Methods for diagnosing endometriosis and for classifying stages of endometriosis

Abstract

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

Description

[0001] The present invention relates to a method for diagnosing whether a subject has endometriosis by determining the amount or concentration of c-Kit in a sample of the subject and comparing the determined level with a reference value, 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. Background Art

[0002] Endometriosis is a chronic disease defined by the growth of endometrial glands and extrauterine stroma-like lesions (Liu et al., 2011). Lesions may be peritoneal, superficial implants or cysts on the ovaries, or deeply infiltrating disease. It originates from eutopic endometrial cells characterized by increased proliferation and adhesion properties (Liu et al., 2011). Increased cell viability in eutopic endometrium is a result of decreased apoptosis and increased cell proliferation (Johnson et al., 2005). Endometriosis affects 5%-8% of all women of childbearing age and 70% of women with chronic pelvic pain. It is estimated that 176 million women worldwide suffer from endometriosis (Adamson et al. J Endometr. 2010; 2: 3-6). For many of these women, the diagnosis of endometriosis is often delayed, resulting in unnecessary suffering and reduced quality of life. In patients aged 18-45 years, there is a delay of 7-10 years. Because most women with endometriosis report experiencing symptoms during adolescence, early referral, diagnosis, disease identification, and treatment can reduce pain and prevent disease progression. Barriers to early diagnosis include the high cost of diagnosis and treatment for adolescent patients and the presentation of confounding symptoms such as cyclical and noncyclical pain (Parasar et al. Curr Obstet Gynecol Rep. 2017;6:34-41).

[0003] The gold standard for diagnosing endometriosis is laparoscopic visualization and subsequent histological confirmation. To date, there is no non-invasive method for diagnosing endometriosis (Hsu et al. Clin Obstet Gynecol 2010: 53: 413-419). During diagnostic laparoscopy, gynecologists who have received training and skills in laparoscopic surgery for endometriosis should perform a systematic examination of the pelvis (NICE guideline NG73, 2017). Surgical visualization requires good expertise, training and skills to make a reliable diagnosis. Diagnosis requires laparoscopic surgery, which doctors try to avoid as much as possible, resulting in a delay of 7-10 years in diagnosis. The lack of non-invasive diagnostic tests is the main reason for 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, there is an unmet medical need for non-invasive tests for diagnosing endometriosis, particularly for diagnosing early, minimal, and mild endometriosis (revised American Society for Reproductive Medicine rASRM stage I-II).

[0004] Non-invasive diagnosis of endometriosis will allow earlier diagnosis and treatment, potentially improving quality of life and reducing 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 can facilitate earlier diagnosis and intervention, ultimately improving quality of life and preserving fertility (Parasar et al. Curr Obstet Gynecol Rep. 2017; 6: 34-41).

[0005] Blood biomarkers are essential to reduce the time delay in diagnosing endometriosis requiring laparoscopy. CA-125 is one of the most commonly used blood biomarkers, however, its diagnostic utility is limited to rASRM stages III and IV of endometriosis (Nisenblat et al., Cochrane Database of Systematic Reviews. 2016;5:CD012179).

[0006] c-Kit is a proto-oncogene that encodes a 145kd 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 a vital role in a variety of biological functions (Sharkey et al., 1994). Serum levels of c-Kit are increased in proliferative mast cell disorders, indicating the presence of a c-Kit shedding pathway in mast cells (Cruz et al. J Biol Chem. 2004). It is essential for the survival, differentiation and mobilization of a variety of cell types, including myeloid cells, erythrocytes, megakaryocytes, lymphocytes, germ cells and melanocyte progenitors (Besmer et al., 1991; Lyam et al., 1998; Papayannopoulou et al., 1991). Several studies have emphasized that dysregulation of c-Kit function is associated with the development of a variety of diseases, including cancer (Vliagoftis et al., 1997; Miettinen et al., 2005). For example, in mast cells, c-Kit regulates differentiation, activation, and homeostasis (Gilfillan et al., 2011), and mutations within the c-Kit receptor can interfere with the signaling cascade and induce constitutive receptor activation independent of SCF, leading to mastocytosis (Cardet et al., 2013). In addition, SCF binding to c-Kit in endothelial cells disrupts endothelial adherens junctions, thereby enhancing vascular leakage (Kim et al., 2014). Alterations in c-Kit signaling have also been implicated in tumorigenesis and the development of a 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 may differ when measured in tissue or serum. For example, complement components C7 and C4 have been shown to be overexpressed in ectopic endometrium of women with endometriosis compared to eutopic endometrium of control women without endometriosis (Ahn et al. Fertil Steril 2016; Eyster et al. Fertil Steril 2007). However, no increase in serum complement component C7 protein and complement component C4 protein was found in circulating blood (Hever et al. PNAS 2007). Brain-derived neurotrophic factor (BDNF) mRNA was expressed at higher levels in ovarian endometriotic lesions than in eutopic endometrium (Wang et al. Journal of Ovarian Research 2022). However, there was no significant difference in serum BDNF in women with endometriosis compared to control women without endometriosis (Perricos et al. Exp Biol Med (Maywood) 2018). Furthermore, in breast cancer, although significant differences have been observed in the blood of normal subjects and patients for molecular markers such as CEA(O), ERβ, CK19, and c-Myc, these markers were not significantly different in tissue samples.

[0008] Therefore, locally altered expression of biomarkers in tissue will not translate 1:1 into significantly different levels of these biomarkers in circulating blood.

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

[0010] Accordingly, the present invention provides tools and methods to meet these needs. Summary of the invention

[0011] In a first aspect, the present invention relates to a method for diagnosing endometriosis in a subject,

[0012] The method comprises the following steps:

[0013] a) determining the level of c-Kit in a biological fluid sample from a subject,

[0014] b) comparing the level of c-Kit to at least one appropriate reference value for c-Kit level,

[0015] c) if the comparison in step b) indicates that the subject has an increased level of c-Kit compared to an appropriate reference value, identifying the subject as having endometriosis.

[0016] In a second aspect, the present invention relates to a method for classifying the stage of endometriosis in a subject, the method comprising the steps of:

[0017] a) determining the level of c-Kit in a biological fluid sample from a subject,

[0018] b) comparing the level of c-Kit to at least one appropriate reference value for c-Kit level,

[0019] 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 at least one appropriate reference value for the level of c-Kit.

[0020] In a third aspect, the present invention relates to a method for monitoring the progression of endometriosis in a subject, the method comprising the steps of:

[0021] i. determining the level of c-Kit in a biological fluid sample from a subject according to steps a) to b) of the above method for diagnosing and classifying endometriosis,

[0022] ii. repeating step i. for specific time intervals using a biological fluid sample obtained from the subject during or after treatment; and

[0023] iii. comparing the level of c-Kit identified in i. with the level of c-Kit identified in ii, wherein a change in the level of c-Kit from i. to ii is indicative of a change in the progression of endometriosis in the subject.

[0024] 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 the steps of:

[0025] i. determining the level of c-Kit in a biological fluid sample from a subject according to steps a) to b) of the above method for diagnosing and classifying endometriosis,

[0026] ii. repeating step i. for specific time intervals using a biological fluid sample obtained from the subject during or after treatment; and

[0027] iii. comparing the level of c-Kit determined in step i. with the level of c-Kit determined in step ii., and if the level of c-Kit decreases after treatment, identifying that the treatment regimen has a therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Hereinafter, embodiments of the present invention will be further described with reference to the accompanying drawings.

[0029] Figure 1 : Box plot analysis: c-kit levels [ng / mL] in serum samples of control groups (non-pathological controls, symptomatic controls (no benign findings) vs. endometriosis case groups (stage I, endometriosis stage II, endometriosis stage III, and endometriosis stage IV) and controls with benign findings. Serum c-Kit levels were measured using the Human CD117 / c-Kit Quantikine ELISA kit (R&D Systems, USA).

[0030] Figure 2 (A and B): Box plot analysis: c-Kit levels [ng / mL] in serum samples of women with endometriosis (stages I-II-III-IV) compared to controls without endometriosis. c-Kit (A) and CA-125 (MUC16) (B) levels were measured using Olink proteomics technology. A: N negative (Ctrl): 16; N positive (Endo): 24; AUC 0.77 (0.62-0.92); B: N negative (Ctrl): 16; N positive (Endo): 24; AUC 0.76 (0.61-0.92) DETAILED DESCRIPTION

[0031] The present invention is based on the surprising discovery that elevated c-Kit levels in serum are associated with endometriosis. C-Kit levels are increased in all stages I, II, III and IV, with stage II showing the highest levels of c-Kit, while stages I, III and IV show comparable increased levels, which confers the diagnostic potential of serum c-Kit for early detection of endometriosis.

[0032] The present inventors have shown for the first time that c-Kit measured in serum is increased in women with endometriosis compared to controls.C-Kit levels are specifically increased in endometriosis stage I, particularly stage II (minor / mild endometriosis).

[0033] There is an unmet medical need for a non-invasive test for reliable diagnosis and / or classification of endometriosis, particularly early-stage endometriosis. The C-Kit has the advantage of being a non-invasive blood-based test that can identify women with early-stage endometriosis.

[0034] The present inventors investigated the levels of c-Kit in serum obtained from women with endometriosis. Surprisingly, they found that increased c-Kit levels could be detected in serum samples of women with endometriosis. In particular, the fact that an increase in c-Kit levels could already be detected in women with early endometriosis makes this marker a useful tool for the early diagnosis of endometriosis. Therefore, an assay capable of determining the levels of c-Kit in such biological fluids may be useful for endometriosis diagnosis and / or classification, prognosis and patient stratification for treatment.

[0035] The data presented herein indicate that determination of c-Kit levels in serum provides a means for diagnosing endometriosis, risk stratifying patients with endometriosis, and classifying the stage of endometriosis in a subject (e.g., when c-Kit levels in a subject are determined periodically or by comparing the determined values ​​to values ​​for a known stage). This also allows monitoring of endometriosis progression and / or evaluation of treatment options.

[0036] The data presented herein also demonstrate that measurement of c-Kit levels in serum provides a more accurate means of detecting early-stage endometriosis and control samples than CA-125.

[0037] definition

[0038] The word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0039] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0040] Concentration, level, amount and other numerical data can be expressed or presented in "range" format herein.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly listed as range limit, but also including all individual numerical values ​​or sub-ranges covered by the range, just as each numerical value and sub-range are clearly listed.As an illustration, the numerical range of "150mg to 600mg" should be interpreted as not only including the clearly listed values ​​of 150mg to 600mg, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values ​​are included in this numerical range, such as 150, 160, 170, 180, 190, ... 580, 590, 600mg and sub-ranges, such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc.This same principle applies to the scope of only quoting one numerical value.In addition, no matter how wide the range or feature is, such explanation is applicable.

[0041] When used in connection with a numerical value, the term "about" is intended to encompass a range of values ​​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.

[0042] In general, the method described is an in vitro method performed using a sample that has been obtained from a subject (i.e., a sample is provided for the method, and the steps taken to obtain the sample from the subject are not included as part of the method). Therefore, the method may include the step of providing a biological fluid sample from a subject. As used herein, "providing", "obtaining" or "obtaining" can be any way of having a sample by a "direct" or "indirect" manner. Directly obtaining a sample means performing a process (e.g., performing a physical method such as extraction) to obtain a sample. Indirectly obtaining a sample refers to receiving a sample from another party or source (e.g., a third-party laboratory that directly obtains a sample).

[0043] The methods provided herein include providing a biological fluid sample (eg, a blood sample) from a subject. The sample tested in the methods described herein is also referred to as a "test sample."

[0044] As used herein, the terms "biological (fluid) sample", "test sample", "sample" and variations thereof refer to a sample obtained or derived from a subject. For the purposes described herein, a sample is or includes a biological fluid (also referred to herein as a body fluid) sample.

[0045] Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, interstitial fluid, capillary blood, peritoneal fluid, menstrual fluid, urine, saliva, and lymph. Analysis of samples can be accomplished on a chemical basis. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or a change in the amount, concentration, or level of a specific indicator.

[0046] The samples are in vitro samples, which will be analyzed outside the body and will not be transferred back into the body.

[0047] The blood sample can be a whole blood sample, or a processed blood sample, such as serum, plasma, etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc.) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques for phlebotomy. 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 biological fluid samples from a subject are generally low-invasive or non-invasive.

[0048] A whole blood sample is defined as a blood sample extracted from the body and (substantially) without removing any component (such as platelets or plasma). In other words, the relative proportions of the components in the whole blood sample are substantially the same as the blood in the body. In this article, "substantially the same" allows very small changes in the relative proportions of the whole blood components, such as changes of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, etc. Whole blood contains both the cellular part and the liquid part of the blood. Therefore, a whole blood sample can also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (that is, at least the necessary white blood cells, red blood cells, platelets are included in the blood) are complete.

[0049] In a preferred embodiment, the biological fluid sample is serum.

[0050] Methods for analyzing (and optionally isolating, enriching or extracting) protein biomarkers from blood, plasma, serum, saliva, and urine samples have been described previously, see, for example, Heitzer, E., Haque, IS, Roberts, CES, et al. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet 20, 71-88 (2019).

[0051] 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 the system. In the art, the term "biomarker" is sometimes also applied to the means for detecting the endogenous substance (e.g., antibodies, nucleic acid probes, etc., imaging systems). In the context of the present invention, the term "biomarker" should only apply to substances, not to detection means. Therefore, a biomarker can be any type of molecule present in a living organism, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, plasmin, etc.), metabolites or hormones (blood sugar, insulin, estrogen, etc.), molecules with certain modification features of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances that have been internalized by an organism or metabolites of such substances. A biomarker is an organic biological molecule (e.g., protein, polypeptide, peptide, isomeric forms thereof, immunologically detectable fragments thereof, corresponding nucleic acid molecules (e.g., mRNA, cDNA, etc.)), which is differentially present in a sample taken from a subject with a disease compared to a subject without the disease. If the mean or median level of a biomarker in different groups is calculated to be statistically relevant, then the biomarker is present differently. Common tests for statistical significance include, among others, t-tests (e.g., Student's t-tests), ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, receiver operating characteristics (ROC curves), accuracy, and odds ratios. Biomarkers, alone or in combination, provide a measure of the relative risk that a subject belongs to one phenotypic state or another phenotypic state.

[0052] Therefore, they can be used as markers of disease (diagnosis), drug treatment efficacy, and drug toxicity.

[0053] Typically, the biomarkers referred to herein are measured at the protein level.

[0054] The methods provided herein refer to "determining" the level of one or more proteins. As will be clear to those skilled in the art, the level of one or more proteins is typically "determined" by measuring the level of the protein in a sample. Therefore, the term "determining" may be replaced herein with the term "measuring" or "determining by measuring."

[0055] As used herein, the term "determine" or "assessment" also refers to assessing / determining whether a patient has endometriosis. Thus, as used herein, assessment / determination includes diagnosing endometriosis, assessing the risk of a subject having endometriosis, selecting a therapy for endometriosis, monitoring a patient having endometriosis or being treated for endometriosis by determining the amount or concentration of c-Kit in a patient sample and comparing the determined amount or concentration with a reference. Typically, the assessment referred to in accordance with the present invention is an assessment of the presence of endometriosis.

[0056] The terms "measurement", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurement.

[0057] Conventional "determining" methods can include sending clinical samples to commercial laboratories to measure the biomarker levels in biological fluid samples, or using commercially available assay kits to measure the biomarker levels in biological fluid samples. Exemplary kits and suppliers will be apparent to those skilled in the art. In various examples, ELISA assays or lateral flow devices can be used to determine, detect and / or quantify biomarkers, such as for point-of-care use, and spot check colorimetric testing.

[0058] The term "level" or "amount" as used herein encompasses the absolute amount of a biomarker as referred to herein, the relative amount or concentration of the biomarker, and any value or parameter associated therewith or derivable therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values ​​in a mass spectrum or NMR spectrum. In addition, encompassed are all values ​​or parameters obtained by indirect measurements specified elsewhere in this specification, such as, for example, the response amount measured from a biological readout system in response to the peptide or the intensity signal obtained from a specifically bound ligand. It should be understood that values ​​associated with the above amounts or parameters can also be obtained by all standard mathematical operations.

[0059] The level of a biomarker present in a biological fluid sample can be determined, for example, by measuring the amount of a protein biomarker present in the sample. Assays for measuring the amount of a particular protein are well known in the art and include direct or indirect measurements. The level of a protein biomarker in a sample can also be determined by determining the level of protein biomarker activity in the sample. Thus, a protein "level" encompasses both the amount of the protein itself or its level of activity.

[0060] 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, for example, a process selected from the group consisting of mass spectrometry, immunoassay, enzyme assay, spectrophotometry, colorimetric assay, fluorescence assay, bacterial assay, protein microarray, compound separation technology, or other known techniques for determining the presence and / or quantity 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 (using, for example, a lateral flow device (LFD) utilizing a membrane-bound antibody specific for a protein biomarker).

[0061] Preferably, the levels of protein biomarkers in the biological fluid sample are measured by ELISA or lateral flow.

[0062] The term "regularly" as used herein refers to the periodic determination of the c-Kit level in the same subject after a predetermined time. Since each subject is different, the rate of progression of the disease may vary from subject to subject. However, in one subject, disease progression may progress from stage I to stage IV in a few years, while in another subject, disease progression may stagnate in one stage for several years. Therefore, in order to obtain a continuous picture of the development of c-Kit levels in a subject and to be able to assign the obtained c-Kit levels to the corresponding disease stages I, II, III and IV, the intervals should be selected in such a way that stages of disease progression are not skipped. Preferably, the c-Kit level of the subject is determined every 6, 7, 8, 9, 10, 11 or 12 months.

[0063] The term "c-Kit" is a gene encoding the receptor tyrosine kinase protein CD117, also known as c-kit and SCF R. It is a cellular receptor for stem cell factor (SCF), also known as c-kit ligand, mast cell growth factor and steel factor. This receptor-ligand system plays an important role in germ cell development, melanogenesis, hematopoiesis and tumorigenesis. CD117 is expressed on lymphocytes, bone marrow cells, erythrocyte and megakaryocyte progenitor cells, NK cells, germ cells, melanocytes, glial cells, vascular smooth muscle cells, placenta and epithelial cells. Altered levels and mutations of CD117 are associated with several types of cancer, including lung cancer, breast cancer, gastrointestinal stromal tumors and germ cell tumors. Multiple transcript variants of this gene have been found to encode different subtypes. SCF exerts its biological effects by binding to the receptor c-kit, which is expressed in mast cells, hematopoietic stem cells and germ line cells [Cho NH. et al. (2004) FertilSteril 81: 403-7]. The amino acid sequence of human c-Kit is available via UniProt (see UniProtKB-P10721 (KIT_HUMAN) HUMAN). Three isoforms are described for c-Kit, with the identifiers P10721-1, P10721-2 and P10721-4.

[0064] "CA-125", 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 cell ovarian cancer, which originates from the luminal epithelium, including the endometrium, fallopian tubes, ovaries, and peritoneum. The diagnostic use of CA-125 is limited to stages III and IV (moderate and severe endometriosis) of endometriosis with moderate sensitivity.

[0065] "Symptoms" of a disease refer to the effects of the disease that are perceptible to the tissue, organ, or organism suffering from the disease, and include, but are not limited to, pain, weakness, tenderness, strain, stiffness, and cramping of a tissue, organ, or individual. "Signs" or "signals" of a disease include, but are not limited to, changes or alterations in specific indicators (such as biomarkers or molecular markers), such as presence, absence, increase or rise, decrease or fall, or the development, presence, or worsening of symptoms. Symptoms of pain include, but are not limited to, unpleasant sensations that may be persistent or varying degrees of burning, throbbing, itching, or tingling.

[0066] The terms "disease" and "illness" are used interchangeably herein and refer to abnormal conditions, especially abnormal medical conditions, such as diseases or injuries, in which tissues, organs or individuals are no longer able to effectively perform their functions. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such a disease. Therefore, the presence of such symptoms or signs can indicate a tissue, organ or individual suffering from a disease. Changes in these symptoms or signs can indicate the progression of the disease. The progression of a disease is typically characterized by an increase or decrease in such symptoms or signs, which can indicate a "deterioration" or "improvement" of the disease. The "deterioration" of a disease is characterized by a decrease in the ability of a tissue, organ or organism to effectively perform its function, while the "improvement" of a disease is typically characterized by an increase in the ability of a tissue, organ or organism to effectively perform its function. Tissues, organs or individuals with a "risk of developing" a disease are in a healthy state, but show the possibility of developing the disease. Typically, the risk of developing a disease is associated with early or faint signs or symptoms of the disease. In this case, 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 disorders, endocrine diseases, intestinal diseases, nervous system disorders, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0067] "Endometriosis" is a chronic, hormone-dependent inflammatory disease characterized by lesions of endometrial-like tissue outside the uterus. The clinical manifestations of endometriosis vary significantly from patient to patient. Patients with endometriosis often present with symptoms such as intermenstrual bleeding, painful menstruation (dysmenorrhea), painful sexual intercourse (dyspareunia), painful defecation (dysdefecation), and painful urination (dysuria). Pelvic pain caused by endometriosis is usually chronic (lasting ≥6 months) and is accompanied by dysmenorrhea (50 to 90% of cases), dyspareunia, deep pelvic pain and lower abdominal pain, with or without back pain and lumbar pain. The pain may occur unpredictably and intermittently throughout the menstrual cycle, or it may be continuous and may be dull, throbbing, or severe, and may be aggravated by physical activity. Bladder-related symptoms and bowel-related symptoms (nausea, bloating, and early satiety) are usually cyclical. The pain usually worsens over time and the characteristics may change; in rare cases, women report a burning sensation or hypersensitivity, symptoms that suggest a neuropathic component. Often, endometriosis may be asymptomatic and only comes to the attention of the clinician during the evaluation of infertility (Sinaii et al. Fertil Steril. 2008; 89(3): 538-545). In women with endometriosis, the monthly fertility rate is reduced (2-10%) compared with that of fertile couples (15-20%). Although endometriosis can impair fertility, it does not usually completely prevent conception (Fadhlaoui et al. Front Surg. 2014; 1: 24).

[0068] The most common sites affected by endometriosis are the pelvic organs and peritoneum, but other parts of the body, such as the lungs, are occasionally affected. The extent of the disease varies from a few small lesions in otherwise normal pelvic organs to large ovarian endometriomas (endometriomas) and / or extensive fibrosis and adhesion formation that cause significant distortion of the pelvic anatomy. Based on location, endometriotic lesions can be divided into peritoneal endometriosis, ovarian endometriomas (endometriomas), deep nodules (deep infiltrating endometriosis), and adenomyosis (Kennedy et al. Hum Reprod. 2005; 20(10): 2698-2704). Deep infiltrating endometriosis is considered to be any manifestation of endometriosis located outside the superficial tissues of the rectovaginal septum and vaginal vault, pelvic wall, parametrial tissue, bowel, uterus, or bladder (Halis et al. (2010). Deutsches Endometrium. International, 107(25), 446). Endometriosis may also involve the diaphragm (diaphragmatic endometriosis) or the thorax (thoracic endometriosis) (Nezhat et al. JSLS 2019).

[0069] The term "rASRM stage" or "rASRM staging" refers to a revised classification system established by the American Society for Reproductive Medicine (ASRM) that describes the severity of endometriosis based on surgical (laparoscopy) findings. The classification is based on the morphology of peritoneal and pelvic implants, such as red, white, and black lesions, and the percentage of involvement of each lesion should be included. The number, size, and location of endometrial implants, plaques, endometriomas, and adhesions should be noted. Endometriosis in the intestine, urinary tract, fallopian tubes, vagina, cervix, skin, or other locations should be recorded in accordance with the ASRM guidelines. According to the ASRM guidelines, the stages of endometriosis are stage I, stage II, stage III, and stage IV, which are determined based on a score, and they correspond to mild, mild, moderate, and severe endometriosis. rASRM stages I and II endometriosis (mild to mild endometriosis) are defined by the presence of superficial peritoneal endometriosis with possible presence of small deep lesions, absence of endometriomas, and / or mild membranous adhesions. rASRM stages III and IV endometriosis (moderate to severe endometriosis) are defined by the presence of superficial peritoneal endometriosis, deeply infiltrating endometriosis with moderate to extensive adhesions between the uterus and bowel, and / or endometrioma cysts (involving the ovaries and fallopian tubes) with moderate to extensive adhesions.

[0070] The term "VAS", or visual analog scale, is a tool for rating pain intensity. The VAS consists of a 10 cm long horizontal line with two ends labeled "no pain" and "worst pain imaginable". Each patient ticks her pain level on the line, and the distance from the leftmost "no pain" to the tick mark is measured in centimeters to give a pain score from 0 to 10. "No pain" corresponds to a pain score of 0, and "worst pain imaginable" corresponds to a pain score of 10. In women with endometriosis, dysmenorrhea is associated with the highest pain perception, with a mean VAS score of about 6 (Cozzolino et al. Rev Bras Ginecol Obstet 2019;41(3):170-175).

[0071] Subjects may be referred to as patients in this article. The terms "subject", "individual" and "patient" are used interchangeably herein and refer to animals, preferably mammals, and more generally to humans. Patients are preferably human women. Diagnosis for endometriosis needs to be performed at a young age because it begins with the start of menstruation. Therefore, patients are preferably young or adolescent human women aged between 12-24 years old. In one embodiment of the present invention, patients are young or adolescent human women. Subjects may be symptomatic (e.g., subjects show symptoms associated with endometriosis), or subjects may be asymptomatic (e.g., subjects do not show symptoms associated with endometriosis). Subjects may be diagnosed with endometriosis, be at risk of developing endometriosis, or show symptoms of endometriosis. Subjects may suffer from or be suspected of having (e.g., showing symptoms or medical history indicating or suggesting) endometriosis.

[0072] Thus, in some instances, the subject has endometriosis (and the method diagnoses, identifies (or detects) that the subject has endometriosis). As used herein, the terms "diagnose," "identify," and "detect" are used interchangeably.

[0073] In certain examples, the subject has early-stage (stage I or II) endometriosis.

[0074] Patients examined by the method of the present invention should be patients with suspected endometriosis. As used herein, the term "suspected endometriosis" means that the patient should show clinical parameters, signs and / or symptoms of endometriosis. Therefore, patients according to the present invention are usually patients suffering from endometriosis or suspected of having endometriosis. Patients suspected of having endometriosis have the following signs and symptoms: dysmenorrhea (menstrual pain), dysuria (painful urination), dysdefecation (difficult or painful defecation), dyspareunia (pain during or after sexual intercourse) and chronic abdominal / pelvic pain unrelated to the menstrual cycle, heavy menstrual bleeding, long menstrual cycles, infertility, fatigue, cyclical lung problems (pneumothorax), cyclical cough, chest pain or coughing up blood (hemoptysis), shoulder tip pain, painful rectal bleeding or blood in the urine (hematuria) and cyclical scar swelling and pain (EHRE Information on Endometriosis, 2022 www.eshre.eu / guidelines).

[0075] Alternatively, c-Kit levels are routinely determined as part of a screening test without suspicion of endometriosis but enabling the detection of asymptomatic endometriosis at an early stage.

[0076] By detecting increased levels of c-Kit in a subject, the suspicion that the subject suffers from endometriosis will be confirmed, and the subject is at high risk of suffering from endometriosis. In particular, in the case where the subject already exhibits clinical parameters, signs and / or symptoms of endometriosis, the determination of increased c-Kit levels will confirm the presence of endometriosis.

[0077] As used herein, the term "comparison" refers to comparing the amount / level of a biomarker in a sample from a subject with a reference amount or reference value of a biomarker specified elsewhere in this specification. It should be understood that comparison as used herein generally refers to the comparison of corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, and comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or computer-assisted. Therefore, the comparison can be performed by a computing device. For example, the value of the amount measured or detected from a sample of a biomarker from a subject can be compared with a reference amount, and the comparison can be performed automatically by a computer program executing an algorithm for comparison. The computer program performing the evaluation will provide the required assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with a value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e., automatically provide the required assessment in an appropriate output format. For computer-assisted comparison, the value of the measured amount can be compared with a value corresponding to a suitable reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison, ie automatically provide the required assessment in a suitable output format.

[0078] As used herein, the term "(appropriate) reference value" or "reference sample" or "control (sample)" refers to a sample that is analyzed in substantially the same manner as the target sample and its information is compared with the information of the target sample. Therefore, the reference sample provides a standard for evaluating the information obtained from the target sample. The control sample can be derived from the body fluids of a healthy individual, in particular serum or plasma for non-invasive testing, thereby providing a standard for the health status of a tissue, organ or individual. The difference between the state of a normal reference sample and the state of the target sample can indicate the presence or further progression of such a disease or illness. The control sample can be derived from an abnormal or diseased tissue, organ or individual, thereby providing a standard for the diseased state of a tissue, organ or individual. The difference between the state of a normal or abnormal reference sample and the state of the target sample can indicate the absence or improvement of such a disease or illness.

[0079] The reference sample may also be derived from the same tissue, organ or individual as the target sample, but has been collected at an earlier time point. The difference between the state of the reference sample collected earlier and the state of the target sample may indicate the progression of the disease, i.e., the improvement or worsening of the disease over time, and may thereby allow classification of the stage of endometriosis as stage I, II, III or IV.

[0080] The determined value may be compared with more than one (appropriate) reference value, which may be of different kinds. For example, the determined value may be compared with one or more values ​​obtained from the same subject at an earlier time point, and in parallel it may be compared with one or more values ​​obtained from other subjects (with endometriosis of known stage).

[0081] The control sample can be an internal or external control sample. Using an internal control sample, the marker level is assessed in the test sample and in one or more other samples taken from the same subject to determine whether there is any change in the level of the marker. For an external control sample, the presence or amount of the marker in a sample derived from an individual is compared with its presence or amount in an individual known to suffer from a given condition or known to be at risk of suffering from a given condition or an individual known to not have a given condition (i.e., a "normal individual").

[0082] Those skilled in the art will appreciate that such external control samples may be obtained from a single individual or from a reference population that is age-matched and free of confounding disease. Typically, samples of 100 well-characterized individuals from an appropriate reference population are used to establish a "reference value". However, a reference population may also be selected to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a preferred reference population for establishing a control value.

[0083] For example, the marker concentration in the patient sample can be compared with a concentration known to be associated with a specific course of a certain disease. For example, it can be compared with a concentration known to be associated with a certain stage of endometriosis. Typically, the marker concentration of a sample is directly or indirectly related to the diagnosis, and the marker concentration is used, for example, to determine whether an individual is at risk of suffering from the disease. Alternatively, the marker concentration can be compared with a marker concentration obtained from the same subject at an earlier time point. Alternatively, in judging the risk of disease progression or in the follow-up of a patient, the marker concentration of a sample can be compared with a marker concentration known to be associated with a therapeutic response to a certain disease, a diagnosis of a certain disease, an assessment of the severity of a certain disease, or a guidance for selecting appropriate drugs for a certain disease. Depending on the intended diagnostic use, an appropriate control sample is selected and a control or reference value of the marker is established therein. As the technician is also aware, the absolute label value established in the control sample will depend on the assay used.

[0084] For the methods described herein, the most common control samples and / or reference values ​​obtained therefrom are obtained from, but not limited to, "non-pathological controls" and "symptomatic controls". The corresponding subjects from which these samples were obtained are "non-pathological subjects" and "symptomatic subjects", respectively.

[0085] "Non-pathological control" refers to a control sample from a subject who does not have endometriosis and does not exhibit any symptoms that may be associated with endometriosis (eg, menstrual / abdominal pain, uterine / ovarian cysts or cancer, etc.).

[0086] "Symptomatic controls" refers to control samples from subjects who have symptoms typically associated with endometriosis (e.g., menstrual / abdominal pain, infertility, etc.), but in which endometriosis can be ruled out based on laparoscopy and no tissue changes (e.g., uterine / ovarian cysts or cancer) are observed.

[0087] "Controls with benign findings" refers to a group of samples from subjects with tissue changes (e.g., uterine / ovarian cysts, cancer, fibroids) that are not similar to endometriosis. In addition, these subjects can be symptomatic (e.g., menstrual / abdominal pain, infertility, etc.) or asymptomatic, which can also change over time.

[0088] The control sample can be assayed at the same time, before or after, separately or simultaneously with the test sample. The control value for comparison with the test sample can be a value calculated as the average or median of more than one (e.g., two or more, five or more, ten or more, a group, etc.) of the control samples. Alternatively, the control sample can be a sample (i.e., a mixture thereof) derived from more than one (e.g., two or more, five or more, ten or more, a group, etc.) individuals (or symptomatic controls) who do not have endometriosis.

[0089] In one example, the control sample is thus obtained from a control subject not suffering from endometriosis (a "non-pathological control"). In a further example, the control sample is obtained from a subject who serves as a "symptomatic control".

[0090] In order to classify the stage of endometriosis, a comparison of the test sample with several different control samples may or must be performed to be able to assign the result to a certain stage. For example, a comparison with a non-pathological sample and a stage II sample. Or a comparison with a non-pathological sample, a stage II sample, and a stage IV sample. This may also be combined with surgery to confirm or determine a certain stage of the disease.

[0091] Alternatively, the level of a biomarker (e.g., a protein) in a biological fluid sample can be compared to a predetermined reference level of a target biomarker. As used herein, a "predetermined reference level" refers to a biomarker level obtained from a reference database that can be used to generate a predetermined cutoff value, i.e., a score that is statistically predictive of endometriosis. In one example, the predetermined reference level is the average or median level of the biomarker in at least one individual from the same species who does not suffer from endometriosis. In one example, the predetermined reference value can be calculated as the average or median taken from a group or group of individuals who do not suffer from endometriosis. For example, the predetermined reference value can be calculated as the average or median taken from a group or group of individuals as a "symptomatic control". The individual or group of individuals can be the same age or in the same health state or condition as the subject from whom the test sample was obtained.

[0092] In one example, the predetermined reference level is thus the mean level of the biomarker in control subjects not suffering from endometriosis. In a further example, the predetermined reference level is the mean level of the biomarker in subjects serving as "symptomatic controls".

[0093] Typically, in methods for diagnosing endometriosis in a subject, a 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 instances, the control or predetermined reference is used as a baseline to determine whether the test subject has endometriosis.

[0094] 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 for the methods described herein for classifying the stage of endometriosis, determining the progression of a subject, determining the therapeutic efficacy of a treatment regimen for endometriosis, and / or determining the compliance or adherence of a subject to a prescribed treatment regimen for endometriosis. To this end, the sample is taken from the same biological fluid of the same subject, wherein the biological fluid is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, preferably the biological fluid sample is serum.

[0095] In such examples, a control sample or predetermined reference level is used to determine any changes in the level of a biomarker in the same subject over a time interval. Thus, the predetermined reference level or control sample may be from the same subject from which the test sample was obtained, for example, at an earlier time point. This earlier time point may be before they were diagnosed with endometriosis.

[0096] The predetermined level can be a single cutoff value, such as median or mean. It can be a cutoff value (or threshold) of a range, such as a confidence interval. It can be established based on a comparison group, such as one of the risk in a defined group is higher or lower than one time (e.g., about 2 times, 4 times, 8 times, 16 times or more) than the risk in another defined group. For example, it can be a range, wherein a subject population (e.g., a control subject) is equally (or unequally) divided into groups, such as a low-risk group, a medium-risk group and a high-risk group, or divided into quartiles, the lowest quartile is the subject with the lowest risk, and the highest quartile is the subject with the highest risk, or divided into n quartiles (i.e., n regularly spaced intervals), the lowest in n quartiles is the subject with the lowest risk, and the highest in n quartiles is the subject with the highest risk. In addition, reference can be a reference to the calculation of the relative amount or absolute amount of the biomarker of the individual population of the subject to be studied, most preferably mean or median. How to calculate a suitable reference value, preferably mean or median, is well known in the art.

[0097] Thus, in some cases, a level of a protein biomarker in a subject that is greater than or equal to the level of the biomarker in a control sample or a predetermined reference level indicates a clinical state (e.g., indicating endometriosis). In other cases, a level of a biomarker in a subject that is less than or equal to the level of the biomarker in a control sample or a predetermined reference level indicates a certain stage of endometriosis.

[0098] Typically, but not necessarily, a greater than or less than sufficient to distinguish a subject from a control subject is a statistically significant greater than or a statistically significant less than. In the case where the level of a biomarker in a subject is equal to the level of a biomarker in a control subject, indicating the stage of endometriosis, "equal" means approximately equal (e.g., not statistically different).

[0099] The predetermined value can depend on the particular subject population (e.g., human subjects) selected. For example, an apparently healthy population will have a different "normal" range for a protein biomarker than a subject population that has or may have endometriosis. Thus, the predetermined value selected can take into account the category (e.g., healthy, diseased, disease stage) to which the subject (e.g., human subject) belongs.

[0100] One of ordinary skill in the art can select appropriate ranges and categories using only routine experimentation.

[0101] Suitably, the level of a specific biomarker detected in a sample (e.g., a test sample, a 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, wherein the reference protein itself is not a marker (it is, for example, a constitutively expressed protein). This normalization allows the level of a biomarker in one sample to be compared with another sample, or to be compared between samples from different sources. The normalized level can then be optionally compared with a reference value or a control. For example, when measuring a protein biomarker in a whole blood sample, the biomarker can be expressed as an absolute concentration, or, alternatively, it can be normalized for known proteins such as albumin, immunoglobulins, or plasma protein concentrations that are constitutively expressed in whole blood.

[0102] For example, when measuring a protein biomarker in a serum (or plasma) sample, the biomarker can be expressed as an absolute concentration or, alternatively, can be normalized to a known protein that is constitutively expressed in serum (or plasma).

[0103] The levels of biomarkers in a test sample can be compared to the levels of the same biomarkers in a control sample or to a predetermined reference level of the same biomarker to identify an increase or decrease in the level of one or more biomarkers in the subject's sample.

[0104] In the methods described herein, if the comparison (between the biomarker level in the control sample / predetermined reference value and the 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.

[0105] The term "classification" as used herein 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 of stages I, III and IV are approximately the same and are increased compared to the control. The c-Kit level of stage II is increased compared to stages I, III and IV. This allows the status of endometriosis to be classified as meeting the criteria of falling into one of the four stages based on the determined c-Kit level based on the comparison of the sample under study with the reference value or with at least one sample studied at an earlier time. Based on the reference value / reference sample, the difference between the level of c-Kit in the target sample and the reference sample can be used to assign the stage of endometriosis to a certain stage according to r-ASRM. For example, if the c-Kit reference value is derived from a sample of a patient with stage II endometriosis, a decreased level of c-Kit in the target sample will mean 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 c-Kit value obtained 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 an increased level in the target sample will mean that the patient has stage II endometriosis. Classification can also be performed if ranges of c-Kit levels assigned to certain stages of endometriosis are defined. Thus, an obtained value falling within the range assigned to, for example, stage I will mean that the patient has stage I endometriosis.

[0106] In addition, it should be understood that if the risk of worsening health conditions is predicted, the prediction is usually made within a prediction window of 6 months and two years. More typically, for non-invasive tests that depend on symptoms (such as pelvic pain), the prediction window is a time window of about 6 months to 12 months.

[0107] As will be appreciated by those skilled in the art, although the assessment made according to the present invention is preferred, it may not generally be correct for 100% of the subjects studied. The term generally requires the ability to correctly assess a statistically significant portion of subjects. Those skilled in the art can use various well-known statistical evaluation tools (e.g., determining confidence intervals, determining p-values, Student's t-tests, Mann-Whitney tests, etc.) to effortlessly determine whether a portion is statistically significant. For detailed information, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. The confidence intervals generally envisioned are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0108] The term "decreased" or "reduced" level of an indicator refers to a reduction in the level of such an indicator in a sample compared to a reference (value) or reference sample. The terms "reduce", "reduced", "reduced", "reduction" or "down-regulated", "decline" are generally used herein to mean a reduction in a statistically significant amount. However, for the avoidance of doubt, "reduced," "reduced," "lowered," or "reduced" means as compared to a reference level / control by at least 10%, for example, a decrease of 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 up to and including a 100% decrease (i.e., absent levels compared to a reference / control sample), or as compared to a reference level / control by any decrease between 10%-100%, or as compared to a reference level / control by 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 decrease between 1.0-fold and 10-fold or greater.

[0109] The term "elevated" or "increased" level of an indicator / (bio)marker refers to a higher level of such an indicator in a sample compared to a reference (value) or reference sample. For example, a protein with an elevated level may be detected in higher amounts in a fluid sample of an individual suffering from a given disease than in the same fluid sample of an individual not suffering from the disease. The terms "increased", "increase" or "up-regulated", "higher" are generally used herein to refer to an increase in a statically significant amount; for the avoidance of any doubt, the term "increased" or "increase" means an increase of at least 10% compared to a reference level / control, such as an increase of 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 up to and including a 100% increase, or any increase between 10%-100% as compared to a reference level / control, or 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 as compared to a reference level / control, or any increase between 1.0-fold and 10-fold or greater.

[0110] As used herein, the term "immunoglobulin (Ig)" refers to an immunity-conferring glycoprotein of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, class I and class II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (about 2 M), CD3, CD4, and CDS.

[0111] Generally, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavities. Human antibodies are divided into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by the Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibodies, i.e., these chains are present in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each playing a different role and directing appropriate immune responses against different types of antigens. Different heavy chains differ in size and composition; and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal areas, such as the intestines, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, to prevent pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4: 389-417). IgD functions primarily 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 participates in allergic reactions by binding to allergens, triggering the release of histamine from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens, and is the only antibody isotype that can cross 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 IgG (about 4%). The biological characteristics of the different IgG classes are determined by the structure of the corresponding hinge region. IgM is expressed on the surface of B cells in monomeric and secretory pentamer forms with very high avidity. IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity, before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118: 429-437).Antibodies exist not only in monomeric form, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of bony fish), or pentamers of five Ig units (e.g., IgM of mammals). Antibodies are usually composed of four polypeptide chains, including two identical heavy chains and two identical light chains, which are connected via disulfide bonds and resemble "Y"-shaped macromolecules. Each chain contains many immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domain consists of a 2-layer sandwich structure, in which 7 to 9 antiparallel chains are arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) domains, and one of which is a variable domain (VH). The light chain usually contains a constant Ig domain (CL) and a variable Ig domain (VL). For example, the human IgG heavy chain consists of four linked Ig domains in the order VwCH1-CH2-CH3 (also called VwCyl-Cy2-Cy3) from the N-terminus to the C-terminus, while the human IgG light chain consists of two linked immunoglobulin domains in the order VL-CL from the N-terminus to the C-terminus, which are of the K type or λ type (VK-CK or VA.-CA.). For example, the constant chain of human IgG contains 447 amino acids. In the present specification and claims, the numbering of amino acid positions in immunoglobulins is the numbering of the "EU index", as in the following documents: Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS and Foeller, C., (1991) Sequences of proteins of immunological interest, 5th edition US Department of Health and Human Service, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue numbering of human IgG IEU antibodies. Thus, the CH domains in the context of IgG are as follows: "CHI" 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; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0112] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term specifically refers to an antibody having heavy chains that include an Fc region.

[0113] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each with a single antigen-binding site, and a residual "Fc fragment" (also called "Fc portion" or "Fc region"), whose name reflects its ability to crystallize readily. 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; in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH2-4) in each polypeptide chain. In addition, smaller immunoglobulin molecules occur naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of the Ig molecule, while a "F(ab')2 fragment" is understood to include two Fab' fragments chemically linked or linked via a disulfide bond. While "single domain antibodies (sdAb)" (Desmyter et al. (1996) Nat. Structure Biol. 3: 803-811) and "nanobodies" include only a single VH domain, "single chain Fv (scFv)" fragments include a heavy chain variable domain joined to a light chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). A divalent single chain variable fragment (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be achieved by creating a single peptide chain with two VH and two VL regions, thereby creating a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create a scFv with a joint that is too short for two variable regions to fold together, thereby forcing scFv dimerization. Typically, a joint with a length of 5 residues is used to generate these dimers. This type is referred to as a "diabody". A shorter joint (one or two amino acids) between VH and VL domains leads to the formation of a monospecific trimer, so-called "triabodies" or "tribadies". Bispecific diabodies are formed by expressing a chain having VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA arrangements, respectively. Single-chain diabodies (scDb) include VHA-VLB and VHB-VLA fragments, which are connected (VHA-VLB-P-VHB-VLA) by 12-20 amino acids, preferably 14 amino acid linker peptides (P)."Bispecific T cell engagers (BiTEs)" are fusion proteins consisting of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor and the other to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22: 238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies that are additionally stabilized by a C-terminal disulfide bond.

[0114] Therefore, 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; sdAb, nanobodies, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDb, BiTE and DART.

[0115] The term "binding affinity" generally refers to the strength of the sum 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 specified, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance-based assays (such as BIAcore assays as described in PCT Application Publication No. WO2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competition assays (e.g., RIAs). Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen quickly and tend to remain bound for longer periods of time. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0116] "Sandwich immunoassays" are widely used to detect target analytes. In this assay, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, sandwich assays require that the capture and detection antibodies bind to different non-covering epitopes on the target analyte. This sandwich complex is measured by an appropriate means and the analyte is quantified thereby. In a typical sandwich assay, a first antibody bound to a solid support or capable of binding to a solid phase and a second antibody detectably labeled each bind to a different non-covering epitope with the analyte. A first analyte-specific binding agent (e.g., an antibody) is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be in the form of a tubule, magnetic beads, microplate tray, or any other surface suitable for performing an immunoassay. Binding methods are well known in the art and typically consist of cross-linked covalent binding or physical adsorption, with the polymer-antibody complex being washed when the test sample is prepared. An aliquot of the sample to be tested is then added to the solid phase complex and incubated under appropriate conditions (e.g., from room temperature to 40°C, such as between 25C and 37C, inclusive) for a sufficient period of time (e.g., 2-40 minutes or overnight if more convenient) to allow binding between the first or capture antibody and the corresponding antigen. After the incubation period is complete, the solid phase, which includes the first or capture antibody and the antigen bound thereto, can be washed and incubated with a secondary antibody or labeled antibody that binds to another epitope on the antigen. The second antibody is linked to a reporter molecule that is used to indicate binding between the second antibody and the first antibody-target antigen complex.

[0117] A very versatile alternative sandwich assay format involves the use of a solid phase coated with a first partner of a binding pair, such as paramagnetic streptavidin-coated microparticles. Such microparticles are mixed and incubated with: an analyte-specific binding agent (e.g., a biotinylated antibody) that binds to a second partner of the binding pair; a sample suspected of or including an analyte to which the second partner of the binding pair binds; and a detectably labeled second analyte-specific binding agent. As will be apparent to one skilled in the art, the components are incubated under appropriate conditions for a sufficient period of time to allow the labeled antibody (via the analyte), the analyte-specific binding agent that binds to the second partner of the binding pair, and the first partner of the binding pair to bind to the solid phase microparticles. Optionally, the assay may include one or more washing steps.

[0118] The term "detectably labeled" encompasses labels that are detectable either directly or indirectly.

[0119] Directly detectable labels provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first label or the second label, for example to cause 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 suitable for labeling. In one embodiment, detectably labeled refers to a label that provides or induces 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, respectively.

[0120] The large number of available labels (also called dyes) can be generally divided into the following categories, all of which collectively and each of which represent embodiments as described in the present disclosure:

[0121] (a) Fluorescent dyes

[0122] 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).

[0123] Fluorescent labels or fluorophores include rare earth chelates (europium chelates); fluorescein type labels, including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine labels, including TAMRA; dansyl; Lissamine; cyanine; phycoerythrin; Texas Red; and analogs thereof. Using the techniques disclosed herein, fluorescent markers can be conjugated to aldehyde groups contained in target molecules. Fluorescent dyes and fluorescent labeling reagents include such fluorescent dyes and reagents available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0124] (b) Luminescent dyes

[0125] Luminescent dyes or labels can be further divided into the following subcategories: chemiluminescent dyes and electrochemiluminescent dyes.

[0126] Different classes of chemiluminescent labels include luminol, acridine compounds, coelenterazine and analogs, dioxetanes, peroxyoxalic acid-based systems and their derivatives. For immunodiagnostic procedures, mainly acridine-based labels are used (for a detailed review, see Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0127] The main related labels used as electrochemiluminescent labels are ruthenium-based and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful in analytical applications as a highly sensitive and selective method. This method combines the analytical advantages of chemiluminescent analysis (no background light signal) with a more convenient control of the reaction by adopting electrode potentials. Typically, ruthenium complexes, especially [Ru(Bpy)3]2+ (releasing photons at about 620nm) regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface are used as ECL labels.

[0128] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurements of the presence and concentration of target analytes. The technique employs a label or other reactant that is induced to emit light when electrochemically oxidized or reduced under appropriate chemical conditions. This electrochemiluminescence is triggered by a voltage applied to a working electrode at a specific time and in a specific manner. The light emitted by the label, after measurement, can indicate the presence or amount of the analyte. For a more complete description of such ECL techniques, reference is made to the following documents: U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Published Application WO90 / 05296, PCT Published Application WO92 / 14139, PCT Published Application WO90 / 05301, PCT Published Application WO96 / 24690, PCT Published Application US95 / 03190, PCT Application US97 / 16942, PCT Published Application US 96 / 06763, PCT published applications WO95 / 08644, PCT published applications WO96 / 06946, PCT published applications WO96 / 33411, PCT published applications WO87 / 06706, PCT published applications WO96 / 39534, PCT published applications WO96 / 41175, PCT published applications WO96 / 40978, PCT / US97 / 03653 and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519). Also cited is a review of ECL analysis applications published by Knight et al. in 1994 (Analyst, 1994, 119: 879-890) and the literature cited in the article. In one embodiment, the method described in accordance with the present description is implemented using electrochemiluminescent labels.

[0129] Recently, iridium-based ECL labels have also been described (WO2012107419).

[0130] (c) Radioactive labeling uses radioactive isotopes (radionuclides) such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 131Bi.

[0131] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (US 2010 / 0111861; US ​​5,342,606; US 5,428,155; US 5,316,757; US 5,480,990; US 5,462,725; US 5,428,139; US 5,385,893; US 5,739,294; US 5,750,660; US 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).

[0132] The methods described herein may further include selecting and optionally administering a treatment regimen for the subject based on the diagnosis (i.e., based on the comparison of the level of the biomarker with a reference value / level / control). Treatment may include, for example, surgery, and in some cases therapy, or a combination thereof. However, in some cases, immediate treatment may not be required, and the subject may be selected for active monitoring.

[0133] As used herein, the terms "active surveillance," "monitoring," and "watchful waiting" are used interchangeably herein and mean closely monitoring the patient's condition without administering any treatment until symptoms appear or change.

[0134] As used herein, the terms "treat", "treating" and "treatment" are considered to include interventions performed with the purpose of altering the pathology of a condition, illness or symptom (i.e., in this case, endometriosis). Thus, "treatment" refers to therapeutic treatment, wherein the purpose is to slow down (mitigate) the target condition, illness or symptom. Thus, "treatment" encompasses reducing, slowing down or inhibiting the symptoms of endometriosis, for example, by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the symptoms before treatment. In the case of endometriosis, appropriate treatment may include analgesics, hormone therapy (such as hormonal contraceptives), gonadotropin-releasing hormone (GnRH) agonists and / or surgery. (Longo, DL et al. 2020. Sc. DN Engl J Med, 382, ​​pp. 1244-56).

[0135] As used herein, the term "surgery" applies to surgical procedures performed to remove endometrial tissue, such as laparoscopy or nerve-sparing surgery.

[0136] As used herein, the term "therapy" includes drug-based therapy, radiation, hormone therapy, cryosurgery, chemotherapy, immunotherapy, biological therapy, and high-intensity focused ultrasound. Drug-based therapy for endometriosis can be, for example, by inhibiting or targeting neurogenic inflammation and / or analgesics and / or hormone therapy.

[0137] The type of treatment will vary depending on the particular form and / or stage of endometriosis the subject has or is suspected of having.

[0138] Example

[0139] The present inventors have surprisingly identified a new protein biomarker, c-Kit, which is increased in biological fluids, in particular serum, of women with endometriosis, in particular women with early stages of endometriosis.

[0140] The biomarker c-Kit can be used to diagnose endometriosis in a subject or classify the stage of endometriosis compared to a control (eg, a non-pathological subject or a symptomatic subject).

[0141] In particular, serum c-Kit can be used as a blood biomarker for early diagnosis and risk stratification of endometriosis. In addition, serum c-Kit can be used to select patients with stage I and II of the disease for early medical management of endometriosis. Therefore, it can significantly reduce the delay in diagnosis of endometriosis, improve the lives of patients, and reduce the economic burden.

[0142] Biomarkers may be advantageously used in any of the methods, kits, assays or uses described herein.

[0143] Method for diagnosing endometriosis in a subject

[0144] In a first aspect, the present invention relates to a method for diagnosing endometriosis in a subject, the method comprising the steps of:

[0145] a) determining the level of c-Kit in a biological fluid sample from a subject,

[0146] b) comparing the level of c-Kit to at least one appropriate reference value for c-Kit level,

[0147] c) if the comparison in step b) indicates that the subject has an increased level of c-Kit compared to an appropriate reference value, identifying the subject as having endometriosis.

[0148] In an embodiment, an elevated level or amount or concentration of c-Kit in a fluid sample of a subject indicates the presence of endometriosis in the subject. In particular, if the amount or concentration of c-Kit in the fluid sample of the subject is higher than the amount or concentration of c-Kit according to a reference value, the amount or concentration of c-Kit in the fluid sample of the subject indicates the presence of endometriosis in the subject.

[0149] In certain embodiments, at least one suitable reference value is

[0150] i. The level of c-Kit in non-pathological subjects or symptomatic subjects, or

[0151] ii. the mean level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects or a combination thereof,

[0152] or where at least one of the appropriate reference values ​​is

[0153] iii. a predetermined value of the level of c-Kit in a non-pathological subject or a symptomatic subject, or

[0154] iv. a predetermined mean value of the level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects or a combination thereof.

[0155] In certain embodiments, the inventors can detect an increase in serum c-Kit in the early stage of endometriosis, followed by a decrease in serum c-Kit in the late stage of endometriosis. However, the amount or concentration of c-Kit in the late stage is still higher than the control level.

[0156] In particular, an increase in the amount of c-Kit of 50% or more indicates the presence of endometriosis or a risk of developing endometriosis. In particular, an increase in the amount of c-Kit of 100% or more indicates the presence of endometriosis. In particular, an increase in the amount of c-Kit of 150% or more indicates the presence of endometriosis. In particular, an increase in the amount of c-Kit of 200% or more indicates endometriosis.

[0157] Suitably, the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, preferably the biological fluid sample is serum.

[0158] In embodiments, the sample is an in vitro sample, ie it is to be analyzed in vitro and not transferred back into the subject. In embodiments, the method of the invention is an in vitro method.

[0159] In certain embodiments, the subject is a human subject. In certain embodiments, the patient is a female human subject. In certain embodiments, the subject is a young or adolescent human female. In particular, the subject is a subject who is capable of having endometriosis due to a medical condition.

[0160] In an embodiment, the diagnosis is made independently of the rASRM staging. In particular, the assessment is made without laparoscopy. In particular, the assessment is made without using laparoscopy and / or rASRM staging to assess the presence or severity of endometriosis in the patient.

[0161] In an embodiment, the diagnosed endometriosis is selected from the group consisting of: stage I endometriosis according to rASRM staging, stage II endometriosis according to rASRM staging, stage III endometriosis according to rASRM staging, stage IV endometriosis according to rASRM staging. In a specific embodiment, the diagnosed endometriosis is stage I, stage II, stage III or stage IV endometriosis.

[0162] In an embodiment, the endometriosis is early-stage endometriosis, in particular stage I endometriosis according to rASRM staging or stage II endometriosis according to rASRM staging.

[0163] In certain embodiments, the diagnosed endometriosis is stage III or stage IV endometriosis.

[0164] In an embodiment, the endometriosis assessed is selected from the group consisting of peritoneal endometriosis, endometrioma, deep infiltrating endometriosis (DIE) and adenomyosis.

[0165] In certain embodiments, the diagnosed endometriosis is peritoneal endometriosis. In other certain embodiments, the diagnosed endometriosis is peritoneal endometriosis of stage I or stage II according to the rASRM staging system.

[0166] In another embodiment, the method further comprises selecting a treatment regimen for the subject based on a comparison of the level of c-Kit with a control sample or with a predetermined reference level. In a specific embodiment, the method further comprises administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen comprises drug-based therapy and / or surgical treatment (laparoscopy). Drug-based therapy for endometriosis can be performed, for example, by pain medication, hormone therapy, and / or surgery.

[0167] Depending on whether the diagnosis suggests a severe disease stage, one skilled in the art will clearly know how to select the most appropriate and promising treatment option.

[0168] In an embodiment according to the present invention, the protein level of c-Kit is optionally determined using a method selected from the group consisting of: ELISA assay, immunoblotting, lateral flow assay, protein microarray, and mass spectrometry.

[0169] In an embodiment, the amount of c-Kit is determined using an antibody, in particular a monoclonal antibody. In an embodiment, step a) of determining the amount of c-Kit in a sample of a patient comprises performing an immunoassay. In an embodiment, the immunoassay is performed in a direct or indirect form. In an embodiment, such an immunoassay is selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), or an immunoassay based on detection of luminescence, fluorescence, chemiluminescence, or electrochemiluminescence.

[0170] In certain embodiments, step a) of determining the level of c-Kit in a sample from a subject comprises the following steps

[0171] i) incubating the sample from the subject with one or more antibodies that specifically bind to c-Kit, thereby forming a complex between the antibody and c-Kit, and

[0172] ii) quantifying the complex formed in step i), thereby quantifying the amount of c-Kit in the sample from the subject.

[0173] In a specific embodiment, in step i), the sample is incubated with two antibodies that specifically bind to c-Kit. As will be apparent to a person skilled in the art, the sample may be contacted with the first antibody and the second antibody in any desired order, i.e. first with the first antibody and then with the second antibody; or first with the second antibody and then with the first antibody; or simultaneously with the first antibody and the second antibody, 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 appreciated by a person skilled in the art, it is no more than routine experimentation to establish a time and conditions that are appropriate or sufficient to form a complex between a specific anti-c-Kit antibody and a c-Kit antigen / analyte (= anti-c-Kit complex) or to form a secondary or sandwich complex comprising a first antibody against c-Kit, c-Kit (analyte) and a second anti-c-Kit antibody (= anti-c-Kit antibody / c-Kit / second anti-c-Kit antibody complex).

[0174] Detection of the anti-c-Kit antibody / c-Kit complex can be performed by any suitable means. Detection of the first anti-c-Kit antibody / c-Kit / second anti-c-Kit antibody complex can be performed by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0175] In certain embodiments, a sandwich will be formed comprising a first antibody to c-Kit, c-Kit (analyte), and a second antibody to c-Kit, wherein the second antibody is detectably labeled.

[0176] In one embodiment, a sandwich will be formed comprising a first antibody to c-Kit, c-Kit (analyte), and a second antibody to c-Kit, wherein the second antibody is detectably labeled and wherein the first anti-c-Kit antibody is capable of binding to or to a solid phase.

[0177] In embodiments, the second antibody is detectably labeled, directly or indirectly. In specific embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0178] In an embodiment, the method further comprises assessing dysmenorrhea and / or lower abdominal pain in the patient. In an embodiment, the presence of dysmenorrhea and / or lower abdominal pain is assessed according to a VAS scale. In an embodiment, a dysmenorrhea VAS score of 4 or higher indicates moderate or severe dysmenorrhea. In an embodiment, a score of 3 or lower indicates no dysmenorrhea or mild dysmenorrhea.

[0179] In embodiments, the method further comprises determining the level of CA-125 in a biological fluid sample from the subject.

[0180] Method for classifying the stage of endometriosis in a subject

[0181] In a second aspect, the present invention relates to a method for classifying the stage of endometriosis in a subject, the method comprising the steps of:

[0182] a) determining the level of c-Kit in a biological fluid sample from a subject,

[0183] b) comparing the level of c-Kit to at least one appropriate reference value for c-Kit level,

[0184] 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 at least one appropriate reference value for the level of c-Kit.

[0185] In an embodiment, an elevated or decreased level or amount or concentration of c-Kit in a fluid sample of a subject may be indicative of the stage of endometriosis in the subject. In particular, if the level of c-Kit in the fluid sample of the subject is higher or lower than the level of c-Kit according to a reference value for a known stage of endometriosis, the level of c-Kit in the fluid sample of the subject is indicative of the stage of endometriosis in the subject. If necessary, comparison is made with several reference values ​​in order to classify the stage of endometriosis. Furthermore, this can be combined with surgical or other parameters used to determine the stage of endometriosis to allow for a more precise classification.

[0186] In particular, if c-Kit is detectable at higher levels in a fluid sample of a subject assessed to have endometriosis than in the same fluid sample of a subject not suffering from endometriosis, this is indicative of any stage of endometriosis, a slight increase would be indicative of stages I, III and IV, and a more robust increase would be indicative of stage II. By further comparing the obtained value with reference values ​​for known stages of endometriosis, it is possible to assign the obtained value to a certain stage of endometriosis.

[0187] In certain embodiments, at least one suitable reference value is

[0188] i. The level of c-Kit in non-pathological subjects, in symptomatic subjects, or in subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), or

[0189] ii the mean level of c-Kit in a group of subjects who are non-pathological subjects, symptomatic subjects, or a combination thereof, or in a group of subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM),

[0190] or where at least one of the appropriate reference values ​​is

[0191] iii. a predetermined value for the level of c-Kit in non-pathological subjects, or in symptomatic subjects, or in subjects with stage I, II, III, or IV endometriosis, according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), or

[0192] iv. A predetermined mean value of the level of c-Kit in a group of non-pathological subjects, symptomatic subjects, or a combination thereof, or in a group of subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM).

[0193] In certain embodiments, the inventors can detect an increase in serum c-Kit in the early stage of endometriosis, followed by a decrease in serum c-Kit in the late stage of endometriosis. However, the amount or concentration of c-Kit in the late stage is still higher than the control level.

[0194] For example, the levels of c-Kit obtained are higher than in non-pathological subjects but lower than in subjects with stage II endometriosis, indicating stage I, III or IV endometriosis.

[0195] For example, the level of c-Kit obtained is higher than in a subject with stage I endometriosis and higher than in a subject with stage III endometriosis, indicating stage II endometriosis.

[0196] In one example, the method is an in vitro method.

[0197] Details of biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values ​​etc. are provided elsewhere and apply equally to this and all other aspects.

[0198] Method for monitoring the progression of endometriosis in a subject

[0199] In a third aspect, the present invention relates to a method for monitoring the progression of endometriosis in a subject, the method comprising the steps of:

[0200] i. determining the level of c-Kit in a biological fluid sample from a subject according to steps a) to b) of the method described herein above,

[0201] ii. repeating step i. for specific time intervals using a biological fluid sample obtained from the subject during or after the treatment; and

[0202] iii. comparing the level of c-Kit identified in i. with the level of c-Kit identified in ii, wherein a change in the level of c-Kit from i. to ii is indicative of a change in the progression of endometriosis in the subject.

[0203] In embodiments, a patient with endometriosis is monitored to determine whether the amount or concentration of c-Kit in a sample from the patient changes over time. In particular, a patient with endometriosis is monitored to determine whether the amount or concentration of c-Kit increases, decreases, or remains unchanged over time. In embodiments, a patient with endometriosis is monitored if an increase in the amount of c-Kit in a sample from the patient is determined.

[0204] The method can be used to monitor the progression of any of the types of endometriosis described herein.

[0205] Typically, such monitoring methods are performed on subjects who have not been treated for endometriosis (ie, they have not previously received treatment (therapy or surgery) for endometriosis). Such subjects are described herein as "untreated" subjects.

[0206] However, such monitoring methods also encompass methods performed on subjects who are already being treated for endometriosis.

[0207] Monitoring the progression of endometriosis in a subject over time helps to identify disease progression (e.g., worsening of a disease state or disease symptoms) as early as possible. Such monitoring naturally involves repeated sample collection 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 development, progression, or improvement of endometriosis in the subject over time, wherein changes in the amount of the level of the tested biomarker in the biological fluid sample (e.g., serum) indicate changes in the progression of the subject's endometriosis.

[0208] Some studies have reported that serum CA-125 levels decrease after drug treatment after surgery for endometriosis (Chen et al. 1998. Acta Obstet Gynecol Scand. 77:665-70; Jacobs et al. 1989. Hum Reprod. 4:1-12).

[0209] When results from two or more time intervals are compared for the same subject, disease progression may be indicated by an increase in the level of c-Kit detected over time.

[0210] In other words, if the method is performed multiple times, when the level of c-Kit detected at a later time interval is higher than the level of c-Kit detected at an earlier time interval, disease progression can be indicated. An "increase" in the level of c-Kit encompasses detection of c-Kit at a later time interval where c-Kit was not detected (i.e., it was not present at a detectable level) when the method was previously performed (i.e., at an earlier time interval) on the same subject (and equivalent biological fluid sample type). This is particularly relevant when monitoring the progression of endometriosis in untreated subjects.

[0211] Suitable time intervals for monitoring disease progression can be readily determined by one skilled in the art and will depend on the specific form of endometriosis being monitored. As non-limiting examples, the method may be repeated at least weekly, monthly, six months, or at least annually, or whenever clinically indicated, i.e., in the event of a significant change in endometriosis symptoms.

[0212] In one example, the method is an in vitro method.

[0213] Details of biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values ​​etc. are provided elsewhere and apply equally to this and all other aspects.

[0214] Method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject

[0215] 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 the steps of:

[0216] i. determining the level of c-Kit in a biological fluid sample from a subject according to steps a) to b) of the method described herein above,

[0217] ii. repeating step a) for specific time intervals using a biological fluid sample obtained from the subject during or after treatment; and

[0218] iii. comparing the level of c-Kit determined in step a) with the level of c-Kit determined in step b), and if the level of c-Kit decreases after treatment, identifying the treatment regimen as having a therapeutic effect.

[0219] In one example, a change in the level of c-Kit that indicates a therapeutic effect is a decrease in the level of c-Kit following treatment. A "decrease" in the level of c-Kit encompasses the fact that when c-Kit was previously detected (i.e., at an earlier time interval) when the method was performed on the same subject (and equivalent biological fluid sample type), c-Kit is not detected (i.e., it is not present at a detectable level) at a later time interval.

[0220] Step i. may first be performed according to the method using a biological fluid sample obtained from the subject at a time point before the start of the treatment regimen for endometriosis. Alternatively, step i. may first be performed using a biological fluid sample obtained from the subject at the same time as the start of the treatment regimen or at a time point after the start of the treatment regimen for endometriosis. Thus, the method may be used to determine the therapeutic effect of a treatment regimen for endometriosis from the beginning (i.e., from the start of the regimen) or from a time point after the start of the treatment regimen (i.e., determining the therapeutic effect of the treatment regimen for endometriosis during the treatment regimen itself).

[0221] In an embodiment, an unchanged or increased amount or concentration of c-Kit in a sample of a subject being treated for endometriosis indicates that the therapy is ineffective, i.e., an unchanged or increased amount or concentration of c-Kit in a sample of a subject being treated for endometriosis indicates persistent or recurrent endometriosis. In particular, if the amount of c-Kit increases to 50% or more, the treatment of endometriosis is ineffective. In particular, if the amount of c-Kit increases to 100% or more, the treatment of endometriosis is ineffective. In particular, if the amount of c-Kit increases to 150% or more, the treatment of endometriosis is ineffective. In particular, if the amount of c-Kit increases to 200% or more, the treatment of endometriosis is ineffective.

[0222] Alternatively, unchanged levels of c-Kit may mean that the disease is stagnant or that it has progressed from stage I to stage III or IV, where c-Kit levels are comparable to stage I.

[0223] Improvement in a disease state or symptom (e.g., during treatment) may also be indicated by stable levels of c-Kit over time (compared to the level of c-Kit observed over an equivalent time period in the absence of treatment or compared to an equivalent control).

[0224] A treatment regimen may be identified as having a therapeutic effect if it results in a delay in disease progression or a delay in the development of symptoms (eg, during the treatment period).

[0225] A treatment regimen can also be identified as having a therapeutic effect if it results in an improvement in the disease state or symptoms (eg, during the treatment period). Methods for determining whether a treatment regimen has a therapeutic effect are well known in the art.

[0226] The treatment period refers to the time interval over which treatment occurs (eg, 1 month, 3 months, 6 months, 1 year, 2 years, etc.).

[0227] As will be clear to one skilled in the art, the direction of change in c-Kit levels indicative of a therapeutic effect may depend on the disease state of the subject prior to treatment and the control / reference used.

[0228] Changes in the levels of c-Kit may also indicate compliance or adherence to prescribed therapy following treatment.

[0229] The trends used to identify that a subject has followed or adhered to a prescribed treatment regimen are equivalent to those described in detail above with respect to determining the therapeutic effect of a treatment regimen for endometriosis. This is because a "prescribed treatment regimen" is a recommended treatment regimen and therefore generally has a therapeutic effect (and therefore, observation of a therapeutic effect on biomarker levels is an indication of a subject's compliance or adherence to the prescribed treatment regimen).

[0230] In an embodiment, the subject is monitored several times at different time points. In an embodiment, the subject is monitored several times over a period of weeks, months or years. In a specific embodiment, the subject is monitored monthly or annually. In an embodiment, a subject with endometriosis is monitored monthly or annually after diagnosis of endometriosis. In an embodiment, a subject being treated for endometriosis is monitored once after therapy, in particular once after surgical therapy. In particular, a subject being treated for endometriosis is monitored monthly or annually to determine the efficacy of the treatment and / or the recurrence of endometriosis.

[0231] The method can also be used as a screening tool to determine whether a particular regimen or treatment has a therapeutic effect on endometriosis. The regimen or treatment tested can be a new regimen or treatment, a modified regimen or treatment, or a known regimen or treatment that requires further testing. In this context, a treatment is, for example, a drug or agent that is useful or suspected of being useful in the treatment of endometriosis.

[0232] In an embodiment, the therapy for endometriosis is selected from the group consisting of: drug-based therapy or surgical therapy. In an embodiment, the treatment regimen includes surgical therapy, radiotherapy, immunotherapy, hormone therapy, ultrasound therapy or a combination thereof. In a preferred embodiment, the surgical therapy for endometriosis is laparoscopy or nerve-sparing surgery. In an embodiment, the drug-based therapy for endometriosis is suppression or targeting of neurogenic inflammation and / or analgesics and / or hormone therapy.

[0233] In certain embodiments, if an unchanged or increased amount or concentration of c-Kit is determined in a sample from a patient being treated for endometriosis, therapy is adjusted.

[0234] Details of biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, etc. are provided elsewhere and are equally applicable in this regard.

[0235] Therefore, all aspects of the method for determining the therapeutic efficacy of a treatment regimen for endometriosis described in detail above are equally applicable here.

[0236] In one example, the method is an in vitro method.

[0237] Details of biomarkers, combinations, samples, method steps, subjects, types of endometriosis, treatments, reference values ​​etc. are provided elsewhere and apply equally to this and all other aspects.

[0238] Computer-implemented method for assessing patients with endometriosis

[0239] In a fourth aspect, the present invention relates to a computer-implemented method for assessing a patient suspected of having endometriosis, comprising the steps of:

[0240] (a) receiving a value for the level of a first biomarker in a sample from a subject, the first biomarker being c-Kit,

[0241] (b) receiving a value for the level of a second biomarker in a sample from the subject, wherein the second biomarker is CA125,

[0242] (c) receiving a value for the level of dysmenorrhea according to VAS and / or lower abdominal pain according to VAS,

[0243] (d) comparing the values ​​for the levels of steps (a) to (c) with a 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

[0244] (e) assessing the subject based on the comparison and / or calculation performed in step (d).

[0245] The term "computer-implemented" as used herein means that the method is performed in an automated manner on a data processing unit, which is typically included in a computer or similar data processing device. The data processing unit should receive a value for the amount of a biomarker. Such a value can be an amount, a relative amount, or any other calculated value reflecting an amount as described in detail elsewhere herein. Therefore, it should be understood that the above method does not require the determination of the amount of a biomarker, but rather uses a value for an already predetermined amount.

[0246] In principle, the present invention also contemplates a computer program, a computer program product or a computer-readable storage medium having a tangible embedded computer program, wherein the computer program comprises instructions which, when executed on a data processing device or a computer, perform the above-mentioned method of the present invention. Specifically, the present disclosure further covers:

[0247] - a computer or a computer network comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification,

[0248] - a computer loadable data structure, which is suitable for performing a method according to one of the embodiments described in this description when the data structure is being executed on a computer,

[0249] - a computer script, wherein the computer program is adapted to carry out a method according to one of the embodiments described in the present description when the program is executed on a computer,

[0250] - a computer program comprising program means for executing a method according to one of the embodiments described in the present description when the computer program is being executed on a computer or on a computer network,

[0251] - a computer program comprising program means according to the preceding embodiment, wherein the program means is stored on a computer-readable storage medium,

[0252] a storage medium, on which a data structure is stored, and wherein the data structure is adapted to carry out a method according to one of the embodiments described in the present description after having been loaded into a main storage device and / or a working storage device of a computer or a computer network,

[0253] - a computer program product having program code means, wherein the program code means can be stored or stored on a storage medium for carrying out a method according to one of the embodiments described in the present description when the program code means is executed on a computer or on a computer network,

[0254] - a data stream signal, typically encrypted, comprising data of the parameters defined elsewhere herein, and - a data stream signal, typically encrypted, comprising the assessment provided by the method of the invention.

[0255] Combined with CA-125, symptoms or clinical data

[0256] The methods according to the present invention may be combined with other tests, biomarkers, clinical data or other information useful for diagnosing or classifying endometriosis in order to obtain the most reliable results.

[0257] Despite its rather weak diagnostic performance, CA-125 is still routinely used as a biomarker for endometriosis. Therefore, in the context of the methods described herein, it may be advantageous to combine the determination of the level of c-Kit with CA-125 obtained from a subject.

[0258] In addition to CA-125, other symptoms or clinical data for diagnosing or classifying endometriosis can also be used in conjunction 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.

[0259] Kits and Devices

[0260] In another aspect, a kit for diagnosing endometriosis in a subject or classifying the stage of endometriosis in a subject is provided. 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 the biomarkers disclosed herein).

[0261] The kits described herein generally include a detectably labeled agent that specifically binds to a c-Kit protein.

[0262] Such a kit may further include a detectably labeled agent that specifically binds CA-125.

[0263] The kits described herein can take a variety of forms. Typically, the kit will include reagents suitable for determining the levels of a plurality of biomarkers (eg, c-Kit and optionally CA-125) in a sample.

[0264] Optionally, the kit may include one or more control samples or references. Typically, the comparison between the level of a biomarker in a subject and the level of a biomarker in a control sample indicates a clinical state (e.g., a diagnosis of endometriosis). In addition, in some cases, the kit will include written information (marking) providing a reference (e.g., a predetermined value), wherein the comparison between the level of a biomarker in a subject and the reference (predetermined value) indicates a clinical state (e.g., a diagnosis of endometriosis). In some cases, the kit includes software that can be used to compare biomarker levels or occurrences with a reference (e.g., a prediction model). Typically, the software will be provided in a computer-readable format (such as a compact disc), but can also be downloaded via the Internet. However, the kit is not limited thereto, and other changes will be apparent to those of ordinary skill in the art.

[0265] The components of the kit may be contained in a container suitable for transport. Details on biomarkers are given above and apply equally here. Suitably, the biomarker may be a protein.

[0266] In some examples, the kit includes a detectably labeled agent on a continuous (e.g., solid) surface (such as a lateral flow surface). Alternatively, in examples including more than one detectably labeled agent, the detectably labeled agents can be located in different (i.e., spatially separated) regions on a (e.g., solid) surface (such as a multi-walled microtiter plate (e.g., for ELISA assays)). Other suitable surfaces and containers well known in the art may also form part of the kits described herein.

[0267] In one example, the kit further includes one or more reagents for detecting the detectably labeled agent. Suitable reagents are well known in the art and include, but are not limited to, standard reagents and buffers required for carrying out any suitable detection method that can be used (and are well known in the art).

[0268] In one example, the kit includes one or more of the following: a multiwell plate, a ball bearing, an extraction buffer, an extraction bottle, and a lateral flow device.

[0269] Also provided are assay devices for diagnosing endometriosis in a subject.

[0270] Generally, the device includes a surface having disposed thereon at least one detectably labeled agent that specifically binds to a c-Kit protein.

[0271] Such a device may further comprise a detectably labeled agent that specifically binds CA-125.

[0272] If two detectably labeled agents are used, they can be located in different regions on the surface. In other words, two detectably labeled agents can be located in different (i.e., spatially separated) regions on a (e.g., solid) surface (such as a multi-well microtiter plate). Detectably labeled agents that specifically bind to a target biomarker are described in detail elsewhere herein.

[0273] The assay device includes a surface on which a detectably labeled agent is located. Suitable surfaces include continuous (e.g., solid) surfaces, such as lateral flow surfaces, dot blot surfaces, test paper surfaces, or surfaces suitable for surface plasmon resonance. Other suitable surfaces include microtiter plates, multiwell plates, etc. Other suitable surfaces well known in the art may also form part of the assay device described herein.

[0274] Thus, suitable assay device formats include, but are not limited to, those suitable for performing any of a lateral flow, dot blot, ELISA or surface plasmon resonance assay to detect the presence, level or absence of a biomarker of interest.

[0275] Data storage

[0276] Biomarker levels and / or reference levels can be stored in a suitable data storage medium (e.g., a database) and can therefore also be used for future diagnosis. This also allows the prevalence of the disease to be effectively diagnosed, because once it is confirmed that the subject from whom the corresponding reference sample is obtained (in the future) does suffer from endometriosis, a suitable reference result can be identified in the database. As used herein, a "database" includes data (e.g., analyte and / or reference level information and / or patient information) collected on a suitable storage medium. In addition, the database may further include a database management system. Preferably, the database management system is a network-based, hierarchical or object-oriented database management system. In addition, the database may be a federated or integrated database. More preferably, the database will be implemented as a distributed (federated) system, such as a client-server system. More preferably, the database is constructed to allow a search algorithm to compare a test data set with a data set included in a data set. Specifically, by using this algorithm, a similar or identical data set indicating endometriosis can be searched in the database (e.g., a query search). Therefore, if the same or similar data set can be identified in the data set, the test data set is associated with endometriosis. Therefore, the information obtained from the data set can be used to diagnose endometriosis or based on a test data set obtained from a subject.More preferably, the data set comprises characteristic values ​​of all analytes included in any of the above groups.

[0277] The methods described herein may further include communicating the results or diagnoses (or both) to, for example, a technician, a doctor, or a patient. In some instances, a computer will be used to communicate the results or diagnoses (or both) to relevant parties (e.g., a doctor and his or her patient).

[0278] In some instances, the result or diagnosis (or both) is communicated to the subject as quickly as possible after obtaining the diagnosis. The result or diagnosis (or both) can be communicated to the subject by the subject's attending physician. Alternatively, the result or diagnosis (or both) can be sent to the subject by email or communicated to the subject by phone. A computer can be used to communicate the result or diagnosis by email or phone. In some instances, a combination of computer hardware and software familiar to the technician in the field of telecommunications can be used to automatically generate a message containing the result or diagnosis and deliver it to the subject.

[0279] use

[0280] Also provided herein is the use of the biomarker c-Kit as a biofluid biomarker for endometriosis.

[0281] In a preferred example, c-Kit can generally be used as a biomarker for endometriosis. In this article, "endometriosis" generally refers to all forms of endometriosis, including but not limited to peritoneal endometriosis, endometrioma, deep infiltrating endometriosis and adenomyosis.

[0282] c-Kit can also be used in combination with CA-125.

[0283] Details of biomarkers, samples, methods, subjects, types of endometriosis, etc. are provided elsewhere and are equally applicable here.

[0284] Companion Diagnostics

[0285] The methods, kits, assay devices and uses provided herein can be used as part of a companion diagnostic, for example as part of a medical device (typically an in vitro device) that provides information necessary for the safe and effective use of the corresponding drug or biologic, wherein the corresponding drug or biologic is used to treat or prevent endometriosis.

[0286] In a further embodiment, the present invention relates to the following aspects:

[0287] 1. A method for diagnosing endometriosis in a subject, the method comprising the steps of:

[0288] a) determining the level of c-Kit in a biological fluid sample from said subject,

[0289] b) comparing said level of c-Kit to at least one appropriate reference value for c-Kit levels,

[0290] 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 an appropriate reference value.

[0291] 2. The method according to aspect 1, wherein the at least one appropriate reference value is

[0292] i. The level of c-Kit in non-pathological subjects or symptomatic subjects, or

[0293] ii. the mean level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects or a combination thereof,

[0294] or where at least one of the appropriate reference values ​​is

[0295] iii. a predetermined value of the level of c-Kit in a non-pathological subject or a symptomatic subject, or

[0296] iv. a predetermined mean value of the level of c-Kit in a group of non-pathological subjects or a group of symptomatic subjects or a combination thereof.

[0297] 3. A method for classifying the stage of endometriosis in a subject, the method comprising the steps of:

[0298] a) determining the level of c-Kit in a biological fluid sample from said subject,

[0299] b) comparing said level of c-Kit to at least one appropriate reference value for c-Kit levels,

[0300] 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 at least one appropriate reference value for the level of c-Kit.

[0301] 4. A method for classifying the stage of endometriosis in a subject, the method comprising the steps of:

[0302] a) periodically determining the level of c-Kit in a biological fluid sample from said subject,

[0303] b) comparing said level of c-Kit to at least one value of the level of c-Kit determined at an earlier time in said subject,

[0304] 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 at least one value of the c-Kit level determined at an earlier stage of the subject.

[0305] 5. The method according to aspect 3 or 4, wherein the at least one appropriate reference value is

[0306] i. The level of c-Kit in non-pathological subjects, in symptomatic subjects, or in subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), or

[0307] ii. the mean level of c-Kit in a group of subjects who are non-pathological subjects, symptomatic subjects, or a combination thereof, or in a group of subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM),

[0308] or where at least one of the appropriate reference values ​​is

[0309] iii. a predetermined value for the level of c-Kit in non-pathological subjects, or in symptomatic subjects, or in subjects with stage I, II, III, or IV endometriosis, according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM), or

[0310] iv. A predetermined mean value of the level of c-Kit in a group of non-pathological subjects, symptomatic subjects, or a combination thereof, or in a group of subjects with stage I, II, III, or IV endometriosis according to the revised scoring system of the American Society for Reproductive Medicine (r-ASRM).

[0311] 6. The method according to any preceding aspect, 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.

[0312] 7. The method according to any preceding aspect, wherein the subject is a human, preferably a female human.

[0313] 8. The method according to any preceding aspect, wherein the protein level of c-Kit is optionally determined using a method selected from the group consisting of: ELISA assay, immunoblotting, lateral flow assay, protein microarray and mass spectrometry.

[0314] 9. The method according to any preceding aspect, further comprising administering to the subject a selected treatment regimen, optionally wherein the selected treatment regimen comprises surgery, radiation therapy, immunotherapy, hormone therapy, ultrasound therapy, or a combination thereof.

[0315] 10. The method according to any preceding aspect, wherein the diagnosis or classification is performed independently of the American Society for Reproductive Medicine scoring system.

[0316] 11. The method according to any one 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 of the American Society for Reproductive Medicine (r-ASRM).

[0317] 12. The method according to any one 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 of the American Society for Reproductive Medicine (r-ASRM).

[0318] 13. The method according to any preceding aspect, wherein endometriosis is selected from the group consisting of peritoneal endometriosis, endometrioma, deep infiltrating endometriosis and adenomyosis.

[0319] 14. The method according to any preceding aspect, further comprising selecting a treatment regimen for the subject based on a comparison of the level of c-Kit with a control sample or with a predetermined reference level.

[0320] 15. The method according to aspect 14, further comprising administering to the subject a selected treatment regimen, optionally wherein the selected treatment regimen comprises drug-based therapy and / or surgical treatment (laparoscopy).

[0321] 16. A method for monitoring the progression of endometriosis in a subject, the method comprising the steps of:

[0322] i. The method according to any one of aspects 1 to 15, steps a) to b) determining the level of c-Kit in a biological fluid sample from the subject,

[0323] ii. repeating step i. for specific time intervals using a biological fluid sample obtained from the subject during or after treatment; and

[0324] iii. comparing the level of c-Kit identified in i. with the level of c-Kit identified in ii, wherein a change in the level of c-Kit from i. to ii indicates a change in the progression of endometriosis in the subject.

[0325] 17. A method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject, the method comprising the steps of:

[0326] i. The method according to any one of aspects 1 to 15, steps a) to b) determining the level of c-Kit in a biological fluid sample from the subject,

[0327] ii. repeating step i. for specific time intervals using a biological fluid sample obtained from the subject during or after treatment; and

[0328] iii. comparing the level of c-Kit determined in step i. with the level of c-Kit determined in step ii., and if the level of c-Kit decreases after treatment, identifying that the treatment regimen has a therapeutic effect.

[0329] 18. The method according to any preceding aspect, further comprising assessing dysmenorrhea according to a visual analogue scale (VAS) and / or assessing lower abdominal pain according to a VAS.

[0330] 19. The method according to any preceding aspect, further comprising determining the level of CA-125 in the biological fluid sample from the subject.

[0331] 20. The method according to clause 18, comprising calculating

[0332] - the ratio of the amount or concentration of c-Kit to the amount or concentration of CA-125, or

[0333] -The ratio of the amount or concentration of c-Kit to dysmenorrhea, or

[0334] - the ratio of the amount or concentration of c-Kit to the amount or concentration of CA-125 and dysmenorrhea, or

[0335] -Ratio of the amount or concentration of c-Kit to the lower abdominal pain according to the VAS scale.

[0336] 21. A computer-implemented method for assessing a patient suspected of having endometriosis

[0337] The computer-implemented method comprises the following steps:

[0338] (a) receiving a value for the level of a first biomarker in a sample from the subject, the first biomarker being c-Kit,

[0339] (b) receiving a value for the level of a second biomarker in a sample from the subject, wherein the second biomarker is CA125,

[0340] (c) receiving a value for the level of dysmenorrhea according to VAS and / or lower abdominal pain according to VAS,

[0341] (d) comparing the values ​​of the levels of steps (a) to (c) with a reference of the biomarker and the amount of dysmenorrhea, and / or calculating a score for assessing the subject suspected of having endometriosis based on the levels of the biomarker and the amount of dysmenorrhea; and

[0342] (e) assessing the subject based on the comparison and / or calculation performed in step (d).

[0343] 22. Use of elevated c-Kit levels in a biological fluid sample as a biomarker for endometriosis.

[0344] 23. Use according to aspect 22, wherein the biological fluid sample is blood or blood-derived, preferably serum.

[0345] 24. The use according to aspects 22 and 23, wherein the use is for the diagnosis and / or classification of endometriosis.

[0346] 25. A kit for diagnosing and / or classifying endometriosis in a subject, comprising at least one detectably labeled agent that specifically binds to a c-Kit protein.

[0347] 26. The kit according to aspect 25, further comprising one or more reagents for detecting the detectably labeled agent.

[0348] 27. An assay device for diagnosing and / or classifying endometriosis in a subject, the device comprising a surface having disposed thereon at least one detectably labeled agent that specifically binds to a c-Kit protein.

[0349] Aspects of the present invention are illustrated by the following non-limiting examples. The following examples and drawings are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that the procedures set forth may be modified without departing from the spirit of the present invention.

[0350] Examples

[0351] Example 1: Diagnostic performance of the biomarker c-Kit in women with endometriosis

[0352] For measurement, a total of 250 serum samples from human women were analyzed (for clinical data, refer to the corresponding part of this article). The concentration of the analyte was determined by ELISA (enzyme-linked immunosorbent assay). The case group was composed of patients diagnosed with endometriosis (peritoneal endometriosis, adenomyosis, endometrioma and deep infiltrating endometriosis; rASRM I-IV stages) by laparoscopy and subsequently confirmed by histology, while the control group included healthy women without endometriosis.

[0353] The concentration of c-Kit in human serum was determined using the Human CD117 / c-Kit Quantikine ELISA Kit (R&D Systems, USA (Catalog No.: DSCR00). The kit utilizes quantitative sandwich ELISA technology. Microtiter plates were pre-coated with monoclonal antibodies specific for human c-Kit. Samples were measured at 50-fold dilution. After all reagents were brought to room temperature, 100 μL of each sample and standard were added. Samples were measured in single aliquots and standards in duplicate. During incubation at room temperature for 2.5 hr on a microplate shaker set at 650 rpm, any c-Kit present was bound to the capture antibody immobilized on the microtiter plate. During the wash step (4x During the 300 μL (100 μL) incubation period, unbound material is removed from the plate before 100 μL of an enzyme-linked monoclonal antibody specific for c-Kit is added to the wells. After 1 h of incubation on a shaker and another wash step to remove any unbound detection antibody, 100 μL of substrate solution is added to the plate. Over the next 10 min, color development is proportional to the amount of c-Kit bound in the initial step. Color development is stopped by adding 50 μL of stop solution and the color intensity is measured using a plate reader at 450 nm for detection and at 570 nm for background subtraction. To generate calibrators The curve was prepared by re-dissolving the lyophilized recombinant c-Kit provided with the kit and diluting it in the calibration diluent. The calibration range of the assay was 6.14 pg / mL to 1500 pg / mL. Calibrator 7 (1500 pg / mL) was prepared by diluting the stock solution 6-fold in the calibration diluent, and calibrators 6 to calibrator 1 (6.14 pg / mL) were prepared by continuous 2.5-fold dilution steps in the calibration diluent. Pure calibration diluent was used as blank (0 pg / mL). The calibration curve was fitted using unweighted 4-parameter nonlinear regression (Newton / Raphson).

[0354] The results are as follows Figure 1 as shown in .

[0355] Compared with nonpathological controls, serum c-Kit levels gradually increased in endometriosis stages I and II, but decreased in stage III.

[0356] In Table 1 below, model performance was determined by looking at the area under the curve (AUC). The best possible AUC is 1, while the lowest possible is 0.5. The best cutoff value (maximum sum of sensitivity plus specificity - 1) was selected using the Youden index.

[0357] Table 1: shows the diagnostic performance of the c-Kit biomarker in distinguishing women with histologically confirmed endometriosis from women without endometriosis (controls) using receiver operating characteristic (ROC) analysis, depicting the area under the curve (AUC) of the ROC analysis and the associated 95% confidence intervals. N represents the number of samples tested (cases plus controls, the number in each group varies according to the analyte).

[0358]

[0359]

[0360] Example 2: Diagnostic performance of the biomarker c-Kit compared to CA-125 in women with endometriosis

[0361] In further experiments, the performance of the biomarker c-Kit was compared with the current standard biomarker CA-125.

[0362] The concentration of CA-125 was determined by the cobas e 601 analyzer. The detection of CA 125II by the cobas e 601 analyzer is based on Electro-ChemiLuminescence (ECL) technology. In short, biotin-labeled and ruthenium-labeled antibodies are combined with the corresponding amount of undiluted samples and incubated on the analyzer. Subsequently, streptavidin-coated magnetic particles are added to the instrument and incubated to promote the binding of biotin-labeled immune complexes. After this incubation step, the reaction mixture is transferred to the measuring cell, where the beads are magnetically captured on the surface of the electrode. ProCell M buffer containing tripropylamine (TPA) for subsequent ECL reactions is then introduced into the measuring cell to separate the bound immunoassay complex from the free remaining particles. The voltage induction between the working electrode and the counter electrode then triggers the reaction, causing the ruthenium complex and TPA to emit photons. The resulting electrochemiluminescent signal is recorded by a photomultiplier tube and converted into a numerical value indicating the concentration level of the corresponding analyte.

[0363] The results are Figure 2A and 2B Shown in.

[0364] Figure 2A and 2BThe box plots in were generated for each of the control and endometriosis stages (stage I, II, III, IV) using data from a high-throughput, multiplex immunoassay PCR (OLINK proteomics) analysis. Data are presented using box and whisker plots, including the median (middle quartile), interquartile range (representing the middle 50% of the group's scores), upper quartile (75% of the scores are below the upper quartile), lower quartile (25% of the scores are below the lower quartile). The whiskers show the 5th and 95th percentiles, respectively.

[0365] Serum c-Kit showed better diagnostic performance in detecting early-stage endometriosis (stage I, II) compared with the reference biomarker CA-125.

[0366] In Table 2 below, the performance of c-Kit as a biomarker compared to CA-125 is determined by looking at the area under the curve (AUC) for both biomarkers in early stage endometriosis (stage I and II).

[0367] Table 2: Diagnostic performance of serum c-Kit biomarker and reference biomarker CA-125 in women with endometriosis (stage I and II) and controls. Measurement of CA-125 was performed by OLINK using a proximity extension assay (PEA). C-Kit levels were measured using a CD117 / c-Kit Quantikine ELISA kit. C-Kit levels gradually increased in endometriosis stage I and II controls with benign findings, while decreasing in stages III and IV.

[0368]

[0369]

[0370] The AUC values ​​of the ROC analysis for stage I and II endometriosis compared with controls were AUC = 0.72\AUC = 0.57 for serum c-Kit and AUC = 0.46\AUC = 0.6 for serum CA-125.

[0371] Serum c-Kit showed better diagnostic performance in detecting early-stage endometriosis (stage I, II) compared with the reference biomarker CA-125.

[0372] Clinical data of the cohort:

[0373] 1) Endometriosis Cases:

[0374] a) Total cohort: 101 patients

[0375] b) Age:

[0376]

[0377] c) Hormonal contraceptive pills / devices:

[0378]

[0379] No contraceptive hormones / devices: 57.42%

[0380] Taking birth control hormones / devices: 41.58%

[0381] d) Types of endometriosis and related diseases:

[0382]

[0383] e) Endometriosis stages:

[0384]

[0385] f) Menstrual pain score:

[0386]

[0387] 88.11% of patients had (VAS ≥ 5)

[0388] 2) No cases of endometriosis, severe pain ("symptomatic controls"):

[0389] a) Total cohort: 65 patients

[0390] b) Age:

[0391]

[0392] c) Hormonal contraceptive pills / devices:

[0393]

[0394] No contraceptive hormones / devices: 64.61%

[0395] Taking birth control hormones / devices: 32.30%

[0396] d) Menstrual pain score:

[0397]

[0398] 3) No endometriosis, no pain ("non-pathological control"):

[0399] a) Total cohort: 84 patients

[0400] b) Age:

[0401]

[0402] c) Hormonal contraceptive pills / devices:

[0403]

[0404] No contraceptive hormones / devices: 73.8%

[0405] Taking birth control hormones / devices: 23.8%

[0406] d) Menstrual pain score:

[0407]

Claims

1. A method for diagnosing endometriosis in a subject, the method comprising the steps of: a) determining the level of c-Kit in a biological fluid sample from said subject, b) comparing the level of c-Kit to at least one appropriate reference value for the level of c-Kit, c) if the comparison in step b) indicates that the subject has an increased level of c-Kit compared to an appropriate reference value, identifying the subject as having endometriosis.

2. A method for classifying the stage of endometriosis in a subject, the method comprising the steps of: a) determining the level of c-Kit in a biological fluid sample from said subject, b) comparing the level of c-Kit to at least one appropriate reference value for the level of c-Kit, 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 at least one appropriate reference value for the level of c-Kit.

3. The method according to any preceding claim, 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.

4. The method according to any one of claims 1 to 3, wherein the diagnosis or classification is performed independently of the scoring system of the American Society for Reproductive Medicine 5. The method according to any one of claims 2 to 4, 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 of the American Society for Reproductive Medicine (r-ASRM).

6. The method according to any preceding claim, wherein the subject's endometriosis stage is classified as stage I or stage II endometriosis according to the revised grading system of the American Society for Reproductive Medicine (r-ASRM).

7. The method according to any preceding claim, wherein the endometriosis is selected from the group consisting of peritoneal endometriosis, endometrioma, deep infiltrating endometriosis and adenomyosis.

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

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

10. A method for monitoring the progression of endometriosis in a subject, the method The following steps are involved: i. The method according to any one of claims 1 to 9, steps a) to b) determining the level of c-Kit in a biological fluid sample from the subject, ii. repeating step i. for specific 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 a change in the level of c-Kit from i. to ii. indicates a change in the progression of endometriosis in the subject.

11. A method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject, the method comprising: The following steps are involved: i. The method according to any one of claims 1 to 9, steps a) to b) determining the level of c-Kit in a biological fluid sample from the subject, ii. repeating step a) for specific 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 the level of c-Kit determined in step b), and if the level of c-Kit decreases after treatment, identifying that the treatment regimen has a therapeutic effect.

12. The method according to any one of the preceding claims, further comprising rating dysmenorrhea according to a visual analogue scale (VAS) and / or rating lower abdominal pain according to a VAS.

13. The method of any of the preceding claims, further comprising determining the level of CA-125 in the biological fluid sample from the subject.

14. The method according to claim 13, comprising calculating - 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 to the amount or concentration of CA-125 and dysmenorrhea, or -Ratio of the amount or concentration of c-Kit to the lower abdominal pain according to the VAS scale.

15. A computer-implemented method for assessing a patient suspected of having endometriosis, the computer-implemented method comprising the steps of: (f) receiving a value for the level of a first biomarker in a sample from the subject, wherein the first biomarker is c-Kit; (g) receiving a value for the level of a second biomarker in a sample from the subject, wherein the second biomarker is CA125, (h) receiving a value for the level of dysmenorrhea according to VAS and / or lower abdominal pain according to VAS, (i) comparing the values ​​for the levels of steps (a) to (c) with a reference for the biomarker and the amount of dysmenorrhea, and / or calculating a score for assessing the subject suspected of having endometriosis based on the levels of the biomarker and the amount of dysmenorrhea; as well as (j) assessing the subject based on the comparison and / or calculation performed in step (d).

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