A method for diagnosing endometriosis and classifying the stage of endometriosis

Abstract

The present invention relates to a method for diagnosing whether a subject has endometriosis, classifying the stage of endometriosis, determining the therapeutic effect of a treatment regimen for endometriosis, and monitoring the progression of endometriosis in a subject by determining the amount or concentration of c-Kit in a sample of the subject and comparing the determined level with a reference value.

Description

Technical Field

[0001] The present invention relates to a method for diagnosing whether a subject has endometriosis, a method for assessing the risk of a subject having endometriosis, a method for classifying the stage of endometriosis, a method for determining the therapeutic effect of a treatment regimen for endometriosis, and a method for monitoring the progression of endometriosis in a subject by determining the amount or concentration of c-Kit in a sample of the subject and comparing the determined level to a reference value.

Background Art

[0002] Background of the Invention Endometriosis is a chronic disorder defined by the growth of endometrial glands and extrauterine stromal-like lesions (Liu et al., 2011). The lesions can be peritoneal lesions, superficial implants or cysts of the ovary, or deep infiltrative disease. It results from eutopic endometrial cells characterized by increased proliferative and adhesive properties (Liu et al., 2011). The increased cell survival rate in eutopic endometrium is the result of decreased apoptosis and increased cell proliferation (Johnson et al., 2005). Endometriosis affects 5-8% of all women of reproductive age and 70% of women with chronic pelvic pain. The number of patients with endometriosis is estimated to be 176 million women worldwide (Adamson et al. J Endometr. 2010;2:3-6). Many of these women are often diagnosed late, resulting in unnecessary pain and a decline in quality of life. In patients aged 18-45, the delay is 7-10 years. Since most women with endometriosis present with symptoms during adolescence, early referral, diagnosis, disease identification, and treatment may reduce pain and prevent disease progression. Barriers to early diagnosis include the high cost of diagnosis and treatment in adolescent patients and the presentation of indistinguishable symptoms such as cyclic and acyclic pain (Parasar et al. Curr Obstet Gynecol Rep. 2017;6:34-41).

[0003] The gold standard for the diagnosis of endometriosis is visualization by laparoscopy and subsequent histological confirmation. Until now, there has been no non-invasive method for the diagnosis of endometriosis (Hsu et al. Clin Obstet Gynecol 2010;53:413-419). During diagnostic laparoscopy, a gynecologist with training and skills in laparoscopic surgery for endometriosis needs to perform a thorough pelvic examination (NICE guideline NG73, 2017). Surgical visualization requires sufficient expertise, training, and skills for a reliable diagnosis. The fact that laparoscopy, which has been avoided by physicians for as long as possible, is required for diagnosis leads to a diagnostic delay of 7 to 10 years. The lack of non-invasive diagnostic tests has been a major cause of the long delay between the onset of symptoms and the definitive diagnosis of endometriosis (Signorile and Baldi. J Cell Physiol 2014;229:1731-1735). Therefore, the medical need for non-invasive tests for the diagnosis of endometriosis, especially for early, minimal, and mild endometriosis (revised American Society for Reproductive Medicine rASRM stages I-II), is unmet.

[0004] Non-invasive diagnosis of endometriosis enables early diagnosis and treatment, potentially improving quality of life and reducing the social costs associated with endometriosis, and has therefore been selected as a research priority by the World Endometriosis Society (WES) and the World Endometriosis Research Foundation (WERF) (Fassbender et al., Springer, Peripheral Blood Biomarkers for Endometriosis. 2017). Therefore, non-invasive tools for diagnosing endometriosis could ultimately improve quality of life and facilitate early diagnosis and intervention that may preserve fertility (Parasar et al. Curr Obstet Gynecol Rep. 2017;6:34-41).

[0005] Blood biomarkers are essential for reducing the diagnostic time lag of endometriosis, which requires laparoscopy. CA-125 is one of the most commonly used blood biomarkers, but its diagnostic utility is limited to endometriosis rASRM stages III and IV (Nisenblat et al., Cochrane Database of Systematic Reviews. 2016;5:CD012179).

[0006] c-Kit is an oncogene that encodes a 145 kd transmembrane tyrosine kinase receptor (CD117) (Roskoski et al., 2005). Stem cell factor (SCF) is the cognate ligand of the c-Kit receptor. SCF-induced c-Kit signaling has been shown to play important roles in a diverse range of biological functions (Sharkey et al., 1994). Serum levels of c-Kit are increased in proliferative mast cell disorders, suggesting the presence of a c-Kit release pathway in mast cells (Cruz et al. J Biol Chem. 2004). It is essential for the survival, differentiation, and mobilization of multiple cell types, including bone marrow, erythroid, megakaryocytic, lymphocytic, germ, and melanocyte progenitor cells (Besmer et al., 1991, Lyam et al., 1998, Papayannopoulou et al., 1991). Several studies have highlighted that dysregulation of c-Kit function is associated with the development of various diseases, including cancer (Vliagoftis et al., 1997, Miettinen et al., 2005). For example, in mast cells where c-Kit regulates differentiation, activation, and homeostasis (Gilfillan et al., 2011), mutations within the c-Kit receptor can disrupt the signaling cascade, induce constitutive receptor activation independent of SCF, and potentially cause mastocytosis (Cardet et al., 2013). Furthermore, SCF binding to c-Kit in endothelial cells disrupts endothelial adhesion junctions and enhances vascular leakage (Kim et al., 2014). Alterations in c-Kit signaling are also associated with carcinogenesis and the development of a wide variety of benign and malignant neoplastic disorders, including gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), mast cell leukemia (MCL), and melanoma (Ha et al., 2010, Matsuda et al., 1993, Sakurai et al., 1999, Ayatollahi et al., 2017, Boissan et al., 2000, Montone et al., 1997).

[0007] The level / presence of biomarkers can vary when measured in tissue or serum. For example, complement component C7 and complement component C4 were overexpressed in the ectopic endometrium of women with endometriosis compared to the eutopic endometrium of control women without endometriosis (Ahn et al. Fertil Steril 2016; Eyster et al. Fertil Steril 2007). However, neither an increase in serum complement component C7 protein nor an increase in complement component C4 protein was observed in the circulating blood (Hever et al. PNAS 2007). Brain-derived neurotrophic factor (BDNF) mRNA expression levels were higher in ovarian endometriotic lesions than in eutopic endometrium (Wang et al. Journal of Ovarian Research 2022). However, serum BDNF did not differ significantly in women with endometriosis compared to control women without endometriosis (Perricos et al. Exp Biol Med (Maywood) 2018). Furthermore, in breast cancer, molecular markers such as CEA(O), ERβ, CK19, c-Myc were significantly different in the blood of patients compared to normal controls, while no significant differences were observed in tissue samples for these markers.

[0008] Therefore, the locally altered expression of biomarkers in tissue does not significantly convert to different levels of these biomarkers in the circulating blood in a 1:1 manner.

[0009] There is a high need for non-invasive diagnosis of endometriosis using biomarkers, which would enable reliable and early risk assessment and / or identification of patients presenting with signs and symptoms of endometriosis.

[0010] Therefore, the present invention provides means and methods to meet these needs. SUMMARY OF THE INVENTION

[0011] Summary of the Invention In a first aspect, the present invention relates to a method for diagnosing endometriosis in a subject, the method comprising a) Determining the level of c-Kit in a biological fluid sample derived from the subject; b) Comparing the level of c-Kit with the c-Kit levels of at least one appropriate reference value; c) If the comparison in step b) indicates that the subject has an increased level of c-Kit compared to the appropriate reference value, identifying the subject as having endometriosis comprising.

[0012] In a second aspect, the present invention relates to a method for classifying the stage of endometriosis in a subject, the method comprising: a) Determining the level of c-Kit in a biological fluid sample derived from the subject; b) Comparing the level of c-Kit with the c-Kit levels of at least one appropriate reference value; c) If the comparison in step b) indicates that the subject has an increased or decreased level of c-Kit compared to the c-Kit levels of at least one appropriate reference value, classifying the stage of endometriosis in the subject comprising.

[0013] In a third aspect, the present invention relates to a method for monitoring the progression of endometriosis in a subject, the method comprising: i. Determining the level of c-Kit in a biological fluid sample derived from the subject according to steps a)-b) of the above method for diagnosing and classifying endometriosis; ii. Repeating step i) at regular time intervals using a biological fluid sample obtained from the subject during or after treatment, and iii. Comparing the level of c-Kit identified in i) with the level of c-Kit identified in ii), wherein the change in the level of c-Kit from i) to ii) indicates a change in the progression of endometriosis in the subject comprising.

[0014] In a fourth aspect, the present invention relates to a method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject, the method comprising: i. A step of determining the level of c-Kit in a biological fluid sample derived from a subject according to steps a) to b) of the above method for diagnosing and classifying endometriosis; ii. A step of repeating step i) at regular time intervals using a biological fluid sample obtained from the subject during or after treatment, and iii. A step of comparing the level of c-Kit determined in step i) with that determined in step ii), and specifying that the treatment regimen has a therapeutic effect when the level of c-Kit has decreased after treatment is included.

Brief Description of Drawings

[0015] Hereinafter, embodiments of the present invention will be further described with reference to the accompanying drawings.

[0016]

Figure 1

Figure 2A

Figure 2B

DETAILED DESCRIPTION OF THE INVENTION

[0017] DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the surprising discovery that elevated levels of c-Kit in serum are associated with endometriosis. c-Kit levels increase in all stages I, II, III, and IV, with stage II showing the highest levels of c-Kit, and stages I, III, and IV showing equivalent level increases, which confers diagnostic potential for early detection of endometriosis in serum c-Kit.

[0018] The inventors are the first to show that c-Kit measured in serum increases in women with endometriosis compared to controls. The levels of c-Kit increase specifically in endometriosis stage I, particularly stage II (minimal / mild endometriosis).

[0019] The medical need for a non-invasive test for the reliable diagnosis and / or classification of endometriosis, particularly early endometriosis, is not met. c-Kit has the advantage of a non-invasive blood-based test for identifying women with early endometriosis.

[0020] The inventors investigated the levels of c-Kit in sera obtained from women with endometriosis. Surprisingly, the inventors found that elevated levels of c-Kit can be detected in serum samples from women with endometriosis. In particular, due to the fact that elevated c-Kit levels can already be detected in women in the early stages of endometriosis, this marker becomes a useful tool for diagnosing endometriosis at an early stage. Thus, an assay that enables determination of the level of c-Kit in such biological fluids can be useful for the diagnosis and / or classification, prognosis, and stratification of patients for the treatment of endometriosis.

[0021] The data presented herein indicate that determination of c-Kit levels in sera provides a means for diagnosing endometriosis, for stratifying the risk of having endometriosis, and for classifying the stage of endometriosis in a subject (e.g., when determining c-Kit levels at regular intervals in a subject or by comparing the determined values to values at known stages). This also enables monitoring of the progression of endometriosis and / or evaluation of a treatment regimen.

[0022] The data presented herein also indicate that determination of c-Kit levels in sera provides a means for more accurately detecting early stages of endometriosis and control samples than CA-125.

[0023] Definitions The word "comprise", and variations such as "comprises" and "comprising", are understood to mean the inclusion of the stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0024] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0025] Concentrations, levels, amounts, and other numerical data may be expressed or presented in the form of a "range" in this specification. It should be understood that such a range format is merely used for convenience and brevity, and thus, it should be interpreted flexibly to include not only the numerical values explicitly listed as the boundaries of the range, but also all of the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly listed. By way of illustration, the numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly listed values of 150 mg to 600 mg, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes individual values such as 150, 160, 170, 180, 190, ··· 580, 590, 600 mg, and sub-ranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges that list only one numerical value. Furthermore, such an interpretation should apply regardless of the width of the range or the property being described.

[0026] The term "about", when used in relation to a numerical value, means a value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0027] Generally, the described methods are in vitro methods that are performed using samples already obtained from a subject (i.e., the samples are provided for the method, and the steps performed to obtain the samples from the subject are not included as part of the method). Thus, the method may include the step of providing a biological fluid sample from the subject. As used herein, "providing," "obtaining," or "obtaining" in relation to a sample can be any means by which one comes to own the sample, by "direct" or "indirect" means. Obtaining a sample directly means performing a process to obtain the sample (e.g., performing a physical method such as extraction). Obtaining a sample indirectly refers to receiving the sample from another entity or source (e.g., a third-party laboratory that directly obtained the sample).

[0028] The methods provided herein include providing a biological fluid sample (e.g., a blood sample) from a subject. The sample tested by the methods described herein is also referred to as a "test sample."

[0029] As used herein, the terms "biological (fluid) sample," "test sample," and "sample" are used interchangeably, and variations thereof refer to a sample obtained from or derived from a subject. For the purposes described herein, the sample is or includes a biological fluid (also referred to herein as a body fluid) sample.

[0030] Examples of samples include, but are not limited to, body fluid samples such as blood, serum, plasma, synovial fluid, interstitial fluid, capillary blood, peritoneal fluid, menstrual fluid, urine, saliva, and lymphatic fluid. Analysis of the sample can be achieved on a chemical basis. Chemical analysis includes, but is not limited to, detection of the presence or absence of specific indicators or changes in their amount, concentration, or level.

[0031] The sample is an in vitro sample and will be analyzed in vitro and not returned to the body.

[0032] The blood sample can be a whole blood sample or a processed blood sample, such as serum, plasma, etc. Methods for obtaining a biological fluid sample (e.g., whole blood, serum, plasma, etc.) from a subject are well known in the art. For example, methods for obtaining a blood sample from a subject are well known and include established techniques used in venipuncture. The obtained blood sample can be further processed using standard techniques to obtain, for example, a serum sample or a plasma sample. Advantageously, methods for obtaining a biological fluid sample from a subject are typically minimally invasive or non-invasive.

[0033] A whole blood sample is defined as a blood sample that has been collected from the body and in which components (such as platelets or plasma) have not been (substantially) removed. In other words, the relative ratios of the components in a whole blood sample are substantially the same as those in the blood in the body. In this context, "substantially the same" allows for very small changes in the relative ratios of the components of whole blood, such as changes of up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, etc. Whole blood contains both the cellular and fluid portions of the blood. Thus, a whole blood sample can also be defined as a blood sample that has (substantially) all of its cellular components in plasma and in which the cellular components (i.e., including at least the required white blood cells, red blood cells, and blood platelets) are intact.

[0034] In a preferred example, the biological fluid sample is serum.

[0035] Methods for analyzing (and optionally isolating, concentrating, or extracting) protein biomarkers from blood, plasma, serum, saliva, and urine samples have been previously described. See, for example, Heitzer, E., Haque, I.S., Roberts, C.E.S. et al. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet 20, 71 - 88 (2019).

[0036] In the context of the present invention, the term "biomarker" refers to a substance within a biological system that is used as an indicator of the biological state of that system. In the art, the term "biomarker" may also be applied to means for detecting such endogenous substances (e.g., antibodies, nucleic acid probes, etc., imaging systems). In the context of the present invention, the term "biomarker" shall be applied only to substances and not to detection means. Thus, a biomarker can be any kind of molecule present in a living body, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (blood glucose, insulin, estrogen, etc.), molecules characteristic of a specific modification of another molecule (e.g., the sugar moiety or phosphoryl residue of a protein, the methyl residue of genomic DNA), or a substance internalized by an organism or a metabolite of such a substance. A biomarker is an organic biomolecule (e.g., protein, polypeptide, peptide, its isomers, its immunologically detectable fragments, corresponding nucleic acid molecules (e.g., mRNA, cDNA, etc.)) that is differentially present in a sample taken from a subject having a disease as compared to a subject not having the disease. A biomarker is differentially present when the average or median level of the biomarker in different groups is calculated to be statistically related. Common tests for statistical significance include, inter alia, t-tests (e.g., Student's t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, receiver operating characteristic (ROC curve), precision, and odds ratio. A biomarker, alone or in combination, provides a measure of the relative risk that a subject belongs to one phenotypic state or another.

[0037] Thus, these are useful as markers for disease (diagnosis), therapeutic efficacy of drugs, and drug toxicity.

[0038] Typically, the biomarkers referred to herein are measured at the protein level.

[0039] The methods provided herein refer to "determining" the levels of one or more proteins. As will be apparent to those skilled in the art, the levels of one or more proteins are typically "determined" by measuring the levels of the proteins in a sample. Thus, the term "determine" can be replaced herein with the term "measure" or "determine by measuring".

[0040] The terms "determining" or "assessing" as used herein also refer to assessing / determining whether a patient has endometriosis. Thus, assessments as used herein include determining the amount or concentration of c-Kit in a patient's sample and comparing the determined amount or concentration to a reference to diagnose endometriosis, assessing the risk that a subject has endometriosis, selecting a treatment for endometriosis, and monitoring patients who have or are being treated for endometriosis. Typically, the assessments referred to in accordance with the present invention are assessments of the presence of endometriosis.

[0041] The terms "measure", "measuring" or "determine" preferably include qualitative, semi-quantitative, or quantitative measurements.

[0042] Conventional "determination" methods can include sending a clinical sample to a commercial laboratory to measure the level of a biomarker in a biological fluid sample or using a commercially available assay kit to measure the level of a biomarker in a biological fluid sample. Exemplary kits and suppliers will be apparent to those skilled in the art. In various examples, biomarkers can be determined, detected, and / or quantified using ELISA assays or lateral flow devices for point-of-care use, among others, as well as spot-check colorimetric tests.

[0043] As used herein, the terms "level" or "amount" include the absolute amount of a biomarker referred to herein, the relative amount or concentration of the biomarker, and any value or parameter that can be correlated with or derived from them. Such values or parameters include intensity signal values derived from all specific physical or chemical properties obtained from the peptide by direct measurement, for example, intensity values in a mass spectrum or an NMR spectrum. Further included are values or parameters obtained by indirect measurement as specified elsewhere herein, for example, the amount of response measured by a biological readout system in response to a peptide, or the intensity signal obtained from a specifically bound ligand. It should be understood that values correlated with the above-described amounts or parameters can also be obtained by all standard mathematical operations.

[0044] The level of a biomarker present in a biological fluid sample can be determined, for example, by assaying the amount of the protein biomarker present in the sample. Assays for measuring the amount of a specific protein are well known in the art and include direct or indirect measurements. The level of the protein biomarker in the sample can also be determined by determining the level of the protein biomarker activity in the sample. Thus, the "level" of a protein includes both the amount of the protein itself and its activity level.

[0045] For example, the level of a protein biomarker in a biological fluid sample can be determined (e.g., measured) by any suitable method and material known in the art, including a process selected from the group consisting of, for example, mass spectrometry, immunoassay, enzyme assay, spectrophotometry, colorimetry, fluorometry, bacterial assay, protein microarray, compound separation techniques, or other known techniques for determining the presence and / or amount of an analyte. Examples of related techniques include enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, and lateral flow (e.g., use of a lateral flow device (LFD) utilizing a membrane-bound antibody specific for a protein biomarker).

[0046] Preferably, the level of a protein biomarker in a biological fluid sample is measured by ELISA or lateral flow.

[0047] As used herein, the term "at regular intervals" refers to the periodic determination of c-Kit levels in the same subject after a predetermined time. Since all subjects are different, the rate of disease onset can vary from subject to subject. In one subject, the progression of the disease can progress from stage I to stage IV within a few years, while in another subject, it can remain stagnant at one stage for several years. Therefore, in order to obtain a continuous picture of the progression of c-Kit levels in a subject and be able to assign the resulting c-Kit levels to the corresponding disease stages I, II, III, and IV, the intervals should be selected so that the stages of disease progression are not skipped. Preferably, the c-Kit levels of a subject are determined every 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0048] The term "c-Kit" refers to the gene encoding the receptor tyrosine kinase protein CD117, also known as c-kit and SCFR. This is the cellular receptor for stem cell factor (SCF), which is also called c-kit ligand, mast cell growth factor, and steel factor. This receptor-ligand system plays an essential role in germ cell development, melanogenesis, hematopoiesis, and tumor formation. CD117 is expressed in lymphocytes, bone marrow, erythrocytes, and megakaryocyte progenitor cells, NK cells, germ cells, melanocytes, glial cells, vascular smooth muscle cells, placenta, and epithelial cells. Altered levels and mutations in CD117 are associated with several types of cancer, including lung, breast, gastrointestinal stromal, and germ cell tumors. Multiple transcript variants encoding different isoforms have been found for this gene. SCF exerts its biological effects through binding to the receptor c-Kit, which is, in turn, expressed in mast cells, hematopoietic stem cells, and germ line cells [Cho NH. et al. (2004) Fertil Steril 81:403-7]. The amino acid sequence of human c-Kit can be accessed via UniProt (see UniProtKB-P10721(KIT_HUMAN)_HUMAN). There are three isoforms of c-Kit described with identifiers P10721-1, P10721-2, and P10721-4.

[0049] "CA-125", a carbohydrate antigen 125, sometimes also called cancer antigen 125 or tumor antigen 125, is a mucin-type glycoprotein produced by the MUC16 gene and associated with the cell membrane. CA-125 is a biomarker for epithelial ovarian cancer derived from epithelial cells of the coelomic epithelium, including the endometrium, fallopian tubes, ovaries, and peritoneum. The diagnostic use of CA-125 is limited to endometriosis stages III and IV (moderate and severe endometriosis) with moderate sensitivity.

[0050] The "symptoms" of a disease are indications of the disease that can be noticed by the tissue, organ, or organism having such a disease, and include, but are not limited to, pain, weakness, tenderness, tension, stiffness, and spasms of the tissue, organ, or individual. The "signs" or "signals" of a disease include, but are not limited to, the presence, increase or elevation, decrease or decline of specific indicators such as biomarkers or molecular markers, changes or alterations such as changes (change) or alterations (alteration), or the onset, presence, or worsening of symptoms. The symptoms of pain include, but are not limited to, unpleasant sensations that can be felt as persistent or various burning pains, throbbing pains, itching, or stabbing pains.

[0051] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition, particularly an abnormal medical condition such as a disease or injury where a tissue, organ, or individual can no longer perform its function efficiently. Although not necessarily so, typically, a disease is associated with specific symptoms or signs indicating the presence of such a disease. Thus, the presence of such symptoms or signs can indicate the tissue, organ, or individual suffering from the disease. Changes in these symptoms or signs can indicate the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs that can indicate the "worsening" or "improvement" of the disease. The "worsening" of a disease is characterized by a decrease in the ability of the tissue, organ, or organism to perform its function efficiently, whereas the "improvement" of a disease is typically characterized by an increase in the ability of the tissue, organ, or individual to perform its function efficiently. A tissue, organ, or individual at "risk of developing" a disease is in a healthy state but shows the potential for the disease to manifest. Typically, the risk of developing a disease is associated with early or weak signs or symptoms of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neuropathies, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0052] Endometriosis is a chronic hormone-dependent inflammatory disease characterized by lesions of endometrial-like tissue outside the uterus. The clinical symptoms of endometriosis vary widely among patients. Endometriosis patients often present symptoms such as mid-cycle bleeding, dysmenorrhea (menstrual difficulty), dyspareunia (painful intercourse), painful defecation (defecation disorder), and painful urination (urination disorder). Pelvic pain due to endometriosis is usually chronic (lasting more than 6 months) and is associated with dysmenorrhea (in 50 - 90% of cases), dyspareunia, deep pelvic pain, and lower abdominal pain with or without back and loin pain. The pain can be unpredictable and intermittent or continuous throughout the menstrual cycle, dull, throbbing, or sharp, and can be worsened by physical activity. Symptoms related to the bladder and bowel (nausea, bloating, and early satiety) are typically cyclical. The pain may gradually worsen and its nature may change. Rarely, women complain of a burning sensation or hypersensitivity, symptoms suggesting a neuropathic component. Often, endometriosis can be asymptomatic and only draw the clinician's attention during the evaluation of infertility (Sinaii et al. Fertil Steril. 2008;89(3):538 - 545). In women with endometriosis, the monthly fecundity rate is reduced (2 - 10%) compared to fertile men and women (15 - 20%). Endometriosis weakens fertility but usually does not completely prevent fertilization (Fadhlaoui et al. Front Surg. 2014;1:24).

[0053] The most commonly affected sites of endometriosis are the pelvic organs and the peritoneum, although in some cases other parts of the body such as the lungs may be affected. The extent of the disease varies from several small lesions in otherwise normal pelvic viscera to large ovarian endometriotic cysts (endometriomas) and / or extensive fibrosis and adhesion formation that cause significant distortion of the pelvic anatomy. Based on location, endometriotic lesions can be classified as peritoneal endometriosis, ovarian endometriotic cysts (endometriomas), deep nodules (deep infiltrating endometriosis), and adenomyosis (Kennedy et al. Hum Reprod. 2005;20(10):2698-2704). Deep infiltrating endometriosis is considered any symptom of endometriosis located outside the rectovaginal septum and the surface tissue of the vaginal vault, pelvic wall, parametrium, bowel, uterus, or bladder (Halis et al. (2010). Deutsches Arzteblatt International, 107(25), 446). Endometriosis may also involve the diaphragm (diaphragmatic endometriosis) or the chest (thoracic endometriosis) (Nezhat et al. JSLS 2019).

[0054] The term "rASRM stage" or "rASRM staging" refers to a revised classification system established by the American Society for Reproductive Medicine (ASRM) to describe the severity of endometriosis based on surgical (laparoscopic) findings. The classification is based on the morphology of peritoneal and pelvic implants such as red, white, and black lesions, and should include the incidence rate of each lesion. Attention should be paid to the number, size, and location of endometrial implants, plaques, endometriomas, and adhesions. Endometriosis in the intestine, urinary tract, fallopian tubes, vagina, cervix, skin, or other sites should be recorded according to the ASRM guidelines. The staging of endometriosis according to the ASRM guidelines is stages I, II, III, and IV determined based on a point score, corresponding to minimal, mild, moderate, and severe endometriosis. Endometriosis at rASRM stages I and II (minimal to mild endometriosis) is characterized by superficial peritoneal endometriosis, the possible presence of small deep lesions, the absence of endometriomas, and / or mild adhesions of the membrane. Endometriosis at rASRM stages III and IV (moderate to severe endometriosis) is characterized by superficial peritoneal endometriosis, deep infiltrating endometriosis with moderate to extensive adhesions between the uterus and the intestine, and / or the presence of endometrioma cysts with moderate to extensive adhesions involving the ovaries and fallopian tubes.

[0055] The term "VAS", which is a visual analog scale, is a tool for assessing the intensity of pain. The VAS consists of a 10-cm horizontal line, the ends of which are marked as "no pain" and "the greatest possible pain". Each patient checks their pain level on the line and measures the distance in centimeters from the "no pain" on the far left to the marked point, obtaining a pain score of 0 to 10. "No pain" corresponds to a pain score of 0, and "the greatest possible pain" corresponds to a pain score of 10. In women with endometriosis, dysmenorrhea is associated with the highest perception of pain, and the average VAS score is approximately 6 (Cozzolino et al. Rev Bras Ginecol Obstet 2019;41(3):170-175).

[0056] The subject may sometimes be referred to as a patient in this specification. The terms "subject", "individual", and "patient" are used interchangeably in this specification and refer to an animal, preferably a mammal, more typically a human. The patient is preferably a human female. Since endometriosis develops from the onset of menstruation, it is necessary to diagnose it at a young age. Therefore, the patient is preferably a young or adolescent human female aged 12 to 24 years. In embodiments of the present invention, the patient is a young or adolescent human female. The subject may be symptomatic (e.g., the subject exhibits symptoms associated with endometriosis) or the subject may be asymptomatic (e.g., the subject does not show symptoms associated with endometriosis). The subject may be diagnosed with endometriosis, at risk of developing endometriosis, or exhibit symptoms of endometriosis. The subject may have endometriosis or may be suspected of having it (e.g., showing symptoms or a medical history that implies or suggests it).

[0057] Accordingly, in some instances, the subject has endometriosis (and the method diagnoses, identifies (or detects) that the subject has endometriosis). In this context, the terms "diagnose", "identify", and "detect" can be used interchangeably.

[0058] In certain instances, the subject has endometriosis at an early stage (stage I or stage II).

[0059] The patients to be investigated by the method of the present invention are patients suspected of having endometriosis. As used herein, the term "suspected of having endometriosis" means that the patient exhibits clinical parameters, signs, and / or symptoms of endometriosis. Thus, the patients according to the present invention are typically patients who have or are suspected of having endometriosis. Patients with suspected endometriosis with signs and symptoms: dysmenorrhea (painful menstrual periods), dysuria (painful urination), defecation disorders (difficulty defecating or painful defecation), dyspareunia (pain during or after sexual intercourse), and chronic abdominal pain / pelvic pain unrelated to the menstrual cycle, heavy menstrual bleeding, long menstrual periods, infertility, fatigue, periodic lung problems (pneumothorax), periodic cough, chest pain or blood in sputum (hemoptysis), shoulder tip pain, painful rectal bleeding or presence of blood in urine (hematuria), and periodic swelling and pain of scars (EHRE Information on Endometriosis, 2022 www.eshre.eu / guidelines).

[0060] Alternatively, c-Kit levels are routinely determined as part of a screening test without any suspicion of endometriosis, but can detect asymptomatic endometriosis at an early stage.

[0061] By detecting elevated levels of c-Kit in a subject, the suspicion that the subject has endometriosis is confirmed, and the subject has a high risk of having endometriosis. In particular, when the subject already exhibits clinical parameters, signs, and / or symptoms of endometriosis, the determination of an increase in c-Kit levels confirms the presence of endometriosis.

[0062] As used herein, the term "compare" refers to comparing the amount / level of a biomarker in a sample from a subject to a reference amount or reference value of the biomarker as specified elsewhere herein. Comparing, as used herein, typically refers to comparing corresponding parameters or values. For example, an absolute amount is compared to an absolute reference amount, while a concentration is compared to a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared to the same type of intensity signal obtained from a reference sample. The comparison may be performed manually or with computer assistance. Thus, the comparison can be carried out by a computing device. The measured or detected amount of a biomarker in a sample from a subject and the value of the reference amount can, for example, be compared to each other, and the comparison can be automatically performed by a computer program that executes an algorithm for the comparison. The computer program that performs the evaluation provides the desired assessment in a suitable output format. In a computer-assisted comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the result of the comparison, i.e., automatically provide the desired assessment in a suitable output format. In a computer-assisted comparison, the value of the measured amount may be compared by a computer program to a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the comparison result, i.e., automatically provide the desired assessment in a suitable output format.

[0063] The terms "(appropriate) reference value" or "reference sample" or "control sample", as used herein, refer to a sample that is analyzed in substantially the same manner as the sample of interest and whose information is compared to the information of the sample of interest. Thereby, the reference sample provides a reference by which to evaluate the information obtained from the sample of interest. A control sample may be derived from the body fluid of a healthy individual, particularly serum or plasma for non-invasive testing, thereby providing a reference for the health status of a tissue, organ or individual. A difference between the state of a normal reference sample and the state of the sample of interest may indicate the presence or further progression of such a disease or disorder. A control sample may be derived from a tissue, organ, or individual having an abnormality or disease, thereby providing a reference for the diseased state of the tissue, organ, or individual. A difference between the state of a normal or abnormal reference sample and the state of the sample of interest may indicate the absence or improvement of such a disease or disorder.

[0064] A reference sample may also be derived from the same tissue, organ, or individual as the sample of interest, but taken at an earlier time point. A difference between the state of a previously taken reference sample and the state of the sample of interest may indicate the progression of a disease, i.e., improvement or worsening of the disease over time, which may thereby enable classification of the stage of endometriosis as stage I, II, III or IV.

[0065] A determined value can be compared to two or more (appropriate) reference values that may be of different types. For example, a determined value can be compared to one or more values obtained from the same subject at an earlier time point and, in parallel, to one or more values obtained from other subjects (of known stage of endometriosis).

[0066] The control sample can be an internal or external control sample. An internal control sample is used, i.e., the marker level is evaluated in the test sample as well as in one or more other samples taken from the same subject to determine whether there is a change in the level of the marker. For an external control sample, the presence or amount of a marker in a sample derived from an individual is compared with the presence or amount of the marker in an individual known to have or be at risk of having a given condition; or an individual known not to have a given condition, i.e., a "normal individual".

[0067] It will be understood by those skilled in the art that such an external control sample may be obtained from a single individual or from a reference population of matched age and free of confounding diseases. Typically, a "reference value" is set using samples from 100 individuals well-characterized from an appropriate reference population. However, the reference population can also be selected to consist of 20, 30, 50, 200, 500, or 1000 individuals. Healthy individuals represent a preferred reference population for setting control values.

[0068] For example, the marker concentration of a patient sample can be compared to a concentration known to be associated with a particular course of a particular disease. For example, it can be compared to a concentration known to be associated with a particular stage of endometriosis. Typically, the marker concentration of a sample is directly or indirectly correlated with a diagnosis, and the marker concentration is used, for example, to determine whether an individual is at risk of a particular pain associated with that disease. Alternatively, the concentration of a marker can be compared to the concentration of the marker obtained from the same subject at an earlier time point. Alternatively, the marker concentration of a sample can be compared to, for example, the concentration of a marker known to be relevant in the response to treatment in a particular disease, the diagnosis of a particular disease, the assessment of the severity of a particular disease, the guidance for selecting an appropriate drug for a particular disease, the determination of the risk of disease progression, or the follow-up of a patient. Depending on the intended diagnostic use, an appropriate control sample is selected, and a control value or reference value for the marker is set therein. As will also be apparent to those skilled in the art, the absolute value of the marker set in the control sample depends on the assay used.

[0069] The most common control samples for the methods described herein and / or reference values derived therefrom are obtained from, but not limited to, "non-pathological controls" and "symptomatic controls". The corresponding subjects from which these samples are obtained are "non-pathological subjects" and "symptomatic subjects", respectively.

[0070] A "non-pathological control" refers to a control sample of a subject who does not have endometriosis and does not exhibit any symptoms that may be associated with endometriosis (e.g., menstrual / abdominal pain, uterine / ovarian cysts or cancer, etc.).

[0071] A "symptomatic control" refers to a control sample of a subject who is afflicted with symptoms normally associated with endometriosis (e.g., menstrual / abdominal pain, infertility, etc.), but in whom endometriosis can be excluded based on laparoscopy and no tissue changes (e.g., uterine / ovarian cysts or cancer) can be observed.

[0072] "Controls with benign findings" refers to a group of samples from subjects who have tissue changes (e.g., uterine / ovarian cysts, cancer, fibromas), but which are not similar to endometriosis. Further, these subjects can be symptomatic (e.g., menstrual / abdominal pain, infertility, etc.) or asymptomatic, and this can also change over time.

[0073] Control samples can be assayed separately or simultaneously, before, after, or at the same time as the test samples. The control values used for comparison with the test samples may be values calculated as the average or median of a plurality (e.g., 2 or more, 5 or more, 10 or more, groups, etc.) of control samples. Alternatively, the control samples can be samples that are derived from (i.e., are a mixture of) more than one (e.g., 2 or more, 5 or more, 10 or more, groups, etc.) individuals who do not have endometriosis (or are "symptomatic controls").

[0074] Thus, in one example, the control samples are obtained from control subjects who do not have endometriosis ("non-pathological controls"). In a further example, the control samples are obtained from subjects who are "symptomatic controls".

[0075] For the classification of the stage of endometriosis, comparisons can be made, or must be made, between the test sample and several different control samples in order to be able to assign the results to a specific stage. For example, a comparison with a non-pathological sample and a stage II sample. Or a comparison with a non-pathological sample and stage II and stage IV samples. This can also, in combination with surgery, confirm or determine the specific stage of the disease.

[0076] Alternatively, the level of a biomarker (e.g., a protein) in a biological fluid sample can be compared to a predetermined reference level of the biomarker of interest. As used herein, "predetermined reference level" refers to a predetermined cut-off value, i.e., the level of the biomarker obtained from a reference database that can be used to generate a score for statistically predicting endometriosis. In one example, the predetermined reference level is the average or median level of the biomarker in at least one individual not suffering from endometriosis and derived from the same species. The predetermined reference value can be calculated as the average or median obtained from a group or population of individuals not suffering from endometriosis. For example, the predetermined reference value can be calculated as the average or median obtained from a group or population of individuals that are "symptomatic controls". The individual or group of individuals can be of the same age, or in the same health state or condition as the subject from whom the test sample is obtained.

[0077] Thus, in one example, the predetermined reference level is the average level of the biomarker in a control subject not having endometriosis. In a further example, the predetermined reference level is the average level of the biomarker in a subject that is a "symptomatic control".

[0078] Typically, in a method for diagnosing endometriosis in a subject, the control sample or predetermined reference is obtained from an individual or group of individuals different from the subject being tested (i.e., the subject from whom the test sample is obtained / provided). In such an example, the control or predetermined reference is used as a baseline for determining whether the subject being tested has endometriosis.

[0079] In an alternative example, the control or predetermined reference value may be obtained from the same individual as the test sample, but at an earlier time point. This is particularly relevant to the methods described herein for classifying the stage of endometriosis, determining the progression in a subject, determining the therapeutic effect of a treatment regimen for endometriosis, and / or determining compliance or adherence of a subject to a prescribed treatment regimen for endometriosis. For this purpose, the sample is obtained from the same biological fluid of the same subject, and the biological fluid is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, and preferably, the biological fluid sample is serum.

[0080] In such an example, a control sample or predetermined reference level is used to determine any change in the level of a biomarker over a time interval in the same subject. Thus, the predetermined reference level or control sample can be obtained from the same subject as the test sample obtained, for example, at an earlier time point. This earlier time point can be before the subject is diagnosed with endometriosis.

[0081] The predetermined level can be a single cut-off value such as a median or an average. This can be a range of cut-off (or threshold) values such as a confidence interval. This can be established based on a comparison group, such as when the risk of one defined group is twice as high or low as that of another defined group (e.g., about 2-fold, 4-fold, 8-fold, 16-fold or more). It can be, for example, a range in which a population of subjects (e.g., control subjects) is equally (or unequally) divided into groups such as a low-risk group, a medium-risk group, and a high-risk group, or into quartiles, where the lowest quartile consists of the lowest-risk subjects and the highest quartile consists of the highest-risk subjects, or into n-quartiles (i.e., n equally spaced intervals), where the lowest n-quartile consists of the lowest-risk subjects and the highest n-quartile consists of the highest-risk subjects. Further, the reference can be a calculated reference, most preferably an average or a median, for the relative or absolute amount of a biomarker in a population of individuals including the subject being investigated. How to calculate a suitable reference value, preferably an average or a median, is well known in the art.

[0082] Thus, in some cases, a level of a protein biomarker in a subject being equal to or higher than a level of the biomarker in a control sample or a predetermined reference level indicates a clinical condition (e.g., indicates endometriosis). In other cases, a level of a biomarker in a subject being equal to or lower than a level of the biomarker in a control sample or a predetermined reference level indicates a particular stage of endometriosis.

[0083] Typically, but not necessarily, being sufficiently different to distinguish from a control subject, larger or smaller means being statistically significantly larger or statistically significantly smaller. When a level of a biomarker in a subject being equal to a level of the biomarker in a control subject indicates a stage of endometriosis, "equal" refers to being approximately equal (e.g., having no statistical difference).

[0084] The predetermined value can depend on a particular population of selected subjects (e.g., human subjects). For example, a population that appears healthy has "normal" ranges of protein biomarkers that are different from a population of subjects having or likely having endometriosis. Thus, the selected predetermined value can take into account the category (e.g., healthy, diseased, stage of disease) that the subject (e.g., human subject) falls into.

[0085] Appropriate ranges and categories can be selected by routine experimentation by those of ordinary skill in the art.

[0086] Preferably, the level of a particular biomarker detected in a sample (e.g., test sample, control sample, etc.) can be normalized by adjusting the measured level (amount or activity) of the biomarker using the level of a reference protein in the same sample, where the reference protein is not the marker itself (it is, for example, a constitutively expressed protein). This normalization enables comparison of the level of a biomarker in one sample to another sample, or between samples from different sources. The normalized level can then optionally be compared to a reference value or control. For example, when measuring a protein biomarker in a whole blood sample, the biomarker may be expressed as an absolute concentration or may be normalized to a known protein constitutively expressed in whole blood, such as albumin, immunoglobulin, or plasma protein concentration.

[0087] For example, when measuring a protein biomarker in a serum (or plasma) sample, the biomarker may be expressed as an absolute concentration or may be normalized to a known protein constitutively expressed in serum (or plasma).

[0088] To identify an increase or decrease in the level of one or more biomarkers in a subject's sample, the level of a biomarker in a test sample can be compared to the level of the same biomarker in a control sample or a predetermined reference level of the same biomarker.

[0089] In the methods described herein, if the comparison (comparison between the level of a biomarker in a control sample / predetermined reference value and a subject's test sample) indicates that the subject has an increased level of c-Kit compared to the control sample or predetermined reference level, the subject can be identified as having endometriosis.

[0090] As used herein, the term "classify" refers to the classification of the stage of endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), which consists of four stages I, II, III, and IV (Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril. 1997). As shown in the data provided herein, the c-Kit levels in stages I, III, and IV are approximately the same and are increased compared to the control. The c-Kit level in stage II is increased compared to stages I, III, and IV. This makes it possible to classify the state of endometriosis according to the criteria for classifying it into one of four stages based on the determined c-Kit level, based on a comparison of the sample investigated with a reference value or at least one sample investigated at a previous stage. Depending on the reference value / reference sample, the difference between the c-Kit level in the sample of interest and the reference sample can be used to assign the stage of endometriosis according to r-ASRM to a specific stage. For example, if the c-Kit reference value is derived from a sample of a patient with stage II endometriosis, a decrease in the c-Kit level in the sample of interest means that the patient has stage I, III, or IV endometriosis, or that the patient does not have endometriosis. This depends on the comparison of the obtained c-Kit value with the reference value of an individual not suffering from endometriosis. Or, if the reference value is derived from a sample of a patient with stage I, III, or IV endometriosis and the level of the sample of interest is elevated, it means that the patient has stage II endometriosis. Classification can also be performed if a range of c-Kit levels assigned to a specific stage of endometriosis is defined. Thus, the fact that the value obtained is, for example, within the range assigned to stage I means that this patient suffers from endometriosis stage I.

[0091] Furthermore, it will be appreciated that when the risk of deterioration of the health state is predicted, typically the prediction is made within prediction windows of 6 months and 2 years. More typically, the prediction window relates to a time window of about 6 to 12 months for non-invasive tests that depend on symptoms such as pelvic pain.

[0092] As will be appreciated by those skilled in the art, the assessments made in accordance with the present invention, although preferred, may not usually be correct for 100% of the subjects investigated. This term typically requires that a statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be readily determined by those skilled in the art using various well-known statistical assessment tools, such as determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically assumed confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. p-values are typically 0.2, 0.1, 0.05.

[0093] The terms "reduced" or "decreased" level of an indicator refer to the level of such an indicator in a sample that is reduced compared to a reference or reference sample. The terms "decrease", "decreased", "reduced", "reduction", or "down-regulated", "lower" are all generally used herein to mean a statistically significant amount of decrease. However, to avoid misunderstanding, "reduced", "reduction", "decreased" or "decrease" means a decrease of at least 10%, for example at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of 100% or less (i.e., a level that does not exist compared to the reference / control sample), or any reduction between 10 and 100% compared to the reference level / control, or a reduction of at least about 0.5-fold, or at least about 1.0-fold, or at least about 1.2-fold, or at least about 1.5-fold, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any reduction of 1.0-fold to 10-fold or more compared to the reference level / control.

[0094] The terms "elevated" or "increased" levels of an indicator / (bio)marker refer to the level of such an indicator in a sample that is higher compared to a reference (value) or reference sample. For example, in a body fluid sample of an individual suffering from a given disease, a protein that can be detected in a higher amount in the same body fluid sample of an individual not suffering from the disease has an elevated level. The terms "increased", "increase" or "upregulated", "higher" are all used herein to generally mean a statistically significant amount of increase, and to avoid any misunderstanding, the terms "increased", "increase" mean an increase of at least 10% compared to the reference level / control, for example at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of 100% or less, or any increase between 10 - 100% compared to the reference level / control, or an increase of at least about 0.5-fold, or at least about 1.0-fold, or at least about 1.2-fold, or at least about 1.5-fold, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase of 1.0-fold to 10-fold or more compared to the reference level / control.

[0095] The term "immunoglobulin (Ig)" as used herein refers to immunity conferred by glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulin" is attached to the membrane of effector cells by their transmembrane regions and includes, but is not limited to, molecules such as B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta2 microglobulin (about 2M), CD3, CD4, and CD8.

[0096] Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can thus be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains denoted by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present determines the class of the antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), each playing a different role and directing an appropriate immune response against different types of antigens. The different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal tract, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD mainly functions as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved in allergic reactions through binding to allergens that cause the release of histamine from mast cells and basophils. IgE is also involved in defense against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody-based immunity against invading pathogens and is the only antibody isotype that can pass through the placenta to confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum, with IgG1 being the most abundant (about 66%), followed by IgG2 (about 23%), IgG3 (about 7%), and IgG4 (about 4%).The biological profiles of different IgG classes are determined by the structures of their respective hinge regions. IgM is expressed on the surface of B cells in monomeric form and as a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens at the initial stage of B cell-mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are found not only as monomers but also form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM in teleost fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically made up of four polypeptide chains, two identical heavy chains and two identical light chains, linked via disulfide bonds, and resemble a "Y"-shaped macromolecule. Each of the chains contains several immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domain consists of a two-layer sandwich of 7-9 anti-parallel strands arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CH1, CH2, CH3) domains and one of which is a variable domain (VH). The light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). By way of example, the human IgG heavy chain is composed of four Ig domains linked in the order V H -CH1-CH2-CH3 (also referred to as V H -Cγ1-Cγ2-Cγ3) from the N-terminus to the C-terminus, while the human IgG light chain is composed of two immunoglobulin domains linked in the order VL-CL from the N-terminus to the C-terminus and is either of the kappa or lambda type (VK-CK or Vλ-Cλ). By way of example, the constant chain of human IgG contains 447 amino acids. Throughout this specification and the claims, the numbering of amino acid positions in immunoglobulins is Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C., (1991) Sequences of proteins of immunological interest, 5.th The numbering of the "EU index" as in the U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD. The "Kabat EU index" refers to the residue numbers of human IgG1 EU antibodies. Thus, the CH domains in the context of IgG are as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0097] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form that is not the antibody fragment defined below. Specifically, these terms refer to an antibody having a heavy chain that includes the Fc region.

[0098] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab fragments" (also referred to as "Fab portions" or "Fab regions"), each having a single antigen-binding site, and the remaining "Fc fragment" (also referred to as "Fc portion" or "Fc region"), named to reflect its ability to readily crystallize. The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In IgG, IgA, and IgD isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody, while in IgM and IgE isotypes, the Fc region contains three heavy-chain constant domains (CH2-4) in each polypeptide chain. Furthermore, smaller immunoglobulin molecules exist naturally or have been constructed artificially. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of the Ig molecule, while the "F(ab')2 fragment" is understood to contain two Fab' fragments that are chemically linked or linked via disulfide bonds. "Single-domain antibodies (sdAbs)" (Desmyter et al. (1996) Nat. Struct. Biol. 3:803-811) and "nanobodies" contain only a single VH domain, while "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Bispecific single-chain variable fragments (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH regions and two VL regions, yielding a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is the generation of an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Usually, a linker of 5 residues in length is used to generate these dimers. This type is known as a "diabody".A shorter linker (one or two amino acids) between the VH and VL domains results in the formation of single-specificity trimers, so-called "triabodies" or "tribodies". Bispecific diabodies are formed by expressing on chains having the sequences VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. A single-chain diabody (scDb) contains VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids. A "bispecific T cell engager (BiTE)" is a fusion protein consisting of two scFvs of different antibodies, where one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). A bispecific affinity retargeting molecule ("DART" molecule) is a diabody further stabilized by a C-terminal disulfide bridge.

[0099] Accordingly, the term "antibody fragment" refers to a portion of an intact antibody, preferably including its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments; diabodies; sdAbs, nanobodies, scFvs, di-scFvs, tandem scFvs, triabodies, diabodies, scDbs, BiTEs, and DARTs.

[0100] The term "binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including, but not limited to, assays based on surface plasmon resonance (e.g., BIAcore assays as described in PCT Publication No. WO 2005 / 012359); enzyme-linked immunosorbent assay (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies generally tend to bind antigen slowly and dissociate readily, while high-affinity antibodies generally tend to bind antigen rapidly and remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present invention.

[0101] "Sandwich immunoassay" is widely used for the detection of the analyte of interest. In such an assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, a sandwich assay requires that the antibodies for capture and detection bind to different, non-overlapping epitopes of the analyte of interest. By appropriate means, such a sandwich complex is measured, whereby the analyte is quantified. In a typical sandwich-type assay, a first antibody that is bound to a solid phase or can bind to a solid phase, and a second antibody that is detectably labeled each bind to the analyte at different, non-overlapping epitopes. A binding agent (e.g., an antibody) specific for the first analyte is either covalently bound or passively bound to a solid surface. The solid surface is typically glass or a polymer, and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be a tube, beads, the disk of a microplate, or any other surface suitable for the performance of an immunoassay. The binding process is well known in the art and generally consists of cross-linking covalent bonding or physical adsorption, and the polymer-antibody complex is washed in the preparation of the test sample. Next, an aliquot of the sample to be tested is added to the solid-phase complex and incubated for a period sufficient to allow binding between the first antibody or the capturing antibody and the corresponding antigen (e.g., 2 to 40 minutes or, more conveniently, overnight) and under appropriate conditions (e.g., room temperature to 40 °C, e.g., 25 °C to 37 °C (including both ends)). Following the incubation period, the solid phase containing the first antibody or the capturing antibody and the antigen bound to the antibody can be washed and incubated with a secondary antibody or a labeled antibody that binds to another epitope on the antigen. The second antibody is bound to a reporter molecule used to indicate the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0102] A very widely used alternative sandwich assay format involves the use of a solid phase coated with the first partner of a binding pair, e.g., microparticles coated with paramagnetic streptavidin. Such microparticles are incubated with a binder specific for an analyte bound to the second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte to which the second partner of the binding pair is bound to a binder specific for the analyte, and a binder specific for a second detectable-labeled analyte. As will be apparent to those skilled in the art, these components are incubated for a period sufficient to bind the labeled antibody to the solid phase microparticles via the analyte, the binder specific for the analyte (bound to) the second partner of the binding pair, and the first partner of the binding pair, under appropriate conditions. Optionally, such an assay may include one or more washing steps.

[0103] The term "detectably labeled" encompasses labels that can be detected directly or indirectly.

[0104] A directly detectable label either provides a detectable signal or the label interacts with a second label to modify the detectable signal provided by the first or second label to give, for example, FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al. “Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable to labeling. In one embodiment, detectably labeled refers to a label that either provides or can be induced to provide a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label.

[0105] A number of labels (also referred to as dyes) are available and can generally be classified into the following categories, all of which are grouped together and each of which represents an embodiment according to the present disclosure.

[0106] (a) Fluorescent dyes Fluorescent dyes are described, for example, by Briggs et al., “Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058).

[0107] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels such as FITC, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine-type labels such as TAMRA, dansyl, lysamine, cyanine, phycoerythrin, Texas Red, and analogs thereof. Fluorescent labels can be attached to aldehyde groups contained in target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0108] (b) Luminescent dyes Luminescent dyes or labels can be further subclassified into chemiluminescent dyes and electrochemiluminescent dyes.

[0109] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, selenotellurazine and analogs, dioxetane, peroxysuccinic acid and peroxysuccinic acid derivatives. For immunoassay procedures, mainly acridinium-based labels are used (a detailed overview is shown in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0110] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a sensitive and selective method. It combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying electrode potential. Generally, ruthenium complexes, especially [Ru(Bpy)3]2+ (which emits photons at about 620 nm) regenerated using TPA (tripropylamine) at the liquid phase or liquid-solid interface, are used as ECL labels.

[0111] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is caused by a voltage applied to a working electrode in a particular pattern at a particular time. The light generated by the label is measured and indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, reference is made to U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Application Publication WO 90 / 05296, PCT Application Publication WO 92 / 14139, PCT Application Publication WO 90 / 05301, PCT Application Publication WO 96 / 24690, PCT Application Publication US 95 / 03190, PCT US Patent Application Publication 97 / 16942, PCT Application Publication US 96 / 06763, PCT Application Publication WO 95 / 08644, PCT Application Publication WO 96 / 06946, PCT Application Publication WO 96 / 33411, PCT Application Publication WO 87 / 06706, PCT Application Publication WO 96 / 39534, PCT Application Publication WO 96 / 41175, PCT Application Publication WO 96 / 40978, PCT / US97 / 03653 and U.S. Patent Application Publication 08 / 437,348 (U.S. Patent No. 5,679,519). Also, reference is made to the 1994 review by Knight, et al. (Analyst, 1994, 119:879-890) on the analytical uses of ECL and the references cited therein. In one embodiment, the method according to the present specification is carried out using an electrochemiluminescence label.

[0112] In recent years, iridium-based ECL labels have also been described (WO 2012107419).

[0113] (c) The radioactive label employs a radioisotope (radionuclide), for example, 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0114] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,461; Hnatowich et al., J. Immunol. Methods 65 (1983) 147-157; Meares et al., Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al., Bioconjugate Chem. 1 (1990) 59-65; Meares et al., J. Cancer (1990), Suppl. 10:21-26; Izard et al., Bioconjugate Chem. 3 (1992) 346-350; Nikula et al., Nucl. Med. Biol. 22 (1995) 387-90; Camera et al., Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al., J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Camera et al., J. Nucl. Med. 21 (1994) 640-646; Ruegg et al., Cancer Res. 50 (1990) 4221-4226; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Lee et al., Cancer Res. 61 (2001) 4474-4482; Mitchell, et al., J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al Bioconjugate Chem. 10 (1999) 103-111; Miederer et al., J. Nucl. Med. 45 (2004) 129-137; DeNardo et al., Clinical Cancer Research 4 (1998) 2483-90; Blend et al., Cancer Biotherapy & Radiopharmaceuticals 18 (2003) 355-363; Nikula et al J. Nucl.Med. 40(1999)166 - 76; Kobayashi et al., J. Nucl. Med. 39(1998)829 - 36; Mardirossian et al., Nucl. Med. Biol. 20(1993)65 - 74; Roselli et al., Cancer Biotherapy & Radiopharmaceuticals, 14(1999)209 - 20).

[0115] The methods described herein can further include selecting and optionally administering a treatment regimen for a subject based on a diagnosis (i.e., based on a comparison of biomarker levels to a reference value / level / control). Treatment can include, for example, surgery and, optionally, therapy, or combinations thereof. However, in some cases, immediate treatment may not be required and the subject can be selected for active surveillance.

[0116] As used herein, the terms "active surveillance", "monitoring", and "close observation" are used interchangeably herein and mean closely monitoring a patient's condition without performing any treatment until symptoms appear or change.

[0117] As used herein, the terms "treat", "treating", and "treatment" are construed to include an intervention performed with the intent to change the condition, disorder, or symptom (i.e., in this case, endometriosis). Thus, "treatment" refers to a therapeutic treatment aimed at slowing down (mitigating) the targeted condition, disorder, or symptom. Thus, "treatment" includes a reduction, delay, or inhibition of the symptoms of endometriosis, such as at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, compared to the symptoms before treatment. In the context of endometriosis, appropriate treatment may include analgesics, hormonal therapy (such as hormonal contraceptives), gonadotropin-releasing hormone (GnRH) agonists, and / or surgery. (Longo, D. L. et al., 2020. Sc.D. N Engl J Med, 382, pp. 1244 - 56).

[0118] As used herein, the term "surgery" applies to surgical methods performed for the removal of endometrial tissue, such as laparoscopy or nerve-sparing surgery, for example.

[0119] As used herein, the term "treatment" includes drug-based treatment, radiation, hormone therapy, cryosurgery, chemotherapy, immunotherapy, biological therapy, and high-intensity focused ultrasound. Drug-based treatment for endometriosis can be, for example, the inhibition or targeting of neurogenic inflammation, and / or analgesics and / or hormone therapy.

[0120] The type of treatment varies depending on the specific form and / or stage of endometriosis that the subject has or is suspected of having.

[0121] Embodiments The inventors have surprisingly identified a new protein biomarker, c-Kit, that increases in the biological fluids, particularly serum, of women with endometriosis, especially women in the early stages of endometriosis.

[0122] The biomarker c-Kit can be used to diagnose endometriosis or to classify the stage of endometriosis in a subject as compared to a control (e.g., a non-pathological or symptomatic subject).

[0123] In particular, serum c-Kit can be used as a blood biomarker for the early diagnosis and risk stratification of endometriosis. Further, serum c-Kit can be used to select patients with disease stages I and II for early medical management of endometriosis. Thus, it can significantly reduce the delay in the diagnosis of endometriosis, improve the lives of patients, and reduce the economic burden.

[0124] The biomarker can advantageously be used in any of the methods, kits, assays, or uses described herein.

[0125] Method for diagnosing endometriosis in a subject In a first aspect, the present invention relates to a method for diagnosing endometriosis in a subject, the method comprising: a) determining the level of c-Kit in a biological fluid sample from the subject; b) comparing the level of c-Kit with the c-Kit level of at least one appropriate reference value; c) identifying the subject as having endometriosis if the comparison in step b) indicates that the subject has an increased level of c-Kit compared to the appropriate reference value. Including.

[0126] In embodiments, an increase in the level or amount or concentration of c-Kit in a biological fluid sample of a patient indicates the presence of endometriosis in the subject. In particular, if the amount or concentration of c-Kit in a biological fluid sample of the subject is higher than the amount or concentration of c-Kit according to the reference value, the amount or concentration of c-Kit in the biological fluid sample of the subject indicates the presence of endometriosis in the subject.

[0127] In certain embodiments, at least one appropriate reference value is i. The level of c-Kit in a non-pathological subject or a symptomatic subject, or ii. The average level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects, or a combination thereof, or at least one appropriate reference value is iii. A predetermined value of the level of c-Kit in a non-pathological subject or a symptomatic subject, or iv. A predetermined average value of the level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects, or a combination thereof.

[0128] In certain embodiments, the inventors were able to detect an increase in serum c-Kit in the early stages of endometriosis, followed by a decrease in the later stages of endometriosis. However, the amount or concentration of c-Kit in the later stages is still higher compared to the control levels.

[0129] In particular, an amount of c-Kit increased by 50% or more indicates the presence or risk of developing endometriosis. In particular, an amount of c-Kit increased by 100% or more indicates the presence of endometriosis. In particular, an amount of c-Kit increased by 150% or more indicates the presence of endometriosis. In particular, an amount of c-Kit increased by 200% or more indicates endometriosis.

[0130] Suitably, the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, and preferably the biological fluid sample is serum.

[0131] In embodiments, the sample is an in vitro sample, i.e., analyzed in vitro and not returned to the subject's body. In embodiments, the method of the present invention is an in vitro method.

[0132] In certain embodiments, the subject is a human subject. In certain embodiments, the patient is a human female subject. In certain embodiments, the subject is a young or adolescent human female. In particular, the subject is a subject who is potentially susceptible to endometriosis due to their physical condition.

[0133] In an embodiment, the diagnosis is performed independently of the rASRM stage classification. In particular, the assessment is performed without performing a laparoscopy. In particular, the assessment is performed without using laparoscopy and / or the rASRM stage classification to assess the presence or severity of endometriosis in a patient.

[0134] In an embodiment, the diagnosed endometriosis is selected from the group consisting of stage I endometriosis according to the rASRM stage classification, stage II endometriosis according to the rASRM stage classification, stage III endometriosis according to the rASRM stage classification, and stage IV endometriosis according to the rASRM stage classification. In a particular embodiment, the diagnosed endometriosis is stage I, stage II, stage III, or stage IV endometriosis.

[0135] In an embodiment, the endometriosis is early-stage endometriosis, particularly stage I endometriosis according to the rASRM stage classification or stage II endometriosis according to the rASRM stage classification.

[0136] In a particular embodiment, the diagnosed endometriosis is stage III or stage IV endometriosis.

[0137] In an embodiment, the endometriosis to be assessed is selected from the group consisting of peritoneal endometriosis, endometrioma, deep infiltrating endometriosis (DIE), and adenomyosis.

[0138] In a particular embodiment, the diagnosed endometriosis is peritoneal endometriosis. In another particular embodiment, the diagnosed endometriosis is stage I or stage II peritoneal endometriosis according to the rASRM stage classification.

[0139] In another embodiment, the method further comprises selecting a treatment regimen for a subject based on a comparison of the level of c-Kit with a control sample or a predetermined reference level. In certain embodiments, the method further comprises administering the selected treatment regimen to the subject, and optionally, the selected treatment regimen comprises a drug-based therapy and / or a surgical procedure (laparoscopy). The drug-based therapy for endometriosis can be, for example, analgesic hormone therapy and / or by surgery.

[0140] Depending on whether the diagnosis suggests a rather severe disease stage, those skilled in the art are well aware of how to select the most appropriate and promising treatment regimen.

[0141] In embodiments according to the invention, the protein level of c-Kit is optionally determined using a process selected from the following, namely ELISA assay, immunoblotting, lateral flow assay, protein microarray, and mass spectrometry.

[0142] In embodiments, the amount of c-Kit is determined using an antibody, particularly a monoclonal antibody. In embodiments, step a) of determining the amount of c-Kit in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassays are selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0143] In certain embodiments, step a) of determining the level of c-Kit in a subject sample comprises i) incubating the subject sample with one or more antibodies that specifically bind to c-Kit, thereby generating a complex of the antibody and c-Kit, and ii) quantifying the complex formed in step i), thereby quantifying the amount of c-Kit in the subject sample comprises.

[0144] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to c-Kit. As will be apparent to those skilled in the art, the sample can be contacted with the first anti-c-Kit antibody, the second anti-c-Kit antibody, or both the first and second antibodies in any desired order, i.e., first the first antibody and then the second antibody, or first the second antibody and then the first antibody, or simultaneously, for a time and under conditions sufficient to form a first anti-c-Kit antibody / c-Kit / second anti-c-Kit antibody complex. As will be readily understood by those skilled in the art, establishing the time and conditions appropriate or sufficient for the formation of a complex between a specific anti-c-Kit antibody and the c-Kit antigen / analyte (= anti-c-Kit complex), or for the formation of a secondary or sandwich complex (= anti-c-Kit antibody / c-Kit / second anti-c-Kit antibody complex) comprising a first antibody to c-Kit, c-Kit (analyte), and a second anti-c-Kit antibody, is routine experimentation.

[0145] Detection of the anti-c-Kit antibody / c-Kit complex can be carried out by any suitable means. Detection of the first anti-c-Kit antibody / c-Kit / second anti-c-Kit antibody complex can be carried out by any suitable means. Those skilled in the art are fully conversant with such means / methods.

[0146] In certain embodiments, a sandwich is formed that comprises a first antibody to c-Kit, c-Kit (analyte), and a second antibody to c-Kit, and the second antibody is detectably labeled.

[0147] In one embodiment, a sandwich is formed that comprises a first antibody to c-Kit, c-Kit (analyte), and a second antibody to c-Kit, the second antibody is detectably labeled, and the first anti-c-Kit antibody can bind to or is bound to a solid phase.

[0148] In an embodiment, the second antibody is detectably labeled either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0149] In an embodiment, the method further comprises assessing dysmenorrhea and / or lower abdominal pain in a patient. In an embodiment, the presence of dysmenorrhea and / or lower abdominal pain is assessed using a VAS scale. In an embodiment, a dysmenorrhea VAS score of 4 or more indicates moderate or severe dysmenorrhea. In an embodiment, a score of 3 or less indicates no or mild dysmenorrhea.

[0150] In an embodiment, the method further comprises determining the level of CA-125 in a biological fluid sample from the subject.

[0151] A method for classifying the stage of endometriosis in a subject In a second aspect, the present invention relates to a method for classifying the stage of endometriosis in a subject, the method comprising a) determining the level of c-Kit in a biological fluid sample from the subject, b) comparing the level of c-Kit with the c-Kit level of at least one appropriate reference value, c) classifying the stage of endometriosis in the subject if the comparison in step b) indicates that the subject has an increased or decreased level of c-Kit compared to the c-Kit level of at least one appropriate reference value comprising.

[0152] In an embodiment, an increase or decrease in the level or amount or concentration of c-Kit in a target body fluid sample can indicate the stage of endometriosis in the target. In particular, the level of c-Kit in the target body fluid sample indicates the stage of endometriosis in the target when the level of c-Kit in the target body fluid sample is higher or lower than the level of c-Kit according to a reference value for which the stage of endometriosis is known. Optionally, comparisons with several reference values are made to classify the stage of endometriosis. Further, this can enable a much more accurate classification in combination with surgery or other parameters used to determine the stage of endometriosis.

[0153] In particular, when c-Kit is detectable at a higher level in a body fluid sample of a target being evaluated for the presence of endometriosis than in the same body fluid sample of a target not suffering from endometriosis, this indicates any stage of endometriosis, a slight increase indicates stages I, III, and IV, while a stronger increase indicates stage II. By further comparing the obtained value with a reference value for which the stage of endometriosis is known, the obtained value can be assigned to a certain stage of endometriosis.

[0154] In certain embodiments, at least one suitable reference value is i. the level of c-Kit in a non-pathological subject, a symptomatic subject, or a subject having endometriosis of stage I, stage II, stage III, or stage IV according to the revised American Society for Reproductive Medicine (r-ASRM) scoring system, or ii. the average level of c-Kit in a group of subjects that are non-pathological subjects, symptomatic subjects, or a combination thereof, or subjects having endometriosis of stage I, stage II, stage III, or stage IV according to the revised American Society for Reproductive Medicine (r-ASRM) scoring system, or at least one suitable reference value is iii. a predetermined value of the level of c-Kit in a non-pathological subject, a symptomatic subject, or a subject having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), or iv. a predetermined average value of the level of c-Kit in a group of non-pathological subjects, symptomatic subjects, or combinations thereof, or subjects having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM).

[0155] In certain embodiments, the inventors were able to detect an increase in serum c-Kit in the early stages of endometriosis, followed by a decrease in the later stages of endometriosis. However, the amount or concentration of c-Kit in the later stages is still higher compared to the control levels.

[0156] For example, the obtained c-Kit level, which is higher than that in non-pathological subjects but lower than that in subjects having stage II endometriosis, indicates stage I, III, or IV endometriosis.

[0157] For example, the obtained c-Kit level, which is higher than that in subjects having stage I endometriosis and higher than that in subjects having stage III endometriosis, indicates stage II endometriosis.

[0158] In one example, the method is an in vitro method.

[0159] Details such as biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values, etc. are presented elsewhere and are equally applicable to this and all other aspects.

[0160] A method for monitoring the progression of endometriosis in a subject In a third aspect, the present invention relates to a method for monitoring the progression of endometriosis in a subject, the method comprising i. A step of determining the level of c-Kit in a biological fluid sample derived from a subject according to steps a) to b) of the method described above in this specification; ii. A step of repeating step i at regular time intervals using a biological fluid sample obtained from the subject during or after treatment, and iii. A step of comparing the level of c-Kit identified in i with the c-Kit level identified in ii, wherein a change in the c-Kit level from i to ii indicates a change in the progression of endometriosis in the subject. It includes.

[0161] In an embodiment, a patient suffering from endometriosis is monitored to determine whether the amount or concentration of c-Kit is changing over time in the patient's sample. In particular, a patient suffering from endometriosis is monitored to determine whether the amount or concentration of c-Kit is increasing, decreasing, or not changing over time. In an embodiment, if an increase in the amount of c-Kit in the sample of a patient suffering from endometriosis is determined, the patient is monitored.

[0162] The method can be used to monitor the progression of any type of endometriosis described herein.

[0163] Typically, such a monitoring method is performed on a subject who has not yet been treated for endometriosis (i.e., who has never previously received treatment (treatment or surgery) for endometriosis). Such a subject is described herein as a "naïve" subject.

[0164] However, such a monitoring method also includes a method performed on a subject who has already been treated for endometriosis.

[0165] Monitoring the progression of endometriosis in a subject over time helps to identify the progression of the disease (e.g., worsening of the disease state or disease symptoms) as early as possible. Such monitoring, of course, involves taking repeated samples over time. Thus, the method can be repeated at one or more time intervals for a particular subject, and the results compared to monitor the onset, progression, or improvement of endometriosis in that subject over time. A change in the amount of the level of a biomarker tested in a biological fluid sample (e.g., serum) indicates a change in the progression of endometriosis in the subject.

[0166] Several studies have reported that the levels of serum CA-125 decreased after medical treatment following endometriosis surgery (Chen et al. 1998. Acta Obstet Gynecol Scand. 77:665-70, Jacobs et al. 1989. Hum Reprod. 4:1-12).

[0167] Disease progression can be indicated by an increase in the level of c-Kit detected over time when the results of two or more time intervals are compared for the same subject.

[0168] In other words, when the method is performed multiple times, disease progression can be indicated when the level of c-Kit detected at a later time interval is higher than when detected at an earlier time interval. An "increase" in the level of c-Kit includes the detection of c-Kit at a later time interval when it was not detected (i.e., was not present at a detectable level) when the method was performed previously (i.e., at an earlier time interval) on the same subject (and equivalent types of biological fluid samples). This is particularly relevant when monitoring the progression of endometriosis in naive subjects.

[0169] An appropriate time interval for monitoring the progression of the disease can be readily determined by one of ordinary skill in the art and depends on the particular form of endometriosis being monitored. By way of non-limiting example, the method can be repeated at least weekly, monthly, every six months, or at least annually, or whenever clinically necessary, i.e., in the case of a significant change in the symptoms of endometriosis.

[0170] In one example, the method is an in vitro method.

[0171] Details such as biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values, etc. are presented elsewhere and are equally applicable to this and all other aspects.

[0172] Method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject In a fourth aspect, the present invention relates to a method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject, the method comprising: i. determining the level of c-Kit in a biological fluid sample derived from the subject according to steps a) - b) of the method described above herein; ii. repeating step a) at regular time intervals using a biological fluid sample obtained from the subject during or after treatment; and iii. comparing the level of c-Kit determined in step a) with that determined in step b), and identifying that the treatment regimen has a therapeutic effect if the level of c-Kit has decreased after treatment. comprises.

[0173] In one example, the change in the level of c-Kit indicating a therapeutic effect is a decrease in the c-Kit level after treatment. A "decrease" in the level of c-Kit encompasses the non-detection of c-Kit (i.e., its absence at a detectable level) at a later time interval when the method was performed previously (i.e., at an earlier time interval) on the same subject (and equivalent types of biological fluid samples).

[0174] Step i can be first carried out according to a method using a biological fluid sample obtained from a subject at a time point before the start of the endometriosis treatment regimen. Alternatively, step i may be first carried out using a biological fluid sample obtained from a subject simultaneously with the start of the treatment regimen or at a time point after the start of the endometriosis treatment regimen. Thus, the method can be used to determine the therapeutic effect of an endometriosis treatment regimen from the start (i.e., from the start of the regimen) or from a time point after the start of the treatment regimen (i.e., to determine the therapeutic effect of the treatment regimen for endometriosis during the treatment regimen itself).

[0175] In an embodiment, no change or an increase in the amount or concentration of c-Kit in a sample of a subject undergoing endometriosis treatment indicates that the treatment is ineffective, i.e., no change or an increase in the amount or concentration of c-Kit in a sample of a subject undergoing endometriosis treatment indicates that endometriosis persists or recurs. In particular, when the amount of c-Kit has increased by 50% or more, the treatment of endometriosis is ineffective. In particular, when the amount of c-Kit has increased by 100% or more, the treatment of endometriosis is ineffective. In particular, when the amount of c-Kit has increased by 150% or more, the treatment of endometriosis is ineffective. In particular, when the amount of c-Kit has increased by 200% or more, the treatment of endometriosis is ineffective.

[0176] Alternatively, a non-changing level of c-Kit may mean either that the disease is stagnant or that the c-Kit level has progressed from stage I, comparable to stage I, to stage III or IV.

[0177] Improvement in the condition or symptoms of the disease (e.g., over the treatment period) can also be indicated by a stabilized level of c-Kit over time (compared to the level of c-Kit observed in the absence of treatment over an equivalent period or compared to an equivalent control).

[0178] A treatment regimen can be identified as having a therapeutic effect if it results in a delay in the progression of the disease or the onset of symptoms (e.g., over the course of the treatment period).

[0179] A treatment regimen can also be identified as having a therapeutic effect if it results in an improvement in the state of the disease or symptoms (e.g., over the course of the treatment period). Methods for determining whether a treatment regimen has a therapeutic effect are well known in the art.

[0180] The treatment period refers to the time interval during which treatment is carried out (e.g., 1 month, 3 months, 6 months, 1 year, 2 years, etc.).

[0181] As will be apparent to those skilled in the art, the direction of change in c-Kit levels indicating a therapeutic effect can depend on the pre-treatment state of the subject's disease and the control / reference used.

[0182] Changes in c-Kit levels can also indicate compliance with the prescribed treatment after treatment.

[0183] The tendency to identify whether a subject has complied with the prescribed treatment regimen is equivalent to that described in detail above with respect to determining the therapeutic effect of an endometriosis treatment regimen. This is because the "prescribed treatment regimen" is the recommended treatment regimen and thus typically has a therapeutic effect (thus, the recognition of a therapeutic effect on biomarker levels is an indicator of the subject's compliance with the prescribed treatment regimen).

[0184] In an embodiment, the subject is monitored several times at different time points. In an embodiment, the patient is monitored several times within a time frame of several weeks, several months or several years. In certain embodiments, the subject is monitored once a month or once a year. In an embodiment, a subject suffering from endometriosis is monitored once a month or once a year after the diagnosis of endometriosis. In an embodiment, a subject being treated for endometriosis is monitored once after treatment, particularly once after surgical treatment. In particular, a subject being treated for endometriosis is monitored once a month or once a year to determine the effectiveness of the treatment and / or recurrence of endometriosis.

[0185] The method may also be useful as a screening tool for determining whether a particular regimen or treatment modality has a therapeutic effect on endometriosis. The regimen or treatment modality tested can be a new regimen or treatment modality, a modified regimen or treatment modality, or a known regimen or treatment modality that requires further testing. In this regard, the treatment modality can be, for example, a drug or medicine that is useful or thought to be useful in the treatment of endometriosis.

[0186] In an embodiment, the treatment method for endometriosis is selected from the group consisting of drug-based treatment methods or surgical treatments. In an embodiment, the treatment regimen includes surgical therapy, radiation therapy, immunotherapy, hormone therapy, ultrasound therapy, or a combination thereof. In a preferred embodiment, the surgical treatment for endometriosis is laparoscopy or nerve-sparing surgery. In an embodiment, the drug-based treatment for endometriosis is inhibition or targeting of neurogenic inflammation and / or analgesics and / or hormone therapy.

[0187] In certain embodiments, the treatment is applied if it is determined that the amount or concentration of c-Kit in a sample of a patient being treated for endometriosis has not changed or has increased.

[0188] Details such as biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, etc. are presented elsewhere and are equally applicable to this aspect as well.

[0189] Therefore, all the aspects described in detail above for the method of determining the therapeutic effect of an endometriosis treatment regimen are equally applicable here.

[0190] In one example, the method is an in vitro method.

[0191] Details such as biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values, etc. are presented elsewhere and are equally applicable to this aspect and all other aspects.

[0192] Computer-implemented method for assessing endometriosis patients In a fourth aspect, the present invention relates to a computer-implemented method for assessing a patient suspected of having endometriosis, the method comprising: (a) receiving a value regarding the level of a first biomarker of a sample of a subject, wherein the first biomarker is c-Kit; (b) receiving a value regarding the level of a second biomarker of a sample of a subject, wherein the second biomarker is CA125; (c) receiving a value regarding the level of dysmenorrhea according to VAS and / or lower abdominal pain according to VAS; (d) comparing the values regarding the levels of steps (a)-(c) with the reference of the biomarker and the amount of dysmenorrhea, and / or calculating a score for assessing a subject suspected of having endometriosis based on the levels of the biomarker and the amount of dysmenorrhea; and (e) assessing the subject based on the comparison and / or calculation performed in step (d). comprises.

[0193] As used herein, the term "computer implemented" means that the method is typically executed in an automated manner on a data processing unit included in a computer or similar data processing device. The data processing unit receives a value regarding the amount of a biomarker. Such a value may be an amount, a relative amount, or any other calculated value reflecting an amount as detailed elsewhere in this specification. Thus, it should be understood that the method described above does not require the determination of the amount of a biomarker, but rather uses a value regarding an already pre-determined amount.

[0194] The present invention also contemplates, in principle, a computer program, a computer program product, or a computer-readable storage medium having the computer program tangibly incorporated therein, the computer program including instructions that, when executed on a data processing device or computer, perform the method of the present invention as specified above. Specifically, the present disclosure further includes - a computer or computer network including at least one processor, the processor being adapted to execute a method according to one of the embodiments described herein, - a computer loadable data structure adapted to execute a method according to one of the embodiments described herein while the data structure is being executed on a computer, - a computer script, the computer program being adapted to execute a method according to one of the embodiments described herein while the program is being executed on a computer, - a computer program comprising program means for executing a method according to one of the embodiments described herein while the computer program is being executed on a computer or computer network, - a computer program comprising program means according to a preceding embodiment, the program means being stored on a computer-readable storage medium, -A data structure is stored in a storage medium, and after the data structure is loaded into the main storage device and / or the working storage device of a computer or a computer network, the storage medium adapted to execute a method according to one of the embodiments described herein, -A computer program product having program code means that can be stored in or on a storage medium for executing a method according to one of the embodiments described herein when the program code means are executed by a computer or a computer network, -Typically an encrypted data stream signal, including data of parameters defined elsewhere in this specification, and -Typically an encrypted data stream signal, including the evaluation provided by the method of the present invention.

[0195] CA-125, in combination with symptoms or clinical data The method according to the present invention can be combined with other tests, biomarkers, clinical data, or further information useful for diagnosing or classifying endometriosis in order to obtain the most reliable results.

[0196] Despite its relatively low diagnostic ability, CA-125 is routinely used as a biomarker for endometriosis. Therefore, it may be advantageous to combine determining the level of c-Kit with CA-125 obtained from a subject in the context of the method described herein.

[0197] In addition to CA-125, other symptoms or clinical data used for diagnosing or classifying endometriosis can be used in combination with the determination of c-Kit levels. Such symptoms or clinical data can be, but are not limited to, age, dysmenorrhea, abdominal pain, or other biomarkers.

[0198] Kits and devices In another aspect, a kit is provided for diagnosing or classifying the stage of endometriosis in a subject. The kit includes reagents suitable for determining the levels of a plurality of analytes in a test sample (e.g., reagents suitable for determining the levels of biomarkers disclosed herein).

[0199] The kits described herein typically include a detectable labeled agent that specifically binds to the c-Kit protein.

[0200] Such kits may further include a detectable labeled agent that specifically binds to CA-125.

[0201] The kits described herein can take various forms. Typically, the kit includes reagents suitable for determining the levels of a plurality of biomarkers (e.g., c-Kit and optionally CA-125) in a sample.

[0202] Optionally, the kit may contain one or more control samples or references. Typically, comparison of the biomarker levels in the subject with those in the control sample indicates the clinical condition (e.g., diagnosis of endometriosis). Also, the kit may, in some cases, include written information (indicators) providing a reference (e.g., a predetermined value), and comparison of the biomarker level in the subject with the reference (predetermined value) indicates the clinical condition (e.g., diagnosis of endometriosis). Optionally, the kit includes software useful for comparing biomarker levels or occurrences to a reference (e.g., a predictive model). Usually, the software is provided in a computer-readable format such as a compact disc, but it can also be obtained by downloading via the Internet. However, the kit is not so limited, and other variations will be apparent to those skilled in the art.

[0203] The components of the kit may be housed in a container suitable for transport. Details regarding the biomarker are provided above and apply equally here. Suitably, the biomarker can be a protein.

[0204] In some examples, the kit includes an agent detectably labeled on a continuous (e.g., solid) surface such as a lateral flow surface. Alternatively, in examples including two or more detectably labeled agents, the detectably labeled agents may be disposed in separate (i.e., spatially separated) zones of a (e.g., solid) surface such as a multiwell microtiter plate (e.g., in the case of an ELISA assay). Other suitable surfaces and containers well known in the art may also form part of the kits described herein.

[0205] In one example, the kit further includes one or more reagents for detecting a detectably labeled agent. Suitable reagents are well known in the art and include, but are not limited to, standard reagents and buffers necessary to perform any one of the suitable detection methods that may be used (and are well known in the art).

[0206] In one example, the kit includes one or more of a multiwell plate, a ball bearing, an extraction buffer, an extraction bottle, and a lateral flow device.

[0207] An assay device for diagnosing endometriosis in a subject is also provided.

[0208] Typically, the device includes a surface on which at least one detectably labeled agent that specifically binds to the c-Kit protein is located.

[0209] Such a device may further include a detectably labeled agent that specifically binds to CA-125.

[0210] When two detectably labeled agents are used, they can be arranged in separate zones on the surface. In other words, the two detectably labeled agents can be located in separate (i.e., spatially separated) zones on a (e.g., solid) surface such as a multi-well microtiter plate. Detectably labeled agents that specifically bind to the biomarker of interest are described in detail elsewhere in this specification.

[0211] The assay device includes a surface on which the detectably labeled agent is disposed. Suitable surfaces include continuous (e.g., solid) surfaces such as lateral flow surfaces, dot blot surfaces, dipstick surfaces, or surfaces suitable for performing surface plasmon resonance. Other suitable surfaces include microtiter plates, multi-well plates, and the like. Other suitable surfaces well known in the art may also form part of the assay devices described herein.

[0212] Thus, suitable assay device formats include, but are not limited to, device formats suitable for performing any one of lateral flow, dot blot, ELISA, or surface plasmon resonance assays for detecting the presence, level, or absence of the biomarker of interest.

[0213] Aspects of data storage Biomarker levels and / or reference levels can be stored in a suitable storage medium (e.g., a database) and, thus, can also be used for future diagnostics. This also enables the efficient diagnosis of the disease prevalence, as, if it is (subsequently) confirmed that the subject from whom the corresponding reference sample was obtained had endometriosis, the appropriate reference results can be identified in the database. As used herein, "database" includes data collected in a suitable storage medium (e.g., analyte and / or reference level information and / or patient information). Further, the database may further comprise a database management system. The database management system is preferably a network-based hierarchical or object-oriented database management system. Further, the database may be a federal or integrated database. More preferably, the database can be implemented as a distributed (federal) system, such as, for example, a client-server system. More preferably, the database is configured such that a test data set can be compared to the data sets included in the data collection by a search algorithm. Specifically, by using such an algorithm, the database can be searched for similar or identical data sets indicative of endometriosis (e.g., query search). Thus, if the same or similar data sets can be identified in the data collection, the test data set is associated with endometriosis. Thus, the information obtained from the data collection can be used to diagnose endometriosis or based on the test data set obtained from the subject. More preferably, the data collection includes the characteristic values of all analytes included in any one of the above groups.

[0214] The methods described herein may further include, for example, communicating the results and / or diagnosis (or both) to a technician, a physician, or a patient. In certain examples, a computer is used to communicate the results and / or diagnosis (or both) to interested parties, such as a physician and their patient.

[0215] In some instances, after a diagnosis is obtained, the results or diagnosis (or both) are communicated to the subject expeditiously. The results or diagnosis (or both) may be communicated to the subject by the subject's treating physician. Alternatively, the results or diagnosis (or both) may be sent to the subject by email or communicated to the subject by telephone. A computer may be used to communicate the results or diagnosis by email or telephone. In certain examples, a message containing the results or diagnosis may be automatically generated and distributed to the subject using a combination of computer hardware and software well known to those of ordinary skill in the art of telecommunications.

[0216] Use The use of biomarker c-Kits as a biomarker in biological fluids for endometriosis is also provided herein.

[0217] In a preferred example, c-Kit can generally be used as a biomarker for endometriosis. In this context, "endometriosis generally" refers to all forms of endometriosis including, but not limited to, peritoneal endometriosis, endometrioma, deep infiltrating endometriosis, and adenomyosis.

[0218] Also, c-Kit may be combined with CA-125.

[0219] Details such as biomarkers, samples, methods, subjects, types of endometriosis, etc. are presented elsewhere and are equally applicable to this aspect.

[0220] Companion Diagnosis The methods, kits, assay devices, and uses provided herein can be used as part of a companion diagnosis that provides information essential for the safe and effective use of a corresponding drug or biological substance (the corresponding drug or biological substance is for treating or preventing endometriosis), for example, as part of a medical device, often an in vitro device. Companion Diagnosis In a further embodiment, the present invention relates to the following aspects: 1. A method for diagnosing endometriosis in a subject, comprising: a) determining the level of c-Kit in a biological fluid sample from the subject; b) comparing the level of c-Kit with the level of c-Kit of at least one appropriate reference value; c) identifying the subject as having endometriosis if the comparison in step b) indicates that the subject has an increased level of c-Kit compared to the appropriate reference value. A method comprising the steps of:

[0221] 2. The at least one appropriate reference value is i. the level of c-Kit in a non-pathological or symptomatic subject, or ii. the average level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects, or a combination thereof, or the at least one appropriate reference value is iii. a predetermined value of the level of c-Kit in a non-pathological or symptomatic subject, or iv. a predetermined average value of the level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects, or a combination thereof, according to the method of aspect 1.

[0222] 3. A method for classifying the stage of endometriosis in a subject, comprising: a) determining the level of c-Kit in a biological fluid sample from the subject; b) comparing the level of c-Kit with the level of c-Kit of at least one appropriate reference value; c) classifying the stage of endometriosis in the subject if the comparison in step b) indicates that the subject has an increased or decreased level of c-Kit compared to the level of c-Kit of at least one appropriate reference value. A method comprising the steps of:

[0223] 4. A method for classifying the stage of endometriosis in a subject, comprising: a) Determining the level of c-Kit in a biological fluid sample from a subject at regular intervals; b) Comparing the level of c-Kit with at least one value of the c-Kit level determined at an initial stage in the subject; c) When the comparison in step b) indicates that the subject has an increased or decreased level of c-Kit compared to at least one value of the c-Kit level determined at an initial stage in the subject, classifying the stage of endometriosis in the subject. A method comprising.

[0224] 5. At least one appropriate reference value is i. The level of c-Kit in a non-pathological subject, a symptomatic subject, or a subject having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine, or ii. The average level of c-Kit in a group of subjects that are non-pathological subjects, symptomatic subjects, or a combination thereof, or subjects having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine, Or, at least one appropriate reference value is iii. A predetermined value of the level of c-Kit in a non-pathological subject, a symptomatic subject, or a subject having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine, or iv. A predetermined average value of the level of c-Kit in a group of non-pathological subjects, symptomatic subjects, or a combination thereof, or subjects having endometriosis of stage I, stage II, stage III, or stage IV according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine, the method according to aspect 3 or 4.

[0225] 6. The method according to any of the preceding aspects, wherein the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably the biological fluid sample is serum.

[0226] 7. The method according to any of the preceding aspects, wherein the subject is a human, preferably a female human.

[0227] 8. The protein level of c-Kit is optionally determined using a process selected from the following, namely ELISA assay, immunoblotting, lateral flow assay, protein microarray, and mass spectrometry, according to the method described in any of the preceding aspects.

[0228] 9. Further comprising administering a selected treatment regimen to the subject, and optionally, the selected treatment regimen includes surgery, radiotherapy, immunotherapy, hormone therapy, ultrasound therapy, or a combination thereof, according to the method described in any of the preceding aspects.

[0229] 10. The method according to any of the preceding aspects, wherein the diagnosis or classification is performed independently of the scoring system of the American Society for Reproductive Medicine.

[0230] 11. The method according to any of aspects 3 to 10, wherein the stage of the subject's endometriosis is classified as stage I, stage II, stage III, or stage IV endometriosis according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine.

[0231] 12. The method according to any of aspects 3 to 11, wherein the stage of the subject's endometriosis is classified as stage I or stage II endometriosis according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine.

[0232] 13. The method according to any of the preceding aspects, wherein the endometriosis is selected from the group consisting of peritoneal endometriosis, endometrioma, deep infiltrating endometriosis, and adenomyosis.

[0233] 14. The method according to any of the preceding aspects, further comprising selecting a treatment regimen for a subject based on a comparison of the level of c-Kit with a control sample or a predetermined reference level.

[0234] 15. The method according to aspect 14, further comprising administering the selected treatment regimen to the subject, and optionally, the selected treatment regimen includes a drug-based therapy and / or a surgical procedure (laparoscopy).

[0235] 16. A method for monitoring the progression of endometriosis in a subject, comprising: i. determining the level of c-Kit in a biological fluid sample derived from the subject according to steps a)-b) of the method according to any one of aspects 1-15; ii. repeating step i at regular time intervals using a biological fluid sample obtained from the subject during or after treatment; and iii. comparing the level of c-Kit identified in i with the c-Kit level identified in ii, wherein a change in the c-Kit level from i to ii indicates a change in the progression of endometriosis in the subject. A method comprising the above steps.

[0236] 17. A method for determining the therapeutic effect of a treatment regimen for a subject's endometriosis, comprising: i. determining the level of c-Kit in a biological fluid sample derived from the subject according to steps a)-b) of the method according to any one of aspects 1-15; ii. repeating step i at regular time intervals using a biological fluid sample obtained from the subject during or after treatment; and iii. comparing the level of c-Kit determined in step i with that determined in step ii, and identifying that the treatment regimen has a therapeutic effect when the level of c-Kit decreases after treatment. A method comprising the above steps.

[0237] 18. The method according to any of the preceding aspects, further comprising the assessment of dysmenorrhea according to the Visual Analog Scale (VAS) and / or lower abdominal pain according to the VAS.

[0238] 19. The method according to any of the preceding aspects, further comprising determining the level of CA-125 in a biological fluid sample derived from a subject.

[0239] 20. The ratio of the amount or concentration of -c-Kit to the amount or concentration of CA-125, or - the ratio of the amount or concentration of -c-Kit to dysmenorrhea, or - the ratio of the amount or concentration of -c-Kit, the amount or concentration of CA-125, and dysmenorrhea, or - the ratio of the amount or concentration of -c-Kit to lower abdominal pain according to the VAS scale The method according to aspect 18, comprising calculating.

[0240] 21. A computer-implemented method for assessing a patient suspected of having endometriosis, comprising: (a) receiving a value regarding the level of a first biomarker in a sample of the subject, wherein the first biomarker is c-Kit; (b) receiving a value regarding the level of a second biomarker in a sample of the subject, wherein the second biomarker is CA125; (c) receiving a value regarding the level of dysmenorrhea according to the VAS and / or lower abdominal pain according to the VAS; (d) comparing the values regarding the levels in steps (a) to (c) with the reference of the biomarker and the amount of dysmenorrhea, and / or calculating a score for assessing a subject suspected of having endometriosis based on the levels of the biomarker and the amount of dysmenorrhea; and (e) assessing the subject based on the comparison and / or calculation performed in step (d). A computer-implemented method comprising.

[0241] Use of elevated c-Kit levels in a biological fluid sample as a biomarker for endometriosis.

[0242] 23. The use according to embodiment 22, wherein the biological fluid sample is blood or blood-derived, preferably serum.

[0243] 24. The use according to embodiments 22 and 23 for diagnosing and / or classifying endometriosis.

[0244] 25. A kit for diagnosing and / or classifying endometriosis in a subject, comprising at least one detectably labeled agent that specifically binds to the c-Kit protein.

[0245] 26. The kit according to embodiment 25, further comprising one or more reagents for detecting the detectably labeled agent.

[0246] 27. An assay device for diagnosing and / or classifying endometriosis in a subject, the device comprising a surface on which at least one detectably labeled agent that specifically binds to the c-Kit protein is disposed.

[0247] Aspects of the present invention are illustrated by the following non-limiting examples. The following examples and figures are provided to assist in the understanding of the present invention, and the true scope of the present invention is set forth in the appended claims. It is understood that modifications can be made to the described procedures without departing from the spirit of the present invention.

Example

[0248] Example 1: Diagnostic ability of the biomarker c-Kit in women with endometriosis For the measurement, a total of 250 serum samples from human females were analyzed (see the corresponding section of this specification for clinical data). The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group consisted of patients diagnosed with endometriosis (peritoneal endometriosis, adenomyosis, endometrioma, and deeply infiltrating endometriosis; rASRM stages I-IV) diagnosed by laparoscopy with subsequent histological confirmation, and the control group included healthy women without endometriosis.

[0249] The concentration of c-Kit in human serum was determined using the Human CD117 / c-Kit Quantikine ELISA Kit (R&D Systems, USA (Catalog number: DSCR00)). The kit utilizes quantitative sandwich ELISA technology. The microtiter plate is pre-coated with a monoclonal antibody specific for human c-Kit. Samples are measured at a 50-fold dilution. After bringing all reagents to room temperature, 100 μL of each sample and standard are added. Samples are measured in series and standards are measured in duplicate. While incubating at room temperature for 2.5 hours on a microplate shaker set at 650 rpm, any c-Kit present is bound to the immobilized capture antibody on the microtiter plate. During the washing step (4 × 300 μL), unbound substances are removed from the plate before adding 100 μL of an enzyme-conjugated monoclonal antibody specific for c-Kit to the wells. After incubating on the shaker for 1 hour and performing another washing step to remove unbound detection antibody, 100 μL of substrate solution is added to the plate. Color development occurs within the next 10 minutes in proportion to the amount of c-Kit bound in the first step. Color development is stopped by adding 50 μL of stop solution, and the color intensity is measured at 450 nm for detection and at 570 nm for background subtraction using a plate reader. To generate a calibration curve, the lyophilized recombinant c-Kit provided with the kit is reconstituted and diluted with the standard diluent. The calibration range of the assay is 6.14 pg / mL to 1500 pg / mL. Calibrator 7 (1500 pg / mL) is prepared by a 6-fold dilution of the stock solution in calibrator diluent, and calibrator 6 (6.14 pg / mL) relative to calibrator 1 is prepared by a serial 2.5-fold dilution step in calibrator diluent. Pure calibrator diluent functions as the blank (0 pg / mL). The calibration curve was fitted using 4-parameter nonlinear regression (Newton / Raphson) without weighting.

[0250] The results are shown in Figure 1.

[0251] Serum c-Kit levels gradually increase in stages I and II of endometriosis and decrease in stage III compared to non-pathological controls.

[0252] In Table 1 below, the ability of the model is determined by examining the area under the curve (AUC). The best possible AUC is 1 and the lowest possible AUC is 0.5. The optimal cut-off was selected using the Youden index (maximum sum of sensitivity + specificity - 1).

[0253]

Table 1

[0254] Example 2: Diagnostic ability of biomarker c-Kit compared to CA-125 in women with endometriosis In a further experiment, the ability of biomarker c-Kit was compared with the current standard biomarker CA-125.

[0255] The concentration of CA-125 was determined by a cobas e601 analyzer. Detection of CA125II using the cobas e601 analyzer is based on Elecsys® electrochemiluminescence (ECL) technology. Briefly, biotin-labeled and ruthenium-labeled antibodies are combined with the respective amounts of undiluted sample and incubated in the analyzer. Then, streptavidin-coated magnetic microparticles are added to facilitate the binding of biotin-labeled immune complexes and incubated in the device. After this incubation step, the reaction mixture is transferred to the measurement cell where the beads are magnetically captured on the surface of the electrode. Next, ProCell M buffer containing tripropylamine (TPA) for the subsequent ECL reaction is introduced into the measurement cell to separate the bound immunoassay complex from the remaining free particles. Then, induction of the voltage between the working electrode and the counter electrode initiates a reaction that results in the emission of photons by the ruthenium complex and TPA. The resulting electrochemiluminescence signal is recorded by a photomultiplier tube and converted into a numerical value indicating the concentration level of each analyte.

[0256] The results are shown in FIGS. 2A and 2B.

[0257] The box plots in FIGS. 2A and 2B were generated for each of the controls and the stages (Stage I, Stage II, Stage III, Stage IV) of endometriosis using data obtained from high-throughput multiplex immunoassay-PCR (OLINK proteomics) analysis. The data are presented using box-and-whisker plots that include the median (mid-quartile), interquartile range (representing the central 50% of the group scores), upper quartile (75% of the scores are below the upper quartile), and lower quartile (25% of the scores are below the lower quartile). The whiskers indicate the 5th and 95th percentiles, respectively.

[0258] Serum c-Kit shows better diagnostic ability for the detection of early-stage (Stage I, Stage II) endometriosis compared to the reference biomarker CA-125.

[0259] In Table 2 below, the ability of c-Kit as a biomarker compared to CA-125 is determined by examining the area under the curve (AUC) of both biomarkers for early-stage endometriosis (Stage I and II).

[0260]

Table 2

[0261] The AUC values for the ROC analysis of Stage I and Stage II endometriosis relative to the control were AUC = 0.72\AUC = 0.57 for serum c-Kit and AUC = 0.46\AUC = 0.6 for serum CA-125.

[0262] Serum c-Kit shows better diagnostic ability for the detection of early-stage (Stage I, Stage II) endometriosis compared to the reference biomarker CA-125.

[0263] Clinical data regarding the cohort: 1) Cases of endometriosis: a) Total cohort size: 101 patients b) Age:

[0264]

Table 3

[0265]

Table 4

[0266]

Table 5

[0267]

Table 6

[0268]

Table 7

[0269] 2) Cases without endometriosis, severe pain ("symptomatic control"): a) Total cohort size: 65 patients b) Age:

[0270]

Table 8

[0271]

Table 9

[0272]

Table 10

[0273] 3) No endometriosis, no pain ("non-pathological control"): a) Total cohort size: 84 patients b) Age:

[0274]

Table 11

[0275]

Table 12

[0276]

Table 13

Claims

1. A method for diagnosing endometriosis, a) A step of determining the level of c-Kit in a biological fluid sample derived from the subject, b) A step of comparing the level of the c-Kit with at least one appropriate reference value of the c-Kit level, c) If the comparison in step b) indicates that the subject has a level of c-Kit that has increased compared to the appropriate reference value, the step of identifying the subject as having endometriosis. A method that includes this.

2. A method for classifying the stage of endometriosis, a) A step of determining the level of c-Kit in a biological fluid sample derived from the subject, b) A step of comparing the level of the c-Kit with at least one appropriate reference value of the c-Kit level, c) If the comparison in step b) indicates that the subject has a level of c-Kit that is increased or decreased compared to the c-Kit level of at least one appropriate reference value, the step of classifying the stage of endometriosis in the subject. A method that includes this.

3. The method according to claim 1 or 2, wherein the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, and preferably the biological fluid sample is serum.

4. The method according to claim 1 or 2, wherein the diagnosis or classification is performed independently of the scoring system of the American Society for Reproductive Medicine.

5. The method according to claim 2, wherein the stage of the endometriosis in the subject is classified as stage I, stage II, stage III, or stage IV endometriosis according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine.

6. The method according to claim 1 or 2, wherein the stage of the endometriosis in the subject is classified as stage I or stage II endometriosis according to the revised scoring system (r-ASRM) of the American Society for Reproductive Medicine.

7. The method according to claim 1 or 2, wherein the endometriosis is selected from the group consisting of peritoneal endometriosis, endometrioma, deep invasive endometriosis, and adenomyosis.

8. The method according to claim 1 or 2, further comprising selecting a treatment regimen for the subject based on the comparison of the level of the c-Kit with a control sample or a predetermined reference level.

9. The method according to claim 8, further comprising administering the selected treatment regimen to the subject, wherein the selected treatment regimen optionally includes a drug-based treatment and / or a surgical procedure (laparoscopy).

10. A method for monitoring the progression of endometriosis in a subject, i. A step of determining the level of c-Kit of a biological fluid sample derived from the subject according to steps a) to b) of the method according to claim 1 or 2, ii. A step of repeating step i at regular time intervals using a biological fluid sample obtained from the subject during or after treatment, and iii. A step of comparing the level of the c-Kit identified in i with the level of the c-Kit identified in ii, wherein the change in the c-Kit level from i to ii indicates a change in the progression of endometriosis in the subject. A method that includes this.

11. A method for determining the therapeutic effect of a treatment regimen for endometriosis, i. A step of determining the level of c-Kit of a biological fluid sample derived from the subject according to steps a) to b) of the method according to claim 1 or 2, ii. A step of repeating step a) at regular time intervals using a biological fluid sample obtained from the subject during or after treatment, and iii. A step of comparing the level of c-Kit determined in step a) with that determined in step b), and determining that the treatment regimen is effective if the level of c-Kit decreases after treatment. A method that includes this.

12. The method according to claim 1 or 2, further comprising rating dysmenorrhea and / or lower abdominal pain according to a visual analog scale (VAS).

13. The method according to claim 1 or 2, further comprising determining the level of CA-125 in the biological fluid sample derived from the subject.

14. - The ratio of the amount or concentration of c-Kit to the amount or concentration of CA-125, or - The ratio of the amount or concentration of c-Kit to dysmenorrhea, - The ratio of the amount or concentration of c-Kit to the amount or concentration of CA-125 to dysmenorrhea, or - The ratio of the amount or concentration of c-Kit to lower abdominal pain according to the VAS scale. The method according to claim 13, which includes calculating a.

15. A computer implementation method for evaluating individuals suspected of having endometriosis, (f) A step of receiving a value relating to the level of a first biomarker of the target biological fluid sample, wherein the first biomarker is c-Kit. (g) A step of receiving a value relating to the level of a second biomarker of the target biological fluid sample, wherein the second biomarker is CA125. (h) A step of receiving values ​​for the level of dysmenorrhea and / or lower abdominal pain according to the VAS, (i) a step of comparing the values ​​for the levels in steps (a) to (c) with the reference values ​​for the biomarker and the amount of dysmenorrhea, and / or calculating a score for evaluating the subject suspected of having endometriosis based on the levels of the biomarker and the amount of dysmenorrhea, and (j) A step of evaluating the subject based on the comparison and / or calculation performed in step (d). Computer implementation methods including

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