit described herein optionally comprises suitable means for detection.
• Binding molecule or detection means described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
• examples of well-known carriers include glass, polystyrene, polyvinyl ion, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
• the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
• a partiuclar preferred solid phase may also be the membranes used in Western blots.
• Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes.
• Suitable methods of immobilizing of binding molecules like, anticalins, antibody(ies), aptamer(s), polypeptide(s), etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions or (chemical) crosslinking and the like.
• immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
• detection assays can comprise radioisotopic or non-radioisotopic methods.
• immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay.
• these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).
• the binding molecule against ezrin (phospho-ezrin), IRS-I and, optionally, against aromatase can comprise a detectable label.
• Appropriate labels and methods for labeling are known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, ⁇ -galactosidase, alkaline phosphatase), radioactive isotopes (like 32 P, 33 P, 35 S or 125 I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).
• the "binding molecule” specifically binds to the herein identified endometriosis markers on molecules that comprise, either directly or indirectly a compound which comprises a "detectable substance” as routinely used in diagnostic assays.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
• the detectable substance may be coupled or conjugated either directly to a binding molecule as defined herein, for example to an Fc portion of an antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• bioluminescent materials include luciferase, luciferin, and aequorin; and
• suitable radioactive material include 125 I, 131 I, or 99 Tc.
• biomolecules A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases).
• Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
• kits wherein said at least "one binding molecule capable of specifically amplifying and/or detecting a translation product” is a primer or a probe specific for e.g. ezrin, IRS-I or, optionally, aromatase.
• phospho-ezrin and IRS-I are tested in a given sample in parallel.
• a third marker e.g. aromatose
• One, non-limiting protocol for a detection of e.g. three markers in the same biologiocal sample e.g. the herein described two markers ezrin/phospho ezrin, IRS-I in combination with the marker aromatase can be as follows: 50 ⁇ l of menstrual blood can be employed after chemical erythrocytes lysis through RT incubation with 50 ⁇ l of Versalyse (Beckman-Coulter) for 10 min.
• the samples can then be mixed with 50 ⁇ L PBS, 1%BSA, 0.1% NaN 3 ; incubated 15min at RT with 10 ⁇ L of phospho-Ezrin (e.g. FTTC-conjugated); 10 ⁇ L of Aromatase (e.g. PE-conjugated); and 5 ⁇ L of IRS-I (e.g. ECD-conjugated) and washed at 800 rpm (RT) with 0.5 mL PBS. Thereafter the pellet is resuspended in 0.5 mL of PBS, supplemented with fixative (Beckman-Coulter) and further processed by FACS ® . Yet, from the above it is to be understood that the expression of the herein described marker can also be tested and assayed independently from each other, e.g. in sequential tests.
• phospho-Ezrin e.g. FTTC-conjugated
• Aromatase e.g. PE-conjugated
• the present invention relates to the use of a primer or pair of primers capable of specifically amplifying the nucleic acid molecules of wild type or mutant ezrin or wild-type or mutant IRS-I (or optionally of wild-type or mutant aromatase).
• primer when used in the present invention means a single-stranded nucleic acid molecule capable of annealing the nucleic acid molecule of the present application and thereby being capable of serving as a starting point for amplification.
• Said term also comprises oligoribo- or desoxyribonucleotides which are complementary to a region of one of the strands of a nucleic acid molecule of the present invention.
• primers means a pair of primers that are with respect to a complementary region of a nucleic acid molecule directed in the opposite direction towards each other to enable, for example, amplification by polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• amplifying refers to repeated copying of a specified sequence of nucleotides resulting in an increase in the amount of said specified sequence of nucleotides, and allows the generation of a multitude of identical or essentially identical (i.e. at least 95% more preferred at least 98%, even more preferred at least 99% and most preferred at least 99.5% such as 99.9% identical) nucleic acid molecules or parts thereof.
• Such methods are well established in the art; see Sambrook et al. "Molecular Cloning, A Laboratory Manual", 2 nd edition 1989, CSH Press, Cold Spring Harbor. They include polymerase chain reaction (PCR) and modifications thereof, ligase chain reaction (LCR) to name some preferred amplification methods.
• a primer to be used according to the invention is preferably a primer which binds to a region of a nucleic acid molecule of the invention which is unique for the herein described endometriosis markers ezrin or IRS-I and which is not present in other, non-related sequences..
• a pair of primers according to the invention it is possible that one of the primers of the pair is specific in the above described meaning or both of the primers of the pair are specific.
• the 3'-OH end of a primer is used by a polymerase to be extended by successive incorporation of nucleotides.
• the primer or pair of primers of the present invention can be used, for example, in primer extension experiments on template RNA according to methods known by the person skilled in the art.
• the primer or pair of primers of the present invention are used for amplification reactions on template RNA or template DNA, preferably cDNA or genomic DNA.
• the primer or pair of primers can also be used for hybridization experiments as known in the art.
• the primer or pair of primers are used in polymerase chain reactions to amplify sequences corresponding to a sequence of the nucleic acid molecule of the present invention. It is known that the length of a primer results from different parameters (Gillam, Gene 8 (1979), 81-97; Innis, PCR Protocols: A guide to methods and applications, Academic Press, San Diego, USA (1990)).
• the primer should only hybridize or bind to a specific region of a target nucleotide sequence.
• the length of a primer that statistically hybridizes only to one region of a target nucleotide sequence can be calculated by the following formula: ( 1 A) x (whereby x is the length of the primer).
• a hepta- or octanucleotide would be sufficient to bind statistically only once on a sequence of 37 kb.
• a primer exactly matching to a complementary template strand must be at least 9 base pairs in length, otherwise no stable-double strand can be generated (Goulian, Biochemistry 12 (1973), 2893- 2901). It is also envisaged that computer-based algorithms can be used to design primers capable of amplifying the nucleic acid molecules of the invention.
• the primers of the invention are at least 10 nucleotides in length, more preferred at least 12 nucleotides in length, even more preferred at least 15 nucleotides in length, particularly preferred at least 18 nucleotides in length, even more particularly preferred at least 20 nucleotides in length and most preferably at least 25 nucleotides in length.
• the invention can also be carried out with primers which are shorter or longer.
• the primer or pair of primers is labeled.
• the label may, for example, be a radioactive label, such as 32 P, 33 P or 35 S.
• the label is a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or a dye.
• the present invention is accordingly, directed to a kit for determining the likelihood of endometriosis in a female subject, said kit comprising, inter alia and as example, (a) specific binding molecule(s) binding to protein products of the endometriosis- related marker gene(s) as disclosed herein, said endometriosis-related marker genes being (i) ezrin; and (ii) insulin receptor substrate-1 (IRS-I).
• kit comprising, inter alia and as example, (a) specific binding molecule(s) binding to protein products of the endometriosis- related marker gene(s) as disclosed herein, said endometriosis-related marker genes being (i) ezrin; and (ii) insulin receptor substrate-1 (IRS-I).
• kits may be (a) specific binding molecule(s) binding to protein products of the aromatase gene.
• Said kit may also comprise at least one substance for detection of said binding molecule(s).
• the inventive kit comprises preferably at least two binding molecules binding to protein products of two (different) endometriosis-related marker(s) as defined herein.
• the markers ezrin also phospho-ezrin
• insulin receptor substrate 1 IMS-I
• the kit of the present invention preferably comprises at least two binding molecules binding to ezrin (or phospho- ezrin) and IRS-I (also in phosphorylated form(s)).
• kits that comprise additional specific binding molecules for further markers related to (or taught in the prior art to relate to) endometriosis.
• Such an additional binding molecule may, e.g. be directed against the marker aromatase.
• kits as disclosed above which comprise specific binding molecules specifically binding to aromatase and further binding molecules binding to ezrin (phospho-ezrin) and/or binding molecules binding to IRS-I (also in phosphorylated form(s)).
• the inventive kits as described may also comprise suitable means of detection, like at least one substance for detection of the binding molecules to the endometriosis marker as defined herein.
• kits, methods and uses provided herein is that the ezrin to be detected is phospho-ezrin.
• the present invention relates to the use of
• a binding molecule capable of specifically amplifying and/or detecting the translation product of the (i) ezrin gene or the (ii) insulin receptor substrate- 1 gene (IRS-I gene) for the preparation of a diagnostic composition for the detection of the susceptibility, predisposition, presence and/or potential risk of developing endometriosis.
• inventive uses are not limited to the individual use of binding molecules directed against (detection means for) ezrin or IRS-I but it is also envisaged that these diagnostic tools are to be employed in combination. It is further envisaged in the uses of the present invention that further markers are to be detected and, accordingly, further binding molecules may be used in the preparation of a diagnostic composition for the detection of the susceptibility, predisposition, presence and/or potential risk of developing endometriosis. Such further binding molecules may be a binding molecule specifically binding to or interacting with aromatase or a binding molecule capable of specifically amplifying and/or detecting the translation product of the aromatase gene is employed in the preparation of the herein defined diagnostic composition.
• ezrin in particular phospho-ezrin.
• Ezrin (to be detected in accordance with this invention in endometriosis diagnostic) belongs to the ERM family that in turn is a member of the erythrocyte protein 4.1 super-family, characterized by a 300- residue globular N-terminal domain, named FERM (band/our-point one, ezrin, radixin, moesin homology).
• the ERM family consists of 3 members: ezrin (80 kDa), radixin (80 kDa) and moesin (75 kDa) that serve as regulated cross-linkers between the actin cytoskeleton and the plasma membrane.
• Ezrin, radixin and moesin are found in vertebrates as highly similar paralogs (-75% sequence identity) that differ in their primary tissue distributions, but probably maintain a high degree of functional equivalence.
• the proteins of the ERM family consist of three functional domains: an amino-terminal 300- residue FERM domain that is responsible for the binding to membrane proteins and various signaling proteins, a central 200-residue putative coiled-coil region that, when phosphorylated on Tyr-353 contributes to an interaction with p85, and a 100-residue auto- inhibitory carboxyl-terminal tail domain (also known as the C-ERMAD) that contains the F- actin binding site.
• ERM proteins In resting cells, ERM proteins are in a dormant state characterized by an intramolecular association of the FERM and tail domains.
• the activation ⁇ i.e. release of the FERM-tail interaction) of the ERM proteins is triggered by phosphorylation of a specific threonine in the tail domain (Thr-567 in ezrin as shown in SEQ ID NO: 2 or corresponding phophorylations in further isoforms/variants of ezrinThr-564 in radixin (NM_002906.3), and Thr-558 in moesin (NM_002444.2) by the Rho-associated kinase (ROCK e.g. XM_928061.1).
• ezrin De novo synthesis of ezrin is required for in vitro invasion and is involved in the acquisition of metastatic potential in endometrial cancer cells.
• ezrin In uterine endometrioid adenocarcinomas (UEC) ezrin is localized in the membrane of metastatic cancer cells, in contrast to the cytoplasmatic distribution of most endometrial hyperplastic cells.
• ezrin localization differs depending on the analysed disease: it is detected in cytosolic as well as in membrane fractions in atypical endometrial hyperplasias (aH) and UEC, whereas it is only detectable in the cytosolic fraction in simple endometrial hyperplasias (sH) and in complex endometrial hyperplasias (cH), suggesting that its expression and subcellular distribution could play an important role in development and progression of many diseases of the endometrium (Ohtani et al., 2002).
• Ezrin is known in the art and is also characterized by the sequences SEQ ID NOS: 1 and 2 shown herein above and relating to the human coding sequence of ezrin (SEQ ID NO: 1) and the corresponding protein/translation product (SEQ ID NO: 2). Yet, the present invention is not limited to the detection of these specific sequences. Also the detection of corresponding variants, like allelic variants and mutant forms is envisaged in context of this invention.
• a further novel and inventive marker for endometriosis in accordance with this invention is Insulin Receptor Substrate-1 (IRS-I), also in its phosphorylated form(s).
• ERS-I belongs to the ERS protein family, which in turn is defined by the highly conserved sequence of the pleckstrin homology (PH) as well as the phosphotyrosine binding (PTB) domains. These domains are closely associated with a contact surface placed between them that is stabilized by ionic, hydrophobic, and hydrogen bonding interactions.
• the four members of the IRS protein family (IRS-I, -2, -3 and -4) are highly homologous (35% identity, 59-67% similarity) and co-localize with insulin receptor (IR) at or near the plasma membrane.
• the IRS protein family is part of the insulin-signaling pathway. Therefore, these IRS proteins help mediate the metabolic actions of insulin.
• the IRS-I is the substrate of both insulin and insulin-like growth factor- 1 (IGF-I) receptors and interacts with the insulin receptor via its PTB domain.
• the PH region of IRS-I serves as a dock between the IR and IRS-I, while the PTB domain interacts with the NPXY motif at the juxtamembrane (JM) domain of the IR.
• JM juxtamembrane
• IRS-I signals downstream metabolic and mitogenic cellular events that include the MAPK pathway.
• IRS-I expression leads to an abnormal cell hyperproliferation, thereby enhancing angiogenesis, which induces invasiveness.
• Insulin receptor substrate- 1 is known in the art and is also characterized by the sequences SEQ ID NOS: 3 and 4 shown herein above and relating to the human coding sequence of insulin receptor substrate- 1 (IRS-I) (SEQ ID NO: 3) and the corresponding protein/translation product (SEQ ID NO: 4).
• the present invention is not limited to the detection of these specific sequences but also comprises the determination of mutant or allelic variants of IRS-I in the herein described methods, kits and uses for endometriosis diagnostic.
• ezrin characterizes eutopic endometrial tissue from endometriosis patients, in comparison to eutopic endometrium of healthy (control) individuals that showed a very limited if any presence of this protein.
• the activation of the protein by screening the presence of the phosphorylated (e.g. Thr567 in exemplified SEQ ID NO: 2) form (phospho-ezrin) was also verified. This phospho-form in particular characterized only the eutopic endometrial lesions and was not present in normal endometrium of healthy individuals.
• At least two of three markers i.e. of aromatase, ezrin and IRS-I (in particular ezrin and IRS-I) are studied. All these markers are over-expressed in the eutopic lesions. The combination of these markers provides for a good parameter to detect the progression of endometriosis since they provide simultaneous information on the grade of dependence on estrogen, on the level of the invasiveness and on the rate of proliferation. It is of note that in context of this invention at least ezrin and IRS-I are measured either alone or in combination and this measurement may, optionally be combined with the measurement of aromatase.
• said "measurement" of the endometriosis markers as provided herein comprise the measurement of the expression level of ezrin (in particular phospho-ezrin), IRS-I and, optionally, aromatase protein product(s) as well as the corresponding measurement of translation products of the corresponding genes, i.e. the RNA. Also provided in the methods of this invention is the measurement and assessment of the DNA-coding sequences for ezrin, IRS-I and, optionally, aromatase.
• the present invention provides for the novel and inventive teaching that ezrin (in particular phospho-ezrin) as well as IRS-I (also in phophorylated form(s)) are detectably overexpressed in an endometric situation in comparison to a normal control, said control being endometriosis-free (e.g. a sample derived from a non-affected individual or a group of non-affected individuals).
• ezrin in particular phospho-ezrin
• IRS-I also in phophorylated form(s)
• IRS-I different phosphorylation sites in IRS-I can be efficiently used in a method for detection of endometriosis.
• activating phosphorylations like Tyr612, Tyr632, Tyr662, Tyr732, Tyr896, or Tyr941 of the exemplified IRS-I sequence given in SEQ ID NO: 4. It is of note that this also comprises highly similar or highly homologous phosphorylation sites in other (human) isoforms/variants of IRS-I.
• the detection method of the present invention may also be based on the combination of at least two markers, selected from aromatase, ezrin and IRS-I, preferably selected from ezrin and IRS-I. Particularly preferred are the combination of the detection of the expression status of ezrin (in particular phospho-ezrin) and IRS-I (also phosphorylated forms thereof) in a given sample.
• the present invention may also be performed on menstrual discharge material, for example in form of a colorimetric assay. It is e.g. envisaged that several channels in fluorometric assays are employed to deduce the presence or absence of elevated expression levels of the herein disclosed markers in a given sample.
• Corresponding fluorochromes are known in the art and comprise, as non-limiting examples Cy5, Texas Red, APC, Alexa 488 ®, Alexa 568 ® and the like. If at least two or even all three proteins are detected simultaneously, an increase of sensitivity/and or specificity in endometriosis diagnostic of up to > 99% may be achieved. Corresponding examples are given in the experimental part.
• One advantage of the present invention is that invasive methods of diagnosis can be avoided.
• the gist of the present invention is the fact that it was surprisingly found that elevated expression levels of ezrin and/or IRS-I are highly specific and sensitive markers for the presence of an endometriosis. Already the measurement of this marker alone leads to a reliable and good diagnosis of endometriosis.
• said reliability, sensitivity and specificity may even further be improved if an additional marker, like aromatase, is determined.
• an additional marker like aromatase
• it is preferred the expression level of ezrin/phospho-ezrin, IRS-I (and phosphorylated forms thereof) and aromatase is measured in a given sample, whereby in particular elevated levels (in comparison to control samples or a control standard) of ezrin/phospho-ezrin, IRS-I (and phosphorylated forms thereof) and aromatase is indicative for endometriosis.
• the expression level of ezrin/phospho-ezrin or of IRS-I is measured in a given sample to be tested either alone or in combination. It is furthermore envisaged that said expression level(s) is/are compared to control samples or control standards. It is also envisaged that the measurement of the expression levels of ezrin/phospho-ezrin or of IRS-I (and phosphorylated forms thereof) is combined with the determination of the expression level of aromatase in a given sample to be tested and suspected to be a sample of a endometriosis-suffering subject, i.e. a human, female patient.
• the detection method of choice will be based upon the simultaneous, concomitant or sequential detection or determination of the (potentially elevated) expression level of aromatase together with the determination or detection of (potentially elevated) expression level of ezrin or phosphorylated forms of ezrin (like on Tyr354 and/or Thr567 of the ezrin shown in SEQ ID NO: 2) and/or the determination or detection of (potentially elevated) expression level of IRS-I (also in phosphorylated form, like on Tyrol 2, Tyr632, Tyr662, Tyr732 or Tyr896, Tyr 941 of the IRS-I shown in SEQ ID NO: 4).
• the inventive in vitro detection method is, inter alia, based on the combination of specific binding molecules, like monoclonal antibodies which target ezrin (or phospho-ezrin, like Tyr354 and/or Thr567 as shown in SEQ ID NO: 2) as well as IRS-I (or phospho-IRS-1, like S312, Tyr612, Tyr632, Tyr662, Tyr732, Tyr896 orTyr 941 Respectively, in SEQ ID NO: 4).
• These primary antibodies may be coupled to different secondary antibodies bound to a detectable marker, like a chromogene substance.
• Example 1 Detection of endometriosis-specific markers in biopsies Tissue collection
• Endometrial biopsies are obtained from patients who underwent laparoscopy or hysterectomy at the Department of Gynecology of the General Hospital/Medical University of Vienna, Vienna.
• the tissue is collected in D-MEM + Ham's F12 medium, on ice and immediately processed thereafter.
• Endometrial tissue is minced into small pieces and then incubated with collagenase (Sigma Chemical Co., St Louis, MO, USA) at 37°C for 10 min.
• collagenase Sigma Chemical Co., St Louis, MO, USA
• the separation between epithelial and stromal cells is achieved by sequential sieving through a 150 (100 ⁇ m) and a 37 (40 ⁇ m) cell strainer. Epithelial glands are retained in between the two strainers. The stromal cells are collected after the second strainer.
• Both types of cells are cultured in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) without phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamin (Gibco) and 1% antibiotics-antimycotics (Gibco).
• DMEM-F12 Dulbecco's modified Eagle's medium-F12
• FBS fetal bovine serum
• 2 mM L-glutamin Gibco
• antibiotics-antimycotics Gibco
• the epithelial cells are cultured in Petri dishes coated with fibronectin (Gibco). The cultures are kept free from CD-45 positive leukocytes and epithelial cells must be less than 1% contaminated by stromal vimentin-positive or endothelial Factor VTJI-positive cells.
• Endometrial cells were lysed from all tissue in Frackelton buffer (10 mM Tris, 30 mM Na 4 P 2 O 7 , 5O mM NaCl, and 1% Triton X-100, pH 7.1) supplemented with 10 ⁇ g/ml leupeptin, 2 ⁇ g/ml aprotinin and 1 ⁇ g/ml pepstatin A (protein inhibitors cocktail, Boehringer Ingelheim), 1 mM Phenyl-methyl-sulfonyl-fluoride, 0.5 mM Na 3 VO 4 and 50 mM NaF at 4°C for 20 min. Insoluble material was removed by centrifugation (20,000 g at 4°C for 20 min).
• the cell lysates were resuspended in 5xSDS loading buffer (10 mM Tris-HCl, pH 7.0, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 1% Triton X-100). The normalized samples were used to perform a PAGE, followed by electrophoretic transfer to nitrocellulose membranes.
• TBS-T 10 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.05% Tween 20
• BSA 2% BSA (fraction V; Sigma-Aldrich)
• the membranes were probed with the appropriate primary antibodies against aromatase, IRS-I (both Santa Cruz Biotechnology, Inc., CA), ezrin and phospho-ezrin (Cell Signaling, MA) in TBS-T added up with 3% BSA before incubation with peroxidase-conjugated secondary antibodies, diluted in TBS-T added up with 5% milk powder, and detection by enhanced chemiluminescence (Pierce Chemical Co.).
• the epithelial cells were washed in cold PBS (Gibco), fixed in MetOH 10' at -20 0 C; washed in PBS pH8. They were blocked in 10% goat serum and incubated with ezrin or phospho- ezrin first antibody (40 ⁇ l/ml) in presence of BSA 1% v/v overnight.
• the cells were subsequently washed in TBS pH8 and incubated with the secondary antibody conjugated with FTTC (0.1 ⁇ g/ml; Alexa, CA).
• the cells were washed at first in TBS pH8 and then 30' in milliQ water.
• the coverslip was mounted and the cells analyzed under the confocal microscope.
• FIGs. 1 and 2 show that aromatase (Figure Ia), ezrin (in particular phospho-ezrin) ( Figure Ia, b) and IRS-I (Figure 2) are simultaneously up-regulated in eutopic endometriotic lesions compared to normal endometrium ( Figure 1, 2).
• the presence of phosphorylated ezrin on Thr567 in ezrin as depicted in SEQ ID NO: 2 is accompanied by an increase of the expression of ROCK and RhoA proteins.
• the active form of ROCK was only detectable in eutopic lesions and was completely absent in normal endometrium. Accordingly and without being bound by theory, it is envisaged that during the establishment of endometriosis the ezrin pathway is up- regulated from RhoA through ROCK and culminates in the activation of the protein with consequent enhanced of cell migration.
• RhoA activates the phosphorylation of MAPK ERK Kinase 1 (MEK 1) and increases the mitogen effects of Raf-1 through ERK phosphorylation by acting on PAKl through Rac-1.
• IRS-I that normally mediates insulin-signaling pathway, has been shown as a partner of ERa, which is up-regulated in eutopic lesions.
• ERa keeps the stability of IRS- 1 by inhibiting the process of its ubiquitination.
• IRS-I expression is strongly enhanced in eutopic lesions in comparison with normal endometrium, where its expression is very weak, if any.
• IRS-I shows a very complicated pattern of phosphorylation that can either activate or inhibit its function. Without being bound by theory, phosphorylated sites of IRS-I can be divided in: negative regulator sites modulated by Casein kinase II that enhance the ubiquitination of the protein and therefore interfere with its stability ( Ser99, in exemplified ERS-I as shown in SEQ ID NO: 4);
• IRS-I positively feeds-back the PI3K/ Akt pathway, which yields three distinct effects on the epithelial component of the eutopic lesions:
• Example 2 Detection of aromatase, phospho-ezrin and IRS-I in menstrual discharge as markers for endometriosis
• -one plug is from a patient who underwent laparoscopic ovarian endometriosis
• Frackelton lysis buffer made up of: 10 mM Tris-HCl, pH 7.05, 50 mM NaCl,
• Insoluble material was removed by centrifuging the samples at 20000 g, 20 min, at 4°C.
• the lysates were mixed with 1 X SDS-sample buffer (0.2 g Tris, 2.5 g SDS, 2.5 mL glycerol and 1.2 mg
• sample lysates were loaded on a SDS-Page gel and run for Ih at 200V.
• the proteins were transferred from the PAA gel to a nitrocellulose membrane (Hybond C, Amersham) in IX transfer buffer for Ih at 30V in a semi-dry blotting apparatus. After the transfer, the membrane was stained with Ponceau Red in order to determine the quality of the blotting. Afterwards, the membrane was rinsed once in water and twice in TTBS, until all the red staining was completely removed.
• a nitrocellulose membrane Hybond C, Amersham
• Non specific binding was blocked by incubating the membrane in a solution of TTBS added up with 5% powder milk. Thereafter, the membrane was washed twice with TTBS for 10 min. Finally, it was incubated overnight with aromatase, phospho-Ezrin or Insulin Receptor
• Substrate-1 (IRS-I) primary antibody diluted in TTBS added up with 3% of BSA (fraction
• the membrane was washed three times with TTBS for about 30 min and finally rinsed in TBS.
• the membrane was incubated with 1 mL of previously mixed Luminol/Enhancer Solution and Stable Peroxyde- Solution (Pierce) for 1 min. The excess of the detection buffer was removed; the membrane was positioned between two clean transparent papers and thereafter placed in a film cassette. Finally, it was exposed to an ECL-hyperfilm (Amersham) in the dark room for 2 min.
• Aromatase is highly detectable in both endometriosis patients as well as in one of the two healthy controls.
• Phospho-Ezrin was restricted to endometriosis patients and was higher in the patient currently suffering from ovarian endometriosis (endometrioma) in comparison to the patient who suffered from endometriosis in the past.
• IRS-I was exclusively detectable in the patient currently suffering from endometriosis.
• a third marker i.e. IRS-I
• IRS-I a third marker
• Combining the markers aromatase with the marker ezrin and preferably (p)-Ezrin provides reliable sensitivity and specificity, whereas addition of IRS-I also increases the specificity.
• ezrin/phospho-ezrin alone and or IRS-I also in "activated” phosphorylated form
• the determination of the elevated expression level of ezrin/phopho-ezrin and/or IRS-I (also in phosphorylated form) in comparison to endometriosis-free control samples or control standards provides for a reliable method for the elucidation of the presence of an endometriosis in a human patient; see in particular appended figures 1, 2 and 3. the most important marker in this context is ezrin and in particular phopho-ezrin; see appended figure 3.

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