EP1931993A2 - Means and methods for diagnosing endometriosis - Google Patents

Means and methods for diagnosing endometriosis

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Publication number
EP1931993A2
EP1931993A2 EP06791999A EP06791999A EP1931993A2 EP 1931993 A2 EP1931993 A2 EP 1931993A2 EP 06791999 A EP06791999 A EP 06791999A EP 06791999 A EP06791999 A EP 06791999A EP 1931993 A2 EP1931993 A2 EP 1931993A2
Authority
EP
European Patent Office
Prior art keywords
ezrin
irs
endometriosis
aromatase
expression level
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06791999A
Other languages
German (de)
French (fr)
Inventor
Walter Tschugguel
Cristina Rubiolo
Andrea Kolbus
Ljubomir Paucz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medizinische Universitaet Wien
Original Assignee
Medizinische Universitaet Wien
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medizinische Universitaet Wien filed Critical Medizinische Universitaet Wien
Publication of EP1931993A2 publication Critical patent/EP1931993A2/en
Withdrawn legal-status Critical Current

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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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Abstract

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-1) in the preparation of diagnostic compositions for endometriosis.

Description

Means and methods for diagnosing endometriosis
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.
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. The release of matrix-metalloproteinases (MMPs) from viable red endometrial implants could initiate the implantation of glands in other peritoneal sites, as in a metastatic process (Kokorine et al., 1997). The partial menstrual shedding within the "red" lesion finally induces an inflammatory reaction, provoking a scarification process that encloses the implant, which in turn becomes a "black" lesion because of the presence of intraluminal debris. In some cases, the inflammatory process and subsequent fibrosis totally devascularize the endometriotic foci, and "white" plaques of old collagen are all that remains of the ectopic implant. White opafication and yellow-brown lesions are inactive lesions that could be quiescent for a long time, referred as latent stages of endometriosis (Nisolle et al., 1993).
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. In the rectovaginal septum 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 17β- hydroxysteroid dehydrogenase (17β-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 American Society for Reproductive Medicine (ASRM) appointed 1997 an Endometriosis Classification Subcommittee to formally assess the current revised American Fertility Society (AFS) classification (American Society for Reproductive Medicine, 1997). Using this classification, visual (laparoscopy or laparotomy) and histological determination of the stage or degree of endometrial involvement is based on a weighted point system designated as stage I (minimal), stage II (mild), stage EU (moderate) and stage IV (severe) disease, taking both endometriotic lesions (superficial or deep) and adhesions (filmy or dense) into account. In addition, the appearance of superficial implant types should be described as red, black, or white.
Accordingly, 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.
The prior art has also provided some attempts to diagnostic tools in endometriosis. However, all these prior art methods appear not to provide for an unambiguous assessment of endometriosis.
For example, 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 17βHSD-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.
Kao (2003) Endocrinology 144, 2870-2881 described an expression profile of endometriosis- positive biopsies and no specific teaching on endometriosis diagnostics is provided.
Bedaiwy (2004) Clin. Chem. Acta 340, 41-56 provides in a review an overview of markers employed in the prior art, whereby these markers related to CA125, CA-19-9, SICAM-I, glycodelin-A (PP- 14), cytokines (like IL-6/TNF), placental protein 14 (PP14), P450 aromatase, cytokeratins, or hormone receptors. Bedaiwy (2004; loc. cit.) however concludes "at present, there are no reliable markers of this disease" (endometriosis) and also teaches that CA- 125 is the most extensively studied serum marker and even this marker has only limited diagnostic utility.
Also expression of aromatase in endometrial biopsy specimens was considered as a diagnostic test for endometriosis. 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. Aromatase expression in defective endometrium leads to local biosynthesis of estrogen that in turn was reported to increase the level of the prostaglandins PGE2 through the stimulation of the cyclooxygenase type-2 (COX-2) enzyme in cultured endometrial stromal cells (Huang et al., Abstract, 1996; Huang et al., 1998). PGE2 was also found to be the most powerful known inducer of aromatase activity by increasing cAMP levels in endometriotic stromal cells (Noble et al., 1997). Thus, a positive feedback loop for continuous local production of estrogen and prostaglandins (PGs) is established, favouring the proliferative and inflammatory features of endometriosis. Additionally, aberrant aromatase expression in eutopic endometrial samples of women suffering from estrogen-dependent diseases (Kitawaki et al., 1999) suggests a genetic defect. When defective endometrium with abnormal aromatase expression reaches the pelvic peritoneum by retrograde menstruation, it causes an inflammatory reaction that exponentially increases local aromatase activity, i.e. estrogen formation, directly or indirectly induced by PGE2S and cytokines (Noble et al., 1997; Huang et al., 1998), probably favouring the formation of endometriotic implants.
Additionally, the mRNA of aromatase was detected in eutopic endometrial samples of women with moderate to severe endometriosis, but not in disease-free endometrium and myometrium (Bulun et al., 1993; Bulun et al., 1994; Noble et al., 1996). In a retrospective study on 105 women who underwent laparotomy or laparoscopy, both mRNA and protein expression of aromatase were evaluated by RT-PCR and immunohistochemistry, respectively (Kitawaki et al., 1999), showing that aromatase deregulation at transcriptional level occurs during endometriosis, adenomyosis, and / or leiomyomas but not in specimens obtained from disease-free patients, with a sensitivity and a specificity of 91% and 100%, respectively. However, a non-defined amount of endometrial biopsy specimens was collected retrospectively; therefore a controlled prospective study was needed to avoid any possible selection bias.
In a study previously published and relating to aromatase being aberrantly expressed in eutopic endometrium (Wolfler et al., 2005), it was shown that among 48 consecutive symptomatic and eligible patients, 25 (52.1%) exhibited endometriosis and 23 (47.9%) were disease-free. A multiple logistic regression model revealed that 95.5% of patients, suffering from moderate to severe dysmenorrhoea (visual analogue scale score >4/10), whose eutopic endometrium was found to be positive for aromatase indeed exhibited endometriosis at laparoscopy. However, Dheenadayalu (2002) in Fert. Steril 78, 825-829 taught that aromatase P450 mRNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis. In his review, Bedaiwy (2004; loc. cit.) teaches that 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.
Therefore, no reliable methods for the in vitro diagnosis of endometriosis in patients with symptoms such as dysmenorrhoea, pelvic pain, infertility, dyspareunia and menstrual irregularities are available in the prior art. At present, the confirmation of endometriosis usually depends upon the invasive and unpleasant procedure of laparoscopy.
A reliable diagnostic test to screen for endometriosis is therefore still needed in order to avoid unnecessary laparoscopy and to overcome the limitations of solutions to the above- identified problem. Furthermore, there is a need in the art to verify data and diagnostic results obtained by the measurement of aromatase in potential endometriotic samples.
The solution to the above-identified technical problem is herein provided by the embodiments characterized in the following description and in the appended claims. In particular, 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
(a) assaying in a biological sample to be analysed the expression level of ezrin and/or of insulin receptor substrate- 1 (ERS-I); and
(b) comparing said expression level of said ezrin and/or insulin receptor substrate- 1 (IRS- 1) to a baseline expression level established and/or obtainable by assaying the expression level of ezrin and/or insulin receptor substrate- 1 (ERS-I) in (an) endometriosis-free reference sample(s).
Said inventive method can be carried out in vitro as also documented in the appended examples.
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. Wόlfler (2005; loc. cit.) or Bedaiwy (2004; loc. cit.)). It is also and in particular part of this invention that the methods and means provided herein are used to verify previous or concomitantly/simultaneously obtained diagnostic data, whereby said data were or are obtained by the measurement of other endometriosis markers, like aromatase. Accordingly, 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 (for example by analysis/measurement of aromatase expression levels) 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.
Accordingly, 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
(b) comparing said expression level of said aromatase and said ezrin and/or said insulin receptor substrate-1 (IRS-I) to a baseline expression level established and/or obtainable by assaying the expression level of aromatase and ezrin and/or insulin receptor substrate-1 (IRS- 1) in (an) endometriosis-free reference sample(s).
In the methods and means of the present invention an elevated level of ezrin/phospho-ezrin and/or of IRS-I (also in phosphorylated forms as defined herein), 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.
With the means and methods provided herein it is possible to determine in a reliable fashion and also in non-invasive methods whether a given patient suffers from endometriosis ("presence of endometriosis") or whether a given patient has a high likelihood to suffer from endometriosis, i.e. whether this patient is susceptible for an endometriotic event. That means 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.
In accordance with this invention a higher expression level of ezrin (in particular phospho- ezrin) and/or a higher expression level of IRS-I (also in phosphorylated form) in comparison to the normal or base-line expression level in a given healthy control sample is, as documented in the appended examples, indicative for an endometriotic event in the sample (and, therefore, in the patient) to be tested. 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. As documented in the experimental part 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).
As documented in the appended examples, already the expression level detection of ezrin (in particular phospho-ezrin) in a given sample to be tested provides for conformation whether an endometriosis is present in a given subject; see inter alia, Figure IA. A particular good marker for endometriosis is an ezrin in its phosphorylated form ("phospho-ezrin); as also documented in the appended examples. The examples also substantiate that insulin receptor substrate-1 (in its non- and in its phosphorylated form) is a marker for endometriosis. Whereas the present invention is not limited to the combinational detection of at least two markers as defined herein, i.e. aromatase, ezrin (also phospho-ezrin) and IRS-I (also in phosphorylated form), 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.
Therefore, it is also envisaged that, in addition to the markers ezrin (in particular phospho- ezrin) and/or insulin receptor substrate-1 (IRS-I) also additional markers may be measured and determined. Such potential 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. Yet, as also documented in the appended examples, the present invention is based on the surprising finding that in particular ezrin and IRS-I provide (independently as well as in combination) for (a) reliable marker(s) for endometriosis. As also shown in the appended examples, in particular the detection of phosphorylated ezrin (phospho-ezrin) is useful in context of the diagnostic means and methods provided herein.
Accordingly, 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. As also documented in the appended examples the advantage of the methods provided herein is that these methods can be carried out on a plurality of biological samples, in particular also on blood samples and menstrual discharge/menstrual blood. This provides for a reliable method for the determination of endometriosis in a subject, in particular a human female patient, which is minimally invasive or even non-invasive. However, it is of note that 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 (not suffering from endometriosis or not having a pre- disposition for endometriosis) 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. It is also possible to deduce with known quantitative methods "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. Approved recommendation on the theory of reference values are, inter alia publishes as "The concept of reference values." J Clin Chem Clin Biochem 1987;25:337-42 ; "Selection of individuals for the production of reference values." J Clin Chem Clin Biochem 1987;25:639- 44; "Preparation of individuals and collection of specimens for the production of reference values" J Clin Chem Clin Biochem 1988;26:593-598; "Control of analytical variation in the production transfer and application of reference values "Eur J Clin Chem Clin. Biochem 1991 ;29:531-5; "Statistical treatment of collected reference values: Determination of reference limits" J Clin Chem Clin Biochem 1987;25:645-56; "Presentation of observed values related to reference values" J Clin Chem Clin Biochem 1987;25:657-62 and Solberg HE. Establishment and use of reference values In: Burtis CA, Ashwood ER, Bruns DE, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. St. Louis: Saunders, 2006: 425-48.
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.
It is within the skill of the person skilled in the art to employ for these quantitative and qualitative methods known techniques and automated systems. Automated systems are routinely used in diagnosis; see as non-exhaustive example, a comparison of 6 automated assays for the measurement of the prostate-specific antigen (PSA) published in Kort (2006), Clinical Chemistry 52, 1568-1574. Here, the different automated assays known to the skilled artisan are discussed and the person skilled in the art can easily adopt the corresponding assay system to the measurement of the herein disclosed markers. Examples of such automated systems comprise but are not limited to Beckman-Coulter Access, Abbott ARCHITECT and AxSYM, Bayer Advia Centaur, DPC IMMULITE 2000, and Roche Modular Analytics E170 Assay systems and methods in clinical chemistry to be employed in context of this invention are also described in Fraser, C G (1986) interpretation of Clinical Chemistry Laboratory Data" Blackwell; Mayne, P D (1994) "Clinical Chemistry in Diagnosis and Treatment" 6th edition. Arnold; Marshall, W J (1996) Clinical Chemistry (3rd) Gower d edition; Price, C P (1990) "Principles and Practice of Immunoassay" Stockton Press; Roitt, I M (1993) "Essential Immunology" Blackwell;. Teitz, N W (1996) "Clinical Guide to Laboratory Tests. Saunders"; Walmsley, R N, Watkinson, L R, Koag, E S C (1989). "Cases in Chemical Pathology - a Diagnostic Approach" P. G. Publishing; Whitby, L G, Smith, A F, Beckett, G J (1988) "Cases in Clinical Biochemistry" Blackwell; Burtis and Ashwood, (1999) "Tietz Textbook of Clinical Chemistry" SAUNDERS; Brunzel, (2004) "Fundamentals of Urine & Body Fluid Analysis" Elsevier or Bishop, (2005) "Clinical Chemistry: Principles, Procedures, Correlations", Lippincott Williams & Wilkins. In accordance with this invention 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.
As known in the art, the "eutopic endometrium" is uterine mucosa, which is localized within the normal, physiologic site, the uterine cavity. In contrast, "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. In context of this invention it was surprisingly found that more ezrin (in particular phospho- ezrin) is expressed within primary cultures of eutopic endometrium cells compared to primary cultures of ectopic endometrium cells from endometriosis patients with respect of the total amount of protein measured by Western analysis.
The means, methods and kits of the present invention are useful in the diagnosis of endometriosis. 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. Less commonly they are found in the lung, arm, thigh, and other locations. This misplaced tissue develops into growths or lesions that respond to the menstrual cycle in the same way that the tissue of the uterine lining does: each month the tissue builds up, breaks down, and sheds. Menstrual blood flows from the uterus and out of the body through the vagina, but the blood and tissue shed from endometrial growths has no way of leaving the body. This results in internal bleeding, breakdown of the blood and tissue from the lesions, and inflammation — and can cause pain, infertility, scar tissue formation, adhesions, and bowel problems. The term ..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.
In the methods, means, kits and uses disclosed herein, the ezrin to be assessed/measured is in one embodiment phospho-ezrin. As documented in the appended examples and as illustrated herein below, 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. In the methods as provided herein and also in relation to the further means, kits and uses of the present invention, the ezrin to be assessed/measured is preferably human ezrin and the insulin receptor substrate- 1 (IRS-I) is preferably human IRS-I. In context of this invention, it is also envisaged to verify the phosphorylation or the phosphorylation status of the other marker molecule for endometriosis, namely human insulin receptor substrate- 1/IRS-l. In context of this invention, "activating" phosphorylations of this molecule are particularly useful as positive markers for endometriosis in a human subject. 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. For example, the following SEQ ID NO: 1 may encode one human ezrin: (also accession number NM_003379).
1 atgccgaaac caatcaatgt ccgagttacc accatggatg cagagctgga gtttgcaatc
61 cagccaaata caactggaaa acagcttttt gatcaggtgg taaagactat cggcctccgg
121 gaagtgtggt actttggcct ccactatgtg gataataaag gatttcctac ctggctgaag
181 ctggataaga aggtgtctgc ccaggaggtc aggaaggaga atcccctcca gttcaagttc
241 cgggccaagt tctaccctga agatgtggct gaggagctca tccaggacat cacccagaaa
301 cttttcttcc tccaagtgaa ggaaggaatc cttagcgatg agatctactg cccccctgag
361 actgccgtgc tcttggggtc ctacgctgtg caggccaagt ttggggacta caacaaagaa
421 gtgcacaagt ctgggtacct cagctctgag cggctgatcc ctcaaagagt gatggaccag
481 cacaaactta ccagggacca gtgggaggac cggatccagg tgtggcatgc ggaacaccgt
541 gggatgctca aagataatgc tatgttggaa tacctgaaga ttgctcagga cctggaaatg
601 tatggaatca actatttcga gataaaaaac aagaaaggaa cagacctttg gcttggagtt
661 gatgcccttg gactgaatat ttatgagaaa gatgataagt taaccccaaa gattggcttt
721 ccttggagtg aaatcaggaa catctctttc aatgacaaaa agtttgtcat taaacccatc
781 gacaagaagg cacctgactt tgtgttttat gccccacgtc tgagaatcaa caagcggatc
841 ctgcagctct gcatgggcaa ccatgagttg tatatgcgcc gcaggaagcc tgacaccatc
901 gaggtgcagc agatgaaggc ccaggcccgg gaggagaagc atcagaagca gctggagcgg 961 caacagctgg aaacagagaa gaaaaggaga gaaaccgtgg agagagagaa agagcagatg
1021 atgcgcgaga aggaggagtt gatgctgcgg ctgcaggact atgaggagaa gacaaagaag
1081 gcagagagag agctctcgga gcagattcag agggccctgc agctggagga ggagaggaag
1141 cgggcacagg aggaggccga gcgcctagag gctgaccgta tggctgcact gcgggctaag
1201 gaggagctgg agagacaggc ggtggatcag ataaagagcc aggagcagct ggctgcggag
1261 cttgcagaat acactgccaa gattgccctc ctggaagagg cgcggaggcg caaggaggat
1321 gaagttgaag agtggcagca cagggccaaa gaagcccagg atgacctggt gaagaccaag
1381 gaggagctgc acctggtgat gacagcaccc ccgcccccac caccccccgt gtacgagccg
1441 gtgagctacc atgtccagga gagcttgcag gatgagggcg cagagcccac gggctacagc
1501 gcggagctgt ctagtgaggg catccgggat gaccgcaatg aggagaagcg catcactgag
1561 gcagagaaga acgagcgtgt gcagcggcag ctgctgacgc tgagcagcga gctgtcccag
1621 gcccgagatg agaataagag gacccacaat gacatcatcc acaacgagaa catgaggcaa
1681 ggccgggaca agtacaagac gctgcggcag atccggcagg gcaacaccaa gcagcgcatc
1741 gacgagttcg aggccctgta a
(SEQ ID NO: 1)
The corresponding amino acid sequence is shown in the following SEQ ID NO: 2.
MPKPINVRVTTMDAELEFAIQPNTTGKQLFDQWKTIGLREVWY
FGLHYVDNKGFPTWLKLDKKVSAQEVRKENPLQFKFRAKFYPEDVAEELIQDITQKLF
FLQVKEGILSDEIYCPPETAVLLGSYAVQAKFGDYNKEVHKSGYLSSERLIPQRVMDQ
HKLTRDQWEDRIQVWHAEHRGMLKDNAMLEYLKIAQDLEMYGINYFEIKNKKGTDLWL
GVDALGLNIYEKDDKLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRI
NKRILQLCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQLERQQLETEKKRRETVE
REKEQMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEAD
RMAALRAKEELERQAVDQIKSQEQLAAELAEYTAKIALLEEARRRKEDEVEEWQHRAK
EAQDDLVKTKEELHLVMTAPPPPPPPVYEPVSYHVQESLQDEGAEPTGYSAELSSEGI
RDDRNEEKRITEAEKNERVQRQLLTLSSELSQARDENKRTHNDIIHNENMRQGRDKYK
TLRQIRQGNTKQRIDEFEAL"
(SEQ ID NO: 2)
Yet further human 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). Also 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).
1 atggcgagcc ctccggagag cgatggcttc tcggacgtgc gcaaggtggg ctacctgcgc
61 aaacccaaga gcatgcacaa acgcttcttc gtactgcgcg cggccagcga ggctgggggc
121 ccggcgcgcc tcgagtacta cgagaacgag aagaagtggc ggcacaagtc gagcgccccc
181 aaacgctcga tcccccttga gagctgcttc aacatcaaca agcgggctga ctccaagaac
241 aagcacctgg tggctctcta cacccgggac gagcactttg ccatcgcggc ggacagcgag
301 gccgagcaag acagctggta ccaggctctc ctacagctgc acaaccgtgc taagggccac
361 cacgacggag ctgcggccct cggggcggga ggtggtgggg gcagctgcag cggcagctcc
421 ggccttggtg aggctgggga ggacttgagc tacggtgacg tgcccccagg acccgcattc
481 aaagaggtct ggcaagtgat cctgaagccc aagggcctgg gtcagacaaa gaacctgatt
541 ggtatctacc gcctttgcct gaccagcaag accatcagct tcgtgaagct gaactcggag
601 gcagcggccg tggtgctgca gctgatgaac atcaggcgct gtggccactc ggaaaacttc
661 ttcttcatcg aggtgggccg ttctgccgtg acggggcccg gggagttctg gatgcaggtg
721 gatgactctg tggtggccca gaacatgcac gagaccatcc tggaggccat gcgggccatg
781 agtgatgagt tccgccctcg cagcaagagc cagtcctcgt ccaactgctc taaccccatc
841 agcgtccccc tgcgccggca ccatctcaac aatcccccgc ccagccaggt ggggctgacc
901 cgccgatcac gcactgagag catcaccgcc acctccccgg ccagcatggt gggcgggaag
961 ccaggctcct tccgtgtccg cgcctccagt gacggcgaag gcaccatgtc ccgcccagcc
1021 tcggtggacg gcagccctgt gagtcccagc accaacagaa cccacgccca ccggcatcgg
1081 ggcagcgccc ggctgcaccc cccgctcaac cacagccgct ccatccccat gccggcttcc
1141 cgctgctcgc cttcggccac cagcccggtc agtctgtcgt ccagtagcac cagtggccat
1201 ggctccacct cggattgtct cttcccacgg cgatctagtg cttcggtgtc tggttccccc
1261 agcgatggcg gtttcatctc ctcggatgag tatggctcca gtccctgcga tttccggagt
1321 tccttccgca gtgtcactcc ggattccctg ggccacaccc caccagcccg cggtgaggag
1381 gagctaagca actatatctg catgggtggc aaggggccct ccaccctgac cgcccccaac
1441 ggtcactaca ttttgtctcg gggtggcaat ggccaccgct gcaccccagg aacaggcttg
1501 ggcacgagtc cagccttggc tggggatgaa gcagccagtg ctgcagatct ggataatcgg
1561 ttccgaaaga gaactcactc ggcaggcaca tcccctacca ttacccacca gaagaccccg
1621 tcccagtcct cagtggcttc cattgaggag tacacagaga tgatgcctgc ctacccacca 1681 ggaggtggca gtggaggccg actgccggga cacaggcact ccgccttcgt gcccacccgc
1741 tcctacccag aggagggtct ggaaatgcac cccttggagc gtcggggggg gcaccaccgc
1801 ccagacagct ccaccctcca cacggatgat ggctacatgc ccatgtcccc aggggtggcc
1861 ccagtgccca gtggccgaaa gggcagtgga gactatatgc ccatgagccc caagagcgta
1921 tctgccccac agcagatcat caatcccatc agacgccatc cccagagagt ggaccccaat
1981 ggctacatga tgatgtcccc cagcggtggc tgctctcctg acattggagg tggccccagc
2041 agcagcagca gcagcagcaa cgccgtccct tccgggacca gctatggaaa gctgtggaca
2101 aacggggtag ggggccacca ctctcatgtc ttgcctcacc ccaaaccccc agtggagagc
2161 agcggtggta agctcttacc ttgcacaggt gactacatga acatgtcacc agtgggggac
2221 tccaacacca gcagcccctc cgactgctac tacggccctg aggaccccca gcacaagcca
2281 gtcctctcct actactcatt gccaagatcc tttaagcaca cccagcgccc cggggagccg
2341 gaggagggtg cccggcatca gcacctccgc ctttccacta gctctggtcg ccttctctat
2401 gctgcaacag cagatgattc ttcctcttcc accagcagcg acagcctggg tgggggatac
2461 tgcggggcta ggctggagcc cagccttcca catccccacc atcaggttct gcagccccat
2521 ctgcctcgaa aggtggacac agctgctcag accaatagcc gcctggcccg gcccacgagg
2581 ctgtccctgg gggatcccaa ggccagcacc ttacctcggg cccgagagca gcagcagcag
2641 cagcagccct tgctgcaccc tccagagccc aagagcccgg gggaatatgt caatattgaa
2701 tttgggagtg atcagtctgg ctacttgtct ggcccggtgg ctttccacag ctcaccttct
2761 gtcaggtgtc catcccagct ccagccagct cccagagagg aagagactgg cactgaggag
2821 tacatgaaga tggacctggg gccgggccgg agggcagcct ggcaggagag cactggggtc
2881 gagatgggca gactgggccc tgcacctccc ggggctgcta gcatttgcag gcctacccgg
2941 gcagtgccca gcagccgggg tgactacatg accatgcaga tgagttgtcc ccgtcagagc
3001 tacgtggaca cctcgccagc tgcccctgta agctatgctg acatgcgaac aggcattgct
3061 gcagaggagg tgagcctgcc cagggccacc atggctgctg cctcctcatc ctcagcagcc
3121 tctgcttccc cgactgggcc tcaaggggca gcagagctgg ctgcccactc gtccctgctg
3181 gggggcccac aaggacctgg gggcatgagc gccttcaccc gggtgaacct cagtcctaac
3241 cgcaaccaga gtgccaaagt gatccgtgca gacccacaag ggtgccggcg gaggcatagc
3301 tccgagactt tctcctcaac acccagtgcc acccgggtgg gcaacacagt gccctttgga
3361 gcgggggcag cagtaggggg cggtggcggt agcagcagca gcagcgagga tgtgaaacgc
3421 cacagctctg cttcctttga gaatgtgtgg ctgaggcctg gggagcttgg gggagccccc
3481 aaggagccag ccaaactgtg tggggctgct gggggtttgg agaatggtct taactacata
3541 gacctggatt tggtcaagga cttcaaacag tgccctcagg agtgcacccc tgaaccgcag 3601 cctcccccac ccccaccccc tcatcaaccc ctgggcagcg gtgagagcag ctccacccgc
3661 cgctcaagtg aggatttaag cgcctatgcc agcatcagtt tccagaagca gccagaggac
3721 cgtcagtag
(SEQ ID NO: 3)
The corresponding amino acid sequence is provided (and known in the art) in the following SEQ ED NO: 4.
MASPPESDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARL
EYYENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAADSEAE
QDSWYQALLQLHNRAKGHHDGAAALGAGGGGGSCSGSSGLGEAGEDLSYGDVPPGPAF
KEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKLNSEAAAWLQLMNIRRCGHSE
NFFFIEVGRSAVTGPGEFWMQVDDSWAQNMHETILEAMRAMSDEFRPRSKSQSSSNC
SNPISVPLRRHHLNNPPPSQVGLTRRSRTESITATSPASMVGGKPGSFRVRASSDGEG
TMSRPASVDGSPVSPSTNRTHAHRHRGSARLHPPLNHSRSIPMPASRCSPSATSPVSL
SSSSTSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSVTPDSL
GHTPPARGEEELSNYICMGGKGPSTLTAPNGHYILSRGGNGHRCTPGTGLGTSPALAG
DEAASAADLDNRFRKRTHSAGTSPTITHQKTPSQSSVASIEEYTEMMPAYPPGGGSGG
RLPGHRHSAFVPTRSYPEEGLEMHPLERRGGHHRPDSSTLHTDDGYMPMSPGVAPVPS
GRKGSGDYMPMSPKSVSAPQQIINPIRRHPQRVDPNGYMMMSPSGGCSPDIGGGPSSS
SSSSNAVPSGTSYGKLWTNGVGGHHSHVLPHPKPPVESSGGKLLPCTGDYMNMSPVGD
SNTSSPSDCYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLSTSSGRL
LYAATADDSSSSTSSDSLGGGYCGARLEPSLPHPHHQVLQPHLPRKVDTAAQTNSRLA
RPTRLSLGDPKASTLPRAREQQQQQQPLLHPPEPKSPGEYVNIEFGSDQSGYLSGPVA
FHSSPSVRCPSQLQPAPREEETGTEEYMKMDLGPGRRAAWQESTGVEMGRLGPAPPGA
ASICRPTRAVPSSRGDYMTMQMSCPRQSYVDTSPAAPVSYADMRTGIAAEEVSLPRAT
MAAASSSSAASASPTGPQGAAELAAHSSLLGGPQGPGGMSAFTRVNLSPNRNQSAKVI
RADPQGCRRRHSSETFSSTPSATRVGNTVPFGAGAAVGGGGGSSSSSEDVKRHSSASF
ENVWLRPGELGGAPKEPAKLCGAAGGLENGLNYIDLDLVKDFKQCPQECTPEPQPPPP
PPPHQPLGSGESSSTRRSSEDLSAYASISFQKQPEDRQ"
(SEQ ID NO: 4)
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).
As pointed out above, besides the marker ezrin (in particular phospho-ezrin) and/or IRS-I (also phosphorylated versions thereof), also other 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. Such 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.
1 atggttttgg aaatgctgaa cccgatacat tataacatca ccagcatcgt gcctgaagcc
61 atgcctgctg ccaccatgcc agtcctgctc ctcactggcc tttttctctt ggtgtggaat
121 tatgagggca catcctcaat accaggtcct ggctactgca tgggaattgg acccctcatc
181 tcccacggca gattcctgtg gatggggatc ggcagtgcct gcaactacta caaccgggta
241 tatggagaat tcatgcgagt ctggatctct ggagaggaaa cactcattat cagcaagtcc
301 tcaagtatgt tccacataat gaagcacaat cattacagct ctcgattcgg cagcaaactt
361 gggctgcagt gcatcggtat gcatgagaaa ggcatcatat ttaacaacaa tccagagctc
421 tggaaaacaa ctcgaccctt ctttatgaaa gctctgtcag gccccggcct tgttcgtatg
481 gtcacagtct gtgctgaatc cctcaaaaca catctggaca ggttggagga ggtgaccaat
541 gaatcgggct atgtggacgt gttgaccctt ctgcgtcgtg tcatgctgga cacctctaac
601 acgctcttct tgaggatccc tttggacgaa agtgctatcg tggttaaaat ccaaggttat
661 tttgatgcat ggcaagctct cctcatcaaa ccagacatct tctttaagat ttcttggcta
721 tacaaaaagt atgagaagtc tgtcaaggat ttgaaagatg ccatagaagt tctgatagca
781 gaaaaaagac gcaggatttc cacagaagag aaactggaag aatgtatgga ctttgccact
841 gagttgattt tagcagagaa acgtggtgac ctgacaagag agaatgtgaa ccagtgcata
901 ttggaaatgc tgatcgcagc tcctgacacc atgtctgtct ctttgttctt catgctattt
961 ctcattgcaa agcaccctaa tgttgaagag gcaataataa aggaaatcca gactgttatt
1021 ggtgagagag acataaagat tgatgatata caaaaattaa aagtgatgga aaacttcatt
1081 tatgagagca tgcggtacca gcctgtcgtg gacttggtca tgcgcaaagc cttagaagat
1141 gatgtaatcg atggctaccc agtgaaaaag gggacaaaca ttatcctgaa tattggaagg
1201 atgcacagac tcgagttttt ccccaaaccc aatgaattta ctcttgaaaa ttttgcaaag
1261 aatgttcctt ataggtactt tcagccattt ggctttgggc cccgtggctg tgcaggaaag
1321 tacatcgcca tggtgatgat gaaagccatc ctcgttacac ttctgagacg attccacgtg
1381 aagacattgc aaggacagtg tgttgagagc atacagaaga tacacgactt gtccttgcac
1441 ccagatgaga ctaaaaacat gctggaaatg atctttaccc caagaaactc agacaggtgt
1501 ctggaacact ag
(SEQ ID NO: 5)
Also provided here is the corresponding aromatase amino acid sequence as SEQ ID NO: 6. MVLEMLNPIHYNITSIVPEAMPAATMPVLLLTGLFLLVWNYEGT
SSIPGPGYCMGIGPLISHGRFLWMGIGSACNYYNRVYGEFMRVWISGEETLIISKSSS
MFHIMKHNHYSSRFGSKLGLQCIGMHEKGIIFNNNPELWKTTRPFFMKALSGPGLVRM
VTVCAESLKTHLDRLEEVTNESGYVDVLTLLRRVMLDTSNTLFLRIPLDESAIWKIQ
GYFDAWQALLIKPDIFFKISWLYKKYEKSVKDLKDAIEVLIAEKRRRISTEEKLEECM
DFATELILAEKRGDLTRENVNQCILEMLIAAPDTMSVSLFFMLFLIAKHPNVEEAIIK
EIQTVIGERDIKIDDIQKLKVMENFIYESMRYQPWDLVMRKALEDDVIDGYPVKKGT
NIILNIGRMHRLEFFPKPNEFTLENFAKNVPYRYFQPFGFGPRGCAGKYIAMVMMKAI
LVTLLRRFHVKTLQGQCVESIQKIHDLSLHPDETKNMLEMIFTPRNSDRCLEH
(SEQ ID NO: 6)
It is understood for the person skilled in the art that 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. In particular, also 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. Accordingly, it is of note that 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.
In terms of the present invention 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. For instance, in context of the present invention, "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.
As documented in the appended examples, in one embodiment, 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 3rd 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. Accordingly, 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.
Similarly, the person skilled in the art is readily in a position to measure 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. However, in accordance with the present invention also further developments of the 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:
(a) obtaining a biological sample from said female subject;
(b) assaying said sample for the expression level of at least two endometriosis-related markers; and
(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:
(i) ezrin;
(ii) insulin receptor substrate-1 (IRS-I); and
(iii) aromatase.
Accordingly, in one embodiment of the present invention, 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. It is also envisaged that all three expression levels, i.e. of aromatase, IRS-l/IRS-1 in phosphorylated form and ezrin/phospho-ezrin is determined in context of the endometriosis diagnostic means and methods of this invention.
Again, as pointed out above, the inventive method for the detection of endometriosis (or the susceptibility thereto) 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. Accordingly, 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. As documented in the examples, in particular menstrual blood or menstrual discharge can easily be assessed for the expression level of ezrin (phospho-ezrin), IRS-I, and (optionally) aromatase. Also 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. It is evident for the skilled artisan that 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. However, in a most preferred embodiment of the present invention, 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. As shown in the appended examples, said blood or said menstrual discharge may, e.g. be obtained from extra- or intravaginal plugs.
It is an object of the present invention to provide a simple test for diagnosing endometriosis.
This object can be reached by 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
(i) ezrin;
(ii) insulin receptor substrate-1 (IRS-I); and, optionally,
(iii) aromatase.
As shown in the appended examples in addition to these two markers ezrin and/or IRS-I, also 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. Also in case of IRS-I, 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.
Furthermore, the present invention provides for a kit useful for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, said kit comprising: at least are binding molecule specifically binding to and/or interacting with
(i) ezrin protein product or a fragment thereof; or
(ii) insulin receptor substrate- 1 (IRS-I) protein product or a fragment thereof; or at least one binding molecule capable of specifically amplifying or detecting the translation product of
(i) ezrin gene; or
(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. For example, based on these "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).
Said kit 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. As discussed above also other markers may, in addition, be measured by the inventive method. Accordingly, 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.
The 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. Accordingly, the inventive kits may also comprise at least one substance for the detection of said specifically binding molecules to
(i) ezrin protein product or fragment thereof;
(ii) insulin receptor substrate- 1 (IRS-I) protein product or fragment thereof; and/or
(iii) aromatase protein product or fragment thereof.
The 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.
Specificity of these binding molecules means that the binding molecules bind to or interact specifically and individually with the herein identified markers ezrin (phospho-ezrin), IRS-I and, optionally, aromatase.
The term "specifically" means that said 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.
The person skilled in the art is readily in the position to use and to obtain specific 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. 13 (2000), 1427-1439), trinectins (Phylos Inc., Lexington, Massachusetts, USA; Xu, Chem. Biol. 9 (2002), 933), anticalins, or the like. These compounds are, for example, described in EP 1 017 814. Said European patent also describes the process of preparing such anticalins with the ability to bind a specific target. In accordance with the present invention, the term "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.
As evident form the disclosure provided herein and in particular from the appended examples, 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. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of 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. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, the (antibody) derivatives can be produced by peptidomimetics. Further, 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. Also, transgenic animals may be used to express humanized antibodies to ezrin (phospho-ezrin), IRS-I and, optionally, aromatase. Most preferably, the antibody to be employed in context of this invention is a monoclonal antibody. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kόhler 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. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., US Patent 4,946,778) can be adapted to produce single chain antibodies to ezrin (phospho-ezrin), IRS-I and, optionally, aromatase. Accordingly, in context of the present invention, the term "antibody molecule" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, 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. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term "antibody molecule" also comprises bifunctional antibodies, trifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
The 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.
As pointed out above, in accordance with the present invention by the term "sample" is intended any biological sample obtained from an individual, or other source containing polynucleotides or polypeptides or portions thereof. As indicated, 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. 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.
As mentioned above, 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. As documented in the examples, a partiuclar preferred solid phase may also be the membranes used in Western blots. It is evident that not only the "binding molecule" but also the target molecule (in context of this invention ezrin (phospho-ezrin), IRS-I (also in phosphorylated form(s)) and, optionally, aromatase may be bound to the "solid phase" and may be tested with the "binding molecules" provided in "liquid phase".
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. Examples of immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay. Furthermore, 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, β-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P, 33P, 35S or 125I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).
Therefore, 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. Again, examples of 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. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to an Fc portion of antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, or 99Tc.
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). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immunoassays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymology", Academic Press, Inc. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
The invention also provides for 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.
In accordance with this invention, it is also envisaged that two herein described markers (phospho-ezrin and IRS-I) are tested in a given sample in parallel. It is also envisaged that a third marker (e.g. aromatose) is measured at the same time. 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 μl of menstrual blood can be employed after chemical erythrocytes lysis through RT incubation with 50 μl of Versalyse (Beckman-Coulter) for 10 min. The samples can then be mixed with 50 μL PBS, 1%BSA, 0.1% NaN3; incubated 15min at RT with 10 μL of phospho-Ezrin ( e.g. FTTC-conjugated); 10 μL of Aromatase ( e.g. PE-conjugated); and 5 μL 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.
Furthermore, 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). The term "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. According to the present invention the term "pair of 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).
The term "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", 2nd 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.
When used in the context of primers the term "specifically" means that only the nucleic acid molecules as described herein above are amplified, e.g. nucleic acid molecules encoding the wild-type ezrin (or a mutant/variant form thereof) or e.g. the wild-type IRS-I (or also a mutant or variant form thereof)- The same applies for any additional marker gene to be tested for example for aromatase. Thus, 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.. In connection with 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. Preferably, 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 terms "template DNA" or "template RNA" refers to DNA or RNA molecules or fragments thereof of any source or nucleotide composition, that comprise a target nucleotide sequence as defined above. The primer or pair of primers can also be used for hybridization experiments as known in the art. Preferably, 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)). Preferably, 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: (1A) x (whereby x is the length of the primer). For example a hepta- or octanucleotide would be sufficient to bind statistically only once on a sequence of 37 kb. However, it is known that 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. Preferably, 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, however, can also be carried out with primers which are shorter or longer.
It is also envisaged that the primer or pair of primers is labeled. The label may, for example, be a radioactive label, such as 32P, 33P or 35S. In a preferred embodiment of the invention, the label is a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or a dye. Further labels had been discussed herein above, in particular in context of labeling "binding molecules", like antibodies. The same applies here for these binding molecules, mutatis mutantis.
As mentioned above, 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).
Optionally, also further markers, like aromatase are measured. It is a particularly preferred embodiment of the present invention to employ aromatase as a first marker for (potential) endometriosis and to then verify the obtained data with a further detection of the herein disclosed expression level of either ezrin/phopho-ezrin or IRS-1/phosphorylated forms of IRS- 1. Therefore, also comprised in said kit 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). In context of this invention, at least two endometriosis-related markers as defined herein are depicted in view of the diagnostic in vitro method as disclosed herein and accordingly, the inventive kit comprises preferably at least two binding molecules binding to protein products of two (different) endometriosis- related marker(s) as defined herein. As documented in the appended examples, in particular the markers ezrin (also phospho-ezrin) and insulin receptor substrate 1 (IRS-I) were found to be reliable diagnostic markers for endometriosis. Accordingly, 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)). Yet, the present invention also provides for 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. Furthermore, the present invention also provides for 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)). As mentioned herein, 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.
A preferred embodiment of the kits, methods and uses provided herein is that the ezrin to be detected is phospho-ezrin.
In another embodiment, the present invention relates to the use of
(a) a binding molecule which specifically binds to or interacts with (i) ezrin protein product or a fragment thereof or a binding molecule which specifically binds to or interacts with (ii) insulin-receptor substrate- 1 (IRS-I) or protein product or a fragment thereof or
(b) 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.
In a particular use of the present invention, a binding molecule specifically binding to or interacting with (i) ezrin and binding molecule specifically interacting with (ii) insulin receptor substrate- 1 (IRS-I) or whereby a binding molecule capable of specifically amplifying and/or detecting the translation product of the (i) ezrin gene and a binding molecule capable of specifically amplifying and/or detecting the translation product of the (ii) insulin receptor substrate- 1 gene (IRS-I gene) is employed for the preparation of a diagnostic composition for the detection of the susceptibility, predisposition, presence and/or potential risk of developing endometriosis.
Accordingly, the 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.
As pointed out above and as illustrated below and in the appended examples, of specific diagnostic value for the determination of the endometriosis status of a patient is 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. 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).
De novo synthesis of ezrin is required for in vitro invasion and is involved in the acquisition of metastatic potential in endometrial cancer cells. 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. On Western blot analysis, 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. After insulin has bound to the extracellular alpha- subunit of the insulin receptor, the receptor undergoes autophosphorylation on several of the tyrosine residues that in turn phosphorylates IRS-I. The tyrosine phosphorylation of IRS-I is important in initiating several biological responses such as stimulation of growth responses and stimulation of glucose uptake into the cells. Indeed, the binding of tyrosine- phosphorylated IRS-I to PI 3-kinase causes a three to five fold increase in the PI 3-kinses's enzymatic activity that leads to the activation of several different serine or threonine kinases. Moreover, IRS-I signals downstream metabolic and mitogenic cellular events that include the MAPK pathway.
Without being bound by theory, an abnormal up-regulation of IRS-I expression leads to an abnormal cell hyperproliferation, thereby enhancing angiogenesis, which induces invasiveness.
Insulin receptor substrate- 1 (IRS-I) 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). As mentioned above for ezrin, 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.
In accordance with this invention, it was surprisingly found that an abnormal and specific production of 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. As documented in the non-limiting examples provided herein, in order to correctly link endometriosis to deregulated ezrin expression and function, 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.
In one embodiment of this invention, it is envisaged that 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. As mentioned herein, 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.
Accordingly, 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).
In accordance with this invention, it is also envisaged that different phosphorylation sites in IRS-I can be efficiently used in a method for detection of endometriosis. Of particular interest in this context are "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. Again, 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. Furthermore, said reliability, sensitivity and specificity may even further be improved if an additional marker, like aromatase, is determined. Accordingly, in context of this invention, 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. Accordingly, in context of this invention the expression level of ezrin/phospho-ezrin or of IRS-I (and phosphorylated forms thereof) 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.
The person skilled in the art is readily in a position to adapt assay systems known in the art to the diagnostic means and methods provided herein.
For example, in order to obtain an efficient and accurate colorimetric assay the following steps may be performed:
• precipitation of and/or elimination of the hemoglobin from the sample to be tested, since this protein may, for example in colorimetric assays, lead to false positive results;
• selection of a buffer that maintains a physiological pH and that does not interfere with the assay, for example the colorimetric assay;
• selection of specific primary detection labels or primary colors for each of the individual different binding molecules, like antibodies.
All the methods for improving given assays are well known in the art and can easily be adapted to any assay. Corresponding assays and assay settings are inter alia described in diagnostic methods are well known in the art and are, inter alia, described in Leonard, Diagnostic Molecular Pathology (2003) Saunders; Ansorge, Molecular Diagnostics (2005) Academic Press; Tsongalis Molecular Diagnostics: for the Clinical Laboratorian (2005) Humana Press; Normansell, The Principles an Practice of Diagnostic Immunology (1994) John Wiley & Sons; Henry, Clinical Diagnosis and Management by Laboratory Methods (2001) Saunders; Pagana, Mosby's Manual of Diagnostic and Laboratory Tests (2005) Mosby; Hommes Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual (1990) Wiley-Liss; Anthony A. Killeen, Molecular Pathology Protocols (Methods in Molecular Medicine) (2001) Humana Press; or Roy W. Stevens, Diagnostic Devices Manual and Directory: Immunology and Microbiology Tests (1986) Marcel Dekker.
The Figures show:
Figure 1:
A. Analysis of the expression of aromatase and ezrin by Western blot
Western blot analysis has been performed on 5 total lysates from eutopic endometrium of endometriosis patients (patient endometrium) and on 5 total lysates from eutopic endometrium of healthy controls (normal endometrium). Both aromatase and ezrin protein amount were restricted to patients' endometrium. Indeed, the phosphorylation of ezrin (on Tyr 354 and/or Thr 567 as shown in SEQ ED NO: 2) was shown only in the endometrium of patients, verified to suffer from endometriosis.
B. Analysis of ezrin expression by immunofluorescence
The analysis has been performed on cultured epithelial cells from eutopic lesion ( 5 human patients) and normal endometrium (5 normal human controls, no endometriosis), respectively. In figure 1, data from one representative patient (endometriosis-positive) and from one prepresentative human, endometriosis-free control sample are shown. Ezrin was strongly expressed in epithelial cells from eutopic lesions, whereas only a basal and unspecific signal arose from the ezrin staining of epithelial cells from normal endometrium. Figure 2:
Analysis of the expression of IRS-I by Western blot
Western blot analysis has been performed on 3 total lysates from eutopic endometrium of different endometriosis patients (patient endometrium) and on 3 total lysates from eutopic endometrium of different healthy controls (normal endometrium). IRS-I expression was restricted to patients' endometrium.
Figure 3:
Detection of aromatase (A), phospho-Ezrin (B) and IRS-I (C) in a patient following endometrioma excision (Pl), in a patient following pelviperitoneal lesion excision in the past (P2) and in two healthy controls (Cl and C2);
The Examples illustrate but do not limit the invention.
Examples:
Materials and Methods
All the methods that are needed for this study are well established in a state of the art laboratory.
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.
Cell culture
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. The separation between epithelial and stromal cells is achieved by sequential sieving through a 150 (100 μm) and a 37 (40 μm) 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). 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.
Western Analysis
Endometrial cells were lysed from all tissue in Frackelton buffer (10 mM Tris, 30 mM Na4P2O7, 5O mM NaCl, and 1% Triton X-100, pH 7.1) supplemented with 10 μg/ml leupeptin, 2 μg/ml aprotinin and 1 μg/ml pepstatin A (protein inhibitors cocktail, Boehringer Ingelheim), 1 mM Phenyl-methyl-sulfonyl-fluoride, 0.5 mM Na3VO4 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). After evaluating the protein concentration, 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. After blocking in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.05% Tween 20) with 5% milk powder or 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.).
Immunofluorescence
The epithelial cells were washed in cold PBS (Gibco), fixed in MetOH 10' at -200C; washed in PBS pH8. They were blocked in 10% goat serum and incubated with ezrin or phospho- ezrin first antibody (40 μl/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 μg/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.
Results
The expression pattern of aromatase, ezrin and IRS-I (also in phosphorylated form(s)) within patient and control endometrium was investigated. 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).
Without being bound by theory, 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. In particular, 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.
The Rho family proteins as well as ezrin have been shown to be involved in other processes, apart from cell motility. In particular, 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.
Indeed, cell migration is not the only one of the known features of endometriosis; another consists in the up-regulation of the rate of cell proliferation, linked to enhanced activation of both MAPK and PDK pathways. Presently, the details of the mechanism that links the up- regulation of estrogen production to the abnormal cell proliferation are still unclear. However, estradiol up-regulates, through PDK and its down-stream target Akt, Insulin Receptor Substrate- 1 (IRS-I) already at the transcriptional level and more interestingly IRS-I seems to be involved in the control of RhoA expression, either by increasing or inhibiting it depending on the investigated tissue. Moreover, ezrin phosphorylation of Tyr354 promotes the activation of PDK, showing a possible linkage among ezrin, IRS-I and aromatase at the level of PDK/ Akt phosphorylation. IRS-I that normally mediates insulin-signaling pathway, has been shown as a partner of ERa, which is up-regulated in eutopic lesions. In particular, ERa keeps the stability of IRS- 1 by inhibiting the process of its ubiquitination. In accordance with this invention, it was observed that 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);
• activating sites upon binding of the ligand (Tyr612; Tyr632, Tyr662, Tyr732 Tyr896 and Tyr 941);
• activating sites due to the substitution of an Ophe with a Tyr (Tyr465, Tyr612, Tyr632, Tyr662, Tyr941 and Tyr986);
• sites that bind upon phosphorylation to η-14-3-3 (Ser272-4, Ser374 and Ser641);
• negative sites on the according SEQ ID NO: 4 regulated by:
1. JNK (Ser307);
2. IKK, JNK and mTOR (Ser312);
3. SK-2 (Ser794);
4. PKC (Ser616);
5. mTOR (Ser636);
6. PKCΘ (Serl l05).
IRS-I positively feeds-back the PI3K/ Akt pathway, which yields three distinct effects on the epithelial component of the eutopic lesions:
• to potentiate the antiapoptotic effects through PI3K and Akt;
• to enhance the rate of cell migration through RhoA;
• to increase the rate of cell proliferation by c-src and PAK activation. Example 2: Detection of aromatase, phospho-ezrin and IRS-I in menstrual discharge as markers for endometriosis
Materials and Methods
Sample collection
Four samples were collected from either extra- or intravaginal plugs to gather menstrual discharge. The plugs were immediately frozen (-200C) at patient's home, transported on ice toward our lab where they were stored at -8O0C before being further processed.
Of these four plugs two plugs are from healthy controls;
-one plug is from a patient who underwent laparoscopic ovarian endometriosis
(endometrioma) excision some months prior to plug collection;
-one plug is from a patient who underwent laparoscopic excision of pelviperitoneal lesions >
10 years prior to plug collection.
Western Blot
The protein extraction from menstrual discharge was achieved as described:
1 mL of blood containing debris from menstrual discharge was lysed in 1.5 mL of ice-cold
Frackelton lysis buffer, made up of: 10 mM Tris-HCl, pH 7.05, 50 mM NaCl,
30 mM sodium pyrophosphate, 50 mM NaF, 1% Triton X-100, 100 μM Na3Vo4 *
1 tablet of Protease inhibitor cocktail (Roche)*, 1 mM PMSF,*
Insoluble material was removed by centrifuging the samples at 20000 g, 20 min, at 4°C.
Quantification of the amount of protein contained in each sample was achieved by Bradford assay. Before the test, the Protein Assay Stock Solution (Biorad) was diluted 1:5 in milliQ water and 2 μl of each sample was added to 1 mL of the diluted Bradford solution. After vortexing, the absorbance was determined (595 nm).
Once that the protein concentration of each sample was measured, 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
Bromphenol, dissolved in milliQ water, brought to pH 6.8 and added up with DTT 100 mM just before using), heated to 75°C for 5 min and stored at -200C.
added immediately before using The sample lysates were loaded on a SDS-Page gel and run for Ih at 200V.
After SDS-PAGE, 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.
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
V; Sigma Aldrich) and 0.1 mM NaN3 (Table 1).
The day after the membrane was washed three times in TTBS and subsequently incubated for
Ih with the appropriate secondary antibody (Table 2), diluted in TTBS added up with 5% powder milk.
Thereafter, the membrane was washed three times with TTBS for about 30 min and finally rinsed in TBS.
Table 1: List of primary antibodies
Table 2: List of the secondary antibodies
After the incubation with the primary and the secondary antibody, 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.
Results.
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.
The results provided herein (in particular in the appended Figure 3) document that a detection of the (elevated) expression level of endometriosis by using ezrin/phospho-ezrin and/or IRS-I, either alone or in combination as a marker/marker system, or also in combination with the detection of the (elevated) expression level of aromatase is not only but can also be carried out with high specificity. It is also documented that this analysis can successfully be carried out in samples obtained form menstrual blood. Furthermore, this invention demonstrates that the use of aromatase as the only (biochemical) marker for endometriosis can not be considered as specific enough. Yet, the concomitant use of ezrin/phospho-ezrin increased the specificity of the method. The additional use of a third marker, i.e. IRS-I, provides for a further increase of the specificity. 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. However, in accordance with this invention also ezrin/phospho-ezrin alone and or IRS-I (also in "activated" phosphorylated form) alone is offered as valuable tool in the diagnosis of endometriosis. As shown herein, 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.
Additional References
American Society for Reproductive Medicine. 1997
Revised American society for reproductive medicine classification of endometriosis: 1996.
Fertil Steril; 67: 817-821.
Bruner, 1995 Proc Natl Acad Sci USA; 92: 7362-7366.
Bulun, 1993 J Clin Endocrinol Metab; 76: 1458-1463.
Bulun, 1994 J Clin Endocrinol Metab; 78: 736-743.
Donnez, 1995 Hum Reprod; 11: 224-228.
Donnez, 1996 Hum Reprod; 11: 641-646.
Giudice, 2004 Lancet; 364(9447): 1789-99. Review.
Huang, October 1996 In: Proceedings of the American Society for Reproductive Medicine
Meeting: Boston, MA.
Huang, 1998 J Clin Endcrinol Metab; 83: 538-541.
Khorram, 1993 Am J Obstet Gyn; 169: 1545-1549.
Kitawaki, 1997 Biol Reprod; 57: 514-519.
Kitawaki, 1999 Fertil Steril; 72: 1100-1106.
Kokorine, 1997 Fertil Steril; 68: 246-251.
Nisolle, 1993 Fertil Steril; 59: 681-684.
Nisolle, 1997 Fertil Steril; 68: 585-596.
Noble, 1996 J Clin Endocrinol Metab; 81: 174-179.
Noble, 1997 J Clin Endocrinol Metab; 82: 600-606.
Olive, 1993 N Engl J Med; 328: 1759-1769.
Osteen, 1996 Semin Reprod Endocrinol; 15: 301-308.
Ohtani, 2002 Cancer Lett; 179(1): 79-86.
Shaw RW (ed.). Endometriosis, Current understanding and management, 1995, pp. 112-113,
Cambridge, Blackwell Science.
Wolfler, 2005 Hum Reprod; 20 (6): 1702-08.
Yamamoto, 1993 Am J Obstet Gynecol; 169: 734-738.
Zeitoun, 1999 Fertil Steril; 72: 961-969.

Claims

Claims
1. 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
(a) assaying in a biological sample to be analysed the expression level of ezrin and/or of insulin receptor substrate- 1 (IRS-I); and
(b) comparing said expression level of said ezrin and/or insulin receptor substrate-1 (IRS-I) to a baseline expression level established and/or obtainable by assaying the expression level of ezrin and/or insulin receptor substrate-1 (IRS-I) in (an) endometriosis-free reference sample(s).
2. The method of claim 1, wherein said method 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).
3. The method of claim 1 or 2, wherein said biological sample and/or said endometriosis-free reference sample(s) is/are of human origin.
4. The method of any one of claims 1 to 3, wherein said ezrin is phospho-ezrin and/or wherein said IRS-I is a phosphorylated IRS-I.
5. The method of any one of claims 1 to 4, wherein said ezrin is human ezrin and/or wherein said insulin receptor substrate-1 (IRS-I) is human insulin receptor substrate- 1 (human IRS-I).
6. The method of any one of claims 1 to 5, wherein said expression levels to be measured comprise the measurement of expressed protein(s) or the measurement of translation product(s).
7. 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:
(a) obtaining a biological sample from said female subject;
(b) assaying said sample for the expression level of at least two endometriosis- related markers; and
(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:
(i) ezrin; and
(ii) insulin receptor substrate- 1 (IRS-I).
8. The method of claim 7, further comprising 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).
9. The method of claim 7 or 8, wherein said ezrin is phospho-ezrin.
10. The method of any one of claims 1 to 9, wherein said biological sample is selected from the group consisting of cells, tissues or body fluids and/or wherein said biological sample is a biopsy sample.
11. The method of claim 10, wherein said cells are cells derived from endometrial glands, endometrial stroma, celomic epithelium, Muellerian duct, endometrial vasculature, endometrial lymphatic system, or the endometrial immune system, wherein said tissue is derived from the ovaries, the rectovaginal septum, the peritonaeum parietale, the peritonaeum viscerale of intraabdominal organs, or of extraabdominal sites, and wherein said body fluids are selected from blood, urine, menstrual discharge, vaginal mucus.
12. The method of claim 11, wherein said blood is menstrual blood.
13. The method of claim 11 or 12, wherein said blood or said menstrual discharge is obtained from extra-or intravaginal plugs.
14. A kit useful for determining the susceptibility, predisposition, presence and/or potential risk of developing endometriosis in a female subject, said kit comprising: at least are binding molecule specifically binding to and/or interacting with
(i) ezrin protein product or a fragment thereof; or
(ii) insulin receptor substrate-1 (IRS-I) protein product or a fragment thereof; or at least one binding molecule capable of specifically amplifying or detecting the translation product of (i) ezrin gene; or (ii) insulin receptor substrate-1 (IRS-I) gene.
15. The kit of claim 14 comprising 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).
16. The kit of claim 14 or 15 further comprising at least a binding molecule specifically binding to (iii) aromatase protein product or fragment thereof or further comprising at least one binding molecule capable of specifically amplifying or detecting the translation product of the aromatase gene.
17. The kit of any one of claims 14 to 15 further comprising at least one substance for the detection of said specifically binding molecules to
(i) ezrin protein product or fragment thereof;
(ii) insulin receptor substrate-1 (IRS-I) protein product or fragment thereof; and/or (iii) aromatase protein product or fragment thereof.
18. The kit of any one of claims 14 to 17, wherein said binding molecule is selected from the group consisting of antibodies, affybodies, trinectins, anticalins, and aptamers.
19. The kit of claim 18, wherein said antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, antibody fragments and antibody-fusion proteins.
20. The kit of any one of claims 14 to 16, wherein said at least one binding molecule capable of specifically amplifying and/or detecting an translation product is a primer or a probe.
21. The kit of any one of claims 14 to 20, wherein said ezrin is phospho-ezrin.
22. Use of
(a) a binding molecule which specifically binds to or interacts with (i) ezrin protein product or a fragment thereof or a binding molecule which specifically binds to or interacts with (ii) insulin-receptor substrate-1 (IRS-I) or protein product or a fragment thereof or
(b) 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.
23. The use of claim 22, whereby a binding molecule specifically binding to or interacting with (i) ezrin and binding molecule specifically interacting with (ii) insulin receptor substrate- 1 (IRS-I) or whereby a binding molecule capable of specifically amplifying and/or detecting the translation product of the (i) ezrin gene and a binding molecule capable of specifically amplifying and/or detecting the translation product of the (ii) insulin receptor substrate- 1 gene (IRS-I gene) is employed.
24. The use of claim 22 or 23, wherein furthermore 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 diagnostic composition.
25. The use of any one of claims 22 to 23, wherein said ezrin is phospho-ezrin.
EP06791999A 2005-09-14 2006-09-12 Means and methods for diagnosing endometriosis Withdrawn EP1931993A2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012002929A1 (en) 2012-02-14 2013-08-14 Jürgen Lewald Analyzing a peripheral blood sample of a female subject based on concentration of a steroid hormone that indicates endometriosis, comprising e.g. testosterone, progesterone, cortisol, dehydroepiandrosterone and androstenedione

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009140126A1 (en) * 2008-05-14 2009-11-19 Juneau Biosciences, Llc Method of administering a therapeutic
WO2010124101A2 (en) 2009-04-22 2010-10-28 Juneau Biosciences, Llc Genetic markers associated with endometriosis and use thereof
RU2675525C1 (en) * 2017-12-12 2018-12-19 Публичное акционерное общество "СИБУР Холдинг" Copolymers of coupped dienes and vinylaromatic monomers and method for their production; rubber mixtures based on the specified copolymers

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU760613B2 (en) * 1998-06-04 2003-05-22 Reprogen, Inc. Use of prothymosin in the diagnosis and treatment of endometriosis
ATE374370T1 (en) * 1999-01-25 2007-10-15 Siemens Medical Solutions Diag METHOD FOR DIAGNOSIS OF ENDOMETRIOSIS
FR2826010B1 (en) * 2001-06-14 2005-02-25 Mahmood Salman Al ANTISENSE OLIGONUCLEOTIDES CAPABLE OF INHIBITING THE FORMATION OF CAPILLARY TUBES BY ENDOTHELIAL CELLS
US20040105860A1 (en) * 2002-08-01 2004-06-03 Losordo Douglas W. Cell modulation using a cytoskeletal protein
US20090297523A1 (en) * 2004-07-27 2009-12-03 Yale University Erm family binding agents and their use in diagnosis and treatment of proliferative conditions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007031277A3 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012002929A1 (en) 2012-02-14 2013-08-14 Jürgen Lewald Analyzing a peripheral blood sample of a female subject based on concentration of a steroid hormone that indicates endometriosis, comprising e.g. testosterone, progesterone, cortisol, dehydroepiandrosterone and androstenedione

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