sFRP4 as a blood biomarker for the non-invasive diagnosis of adenomyosis

Abstract

The present invention relates to methods for assessing whether a patient has or is at risk of developing adenomyosis, for selecting patients for treatment of adenomyosis, and for monitoring patients suffering from or undergoing treatment for adenomyosis, by determining the amount or concentration of sFRP4 in a sample from the patient and comparing the determined amount or concentration to a standard.

Description

[Technical Field]

[0001] The present invention relates to methods for assessing whether a patient has or is at risk of developing adenomyosis, for selecting patients for treatment of adenomyosis, and for monitoring patients suffering from or undergoing treatment for adenomyosis, by determining the amount or concentration of sFRP4 in a sample from the patient and comparing the determined amount or concentration with a reference standard. [Background technology]

[0002] background Adenomyosis is a heterogeneous gynecological disease. Patients with adenomyosis may have a variety of clinical symptoms. The most common symptoms are abnormal menstrual bleeding and dysmenorrhea. However, pain, nausea, or difficulty urinating may also occur. Patients with adenomyosis may be asymptomatic and only come to the attention of clinicians during infertility evaluation.

[0003] Adenomyosis is also associated with infertility and is diagnosed in approximately 20% of infertile women undergoing assisted reproductive technology (ART). (Puente JM et al., Reproductive Biology and Endocrinology, 2016;14:60) Women with adenomyosis often have other associated gynecological conditions, such as endometriosis or leiomyomatosis, making diagnosis and evaluation of response to treatment difficult. (Pontis A et al., Gynecol Endocrinol, 2016;32(9):696-700)

[0004] Currently, there are no standard imaging criteria, making it difficult to select the most appropriate treatment for each patient.

[0005] Adenomyosis is defined as the infiltration of benign endometrial glands and stroma into the myometrium, the outer muscle layer of the uterus. In contrast, endometriosis is defined as a disease characterized by the presence of endometrioid epithelium and / or stroma outside the endometrium and myometrium. Depending on morphology and location, there are three distinct types of adenomyosis: internal adenomyosis, external adenomyosis, and adenomyomatous fibroids. Internal adenomyosis can be further classified into focal, diffuse, and superficial adenomyosis (Bazot M et al., Fertil Steril. 2018;109:389-397). Adenomyosis can also be classified into four subtypes I-IV based on magnetic resonance imaging (MRI) findings, depending on whether it is located in the outer or inner layer of the uterus. Common signs and symptoms of adenomyosis include heavy bleeding during menstruation (menorrhagia) and between menstrual periods (metrorrhagia), dysmenorrhea, chronic pelvic pain, dyspareunia, and infertility, which severely impact the quality of life of women. Furthermore, adenomyosis significantly impacts fertility and in vitro fertilization (IVF) outcomes (Chapron C et al., Hum Reprod Update. 2020;26:392-411).

[0006] The prevalence of adenomyosis varies widely, with an average rate of 20% to 25%. Approximately 20% of adenomyosis cases occur in women of reproductive age (under 40 years of age), and the remaining 80% occur in women in their late reproductive years (40-50 years of age). (Pontis A et al., Gynecol Endocrinol, 2016;32(9):696-700)

[0007] For women with bothersome symptoms who have completed childbearing, removal of the uterus (hysterectomy) is the treatment of choice and the only definitive cure for adenomyosis. Uterine-sparing surgical resection of adenomyosis lesions or cysts is rarely performed in patients with localized or cystic localized adenomyosis, but it is not the standard treatment. Uterine artery embolization (UAE) is a new treatment approach, especially in patients resistant to conventional treatments and those who wish to preserve the uterus. However, recurrence of adenomyosis signs and symptoms has been observed in more than 40% of patients (J. Zhou et al., PLOS One, 2016;1-15).

[0008] There are no specific drugs currently available to treat adenomyosis, and no specific guidelines to follow for best management.

[0009] The intended outcome of treating symptomatic adenomyosis is the alleviation of signs and symptoms, preservation or improvement of fertility, while minimizing side effects.

[0010] Several non-hormonal (i.e., nonsteroidal anti-inflammatory drugs (NSAIDs)) and hormonal treatments (i.e., progestins, oral contraceptives, gonadotropin-releasing hormone [GnRH] analogs) are used to control pain symptoms and abnormal uterine bleeding in adenomyosis (S. Vannuccini et al., Fertility and Sterility®, Vol. 109, No. 3, March 2018; A. Pontis et al., Gynecol Endocrinol, 2016;3590:1-5).

[0011] The diagnosis of adenomyosis is currently based on transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI), with overall sensitivity and specificity rates of 83.8%, 63.9%, and 77.3%, 89.8%, respectively (Chapron C et al., Hum Reprod Update. 2020;26:392-411). However, there are currently no standard imaging criteria, making it difficult to select the optimal treatment for each patient. Both TVUS and transabdominal ultrasound characterize adenomyosis by identifying ill-defined foci of intramyometrial microcysts, anteroposterior asymmetry of the myometrium, and abnormal myometrial echocardiography. MRI findings include a large, asymmetric uterus without leiomyomata, junctional thickening, or an abnormal ratio of junctional to myometrial thickness. The junctional junction is the innermost myometrium.

[0012] The only definitive diagnosis of adenomyosis is a histological diagnosis based on pathologic evaluation of the uterus after hysterectomy.

[0013] Only highly skilled operators can diagnose adenomyosis using transvaginal ultrasound (TVUS) or magnetic resonance imaging (MRI). These techniques require a high level of experience and expertise, making them difficult to apply for routine diagnosis. Furthermore, TVUS results are operator-dependent (Dueholm M Bestt Practice & Research Clinical Obstetrics and Gynaecology 2006;20(4):569-582). Detection of adenomyosis by transvaginal ultrasound also requires an appropriate ultrasound device, and results may depend on the specific ultrasound device used, the physician's evaluation of the ultrasound images, and subjective analysis.

[0014] Among the various types of adenomyosis, diffuse adenomyosis is by far more difficult to detect by imaging techniques and requires experienced sonographers. Also, access to imaging equipment is limited, especially among primary health care professionals, requiring trained staff and specialized resources.

[0015] Therefore, a non-invasive, blood-based test would allow for medical evaluation of adenomyosis without the need for imaging equipment, reducing inter-operator variability and allowing for a more standardized diagnosis of the condition. (Chapron C et al., Hum Reprod Update. 2020;26:392-411).

[0016] CA125 has been reported in the differential diagnosis of uterine adenomyosis and fibroids, but the authors found that its diagnostic accuracy was limited (Kicheol Kil et al., Eur J Obstet Gynecol Reprod Biol. 2015 Feb;185:131-5).

[0017] CA125 serum levels were found to be useful in predicting the prognosis of adenomyosis before and after interventional surgical treatment (Y. Mu et al., Int J Clin Exp Med 2015;8(6):9549-9554).

[0018] Secreted frizzled-related protein 4 (sFRP4) is a glycoprotein that belongs to a family of secreted proteins that act as antagonists of Wnt ligands. It is also known by its synonyms FRP-4, frpHE (FRP human endometrium), FRPHE, and FRZB-2 sFRP, which contains a Wnt-binding domain and is a soluble regulator of the Wnt signaling pathway. sFRP4 inhibits the canonical Wnt signaling pathway, which normally induces cell proliferation and reduces apoptosis.

[0019] The sFRP4 gene is normally expressed in various tissues, including the endometrial stroma (highly expressed during the proliferative phase of the menstrual cycle), pancreas, stomach, colon, lung, skeletal muscle, testis, ovaries, kidney, heart, brain, breast, cervix, eye, bone, prostate, and liver. Overexpression of sFRP4 is associated with various pathologies, including bone, skin, kidney, endocrine, and cancer (Pawar N et al., Secreted frizzled related protein 4 (sFRP4) update: A brief review. Cellular Signaling 2018;45:63-70; S. Pohl et al., Tumor Biol. 2015;36:143-152).

[0020] Furthermore, it has been widely reported that sFRP4 is overexpressed in type 2 diabetes (e.g., T. Mahdi et al., Cell Metabolism, 16, 625-633, November 7, 2012).

[0021] Estrogen and progesterone have been found to regulate the expression of sFRP4 during the endometrial cycle (higher expression during the proliferative phase of the menstrual cycle). During ovulation, sFRP4 increases apoptosis, promoting the process.

[0022] The role of sFRP4 in endometrial development and its upregulation in ovarian and endometrial cancer, as well as upregulation of sFRP4 by hCG in animals with endometriosis, have been described in a baboon model (Sherwin JRA et al., Endocrinology, 2010;151(10):4982-4993).

[0023] International Publication No. 2001032920 describes a method for screening the genes and gene products associated with endometriosis by comparing the gene expression pattern in diseased endometrium with that in healthy endometrium.Among other gene products, the expression level of sFRP4 is found to be different compared with that of healthy control group.

[0024] Furthermore, WO2007090872 describes antibodies against secreted frizzled related protein 4 (sFRP4) and their use for the detection of sFRP4.

[0025] Pawar et al. described sFRP4 as a protein marker for detecting endometriosis in serum. In a study involving 21 patients and 22 healthy controls, they reported increased serum levels of sFRP4 in endometriosis patients compared with controls, and significantly increased levels in endometriosis grades 3 and 4, according to the revised American Society for Reproductive Medicine (rASRM). sFRP4 was quantified by ELISA assay (Pawar NM et al., Indian Journal of Clinical Biochemistry 2016;31(Suppl. 1):S1-S129).

[0026] However, there is a great need for non-invasive diagnosis of adenomyosis through the use of biomarkers that allow for reliable and early evaluation of patients presenting with signs and symptoms of adenomyosis.

[0027] Therefore, the present invention provides means and methods to meet these needs. Summary of the Invention

[0028] In a first aspect, the present invention relates to a method for assessing whether a patient has or is at risk of developing adenomyosis, the method comprising determining the amount or concentration of sFRP4 in a sample from the patient and comparing the determined amount or concentration with a reference standard.

[0029] In a second aspect, the present invention relates to a method for selecting patients for treatment of adenomyosis, in particular for drug-based, pain management or surgical treatment, comprising determining the amount or concentration of sFRP4 in a patient sample and comparing the determined amount or concentration with a standard.

[0030] In a third aspect, the present invention relates to a method for monitoring a patient suffering from or undergoing treatment for adenomyosis, comprising determining the amount or concentration of sFRP4 in a sample from the patient and comparing the determined amount or concentration with a standard.

[0031] In a fourth aspect, the present invention relates to a computer-implemented method of assessing whether a patient has or is at risk of developing adenomyosis, the method comprising: receiving, in a processing unit, a value for the level of sFRP4 in a sample from the patient; processing the received value in step (a) with the processing unit, said processing comprising retrieving from a memory one or more threshold values for the level of sFRP4 and comparing the value received in step (a) with the one or more threshold values; and assessing via an output device whether the patient has or is at risk of developing adenomyosis, said assessment being based on the result of step (b). [Brief explanation of the drawings]

[0032] [Figure 1a] Boxplot of sFRP4 in adenomyosis cases (N=124) and controls (N=177). [Figure 1b]Receiver operating curve (ROC) analysis of the biomarker sFRP4. The AUC value of the ROC analysis of adenomyosis versus controls without adenomyosis was 0.62. x-axis = specificity, y-axis = sensitivity. [Figure 2a] Boxplot of CA125 in adenomyosis cases (N=124) and controls (N=177). The AUC value of the ROC analysis of adenomyosis versus controls without adenomyosis was 0.51. x-axis=specificity, y-axis=sensitivity. [Figure 2b] Receiver operating curve (ROC) analysis of the biomarker CA125. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention We show for the first time that sFRP4 measured in blood is increased in women with adenomyosis compared with controls. There is an unmet medical need for a noninvasive blood test for the diagnosis and evaluation of adenomyosis.

[0034] definition The word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0035] 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.

[0036] Concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It is understood that such range formats are used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values explicitly recited as boundaries of the range, but also all individual numerical values or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "150 mg to 600 mg" should be interpreted not only to include the explicitly recited value of 150 mg to 600 mg, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values such as 150, 160, 170, 180, 190, 580, 590, and 600 mg, as well as subranges such as 150 to 200, 150 to 250, 250 to 300, and 350 to 600. This same principle applies to ranges reciting only a single numerical value. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics described.

[0037] The term "about," when used in connection with a numerical value, is meant to encompass numerical values within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0038] As used herein, the term "indicator" refers to a sign or signal of a symptom or is used to monitor a condition. Such a "condition" refers to the biological state of a cell, tissue, or organ, or to the health and / or disease state of an individual. An indicator can be the presence or absence of molecules, including, but not limited to, peptides, proteins, and nucleic acids, or a change in the expression level or pattern of such molecules in a cell, or tissue, organ, or individual. An indicator can be an indication of the occurrence, development, or presence of a disease in an individual, or an indication of the further progression of such a disease. An indicator can also be an indication of the risk of developing a disease in an individual.

[0039] In the context of the present invention, the term "biomarker" refers to a substance in a biological system that is used as an indicator of the biological state of the system. In the art, the term "biomarker" may also be applied to means for detecting the endogenous substance (e.g., antibodies, nucleic acid probes, imaging systems, etc.). In the context of the present invention, the term "biomarker" applies only to the substance, not the means for detection. Thus, a biomarker can be any type of molecule present in a living organism, such as a nucleic acid (e.g., DNA, mRNA, miRNA, rRNA), a protein (e.g., cell surface receptor, cytosolic protein), a metabolite or hormone (e.g., blood glucose, insulin, estrogen), a molecule characteristic of a specific modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or a substance internalized by an organism or a metabolite of such a substance.

[0040] The term "sFRP4," or "secreted frizzled-related protein 4," is also referred to as FRP-4, FRPHE, sFRP-4, or secreted frizzled-related protein. Secreted frizzled-related proteins (sFRPs) comprise a family of five mammalian proteins containing a Wnt-binding domain. They are soluble regulators of the Wnt signaling pathway, first identified as antagonists of the Wnt / β-catenin pathway during embryonic development (Kawano, Y., and Krypta, R., Secreted antagonists of the Wnt signaling pathway. J. Cell Sci. 116, 2627–2634, doi:10.10242 / jcs.00623 (2003)).

[0041] sFRP4 inhibits the canonical Wnt signaling pathway, which normally induces cell proliferation and reduces apoptosis. Furthermore, high serum levels of sFRP have been demonstrated in pathological conditions such as obesity, diabetes, and osteoporosis (Belaya, ZE et al.; Osteoporos 2013; International Patent No. 24:2191-2199). The terms polypeptide, peptide, and protein are used interchangeably throughout this specification.

[0042] The sFRP4 protein is composed of 346 amino acids with a predicted molecular weight of 39.9 kDa and an actual molecular weight of approximately 50-55 kDa. It folds into two independent domains. The N-terminus contains a secretory signal peptide followed by a cysteine-rich domain (CRD) of approximately 120 amino acids. The CRD is 30-50% identical to the extracellular putative Wnt-binding domain of the frizzled (Fzd) receptor and is characterized by the presence of 10 cysteine residues at conserved positions. These cysteines form a disulfide bridge pattern.

[0043] As used herein, sFRP4 also encompasses variants of the aforementioned specific sFRP4 polypeptides. Such variants have at least the same essential biological and immunological properties as the specific sFRP4 polypeptides. In particular, if they can be detected by the same specific assays referred to herein, for example, by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize the sFRP4 polypeptides, they share the same essential biological and immunological properties. Preferred assays are described in the accompanying Examples. Furthermore, the variants referred to in accordance with the present invention shall have a different amino acid sequence due to at least one amino acid substitution, deletion, and / or addition, and the amino acid sequence of the variants shall still be understood to be preferably at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical over the entire length of the amino acid sequence of a specific sFRP4 polypeptide, preferably the amino acid sequence of human sFRP4, more preferably a specific sFRP4, for example, human sFRP4. The degree of identity between two amino acid sequences can be determined as described above. The variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologs. Furthermore, the variants referred to herein include fragments of a specific sFRP4 polypeptide, or variants of the above-mentioned types, so long as these fragments have the essential immunological and biological properties referred to above. Such fragments may be, for example, degradation products of the sFRP4 polypeptide. Further included are variants that differ due to post-translational modifications such as phosphorylation or myristylation.

[0044] Carbohydrate antigen 125 (CA-125), sometimes called cancer antigen 125 or tumor antigen 125, is a mucin-type glycoprotein produced by the MUC16 gene and associated with cell membranes. CA-125 is a biomarker for epithelial cell ovarian cancer, derived from the coelomic epithelium, including the endometrium, fallopian tubes, ovaries, and peritoneum. Its diagnostic use is limited to endometriosis stages III and IV (moderate and severe endometriosis), with moderate sensitivity.

[0045] A "symptom" of a disease is an indication of the disease that is noticeable by a tissue, organ, or organism with such disease, and includes, but is not limited to, pain, weakness, tenderness, tension, stiffness, and spasms of a tissue, organ, or individual. A "signal" or "signal" of a disease includes, but is not limited to, the presence or absence, increase or elevation, decrease or decline, or other change or alteration, of a particular indicator, such as a biomarker or molecular marker, or the onset, presence, or worsening of a symptom. Symptoms of pain include, but are not limited to, an unpleasant sensation that may be experienced as a persistent or variable burning, throbbing, itching, or tingling pain.

[0046] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition, particularly an abnormal medical condition such as an illness or injury in which a tissue, organ, or individual can no longer perform its function efficiently. Typically, but not necessarily, a disease is associated with specific symptoms or signs that indicate the presence of such a condition. Thus, the presence of such symptoms or signs may indicate a tissue, organ, or individual suffering from a disease. Changes in these symptoms or signs may indicate the progression of such a disease. Disease progression is typically characterized by an increase or decrease in such symptoms or signs, which may indicate a "worsening" or "improvement" of the disease. A "worsening" disease is characterized by a decrease in the ability of a tissue, organ, or organism to perform its function efficiently, whereas a "improvement" disease is typically characterized by an increase in the ability of a tissue, organ, or individual to perform its function efficiently. A tissue, organ, or individual at "risk for developing" a disease is one that is healthy but exhibits the potential for the disease to manifest. Typically, the risk of developing a disease is associated with early or weak signs or symptoms of such a disease. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, traumatic diseases, and various types of cancer.

[0047] The term endometriosis is defined as a disease characterized by the presence of endometrial-like epithelium and / or stroma outside the endometrium and myometrium, usually with an associated inflammatory process (International working group of AAGL ESGE ESHRE and WES et al.; ESHRE 2022 Guideline Endometriosis).

[0048] In contrast, the term adenomyosis is defined as the presence of ectopic endometrial tissue (endometrial stroma and glands) within the myometrium (International working group of AAGL ESGE ESHRE and WES et al., 2021). Adenomyosis is not considered a form or subtype of endometriosis and is therefore not included in the guidelines (ESHRE Guideline 2022, p. 8).

[0049] Adenomyosis is further defined as "endometrial invasion into the myometrium (muscle layer of the uterus), resulting in a diffusely enlarged uterus microscopically presenting ectopic, non-neoplastic endometrial glands and stroma surrounded by hypertrophic and hyperplastic myometrium" (Bird C et al., Am J Obstet Gynecol 1972;112:583e593).

[0050] Different classifications of adenomyosis subtypes have been suggested. In 2012, Kishi et al. classified adenomyosis into four subtypes, I to IV, based on magnetic resonance imaging (MRI) findings, depending on whether the lesion is located in the outer or inner layer of the uterus (Y Kishi et al., Am J Obstet Gynecol. 2012;207:114.e1-114.e7).

[0051] In 2018, Bazot et al. proposed three distinct types of adenomyosis: internal adenomyosis, external adenomyosis, and adenomyoma, depending on the morphology, degree of myometrial invasion, and location. Internal adenomyosis was further classified into focal, diffuse, and superficial adenomyosis (Bazot M et al., Fertil Steril. 2018;109:389-397). Women with focal adenomyosis present with limited areas of hypertrophied and distorted endometrium and myometrium, usually embedded within the myometrium. Localized adenomyosis can be further subdivided into adenomyoma, which has more or less distinct borders and primarily solid features, and cystic adenomyosis (also known as juvenile cystic adenomyosis in women under 30 years of age), which is characterized primarily by the presence of a single adenomyotic cyst within the myometrium. Diffuse adenomyosis, a widespread form of the disease, is characterized by foci of endometrial mucosa (glandular and stromal) scattered throughout the myometrial tissue. Polypoid adenomas represent endometrial masses composed primarily of endometrioid glands and a stromal component primarily of smooth muscle. Adenomyomatous polyps of the cervix represent a rare form of adenomyosis. It is important to distinguish these lesions from malignant adenomas (G Grimbizis et al., FERTILITY PRESERVATION 2014;101(2):472). (19)

[0052] The term adenomyosis encompasses internal adenomyosis, external adenomyosis, and adenomyoma. It also encompasses adenomyosis subtypes such as diffuse, focal, and superficial adenomyosis, adenomyotic cysts, adenomyomas, cystic adenomyosis, typical and atypical polypoid adenomyosis and adenomyomatous polyps, retroperitoneal adenomas, and adenomatous nodules (Bazot M et al., Fertil Steril. 2018;109:389-397; G Grimbizis et al., FERTILITY PRESERVATION 2014;101(2):472). Adenomyosis subtypes I–IV are classified based on magnetic resonance imaging (MRI) findings.

[0053] Patients with adenomyosis may have a variety of clinical symptoms. The most common symptoms of adenomyosis are abnormal menstrual bleeding (menorrhagia), painful periods (dysmenorrhea), and uterine enlargement. These symptoms are common to many other gynecological disorders in women. The pain may be sharp, knife-like pelvic pain, or there may be severe menstrual cramps during menstruation. The pain may be unpredictable and intermittent throughout the menstrual cycle, or it may be constant. However, different forms of pain may also exist, and the pain may worsen over time and change in characteristics.

[0054] An enlarged uterus can put pressure on the bladder and rectum, making urination difficult. Nausea has also been reported by women with adenomyosis. Less commonly reported symptoms include dyspareunia and chronic, irregular, or persistent pelvic pain. Similar symptoms have been reported in patients with endometriosis, with some women suffering from both conditions. Adenomyosis is also associated with infertility and is diagnosed in approximately 20% of infertile women undergoing assisted reproductive technology (ART) (S. Vannuccini et al., Fertility and Sterility®, Vol. 109, No. 3, March 2018). Patients with adenomyosis often have other associated gynecological conditions, such as endometriosis or leiomyomatosis, making diagnosis and evaluation of response to treatment difficult.

[0055] The prevalence of adenomyosis varies widely, with an average rate of 20% to 25%. Approximately 20% of adenomyosis cases occur in women of reproductive age (under 40 years of age), while the remaining 80% occur in women in their late reproductive years (40-50 years of age). One-third of women with adenomyosis are asymptomatic (Pontis A et al., Gynecol Endocrinol, 2016;32(9):696-700).

[0056] Preferably, the term "treatment" as used in connection with the methods of assessing whether a patient has or is at risk of developing adenomyosis encompasses exacerbation and worsening of symptoms, either to prevent the disease from worsening altogether or to significantly reduce its impact on the patient, for example, to reduce a patient's lower abdominal pain and heavy menstrual bleeding, reduce uterine size, and improve the chances of conception in infertile women with adenomyosis prior to sterilization treatment.

[0057] Drug-based treatments include non-hormonal medications (i.e., nonsteroidal anti-inflammatory drugs (NSAIDs)), hormonal treatments (i.e., progestins, i.e., norethindrone acetate, oral contraceptives, combined oral contraceptives (COCS), gonadotropin-releasing hormone analogs (GnRH analogs), levonorgestrel-IUS (levonorgestrel-releasing intrauterine system), selective progesterone receptor modulators, aromatase inhibitors, valproic acid, and antiplatelet therapy (S. Vannuccini et al., Fertility and Sterility®, Vol. 109, No. 3, March 2018; A. Pontis et al., Gynecol Endocrinol. 2016; 3590:1-5).

[0058] Surgical treatments include removal of the uterus (hysterectomy) and uterus-conserving surgery, such as surgical removal of adenomatous lesions or cysts (adenomyectomy, cystectomy) or uterine artery embolization (UAE).

[0059] The term "VAS," or visual analog scale, is a tool used to assess pain intensity. The VAS consists of a 10-cm-long horizontal line with two ends marked "no pain" and "worst imaginable pain." Each patient checks their pain level on the line and measures the distance in centimeters from the leftmost "no pain" to the checked mark, resulting in a pain score of 0 to 10. "No pain" corresponds to a pain score of 0, and "worst imaginable pain" corresponds to a pain score of 10. In women with adenomyosis and dysmenorrhea, dysmenorrhea is associated with the highest perception of pain, with a mean VAS score of approximately 6 (Cozzolino et al., Rev Bras Ginecol Obstet. 2019;41(3):170-175).

[0060] As used herein, "patient" refers to any mammalian animal that can benefit from the diagnosis, prognosis, or treatment described herein. In particular, the "patient" is selected from the group consisting of experimental animals (e.g., mice, rats, rabbits, or zebrafish), livestock (e.g., guinea pigs, rabbits, horses, donkeys, cows, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, tortoises, snakes, lizards, or goldfish), or primates, including chimpanzees, bonobos, gorillas, and humans. It is particularly preferred that the "patient" is a human.

[0061] In particular, "patients" suitable for the present invention are women who have symptoms of abnormal uterine bleeding, such as heavy menstrual bleeding, prolonged menstrual bleeding, bleeding between periods, dysmenorrhea, severe menstrual pain, abdominal pressure and bloating, etc. Women (with or without symptoms of adenomyosis) are often unable to conceive and are often infertile women undergoing assisted reproductive technology (ART).

[0062] Furthermore, patients suffering from adenomyosis are usually women of reproductive age between 14 and 50 years old, especially those between 30 and 50 years old.

[0063] The terms "sample" and "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, typically smaller than the entire tissue, organ, or individual intended to represent such tissue, organ, or individual. Upon analysis, the sample yields information regarding the condition of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. Analysis of the sample can be accomplished visually or chemically. Visual analysis includes, but is not limited to, microscopic imaging or radiological scanning of the tissue, organ, or individual, which allows for morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of specific indicators or changes in their amount, concentration, or level. The sample is an in vitro sample and will be analyzed in vitro and will not be returned to the body.

[0064] The term "amount," as used herein, encompasses the absolute amount of a biomarker referred to herein, the relative amount or concentration of said biomarker, and any value or parameter that correlates therewith or can be derived therefrom. Such values or parameters include intensity signal values derived from any specific physical or chemical property obtained from said peptide by direct measurement, such as intensity values in a mass spectrum or NMR spectrum. Furthermore, values or parameters obtained by indirect measurement as specified elsewhere herein, such as the amount of response measured by a biological readout system in response to a peptide, or the intensity signal obtained from a specifically bound ligand, are all encompassed. It should be understood that values that correlate with the above-mentioned amounts or parameters can also be obtained by any standard mathematical operation.

[0065] The term "comparing," as used herein, refers to comparing the amount of a biomarker in a sample from a subject with a reference amount of the biomarker specified elsewhere in this specification. It should be understood that comparing, as used herein, typically refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to a reference absolute amount, while a concentration is compared to a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared to an intensity signal of the same type obtained from a reference sample. The comparison may be performed manually or computer-assisted. Thus, the comparison can be performed by a computing device. The measured or detected amount of a biomarker in a sample from a subject and the value of the reference amount can, for example, be compared with each other, or the comparison can be performed automatically by a computer program executing an algorithm for the comparison. The computer program performing the evaluation provides the desired assessment in a suitable output format. In a computer-assisted comparison, the value of the measured amount may be compared by the computer program with a value corresponding to a suitable reference stored in a database. The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired assessment in a suitable output format. In a computer-assisted comparison, the value of the measured quantity may be compared by a computer program with values corresponding to suitable standards stored in a database, and the computer program may further evaluate the results of the comparison, i.e., automatically provide the desired rating in a suitable output format.

[0066] The phrase "comparing the determined amount or concentration to a standard" is merely used to further explain what would be obvious to one skilled in the art anyway. The standard concentration is established in a control sample.

[0067] The term "reference sample" or "control sample," as used herein, refers to a sample that is analyzed in substantially the same manner as the sample of interest, and its information is compared with that of the sample of interest. The reference sample thus provides a standard that allows the evaluation of information obtained from the sample of interest. The control sample is derived from a healthy or normal tissue, organ, or individual, thereby providing a reference for the health status of the tissue, organ, or individual. The difference between the state of the normal reference sample and the state of the sample of interest can indicate the risk of disease development or the presence or further progression of such a disease or disorder. The control sample can be derived from an abnormal or diseased tissue, organ, or individual, thereby providing a reference for the pathological state of the tissue, organ, or individual. The difference between the state of the abnormal reference sample and the state of the sample of interest can indicate a reduced risk of disease development or the absence or improvement of such a disease or disorder. The reference sample can also be derived from the same tissue, organ, or individual as the sample of interest, but collected at an earlier time point. The difference between the state of the previously collected reference sample and the state of the sample of interest can indicate disease progression, i.e., improvement or worsening of the disease over time.

[0068] Control sample can be internal or external control sample.Using internal control sample, that is, in test sample and one or more other samples taken from the same subject, evaluate marker level to determine whether there is change in the level of said marker.For external control sample, the presence or amount of marker in the sample from individual is compared with the presence or amount of marker in the individual who is known to suffer from or known to be at risk of given condition; or in the individual who is known not to have given condition (i.e., " normal individual ").

[0069] Those skilled in the art will understand that such external control samples can be obtained from a single individual or from a reference population that is age-matched and free of confounding diseases. Typically, samples from 100 well-characterized individuals from an appropriate reference population are used to establish a "reference value." However, the reference population can also be selected to consist of 20, 30, 50, 200, 500, or 1000 individuals. Healthy individuals represent a preferred reference population for establishing a control value.

[0070] For example, the marker concentration in a patient sample can be compared with a concentration known to be associated with a specific course of a specific disease.Usually, the marker concentration of a sample is directly or indirectly correlated with diagnosis, and the marker concentration is used, for example, to determine whether an individual is at risk of a specific disease.Alternatively, the marker concentration of a sample can be compared with a marker concentration known to be associated with, for example, the response to treatment in a specific disease, the diagnosis of a specific disease, the assessment of the severity of a specific disease, the guidance for selecting an appropriate drug for a specific disease, determining the risk of disease progression, or in patient follow-up.Depending on the intended diagnostic application, an appropriate control sample is selected, and a control value or reference value for the marker is set therein.As will be apparent to those skilled in the art, the absolute marker value set in the control sample depends on the assay used.

[0071] The term "reduced" or "diminished" level of an indicator refers to the level of such indicator in a sample that is reduced compared to a reference or reference sample.

[0072] The term "elevated" or "increased" level of an indicator refers to a higher level of such indicator in a sample compared to a reference or reference sample. For example, a protein that is detectable in a higher amount in a fluid sample of an individual suffering from a given disease than in the same fluid sample of an individual not suffering from said disease has an elevated level.

[0073] The terms "measurement", "measuring" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0074] The term "immunoglobulin (Ig)" as used herein refers to immune conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells by their transmembrane regions and include molecules such as, but not limited to, B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin (approximately 2M), CD3, CD4, and CDS.

[0075] Typically, the term "antibody," as used herein, refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, designated by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), each performing a different role and directing the appropriate immune response to different types of antigens. Different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the intestine, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, where it prevents colonization by pathogens (Underdown and Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD primarily functions as an antigen receptor on unexposed B cells and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved in allergic reactions through binding to allergens, which triggers histamine release from mast cells and basophils. IgE is also involved in protection from 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 and confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG (approximately 4%).The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in the early stage of B cell-mediated (humoral) immunity, eliminating pathogens before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers, 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., mammalian IgM). Antibodies are typically made up of four polypeptide chains, including two identical heavy chains and two identical light chains linked via disulfide bonds, resembling a "Y"-shaped macromolecule. Each chain contains several immunoglobulin domains, some of which are constant domains and others are variable domains. Immunoglobulin domains consist of a two-layer sandwich of seven to nine antiparallel strands arranged in two or more sheets. Typically, an antibody heavy chain contains four Ig domains, three of which are constant (CH domains: CH1, CH2, CH3) domains and one of which is a variable domain (VH). Light chains typically contain one constant Ig domain (CL) and one variable Ig domain (VL). For example, a human IgG heavy chain consists of four Ig domains linked N- to C-terminally in the order VwCH1-CH2-CH3 (also referred to as VwCyl-Cy2-Cy3), while a human IgG light chain consists of two immunoglobulin domains linked N- to C-terminally in the order VL-CL, and can be either kappa or lambda (VK-CK or VA-CA). By way of example, the constant chain of human IgG contains 447 amino acids. Throughout this specification and claims, the numbering of amino acid positions in immunoglobulins is based on Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C., (1991) Sequences of proteins of immunological interest, 5.th ed. USDapartment of Health and Human Services, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue numbering of the human IgG lEU antibody. Thus, the CH domains in the context of IgG are as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0076] 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. These terms specifically refer to an antibody having a heavy chain that includes an Fc region.

[0077] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each containing a single antigen-binding site, and a remaining "Fc fragment" (also called "Fc portion" or "Fc region"), named for its ability to readily crystallize. The crystal structure of the human IgG Fe region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In IgG, IgA, and IgD isotypes, the Fe region consists of two identical protein fragments derived from the CH2 and CH3 domains of the antibody's two heavy chains, while in IgM and IgE isotypes, the Fe region contains three heavy-chain constant domains (CH2-CH4) in each polypeptide chain. Additionally, smaller immunoglobulin molecules exist naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally contains the hinge region of an Ig molecule, while a "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or linked via disulfide bonds. "Single-domain antibodies (sdAbs)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "nanobodies" contain only a single VH domain, while "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Bivalent single-chain variable fragments (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH and two VL regions, resulting in a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Typically, a linker with a length of 5 residues is used to generate these dimers. This type is known as a "diabody."Shorter linkers (one or two amino acids) between the VH and VL domains result in the formation of monospecific trimers, so-called "triabodies" or "tribodies." Bispecific diabodies are formed by expressing chains with the sequences VHA-VLB and VHB-VLA, or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDbs) contain VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids. "Bispecific T cell engagers (BiTEs)" are fusion proteins consisting of two scFvs from different antibodies; one scFv binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies further stabilized by a C-terminal disulfide bridge.

[0078] Thus, the term "antibody fragment" refers to a portion of an intact antibody, preferably comprising its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments; diabodies; sdAbs, nanobodies, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDbs, BiTEs, and DARTs.

[0079] 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 indicated, as used herein, "binding affinity" refers to the 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 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 (e.g., BIAcore assays, such as those described in PCT Application Publication WO 2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competitive assays (e.g., RIAs). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0080] "Sandwich immunoassays" are widely used for detecting an analyte of interest. In such assays, 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-overlapping epitopes on the analyte of interest. The sandwich complex is measured by appropriate means, thereby quantifying the analyte. In a typical sandwich-type assay, a first antibody bound to or capable of binding to a solid phase and a detectably labeled second antibody each bind to the analyte at different, non-overlapping epitopes. A binding agent (e.g., an antibody) specific for the first analyte is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer; the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be a tube, bead, microplate disk, or any other surface suitable for performing immunoassays. The binding process is well known in the art and generally consists of cross-linking, covalent binding, or physical adsorption. The polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid-phase complex and incubated for a period of time (e.g., 2-40 minutes or, more conveniently, overnight) sufficient to allow binding between the first or capture antibody and the corresponding antigen, and under appropriate conditions (e.g., room temperature to 40°C, e.g., 25°C to 37°C, inclusive). Following the incubation period, the solid phase containing the first or capture antibody and the antigen bound to the antibody is washed and may be incubated with a secondary or labeled antibody that binds to a different epitope on the antigen. The second antibody is conjugated to a reporter molecule that is used to indicate binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0081] A highly versatile alternative sandwich assay format involves the use of a solid phase coated with a first partner of a binding pair, such as a microparticle coated with paramagnetic streptavidin. Such microparticles are incubated with an analyte-specific binding agent bound to a second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte whose second partner of the binding pair is bound to the analyte-specific binding agent, and a detectably labeled second analyte-specific binding agent. As will be apparent to those skilled in the art, these components are incubated under appropriate conditions for a period of time sufficient to allow the analyte, the analyte-specific binding agent (bound to the second partner of the binding pair), and the labeled antibody to bind to the solid-phase microparticle via the first partner of the binding pair. Optionally, such an assay may include one or more washing steps.

[0082] The term "detectably labeled" encompasses labels that can be detected either directly or indirectly.

[0083] A directly detectable label either provides a detectable signal, or the label interacts with a second label to modify the detectable signal provided by the first or second label, for example, to provide FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (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) provide detectable signals and are generally applicable to labeling. In one embodiment, detectably labeled refers to a label that provides or can be induced to provide a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label, respectively.

[0084] Numerous labels (also referred to as dyes) are available that can be broadly grouped into the following categories, all of which together represent embodiments according to the present disclosure:

[0085] (a) Fluorescent dye 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).

[0086] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels (including FITC, 5-carboxyfluorescein, and 6-carboxyfluorescein), rhodamine-type labels (including TAMRA), dansyl, lissamine, cyanine, phycoerythrin, Texas Red, and analogs thereof. Fluorescent labels can be attached to aldehyde groups contained within target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon) and Pierce Biotechnology, Inc. (Rockford, Illinois).

[0087] (b) Luminescent dye Luminescent dyes or labels can be further sub-classified into chemiluminescent dyes and electrochemiluminescent dyes.

[0088] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, peroxyoxalic acid and peroxyoxalic acid derivatives. For immunodiagnostic procedures, acridinium-based labels are mainly used (a detailed overview is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0089] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a highly sensitive and selective method. ECL combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. Generally, ruthenium complexes, especially [Ru(Bpy)3]2+ (emitting photons at approximately 620 nm) regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface, are used as ECL labels.

[0090] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurements of the presence and concentration of an analyte of interest. Such techniques use a label or other reactant that can be induced to emit light when electrochemically oxidized or reduced in the appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage applied to the working electrode at a specific time and in a specific manner. The light produced by the label is measured and indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, see U.S. Pat. No. 5,221,605; U.S. Pat. No. 5,591,581; U.S. Pat. No. 5,597,910; PCT Publication WO 90 / 05296; PCT Publication WO 92 / 14139; PCT Publication WO 90 / 05301; PCT Publication WO 96 / 24690; PCT Publication US 95 / 03190; PCT Publication US 97 / 16942; PCT Publication US 96

[0010] Reference is made to PCT Publication Nos. WO 95 / 08644, WO 96 / 06946, WO 96 / 33411, WO 87 / 06706, WO 96 / 39534, WO 96 / 41175, WO 96 / 40978, PCT / US97 / 03653, and U.S. Patent Application No. 08 / 437,348 (U.S. Patent No. 5,679,519). Reference is also made to the 1994 review of analytical uses of ECL by Knight et al. (Analyst, 1994, 119:879-890) and the references cited therein. In one embodiment, the methods herein are practiced using electrochemiluminescent labels.

[0091] Recently, iridium-based ECL labels have also been described (WO2012107419).

[0092] (c) The radiolabel employs a radioactive isotope (radionuclide), such as H, C, C, F, P, S, Cu, Gn, Y, Zr, TC, In, I, I, I, I, Xe, Lu, At, or Bi.

[0093] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,461; Hnatowich et al., J. Immunol. Methods 65 (1983) 147-157; Meares et al., Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al., Bioconjugates 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). .

[0094] Embodiment In a first aspect, the present invention provides a method of assessing whether a patient has or is at risk of developing adenomyosis, comprising: a) determining the amount of sFRP4 in a patient sample; b) comparing the determined amount with a standard; The present invention relates to a method, comprising:

[0095] In embodiments, the elevated amount of sFRP4 in patient samples indicates the presence or risk of developing adenomyosis in patients.In particular, the amount of sFRP4 in patient samples indicates the presence or risk of developing adenomyosis in patients, if the amount of sFRP4 in patient samples is higher than the amount of sFRP4 in reference or reference samples.In particular, sFRP4 can be detected in higher amounts in fluid samples from patients who are evaluated for the presence or risk of developing adenomyosis than in the same fluid samples from individuals who do not suffer from or are not at risk of developing adenomyosis.

[0096] In particular, a 50% or greater increase in the amount of sFRP4 indicates the presence of, or a risk of developing, adenomyosis. In particular, a 100% or greater increase in the amount of sFRP4 indicates the presence of, or a risk of developing, adenomyosis. In particular, a 150% or greater increase in the amount of sFRP4 indicates the presence of, or a risk of developing, adenomyosis. In particular, a 200% or greater increase in the amount of sFRP4 indicates the presence of, or a risk of developing, adenomyosis.

[0097] In embodiments, the patient sample is a body fluid sample. In certain embodiments, the sample is a whole blood, serum, or plasma sample. In embodiments, the sample is an in vitro sample, i.e., it will be analyzed in vitro and will not be returned to the body.

[0098] In certain embodiments, the patient is a laboratory animal, a livestock animal, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a female human patient.

[0099] In particular, the evaluation is performed without surgery. In particular, the evaluation is performed without using surgery to evaluate the presence or severity of adenomyosis in a patient.

[0100] In an embodiment, the method of the invention is an in vitro method.

[0101] In embodiments, the amount of sFRP4 is determined using an antibody, particularly a monoclonal antibody. In embodiments, step a) of determining the amount of sFRP4 in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0102] In certain embodiments, step a) of determining the amount of sFRP4 in the patient sample comprises: i) incubating a patient sample with one or more antibodies that specifically bind to sFRP4, thereby generating a complex between the antibody and sFRP4; and ii) quantifying the complex formed in step i), thereby quantifying the amount of sFRP4 in the patient sample. Includes:

[0103] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to sFRP4. As will be apparent to those skilled in the art, the sample can be contacted with the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or the first and second antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex. As will be readily apparent to those skilled in the art, it will take no more than routine experimentation to establish the time and conditions that are appropriate or sufficient for the formation of a complex between a specific anti-sFRP4 antibody and the sFRP4 antigen / analyte (=anti-sFRP4 complex), or for the formation of a secondary or sandwich complex comprising the first anti-sFRP4 antibody, sFRP4 (analyte), and the second anti-sFRP4 antibody (=anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex).

[0104] Detection of the anti-sFRP4 antibody / sFRP4 complex can be carried out by any suitable means. Detection of the first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0105] In certain embodiments, a sandwich is formed comprising a first antibody to sFRP4, sFRP4 (the analyte), and a second antibody to sFRP4, wherein the second antibody is detectably labeled.

[0106] In one embodiment, a sandwich is formed comprising a first antibody against sFRP4, sFRP4 (analyte) and a second antibody against sFRP4, wherein the second antibody is detectably labeled and the first anti-sFRP4 antibody is capable of binding to a solid phase or is bound to a solid phase.

[0107] In embodiments, the second antibody is detectably labeled, either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent or electrochemiluminescent dye.

[0108] In embodiments, the method further comprises assessing the presence of dysmenorrhea and / or abdominal pain in the patient. In embodiments, the presence of dysmenorrhea and / or abdominal pain is assessed by a VAS scale. In embodiments, a dysmenorrhea VAS score of 4 or greater indicated moderate or severe dysmenorrhea.

[0109] In a second aspect, the present invention provides a method for selecting a patient for treatment of adenomyosis, comprising: determining the amount or concentration of sFRP4 in the patient sample; Comparing the determined amount or concentration to a standard The present invention relates to a method, comprising:

[0110] In a further embodiment of the invention, the treatment is in particular a drug-based treatment, a pain management treatment or a surgical treatment.

[0111] In embodiments, when the amount of sFRP4 is determined to be elevated in patient samples, the patient is selected for adenomyosis treatment.Particularly, when the amount of sFRP4 in patient samples is greater than the amount of sFRP4 in standard or standard sample, the patient is selected for adenomyosis treatment.Particularly, when the amount of sFRP4 is greater in the same fluid sample from patient than in the fluid sample from the individual who does not suffer from adenomyosis or is not at risk of developing adenomyosis, or is not selected for adenomyosis therapy, the patient is selected for adenomyosis treatment.

[0112] In particular, if the amount of sFRP4 is elevated by 50% or more, the patient is selected for treatment of adenomyosis. In particular, if the amount of sFRP4 is elevated by 100% or more, the patient is selected for treatment of adenomyosis. In particular, if the amount of sFRP4 is elevated by 150% or more, the patient is selected for treatment of adenomyosis. In particular, if the amount of sFRP4 is elevated by 200% or more, the patient is selected for treatment of adenomyosis.

[0113] In embodiments, the patient is selected for a treatment for adenomyosis selected from the group consisting of drug-based treatments or surgical treatments.

[0114] In embodiments, the drug-based treatment for adenomyosis is inhibiting or targeting neurogenic inflammation and / or analgesics and / or hormonal treatment (e.g., hormonal contraceptives or GnRH analogs).

[0115] In some embodiments, surgical treatments for adenomyosis include, inter alia, hysterectomy, removal of the uterus; uterus-conserving surgery, e.g., surgical removal of adenomatous lesions; cysts (adenomyectomy, cystectomy); uterine artery embolization (UAE) or nerve-sparing surgery.

[0116] In embodiments, the patient sample is a body fluid sample. In certain embodiments, the sample is a whole blood, serum, or plasma sample. In embodiments, the sample is an in vitro sample, i.e., it is analyzed in vitro and is not returned to the body.

[0117] In certain embodiments, the patient is a laboratory animal, a livestock animal, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a female human patient.

[0118] In an embodiment, the method of the invention is an in vitro method.

[0119] In embodiments, the amount of sFRP4 is determined using an antibody, particularly a monoclonal antibody. In embodiments, step a) of determining the amount of sFRP4 in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0120] In certain embodiments, step a) of determining the amount of sFRP4 in the patient sample comprises: i) incubating a patient sample with one or more antibodies that specifically bind to sFRP4, thereby generating a complex between the antibody and sFRP4; and ii) quantifying the complex formed in step i), thereby quantifying the amount of sFRP4 in the patient sample. Includes:

[0121] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to sFRP4. As will be apparent to those skilled in the art, the sample can be contacted with the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or the first and second antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex. As will be readily apparent to those skilled in the art, it will take no more than routine experimentation to establish the time and conditions that are appropriate or sufficient for the formation of a complex between a specific anti-sFRP4 antibody and the sFRP4 antigen / analyte (=anti-sFRP4 complex), or for the formation of a secondary or sandwich complex comprising the first anti-sFRP4 antibody, sFRP4 (analyte), and the second anti-sFRP4 antibody (=anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex).

[0122] Detection of the anti-sFRP4 antibody / sFRP4 complex can be carried out by any suitable means. Detection of the first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0123] In certain embodiments, a sandwich is formed comprising a first antibody to sFRP4, sFRP4 (the analyte), and a second antibody to sFRP4, wherein the second antibody is detectably labeled.

[0124] In one embodiment, a sandwich is formed comprising a first antibody against sFRP4, sFRP4 (analyte) and a second antibody against sFRP4, wherein the second antibody is detectably labeled and the first anti-sFRP4 antibody is capable of binding to a solid phase or is bound to a solid phase.

[0125] In embodiments, the second antibody is detectably labeled, either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent or electrochemiluminescent dye.

[0126] In embodiments, the method further comprises assessing the presence of dysmenorrhea and / or abdominal pain in the patient. In embodiments, the presence of dysmenorrhea and / or abdominal pain is assessed by a VAS scale. In embodiments, a dysmenorrhea VAS score of 4 or greater indicates moderate or severe dysmenorrhea. In embodiments, a score of 3 or less indicates no or mild dysmenorrhea.

[0127] In embodiments, the method includes calculating the ratio of the amount or concentration of sFRP4 to dysmenorrhea, the ratio of the amount or concentration of sFRP4 to lower abdominal pain according to a VAS scale, or the amount or concentration of sFRP4 and the amount or concentration of CA-125.

[0128] In a third aspect, the present invention provides a method of monitoring a patient suffering from or undergoing treatment for adenomyosis, comprising: a) determining the amount or concentration of sFRP4 in a patient sample; b) comparing the determined amount or concentration with a standard; The present invention relates to a method, comprising:

[0129] In embodiments, the patient suffering from adenomyosis is monitored to determine whether the amount or concentration of sFRP4 in the patient's sample changes over time.In particular, the patient suffering from adenomyosis is monitored to determine whether the amount or concentration of sFRP4 increases, decreases, or remains unchanged over time.In embodiments, if the amount of sFRP4 in the patient's sample is determined to be elevated, the patient is monitored.

[0130] In an embodiment, a patient undergoing treatment for adenomyosis is monitored to determine whether the amount or concentration of sFRP4 in the patient's sample changes. In particular, a patient undergoing treatment for adenomyosis is monitored to determine whether the amount or concentration of sFRP4 increases, decreases, or remains unchanged. In particular, a patient undergoing treatment for adenomyosis is monitored to determine whether the amount or concentration of sFRP4 increases, decreases, or remains unchanged due to the treatment applied. In an embodiment, a decrease in the amount or concentration of sFRP4 in a patient undergoing treatment for adenomyosis indicates that the treatment is effective. In an embodiment, an unchanged or increased amount or concentration of sFRP4 in a patient's sample undergoing treatment for adenomyosis indicates that the treatment is ineffective, i.e., an unchanged or increased amount or concentration of sFRP4 in a patient's sample undergoing treatment for adenomyosis indicates that the treatment is persistent or recurrent adenomyosis.

[0131] In certain embodiments, treatment is indicated when the amount or concentration of sFRP4 is determined to be unchanged or increased in a sample from a patient undergoing treatment for adenomyosis.

[0132] In embodiments, patients are monitored several times at different times. In embodiments, patients are monitored several times within a time frame of several weeks, months, or years. In certain embodiments, patients are monitored monthly or annually. In embodiments, patients suffering from adenomyosis are monitored monthly or annually after the diagnosis of adenomyosis. In embodiments, patients undergoing treatment for adenomyosis are monitored once after treatment, particularly once after surgical treatment. In particular, patients undergoing treatment for adenomyosis are monitored monthly or annually to determine the effectiveness of the treatment and / or the recurrence of adenomyosis.

[0133] In some embodiments, the treatment for adenomyosis is selected from the group consisting of drug-based treatment or surgical treatment.In some embodiments, the drug-based treatment for adenomyosis is to inhibit or target neurogenic inflammation and / or analgesics and / or hormonal treatment (for example, hormonal contraceptives or GnRH analogues).In some embodiments, the surgical treatment for adenomyosis comprises, among others, hysterectomy, uterus-conserving surgery, such as surgical removal of adenomatous lesions, cysts, or uterine artery embolization (UAE) or nerve-conserving surgery.

[0134] In embodiments, the patient sample is a body fluid sample. In certain embodiments, the sample is a whole blood, serum, or plasma sample. In embodiments, the sample is an in vitro sample, i.e., it is analyzed in vitro and is not returned to the body.

[0135] In certain embodiments, the patient is a laboratory animal, a livestock animal, or a primate. In certain embodiments, the patient is a human patient. In certain embodiments, the patient is a female human patient.

[0136] In an embodiment, the method of the invention is an in vitro method.

[0137] In embodiments, the amount of sFRP4 is determined using an antibody, particularly a monoclonal antibody. In embodiments, step a) of determining the amount of sFRP4 in a patient sample comprises performing an immunoassay. In embodiments, the immunoassay is performed in either a direct or indirect format. In embodiments, such immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0138] In certain embodiments, step a) of determining the amount of sFRP4 in the patient sample comprises: i) incubating a patient sample with one or more antibodies that specifically bind to sFRP4, thereby generating a complex between the antibody and sFRP4; and ii) quantifying the complex formed in step i), thereby quantifying the amount of sFRP4 in the patient sample. Includes:

[0139] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to sFRP4. As will be apparent to those skilled in the art, the sample can be contacted with the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or the first and second antibodies simultaneously, in any desired order, for a time and under conditions sufficient to form a first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex. As will be readily apparent to those skilled in the art, it will take no more than routine experimentation to establish the time and conditions that are appropriate or sufficient for the formation of a complex between a specific anti-sFRP4 antibody and the sFRP4 antigen / analyte (=anti-sFRP4 complex), or for the formation of a secondary or sandwich complex comprising the first anti-sFRP4 antibody, sFRP4 (analyte), and the second anti-sFRP4 antibody (=anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex).

[0140] Detection of the anti-sFRP4 antibody / sFRP4 complex can be carried out by any suitable means. Detection of the first anti-sFRP4 antibody / sFRP4 / second anti-sFRP4 antibody complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods.

[0141] In certain embodiments, a sandwich is formed comprising a first antibody to sFRP4, sFRP4 (the analyte), and a second antibody to sFRP4, wherein the second antibody is detectably labeled.

[0142] In one embodiment, a sandwich is formed comprising a first antibody against sFRP4, sFRP4 (analyte) and a second antibody against sFRP4, wherein the second antibody is detectably labeled and the first anti-sFRP4 antibody is capable of binding to a solid phase or is bound to a solid phase.

[0143] In embodiments, the second antibody is detectably labeled, either directly or indirectly. In certain embodiments, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent or electrochemiluminescent dye.

[0144] In a fourth aspect, the present invention provides a computer-implemented method for assessing whether a patient has or is at risk of developing adenomyosis, comprising: a) receiving, in a processing unit, a value of the level of sFRP4 in a sample from a patient; b) processing the value received in step (a) with a processing unit, wherein processing includes retrieving one or more thresholds for the level of sFRP4 from a memory and comparing the value received in step (a) with the one or more thresholds; c) assessing via the output device whether the patient has or is at risk of developing adenomyosis, wherein the assessment is based on the results of step (b); The present invention relates to a method, comprising:

[0145] The above method is a computer-implemented method. Preferably, all steps of the computer-implemented method are performed by one or more processing units of a computer (or computer network). Thus, the evaluation of step (c) is performed by a processing unit. Preferably, said evaluation is based on the result of step (b).

[0146] The value(s) received in step (a) are derived from determining biomarker levels from patients suffering from adenomyosis, as described elsewhere herein. Preferably, the values are biomarker concentration values. The values are typically received by the processing unit by uploading or transmitting the values to the processing unit. Alternatively, the values can be received by the processing unit by entering the values via a user interface.

[0147] In one embodiment of the foregoing method, the criterion(s) indicated in step (b) are established from memory. Preferably, the criterion values are established from memory.

[0148] In one embodiment of the aforementioned computer-implemented method of the present invention, the results of the assessment performed in step c) are provided via a display configured to present the results.

[0149] In one embodiment of the aforementioned computer-implemented method of the present invention, the method may comprise the further step of transferring information regarding the assessment performed in step c) to the patient suffering from adenomyosis by using an electronic medical record.

[0150] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0151] Example Example 1: Diagnostic performance of biomarker sFRP4 and biomarker CA-125 in women with adenomyosis and controls in samples from a multicenter study.

[0152] The case group consisted of patients diagnosed with adenomyosis by ultrasound or laparoscopic visualization. The control group included women without adenomyosis or endometriosis. Inclusion criteria for the case and control groups were the presence of pelvic pain / infertility for which laparoscopy or laparotomy was planned and age between 18 and 45 years. Exclusion criteria for the case group were pregnancy / lactation, malignancy, recurrent adenomyosis and endometriosis, and laparoscopy / laparotomy for another reason (≤6 months).

[0153] sFRP4 was measured with the pre-marketed ECLIA assay for sFRP4, a sandwich immunoassay developed for the cobas Elecsys® ECLIA platform (ECLIA assay from Roche Diagnostics, Germany). The assay contains a biotinylated and ruthenium-conjugated monoclonal antibody that specifically binds to sFRP4. 49 μL of each serum sample was used and measured undiluted on a cobas e 601 analyzer (Roche Diagnostics, Germany). The Elecsys® electrochemiluminescence (ECL) technology and assay method for the determination of CA 125 II are briefly described below.

[0154] The concentration of CA-125 was determined using a cobas e 601 analyzer. CA-125 II detection using the cobas e 601 analyzer is based on Elecsys® electrochemiluminescence (ECL) technology. Briefly, biotin- and ruthenium-labeled antibodies are combined with the respective amounts of undiluted sample and incubated in the analyzer. Streptavidin-coated magnetic microparticles are then added to promote binding of the biotin-labeled immunoconjugates and incubated on the analyzer. After this incubation step, the reaction mixture is transferred to the measurement cell, where the beads are magnetically captured on the surface of the electrode. To separate the bound immunoassay complexes from the remaining free particles, ProCell M buffer containing tripropylamine (TPA) is then introduced into the measurement cell for the subsequent ECL reaction. The induction of a voltage between the working and counter electrodes then initiates a reaction that results in the emission of photons by the ruthenium complex and TPA. The resulting electrochemiluminescence signal is recorded by a photomultiplier tube and converted into a numerical value indicating the concentration level of the respective analyte.

[0155] Receiver operating characteristic (ROC) curves were generated (see Figure 1 for sFRP4 and Figure 2 for Ca-125). Model performance was determined by examining the area under the curve (AUC). The best possible AUC is 1, and the worst possible AUC is 0.5. The optimal cutoff was selected using Youden's index (maximum sum of sensitivity + specificity - 1). [Table 1]

[0156] The diagnostic performance of sFRP4 to distinguish adenomyosis cases from controls is higher compared to that of the biomarker CA-125.

Claims

1. 1. A method for assessing whether a patient has or is at risk for developing adenomyosis, comprising: determining the amount or concentration of sFRP4 in the patient sample; comparing said determined amount or concentration to a standard; Including, wherein the sample is a bodily fluid selected from blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid.

2. 1. A method for selecting a patient for treatment of adenomyosis, comprising: determining the amount or concentration of sFRP4 in the patient sample; comparing said determined amount or concentration to a standard; Including, wherein the sample is a bodily fluid selected from blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid.

3. 3. The method of claim 2, wherein the treatment for adenomyosis is a drug-based treatment, a pain management treatment, or a surgical treatment.

4. 1. A method of monitoring a patient suffering from or undergoing treatment for adenomyosis, comprising: determining the amount or concentration of sFRP4 in the patient sample; comparing the determined amount or concentration to the amount or concentration of sFRP4 in a reference sample taken from the patient at an earlier time point to determine the difference between the determined amount or concentration and the amount or concentration in the reference sample; Including, wherein the sample is a bodily fluid selected from blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid.

5. The method of claim 4, wherein an elevated amount or concentration of sFRP4 in the sample from the patient indicates: that the patient suffering from adenomyosis should be monitored; or indicates the presence of persistent or recurrent adenomyosis in a patient undergoing treatment for adenomyosis.

6. The method of any one of claims 1 to 4, wherein the patient is a female patient.

7. 7. The method of claim 6, wherein the patient is a female human patient.

8. 1. A computer-implemented method for assessing whether a patient has or is at risk for developing adenomyosis, comprising: a) receiving, in a processing unit, a value of a level of sFRP4 in a sample from a patient, wherein the sample is a body fluid selected from blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid; b) processing the value received in step (a) with the processing unit, said processing comprising retrieving one or more thresholds for the level of sFRP4 from a memory and comparing the value received in step (a) with the one or more thresholds; c) assessing via an output device whether the patient has or is at risk of developing adenomyosis, said assessment being based on the results of step (b); and A method comprising:

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