Methods for detecting or treating endometrial and ovarian hyperproliferative disorders - Patents.com

Abstract

The present disclosure provides a highly sensitive diagnostic method that does not require invasive laparoscopic surgery. It has been found that monomeric myeloperoxidase (MPO) is found in the cells, tissues and serum of endometriosis patients. This novel biomarker can be used to detect ovarian hyperproliferative disorder and endometrial hyperproliferative disorder. Therefore, an aspect of the present disclosure relates to a method for evaluating a subject, comprising detecting monomeric myeloperoxidase (MPO) in a biological sample from the subject. Also disclosed is a method for treating a subject with endometrial hyperproliferative disorder or ovarian hyperproliferative disorder, comprising administering a treatment for endometrial hyperproliferative disorder or ovarian hyperproliferative disorder to the subject, the level of monomeric MPO having been evaluated in a biological sample from the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 176,852, filed April 19, 2021, and U.S. Provisional Patent Application No. 63 / 277,438, filed November 9, 2021, each of which is incorporated by reference in its entirety.

[0002] I. FIELD OF THE INVETION The present invention relates generally to the fields of molecular biology and therapeutic diagnostics. More specifically, the present invention relates to methods and compositions relating to the prognosis, diagnosis, and treatment of hyperproliferative disorders. [Background technology]

[0003] II. Background Hyperproliferative disorders, such as ovarian cancer and endometriosis, are particularly difficult to detect and diagnose. Endometriosis affects 6-11% of premenopausal women and contributes to pain, infertility, impaired quality of life, and huge annual healthcare-related costs of $70 billion in 2016 (GlobalData 2018). Although the diagnosed prevalence of endometriosis is lower than other chronic diseases, the economic burden of endometriosis is comparable to other chronic diseases, such as diabetes, Crohn's disease, and rheumatoid arthritis (RA). Although the etiology is poorly understood, endometriosis is characterized by the extrauterine transfer of hormone-responsive endometrium. In the case of ovarian cancer, the cancer often goes undetected until it has spread within the pelvis and abdomen.

[0004] At present, laparoscopic surgery is the only means of providing a definitive diagnosis of endometriosis, and most patients struggle to find effective treatment modalities to alleviate symptoms. Many studies have investigated noninvasive biomarkers to diagnose endometriosis and predict prognosis and appropriate treatment, but none have performed well enough to be translated into clinical use. Endometriosis is also associated with noncorrectable risk factors, such as family history, early age at menarche, and low parity. Early identification of symptoms associated with endometriosis in high-risk women can limit the costs associated with disease progression and reduce the number of late-stage endometriosis cases. However, early diagnosis of endometriosis can be difficult because symptoms are often nonspecific, physical examination often has limited clinical signs, and clinical testing is unhelpful and imaging provides limited benefit. Therefore, laparoscopy is required to make a definitive diagnosis. For these reasons, there is an average delay of 7-12 years between the onset of symptoms and the diagnosis of endometriosis, which significantly impacts patients' quality of life.

[0005] A key challenge is accurate diagnosis at an early stage. Hence, there is a need for early non-invasive diagnosis (RNID) that can save these women through proper and early diagnosis of their hyperproliferative disorders. Summary of the Invention

[0006] Abstract The present disclosure meets the above needs by providing a highly sensitive diagnostic method and kit that does not require invasive laparoscopic surgery. It has been found that monomeric myeloperoxidase (MPO) is found in cells, tissues, and serum of endometriosis patients. This novel biomarker can be used to detect ovarian hyperproliferative disorder and endometrial hyperproliferative disorder. Therefore, an aspect of the present disclosure relates to a method for evaluating a subject, comprising detecting monomeric myeloperoxidase (MPO) in a biological sample from the subject. Also disclosed is a method for treating a subject with endometrial hyperproliferative disorder or ovarian hyperproliferative disorder, comprising administering a treatment for endometrial hyperproliferative disorder or ovarian hyperproliferative disorder to the subject, the subject having been evaluated for the level of monomeric MPO in a biological sample from the subject.

[0007] A further aspect of the disclosure relates to a method of diagnosing a subject having an endometrial or ovarian hyperproliferative disorder, comprising the steps of: (a) assessing monomeric MPO in a biological sample from the subject; (b) comparing the measured level to a control level or sample; and (c) diagnosing the subject having an endometrial or ovarian hyperproliferative disorder based on the measured level of monomeric MPO. A further aspect relates to a method for monitoring a subject being treated for an endometrial or ovarian hyperproliferative disorder with a therapeutic agent, comprising the steps of: (a) assessing monomeric MPO in a biological sample from the subject; (b) comparing the measured level to a control level or sample; and (c) determining the effectiveness of the therapeutic agent based on the measured level of monomeric MPO. A further aspect relates to a method for generating a complex, comprising contacting a biological sample with an antibody that binds monomeric MPO.

[0008] A further aspect relates to a kit comprising one or more anti-MPO antibodies or MPO-binding fragments thereof and a size exclusion column for fractionating a sample, the fraction separating polypeptides having a size of 75 kDa and polypeptides having a size of 150 kDa into separate fractions.

[0009] The methods of the disclosure include prognosing, diagnosing, or monitoring a subject having, suspected of having, and / or having symptoms of an endometrial or ovarian hyperproliferative disorder. The subject may be diagnosed as having stage I, II, III, or IV cancer based on the level of monomeric MPO assessed.

[0010] The term hyperproliferative disorder refers to a disorder characterized by abnormal growth or hyperproliferation of tissue. Hyperproliferative disorders can include hyperplasia, dysplasia, and precancerous lesions. Hyperproliferation can be further characterized by tumors. In some aspects, the hyperproliferation is benign, meaning that it is non-cancerous. In some aspects, the hyperproliferation is malignant, meaning that it is cancerous growth.

[0011] The biological sample may include serum, plasma, or tissue samples. In some aspects, the biological sample is a blood sample or a fraction thereof. In some aspects, the biological sample is a biological sample described herein. In certain aspects, the biological sample is a serum sample. In some aspects, the biological sample is a plasma sample.

[0012] Aspects of the present disclosure also relate to assessing iron levels in a biological sample from a subject. Assessment of iron levels may be combined with assessment of monomeric MPO to provide a diagnosis or prognosis for the subject. In some aspects, the method further comprises detecting iron levels in a biological sample from a subject. In some aspects, the detecting iron levels in a biological sample from a subject comprises one or more of a serum iron test, a free iron test, a total iron binding capacity (TIBC) test, or a ferritin test. In aspects of the method of the present disclosure, the subject is a subject who has been evaluated for iron levels. In some aspects, the subject has been evaluated for iron levels by performing one or more of a serum iron test, a free iron test, a total iron binding capacity (TIBC) test, or a ferritin test on a biological sample from the subject. In some aspects, the subject has been determined to have abnormal iron levels. In some aspects, the subject has been determined to have elevated TIBC that is lower than normal. In some aspects, the subject has been determined to have elevated ferritin. In some aspects, the subject has been determined to have elevated serum iron. In some aspects, the subject has been determined to have normal iron levels. The normal range for TIBC is 250-450 mcg / dL. In some aspects, the subject is determined to have a TIBC of less than 250 mcg / dL. The normal range for ferritin is 20-250 ng / mL for adult males, 10-120 ng / mL for adult females aged 18-39, and 12-263 ng / mL for females aged 40 or older. In some aspects, the ferritin is determined to be greater than 120 ng / mL, 250 ng / mL, or 263 ng / mL. In some aspects, the subject is determined to have abnormal transferrin saturation. In some aspects, the subject is determined to have elevated transferrin saturation. The normal range for transferrin saturation is 20-50%. In some aspects, the subject is determined to have a transferrin saturation of greater than 50% or the biological sample has a transferrin saturation of greater than 50%.In some aspects, the subject has been determined to have an elevated serum iron level, has an elevated serum iron level, or a biological sample from the subject has an elevated serum iron level. The normal range for serum iron is 60-170 micrograms per deciliter (mcg / dL), or 10.74-30.43 micromoles per liter (micromoles / L). In some aspects, the subject has been determined to have a serum iron level greater than 170 mcg / dL, has a serum iron level greater than 170 mcg / dL, or a biological sample has a serum iron level greater than 170 mcg / dL.

[0013] In some aspects, the TIBC in the biological sample is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, or 550 The TIBC in the subject has been determined to be, or the TIBC in a biological sample derived from the subject has been determined to be, higher than, lower than, at least, or at most mcg / dL, or any possible range therebetween. In some aspects, ferritin in the biological sample has been determined to be, or has been determined to be, greater than, less than, at least, or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, or 350 ng / mL, or any possible range therebetween. In some aspects, the transferrin saturation in the biological sample is greater than, less than, at least, or at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% (or any possible range therebetween), the transferrin saturation in the subject has been determined to be, or the transferrin saturation in a biological sample derived from the subject has been determined to be.In some aspects, the transferrin saturation in the biological sample has been determined to be, or the transferrin saturation in the subject has been determined to be, greater than, less than, at least, or up to 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 micrograms per deciliter, or any possible range therebetween. In some aspects, the free iron in a biological sample is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, or 550 The free iron in the subject has been determined to be, or the free iron in the biological sample from the subject has been determined to be, greater than, less than, at least, or at most, or any possible range therebetween. In some aspects, the higher than normal level of free iron in the biological sample from the subject is greater than 100 mcg / dL, greater than 110 mcg / dL, greater than 120 mcg / dL, greater than 130 mcg / dL, greater than 140 mcg / dL, greater than 150 mcg / dL, or any possible range therebetween.

[0014] The disclosed method may include or further include size fractionation of the biological sample. In some aspects, the biological sample is a size fractionated sample. The size fractionation in the disclosed method may be performed in a manner to provide a fraction of the biological sample that includes molecules of 75 kDa and does not include molecules of 150 kDa or larger. In some aspects, the biological sample is a size fractionated sample that includes molecules of 75 kDa and does not include molecules of 150 kDa or larger. The size fractionation may separate the sample into fractions that include molecules in the size range of 10-100 kDa, 30-100 kDa, 40-100 kDa, 50-100 kDa, 60-90 kDa, or 60-85 kDa. In some aspects, size fractionation may provide fractions ranging from about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 kDa to about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or 145 kDa (and all possible ranges therebetween). In some aspects, the average size of the fractions of the biological sample is, at least, or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 5, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, or 190 kDa (and all possible ranges therebetween). In some aspects, the fraction does not contain molecules that are, are at least, or are at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 5, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, or 190 kDa (and any range that may occur therebetween). In some aspects, the method includes detecting monomeric MPO in the fraction that contains 75 kDa molecules.

[0015] In an aspect of the present disclosure, the step of detecting monomeric MPO comprises immunological detection of monomeric MPO. In some aspects, the step of detecting or evaluating monomeric MPO comprises an enzyme-linked immunosorbent assay (ELISA) assay. Elisa assays use a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (generally a protein) in a liquid sample using an antibody against the protein being measured. In some aspects, an antigen from a biological sample or a fraction thereof is attached to a surface. Thus, an antibody, such as an anti-MPO antibody, can be applied onto the surface, whereby it can bind to any MPO from the biological sample. This antibody can be linked to a detection molecule, such as an enzyme, and then unbound antibody can be removed. In a final step, the detection molecule can be detected qualitatively or quantitatively. In aspects where the detection molecule is an enzyme, a substrate for that enzyme can be added, which results in a reaction that produces a detectable signal, most commonly a color change that can be measured quantitatively or qualitatively.

[0016] In some aspects, the ELISA is further characterized as a sandwich ELISA. The anti-MPO antibody can be immobilized on a solid support, such as a microtiter plate or a polystyrene microtiter plate. The biological sample or fraction can be added to the solid support to achieve binding between the anti-MPO antibody and MPO in the biological sample or fraction. Unbound molecules can be washed off the solid support. After the MPO antigen is immobilized, a detection antibody that complexes with the antigen can be added. The detection antibody can be covalently linked to a detection molecule, such as an enzyme, or can itself be detected by a secondary antibody linked to a detection molecule, such as an enzyme. Between each step, the plate is typically washed with a mild detergent solution to remove any non-specifically bound proteins or antibodies. After the final washing step, the plate is developed by qualitatively or quantitatively detecting the detection molecule. In the case of enzyme detection, the final step can include adding an enzyme substrate to produce a visual signal that can be detected qualitatively or quantitatively.

[0017] ELISAs can be performed using other forms of ligand binding assays instead of being strictly immunoassays. ELISAs can include any ligation reagent that can be immobilized on a solid phase with a detection reagent that specifically binds to it, and can use detectable molecules to generate a signal that can be appropriately quantified. During washing, only the ligand and its specific binding partner remain specifically bound or "immunoadsorbed" by antigen-antibody interactions to the solid phase, while non-specific or unbound components are washed away.

[0018] "Detectable label, molecule, or moiety" or "detection molecule, label, or moiety" are used interchangeably to refer to compounds and / or elements that can be detected by their unique functional properties and / or chemical characteristics by use of which an antibody can be detected and / or further quantified if desired. Examples of detectable labels include, but are not limited to, radioisotopes, fluorophores, semiconductor nanocrystals, chemiluminescent materials, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin, or haptens), and the like. Specific examples of labels include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester (DMAE), Texas Red, luminol, NADPH, and α- or β-galactosidase. Antibody conjugates include those intended primarily for in vitro use, where the antibody is linked to a secondary binding ligand and / or an enzyme that produces a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference. Molecules containing azide groups can also be used to form covalent bonds with proteins through reactive nitrene intermediates generated by low intensity ultraviolet light (Potter & Haley, 1983).

[0019] In some aspects, detecting monomeric MPO comprises contacting the biological sample or a fraction containing 75 kDa molecules with an anti-MPO antibody or MPO binding molecule under conditions that allow binding of MPO to the anti-MPO antibody. In some aspects, the anti-MPO antibody or binding molecule is linked to a solid support. Suitable solid supports or carriers include any support capable of binding antigens or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The method of the present disclosure may comprise or further comprise washing the solid support to remove unbound molecules. In some aspects, the method comprises or further comprises contacting the biological sample or fraction with a capture antibody or antigen binding molecule. In some aspects, the capture antibody or antigen binding molecule comprises a second anti-MPO antibody or MPO antigen binding fragment. Exemplary antigen-binding fragments include, for example, single chain variable fragments (scFv), F(ab')2, Fab', Fab, Fv, or rIgG. In some aspects, the capture antibody is linked to a detectable label. The method may include or further include a step of quantitatively or qualitatively evaluating the detectable label.

[0020] In aspects of the present disclosure, the subject or patient can be a human subject or a human patient. In some aspects, the subject or patient is a non-human animal. In some aspects, the non-human animal is a bat, monkey, camel, rat, mouse, rabbit, goat, chicken, bird, cat, dog. The subject can be further defined as a high-risk subject. In some aspects, the subject is a subject with one or more symptoms of endometriosis and / or ovarian cancer. Symptoms of ovarian cancer include abdominal bloating or distension, feeling full quickly when eating, weight loss, discomfort in the pelvic area, changes in bowel habits, such as constipation, and needing to urinate frequently. The subject can also be a subject that has been diagnosed with endometrial hyperproliferative disorder or ovarian hyperproliferative disorder. In some aspects, the subject has been treated for endometrial hyperproliferative disorder or ovarian hyperproliferative disorder. In some aspects, the subject is to be treated for endometrial hyperproliferative disorder or ovarian hyperproliferative disorder. In some aspects, the subject is currently undergoing treatment for endometrial hyperproliferative disorder or ovarian hyperproliferative disorder. The subject may be further defined as a human subject. In some aspects, the subject is a woman. In some aspects, the subject is undergoing hormone therapy. The hormone therapy may include contraception or hormone replacement therapy. In some aspects, the female subject is adolescent, peri-menopausal, or menopausal woman. In some aspects, the subject is a subject with one or more symptoms of endometrial hyperproliferative disorder or ovarian hyperproliferative disorder.

[0021] The method may include or may further include a step of quantifying the level of monomeric MPO in the biological sample. In some aspects, the level of monomeric MPO in the biological sample or fraction has been quantified. In some aspects, the level of monomeric MPO is normalized. In some aspects, the level of monomeric MPO is compared to a control. In some aspects, the level of monomeric MPO is determined to be higher than the control. In some aspects, the level of monomeric MPO is determined to be lower than the control. In some aspects, the subject has or has been determined to have a level of monomeric MPO in the biological sample that is higher than the level of monomeric MPO in the control sample. In some aspects, the subject has or has been determined to have a level of monomeric MPO in the biological sample that is lower than the level of monomeric MPO in the control sample. In some aspects, the subject has or has been determined to have a level of monomeric MPO in the biological sample that is not significantly different from the level of monomeric MPO in the control sample. For example, the level of monomeric MPO may be determined to be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 5 standard deviations different from, at least that much, or at most that much different from, the control value or within the control value. In some aspects, the level of monomeric MPO may be determined to be 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100% (and all possible ranges therebetween) above or below, at least that much above or below, or at most that much above or below the control level of monomeric MPO. The control may include a level of monomeric MPO that represents the level of monomeric MPO in a biological sample from a subject with endometriosis. In some aspects, the control may include a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with ovarian cancer. In some aspects, the control may include a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometrial hyperproliferative disorder or ovarian hyperproliferative disorder.In some aspects, the method further comprises diagnosing the subject. In some aspects, the subject is diagnosed as having ovarian cancer based on the determined level of monomeric MPO. In some aspects, the subject is diagnosed as having or includes stage I, II, III, or IV ovarian cancer based on the determined level of monomeric MPO. The method of the present disclosure may also comprise or further comprise treating the subject for ovarian cancer. In some aspects, the subject is diagnosed as having endometriosis based on the determined level of MPO. The method of the present disclosure may comprise or further comprise treating the subject for endometriosis.

[0022] In some aspects, a therapeutic agent is determined to be effective if monomeric MPO is (i) not detectable in a biological sample from the subject; (ii) not significantly different from a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject not having an endometrial or ovarian hyperproliferative disorder; (iii) less than a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject having an endometrial or ovarian hyperproliferative disorder; or (iv) decreased compared to the level of monomeric MPO prior to treatment of the subject with the therapeutic agent. In some aspects, a therapeutic agent is determined to be ineffective if monomeric MPO is (i) detectable in a biological sample from the subject; (ii) increased compared to a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject not having an endometrial or ovarian hyperproliferative disorder; (iii) not significantly different from or greater than a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject having an endometrial or ovarian hyperproliferative disorder; or (iv) not significantly different from or increased compared to the level of monomeric MPO prior to treatment of the subject with the therapeutic agent.

[0023] In some aspects, the method further comprises evaluating the level of monomeric MPO in a biological sample from the subject obtained before treatment.In some aspects, the method further comprises evaluating the level of monomeric MPO in a biological sample from the subject obtained after one or more treatments.For example, the method can comprise or further comprise evaluating the level of monomeric MPO after one dose of a specific treatment, after two doses of a specific treatment, after three doses of a specific treatment, after four doses of a specific treatment, after five doses of a specific treatment, and / or after six doses of a specific treatment.The effectiveness of the treatment can be determined based on the level of monomeric MPO evaluated.

[0024] In some aspects, the level of monomeric MPO is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 in a biological sample from a subject. Determined to be, determined to be at least, or determined to be at most, ng / ml, mcg / ml, mg / ml, (or any possible range therebetween). In some aspects, the subject is diagnosed with a level of monomeric MPO of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 in a biological sample from the subject. A person is diagnosed with stage I cancer if their serum plasma levels are determined to be, at least, or at most ng / ml, mcg / ml, mg / ml, (or any possible range therebetween).In some aspects, the subject is diagnosed with a level of monomeric MPO of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 in a biological sample from the subject. A person is diagnosed with stage II cancer if their serum plasma levels are determined to be, at least, or at most ng / ml, mcg / ml, mg / ml, (or any possible range therebetween). In some aspects, the subject is diagnosed with a level of monomeric MPO of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 in a biological sample from the subject. A person is diagnosed with stage III cancer if their serum creatinine level is determined to be, at least, or at most ng / ml, mcg / ml, mg / ml, (or any possible range therebetween).In some aspects, the subject is diagnosed with a level of monomeric MPO of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, or 500 in a biological sample from the subject. A person is diagnosed with stage IV cancer when the serum creatinine level is determined to be, at least, or at most ng / ml, mcg / ml, mg / ml, (or any possible range therebetween). In aspects of the present disclosure, late stage cancer includes stage IV and / or stage III. In aspects of the present disclosure, early stage cancer includes stage I and / or stage II.

[0025] The control level may comprise a level of monomeric MPO representing the level of monomeric MPO in a subject with endometriosis. In some aspects, the control comprises a level of monomeric MPO representing the level of monomeric MPO in a subject with ovarian cancer. In some aspects, the control comprises a level of monomeric MPO representing the level of monomeric MPO in a subject without endometrial or ovarian hyperproliferative disorder. In some aspects, the hyperproliferative disorder comprises endometriosis. In some aspects, the hyperproliferative disorder comprises ovarian cancer.

[0026] The treatment can be any treatment known in the art or described herein for endometriosis or ovarian cancer.In some aspects, the treatment includes hormone therapy or surgery.Other treatments useful in the methods of the present disclosure include surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or targeted therapy.

[0027] In some aspects, a subject is diagnosed as not having an endometrial or ovarian hyperproliferative disorder if monomeric MPO is (i) not detectable in a biological sample from the subject; (ii) not significantly different from a control comprising a level of monomeric MPO representative of the level of monomeric MPO in a biological sample from a subject not having an endometrial or ovarian hyperproliferative disorder; or (iii) less than a control comprising a level of monomeric MPO representative of the level of monomeric MPO in a biological sample from a subject having an endometrial or ovarian hyperproliferative disorder. In some aspects, a subject is diagnosed with endometriosis if monomeric MPO is (i) greater than a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with neither endometrial nor ovarian hyperproliferative disorder; (ii) not significantly different from a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis; or (iii) less than a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with ovarian cancer. In some aspects, a subject is diagnosed with ovarian cancer if monomeric MPO is (i) greater than a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with neither endometrial nor ovarian hyperproliferative disorder or from a subject with endometriosis; or (ii) not significantly different from a control comprising a level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with ovarian cancer. In some aspects, the step of evaluating monomeric MPO comprises qualitatively detecting the level of monomeric MPO in biological sample.In some aspects, the subject is diagnosed as not having endometrial hyperproliferative disorder or ovarian hyperproliferative disorder when monomeric MPO is not detected in biological sample from the subject.In some aspects, the subject is diagnosed as having endometrial hyperproliferative disorder or ovarian hyperproliferative disorder when monomeric MPO is detected in biological sample from the subject.

[0028] Aspects of the kit of the present disclosure may include or further include one or more negative or positive control samples. In some aspects, the kit includes an ELISA for detecting MPO. In some aspects, the anti-MPO antibody or binding fragment is operably linked to a solid support. In some aspects, the kit includes at least two anti-MPO antibodies, at least two anti-MPO antibody binding fragments, or one anti-MPO antibody and one anti-MPO antibody binding fragment. In some aspects, one or more anti-MPO antibodies or MPO antibody binding fragments are operably linked to a detectable label.

[0029] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cell and molecular biology to indicate that a value includes the standard error for the device or method being employed to determine the value.

[0030] The use of the words "a" or "an" when used in conjunction with the term "comprising" can mean "one," but it also has the meaning of "one or more," "at least one," and "one or more than one."

[0031] As used herein, the terms "or" and "and / or" are used to express that multiple elements are in combination with each other or exclusive of each other. For example, "x, y, and / or z" can represent "x" only, "y" only, "z" only, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It specifically contemplates that x, y, or z can be individually excluded from an embodiment or aspect.

[0032] The words "comprising" (and any form of including, e.g., "comprise" and "comprises"), "having" (and any form of including, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), "characterized by" (and any form of including, e.g., "characterized as"), or "containing" (and any form of including, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0033] The compositions and methods of their use may "comprise," "consist essentially of," or "consist of" any component or step disclosed throughout this specification. The phrase "consisting of" excludes any element, step, or ingredient not recited. The phrase "consisting essentially of" limits the scope of the described subject matter to those materials or steps recited and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described under the term "comprising" may also be realized under the term "consisting of" or "consisting essentially of."

[0034] It is specifically envisioned that any limitation discussed with respect to one embodiment or aspect of the present invention can be applied to any other embodiment or aspect of the present invention.Furthermore, any composition of the present invention can be used in any method of the present invention, and any method of the present invention can be used to make or utilize any composition of the present invention.The aspects of the embodiments shown in the examples are also embodiments that can be implemented under the relationship with the embodiments or aspects discussed elsewhere in different examples or elsewhere in this application, for example, in the summary of the invention, detailed description of the embodiments, claims, and the description of the notes to the figures.

[0035] Any method related to a therapeutic, diagnostic, or physiological purpose or effect may also be described in the claim language "use," e.g., "use of" any compound, composition, or agent discussed herein to achieve or realize a described therapeutic, diagnostic, or physiological purpose or effect.

[0036] Based on any method described herein, the use of one or more sequences or compositions can be adopted.Other embodiments are also discussed throughout this application.Any embodiment or aspect discussed with respect to one aspect of this disclosure also applies to other aspects and embodiments of this disclosure, and vice versa.

[0037] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the present invention, are given by way of example only, and that various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0038] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0039] [Figure 1] Image showing monomeric and dimeric forms of MPO in endometriosis. Western blot analysis was performed using non-reducing gels to detect monomeric and dimeric forms of MPO. Only monomeric form of MPO is detected in endometriotic tissue as indicated by the 75 kDa band. [Diagram 2]Image showing monomeric and dimeric forms of MPO in ovarian cancer. Western blot analysis was performed using non-reducing gels to detect monomeric and dimeric forms of MPO in serum from healthy volunteers, ovarian cancer patients, and selected ovarian cancer cell lines. Only monomeric forms of MPO are detected in ovarian cancer cell lines as indicated by the 75 kDa band. Interestingly, monomeric forms of MPO are also detected in serum obtained from ovarian cancer patients, but not in healthy serum, which only shows dimeric MPO forms as indicated by the 150 kDa band. [Diagram 3] Real-time RT-PCR analysis of MPO in multiple cancer cell lines. MPO mRNA levels were determined in epithelial ovarian cancer (SKOV-3, MDAH-2774, OVCAR-3, OV-21, OV-90), pancreatic cancer (BXPC-3), colon cancer (COLO-3), non-Hodgkin's B-cell lymphoma (DLCL-2), bladder cancer (HTB-4), and endometrial cancer (CRL-1671) cell lines. [Figure 4] Box plots showing dimeric / monomeric serum MPO concentrations in four different conditions [control = healthy controls (n = 15), NC_IGC = non-cancerous inflammatory gynecologic diseases (n = 14), ES_EOC = early stage epithelial ovarian cancer (n = 12), LS_EOC = late stage epithelial ovarian cancer (n = 16)], with the x-axis log10 scaled. [Diagram 5] Receiver operating curves and area under the curve (AUC) comparing the predictive power of serum dimeric MPO 84.5% and monomeric MPO 100% in discriminating samples between NC_IGC=noncancerous inflammatory gynecological diseases and ES_EOC=early stage epithelial ovarian cancer (p=0.050) and between ES_EOC=early stage epithelial ovarian cancer and LS_EOC=late stage epithelial ovarian cancer (p=0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention In this application, we report that ovarian cancer cells express only the monomeric form of MPO, which differentiated between early and late stages of the disease. Total RNA and protein were isolated from various human ovarian cancer cells, serum from patients with ovarian cancer at various stages, serum from non-cancerous inflammatory gynecological diseases, and healthy volunteers. In all samples, a combination of ELISA and non-reduced Western blot was used to determine the levels of monomeric and dimeric MPO. Real-time PCR was used to measure MPO mRNA levels in various cancer cell lines. Receiver operating curves (ROC) were used to compare the predictive power of serum dimeric and monomeric MPO as well as the discrimination between samples. We demonstrated that monomeric MPO is the predominant form of MPO in ovarian cancer cell lines. Interestingly, monomeric MPO was also detected in serum obtained from patients with ovarian cancer at various stages, but not in healthy individuals. More importantly, monomeric MPO differentiated between early and late stages of the disease. The new findings from this study strongly indicate that MPO is a critically needed biomarker for the early detection of this disease.

[0041] II. Treatment of Hyperproliferative Disorders Methods and compositions for treating hyperproliferative disorders can be provided, particularly utilizing biomarker expression or activity levels. Different treatments can be prescribed or recommended for different patients based on the profile of biomarker expression or activity levels. In some aspects, the hyperproliferative disorder comprises ovarian cancer or cancer originating from ovarian epithelium, germ cell, or stroma, fallopian epithelium, ovary, cervix, fallopian tube, or uterus. In some aspects, the hyperproliferative disorder is primary peritoneal cancer, fallopian tube cancer, teratoma, dysgerminoma, or yolk sac tumor. Cancer is further characterized as comprising cancer stage, TNM, and / or having the characteristics described below.

[0042] A. Cancer Staging The most common staging system is the American Joint Committee on Cancer (AJCC) TNM (Tumor / Node / Metastasis) system. The TNM system assigns numbers based on three categories: "T" indicates the extent of invasion of the bowel wall, "N" indicates the extent of lymph node involvement, and "M" indicates the extent of metastasis. Higher stages of cancer are usually designated with numbers I, II, III, IV based on the TNM score, which is grouped by prognosis, with higher numbers indicating more advanced cancer and a worse potential outcome. The system is detailed in the table below. TIFF2024515726000001.tif92164TIFF2024515726000002.tif227164TIFF2024515726000003.tif83164

[0043] The "cancer" referred to in the methods described herein may include or exclude any of the above stages or TNM categories. The "cancer" referred to in the methods described herein may include or exclude any of the above stages or TNM categories. For example, the cancer may be or exclude stage 0, I, IA, IB, IC, II, IIA, IIB, IIIA1, IIIA2, IIIB, IIIC, IVA, or IVB cancer. The patient may have and / or have been determined to have stage 0, I, IA, IB, IC, II, IIA, IIB, IIIA1, IIIA2, IIIB, IIIC, IVA, or IVB cancer. Further, the cancer may be at stages N0 and / or M0; T1, N0, and / or M0; T1, N1, and / or M0; T2, N0, and / or M0; T1, N2, and / or M0; T2, N1, and / or M0; T3, N0, and / or M0; T1, N3, and / or M0; T2, N2, and / or M0; T3, N1, and / or M0; T4a, N0, and / or It can be M0; T2, N3, and / or M0; T3, N2, and / or M0; T4a, N1, and / or M0; T3, N3, and / or M0; T4a, N2, and / or M0; T4b and / or N0; N1 and / or M0; T4a, N3, and / or M0; T4b and / or N2; N3 and / or M0; any T; any N, and / or M1.

[0044] B. Treatment The method of the present disclosure relates to treating subjects and patients with cancer therapy.Cancer therapy can be the therapy described below, and can be administered to patients who are determined to have a particular biomarker profile.For example, in some aspects, the therapy described below is administered to patients who are determined to have poor prognosis, unfavorable prognosis, or to patients who are determined to be at high risk.In some aspects, the therapy described below is administered to patients who are determined to have favorable prognosis, or to patients who are determined to be at low risk.Combinations of the therapies described below are also envisioned.

[0045] The cancer treatment can be surgery, radiation, chemotherapy, hormonal therapy, or targeted therapy. Radiation can be further characterized as external beam radiation therapy or brachytherapy. The chemotherapy can be a platinum compound and / or a taxane. Platinum compounds include cisplatin and carboplatin. Taxanes include paclitaxel and docetaxel. In some aspects, the chemotherapy includes a combination of a chemotherapy platinum compound and a taxane. Additional chemotherapy includes albumin-bound paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, ifosfamide, irinotecan, liposomal doxorubicin, melphalan, pemetrexed, topotecan, and vinorelbine.

[0046] Common methods of administering chemotherapy include placing an intravenous (IV) tube into a vein with a needle, or swallowing it (orally) in a pill or capsule. Chemotherapy regimens usually involve a certain number of cycles administered over a set period of time. Patients may be given one drug at a time or a combination of different drugs at the same time.

[0047] Antimetabolites can be used in cancer treatment because they interfere with DNA production and therefore cell division and tumor growth. Cancer cells divide more times than other cells, so inhibiting cell division harms tumor cells more than other cells. Antimetabolites masquerade as purines (azathioprine, mercaptopurine) or pyrimidines, compounds that become the building blocks of DNA. They prevent these substances from being incorporated into DNA during the S phase (of the cell cycle), halting normal development and division. They also affect RNA synthesis. However, because thymidine is used in DNA and not RNA (uracil is used instead), inhibition of thymidine synthesis through thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis. Due to their efficiency, these drugs are the most widely used cytostatics. In the ATC system, they are classified as L01B.

[0048] Thymidylate synthase inhibitor is a chemical agent that inhibits the enzyme thymidylate synthase and has the potential as anti-cancer chemotherapy.As the target of anti-cancer chemotherapy, thymidylate synthase can be inhibited by thymidylate synthase inhibitors, such as fluorouracil, which is a fluorinated pyrimidine, or certain folic acid analogs, the most famous of which is raltitrexed (brand name Tomudex).Additional agents include pemetrexed, nolatrexed, ZD9331, and GS7904L.

[0049] In a further aspect, prodrugs that can be converted to thymidylate synthase inhibitors in the body can be used, such as capecitabine (INN), an orally administered chemotherapy drug used in the treatment of many cancers. Capecitabine is a prodrug that is enzymatically converted to 5-fluorouracil in the body.

[0050] If the cancer has spread to the lymph nodes, the administration of chemotherapy drugs fluorouracil or capecitabine may prolong life. Chemotherapy drugs for this condition may include capecitabine, fluorouracil, irinotecan, leucovorin, oxaliplatin, and UFT. Another type of drug that is sometimes used is an epidermal growth factor receptor inhibitor.

[0051] In certain aspects, alternative treatments can be prescribed or recommended based on biomarker profiles.In addition to traditional chemotherapy for gastric cancer patients, cancer therapy also includes various combination therapies using both compound-based and radiation-based treatments.Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agent, taxol, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the above compounds.

[0052] Like chemotherapy, radiation therapy can be used in the neoadjuvant and adjuvant settings for some stages of cancer.

[0053] Targeting therapy can also be used in the methods described herein.Targeting therapy includes angiogenesis inhibitors, such as bevacizumab, and / or PARP inhibitors, such as olaparib, rucaparib, and / or niraparib.NTRK targeting drugs, such as larotrectinib and entrectinib, are also included.

[0054] Hormonal therapies include luteinizing hormone releasing agonists, tamoxifen, and aromatase inhibitors.

[0055] Immunotherapy designed to enhance the body's natural defenses against cancer may also be used. Immunotherapy is usually based on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of the tumor cell. The antibody may function alone as an effector of therapy or it may actually recruit other cells that result in cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function only as a targeting agent. Alternatively, the effector may be a lymphocyte with a surface molecule that interacts either directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0056] Usually, tumor cells should have some markers that are available for targeting, i.e., that are not present on most other cells. There are many tumor markers, and any of these may be suitable for targeting.

[0057] C. Monitoring In certain aspects, the biomarker-based methods may be combined with one or more other cancer diagnostic or screening tests, frequently when a patient is determined to have a high risk of recurrence or a poor prognosis based on the above biomarkers.

[0058] In some aspects, the method of the present disclosure further comprises one or more monitoring tests. The monitoring protocol can include any method known in the art. In particular, the monitoring includes obtaining a sample and testing the sample for diagnosis. For example, the monitoring can include endoscopy, biopsy, laparoscopy, colonoscopy, blood test, genetic test, endoscopic ultrasound, X-ray, barium enema X-ray, chest x-ray, barium swallow, CT scan, MRI, PET scan, or HER2 test. In some aspects, the monitoring test includes radiological imaging. Examples of radiological imaging that are useful in the method of the present disclosure include liver ultrasound, computed tomography (CT) abdominal scan, liver magnetic resonance imaging (MRI), whole body CT scan, and whole body MRI.

[0059] D.ROC analysis In statistics, a receiver operating characteristic (ROC) or ROC curve is a graphical plot that represents the performance of a binary classification system as its discrimination threshold is varied. ROC analysis can be applied to determine the cutoff or threshold settings for biomarker expression. For example, a patient whose biological sample is determined to have a biomarker expression value above a particular cutoff threshold but below a higher cutoff threshold can be determined to have endometriosis. A patient whose biological sample is determined to have a biomarker expression level that exceeds the cutoff threshold for endometriosis can be determined to have cancer. The curve is created by plotting the true positive rate against the false positive rate at various threshold settings. (The true positive rate is also known as sensitivity in biomedical informatics, or recall in machine learning. The false positive rate is also known as fall-out and can be calculated as 1-specificity). The ROC curve is therefore sensitivity as a function of fall-out. Typically, when the probability distributions for both detection and false alarms are known, an ROC curve can be constructed by plotting the cumulative distribution function of the detection probability (the area under the probability distribution from -infinity to +infinity) on the y-axis against the cumulative distribution function of the false alarm probability on the x-axis.

[0060] ROC analysis provides a tool to select the presumed optimal model independent of (and prior to) the cost context or class distribution and to discard suboptimal models. ROC analysis relates in a direct and natural manner to the cost / benefit analysis of diagnostic decision making.

[0061] ROC curves were originally developed by electrical engineers and radar technicians during World War II to detect enemy objects on the battlefield, and were soon introduced into psychology to understand the perceptual detection of stimuli. Since then, ROC analysis has been used in medicine, radiology, biometrics, and other domains for decades, and is increasingly being used in machine learning and data mining research.

[0062] ROC is also known as relative operating characteristic curve, because it is a comparison of two operating characteristics (TPR and FPR) while changing its criteria.ROC analysis curve is known in the art and described in Metz CE (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine 8:283-298;Youden WJ (1950) An index for rating diagnostic tests. Cancer 3:32-35;Zweig MH, Campbell G (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical Chemistry 39:561-577;and Greiner M, Pfeiffer D, Smith RD (2000) Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45:23-41, which are incorporated herein by reference in their entirety. ROC analysis can be used to generate cut-off values ​​for prognostic and / or diagnostic purposes.

[0063] III. Protein Assays Various techniques can be used to measure the expression levels of polypeptides and proteins in biological samples to determine biomarker expression levels. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Those skilled in the art can easily adapt known protein / antibody detection methods for use in determining the protein expression levels of biomarkers.

[0064] In one aspect, antibodies, or antibody fragments or derivatives, can be used in methods such as Western blot, ELISA, or immunofluorescence techniques to detect biomarker expression. In some aspects, either the antibody or protein is immobilized on a solid support. Suitable solid supports or carriers include any support that can bind antigens or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite.

[0065] Those skilled in the art will know many other suitable carriers for binding antibodies or antigens and will be able to adapt such supports for use in the present disclosure. The support can then be washed with an appropriate buffer and then treated with detectably labeled antibody. The solid support can then be washed a second time with buffer to remove unbound antibody. The amount of label bound to the solid support can then be detected by conventional means.

[0066] Immunohistochemistry methods are also suitable for detecting the expression level of biomarkers. In some aspects, antibodies or antisera, including polyclonal antisera and monoclonal antibodies, specific to each marker can be used to detect expression. Antibodies can be detected by direct labeling of the antibody itself, for example, with radioactive labels, oral labels, hapten labels, such as biotin, or enzymes, such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies are used with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific to the primary antibodies. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

[0067] Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS) and antibody arrays. Such immunoassays typically involve the measurement of complex formation between a protein and its specific antibody. These assays and their quantification against purified, labeled standards are well known in the art. Two-site, monoclonal antibody-based immunoassays using antibodies reactive to two non-interfering epitopes or competitive binding assays can be used.

[0068] Numerous labels are available and widely known in the art. Radioisotope labels include, for example, 36S, 14C, 125I, 3H, and 131I. Antibodies can be labeled with radioisotopes using techniques known in the art. Fluorescent labels include, for example, rare earth chelates (europium chelates) or labels such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red, and are available. Fluorescent labels can be conjugated to antibody variants using techniques known in the art. Fluorescence can be quantified using a fluorometer. A variety of enzyme-substrate labels are available, and U.S. Patent Nos. 4,275,149 and 4,318,980 provide reviews of some of these. Enzymes generally catalyze a chemical change in a chromogenic substrate, which can be measured using a variety of techniques. For example, enzymes can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate becomes electrically excited by a chemical reaction and can then emit light that can be measured (e.g., using a chemiluminometer) or donate energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dihydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, Methods in Enzymology (Ed. J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

[0069] In some aspects, the detection label is indirectly conjugated to the antibody. Those skilled in the art know various techniques to achieve this. For example, the antibody can be conjugated to biotin, and any of the three major categories of labels above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and therefore the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label and the antibody, the antibody is conjugated to a small hapten (e.g., digoxin), and one of the different types of labels above is conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). In some aspects, the antibody does not need to be labeled, and its presence can be detected using a labeled antibody that binds to the antibody.

[0070] IV. Sample preparation In certain aspects, the method includes obtaining a sample from a subject. The obtaining methods provided herein may include biopsy, such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, excision biopsy, excision biopsy, punch biopsy, excision biopsy, or skin biopsy. In certain aspects, the sample is obtained from a biopsy from ovarian or endometrial tissue by any of the biopsy methods mentioned above. In other aspects, the sample is obtained from any of the tissues provided herein, including but not limited to ovarian epithelium, fallopian epithelium, ovary, cervix, fallopian tube, or uterus, and non-cancerous or cancerous tissue and non-cancerous or cancerous tissue. Alternatively, the sample may be obtained from any other source, including but not limited to blood, serum, plasma, sweat, hair follicle, buccal tissue, tears, saliva, stool, or saliva. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, may obtain the biological sample for testing. Still further, the biological sample may be obtained without the assistance of a medical professional.

[0071] Samples may include, but are not limited to, tissues, cells, or biological material derived from cells or from cells of a subject. Biological samples may be heterogeneous or homogeneous populations of cells or tissues. Biological samples may be obtained using any method known in the art that can provide samples suitable for the analytical methods described herein. Samples may be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, cheek swabbing, saliva collection, urine collection, stool collection, saliva, tears, or semen collection.

[0072] The sample may be obtained by methods known in the art. In certain aspects, the sample is obtained by biopsy. In other aspects, the sample is obtained by swabbing, endoscopy, scraping, blood sampling, or any other method known in the art. In some aspects, the sample may be obtained, stored, or transported using the components of the kit of the method. In some examples, multiple samples, such as multiple plasma or serum samples, may be obtained for diagnosis by the methods described herein. In other examples, multiple samples, such as one or more samples from one tissue type (e.g., ovary or related tissue) and one or more samples from another tissue type (e.g., serum), may be obtained for diagnosis by the methods. Samples may be obtained at different times and stored and / or analyzed by different methods. For example, samples may be obtained and analyzed by conventional staining methods or any other cytological analysis methods.

[0073] In some aspects, biological sample can be obtained by a doctor, nurse, or other medical professional, such as a medical technician, an endoscopist, a cytologist, a phlebotomist, a radiologist, or a pulmonologist.The medical professional can indicate the appropriate test or assay to be performed on the sample.In certain aspects, a molecular profiling provider can advise on which assay or test is most appropriate.In a further aspect of the method, the patient or subject can obtain biological sample for testing without the assistance of a medical professional, for example, obtain a whole blood sample, a urine sample, a stool sample, a buccal sample, or a saliva sample.

[0074] In other examples, samples are obtained by invasive procedures including, but not limited to, biopsy, needle aspiration, blood drawing, endoscopy, or blood sampling. Needle aspiration methods may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0075] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, the entire contents of which are incorporated herein by reference, describe general methods of biopsy and cytological methods.

[0076] In some aspects of the method, the molecular profiler can obtain biological sample directly from the subject, from a medical professional, from a third party, or from a kit provided by the molecular profiler or a third party.In some examples, the biological sample can be obtained by the molecular profiler after the subject, the medical professional, or a third party obtains the biological sample and sends it to the molecular profiler.In some examples, the molecular profiler can provide suitable containers and excipients for storing the biological sample and transporting it to the molecular profiler.

[0077] In some aspects of the methods described herein, a medical professional need not be involved in either the initial diagnosis or sample acquisition. Instead, an individual may obtain a sample through the use of an over-the-counter (OTC) kit. The OTC kit may include a means for obtaining a sample as described herein, a means for storing the sample for testing, and instructions for proper use of the kit. In some examples, the molecular profiling service is included in the purchase price of the kit. In other examples, the molecular profiling service is charged separately. A sample suitable for use by a molecular profiling service may be any material that includes tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining the suitability and / or sufficiency of a sample are provided.

[0078] In some aspects, the subject may be referred to a specialist, such as an oncologist, surgeon, or endocrinologist.The specialist may also obtain biological samples for testing or refer the individual to a testing center or laboratory for submitting biological samples.In some instances, a medical professional may refer the individual to a testing center or laboratory for submitting biological samples.In other instances, the subject may provide the sample.In some instances, a molecular profiling company may obtain the sample.

[0079] V. Administration of Therapeutic Compositions The therapy provided herein may include a combination of therapeutic agents, such as administering a first cancer therapy and a second cancer therapy. The therapy may be administered in any suitable manner known in the art. For example, the first and second cancer treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some aspects, the first and second cancer treatments are administered in separate compositions. In some aspects, the first and second cancer treatments are administered in the same composition.

[0080] Aspects of the present disclosure relate to compositions and methods that include therapeutic compositions. Different therapies can be administered in one composition or in two or more compositions, for example, two compositions, three compositions, or four compositions. Various combinations of drugs can be used.

[0081] The therapeutic agents of the present disclosure may be administered by the same or different administration routes. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0082] The treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered and the particular route and formulation are within the ability of those skilled in the clinical art to determine. A unit dose need not be administered in a single injection, but may include continuous infusion over a period of time. In some aspects, a unit dose includes a single administrable dose.

[0083] The precise amount of therapeutic composition depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (relief of symptoms or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy to which the subject may be subjected.

[0084] VI. Pharmaceutical Compositions In certain aspects, the composition or agent used in the method, such as a chemotherapeutic agent or a biomarker modulator, is suitably contained in a pharma- ceutically acceptable carrier. The carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the agent. In some aspects of the present disclosure, the agent can be formulated into a preparation for local (i.e., to a specific location in the body, such as skeletal muscle or other tissue) or systemic delivery, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, in solid, semi-solid, gel, liquid, or gaseous form that allows oral, parenteral, or surgical administration. Certain aspects of the present disclosure also contemplate local administration of the composition by coating a medical device, etc.

[0085] Carriers suitable for parenteral and local delivery through injection, infusion or irrigation include distilled water, physiological phosphate buffered saline, standard or lactate Ringer's solution, dextrose solution, Hank's solution or propanediol.In addition, sterile fixed oil can be used as solvent or suspension medium.For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides.In addition, fatty acids, such as oleic acid, find use in the preparation of injections.Carriers and drugs can be formulated as liquids, suspensions, polymerizable or non-polymerizable gels, pastes or ointments.

[0086] Carriers may also include delivery vehicles for sustaining (i.e., extending, delaying or modulating) the delivery of an agent or for improving the delivery, uptake, stability or pharmacokinetics of a therapeutic agent. Such delivery vehicles may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0087] In certain aspects, the actual dosage of the composition administered to a patient or subject may be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, acute symptoms of the patient, and route of administration. In any event, the practitioner responsible for administration will determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject.

[0088] The solution of pharmaceutical composition can be prepared in water, suitably mixed with surfactant, such as hydroxypropylcellulose.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oil.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0089] In certain aspects, pharmaceutical compositions are advantageously administered in the form of injectable compositions, either as liquid solutions or suspensions, and can also be prepared in suitable solid forms or solutions or suspensions in liquids prior to injection.These preparations can also be emulsified.A typical composition for such purposes includes a pharmaceutically acceptable carrier.For example, the composition can include 10 mg or less, 25 mg, 50 mg, or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, etc.

[0090] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antifungal agents, antioxidants, chelating agents and inert gases. The pH and exact concentrations of the various components of the pharmaceutical composition are controlled according to well-known parameters.

[0091] Further formulations are suitable for oral administration. Oral formulations contain typical excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0092] In further aspects, the pharmaceutical composition may include classical pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be through any common route, so long as the target tissue is available through that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for treating skin cancer, to prevent chemotherapy-induced alopecia or other skin hyperproliferative disorders. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions may be administered as pharmacologic acceptable compositions, usually including physiologically acceptable carriers, buffers or other excipients. For the treatment of pulmonary conditions, aerosol delivery may be used. The volume of the aerosol is between about 0.01 ml and 0.5 ml.

[0093] The effective amount of pharmaceutical composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of pharmaceutical composition calculated to produce the desired response discussed in relation to its administration, i.e., appropriate route and treatment regimen. The amount administered, both according to the number of treatments and the number of unit doses, depends on the protection or effect sought.

[0094] The precise amount of the pharmaceutical composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

[0095] VII. Kits Certain aspects of the present invention also relate to kits that include the compositions of the present invention or the compositions for carrying out the methods of the present invention.In some aspects, the kits can be used to evaluate one or more biomarkers.In certain aspects, the kits include at least or include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules, antibodies, or inhibitors, or any value or range and combination that may occur therebetween. In some aspects, there is a kit for assessing biomarker activity or levels in a cell.

[0096] Kits may contain components that may be individually packaged or placed into containers, such as tubes, bottles, vials, syringes, or other suitable containment means.

[0097] Individual components may also be provided in the kit in concentrated amounts, and in some aspects components that are in solution with other components are provided individually at the same concentration. Concentrations of components may be provided at 1x, 2x, 5x, 10x, or 20x or more.

[0098] Included as part of this disclosure are kits for using the probes, antibodies, synthetic nucleic acids, non-synthetic nucleic acids, and / or inhibitors of the present disclosure for prognostic or diagnostic applications. Specifically contemplated are any such molecules that correspond to any of the biomarkers identified herein, including antibodies that bind to such biomarkers, as well as nucleic acid primers / primer sets and probes that are identical to or complementary to all or a portion of the biomarker, which may include non-coding sequences of the biomarker and coding sequences of the biomarker.

[0099] In certain aspects, negative and / or positive control nucleic acids, antibodies, probes, and inhibitors are included in some kit aspects. Additionally, the kits may include samples that are negative or positive controls for methylation of one or more biomarkers.

[0100] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein and that different aspects may be combined. It is contemplated that the claims as originally filed will cover any polynomial reciting claim or combination of claims filed. EXAMPLES

[0101] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples are techniques found by the inventors to function well in the practice of the invention and therefore can be considered to constitute preferred modes of its practice. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made in the specific examples disclosed without departing from the spirit and scope of the invention and still achieve the same or similar results.

[0102] Example 1: Serum monomeric MPO in endometriosis and ovarian cancer As shown in Figure 1, monomeric forms of MPO were detected in serum obtained from endometriosis patients but not in healthy serum, which showed only the dimeric MPO form represented by the 150 kDa band. As expected, MPO is not detectable in normal tissues.

[0103] FIG. 2 shows that there is more monomeric than dimeric MPO in all ovarian cancer cells compared to normal macrophages.

[0104] Example 2: Monomeric myeloperoxidase: a potential biomarker for early detection of ovarian cancer A. Introduction Ovarian cancer is the leading cause of cancer death in women, with the underlying causes still unknown

[14] . Ovarian cancer arises in the epithelium of inclusion cysts, whereas high-grade serous ovarian cancer, the most common type of ovarian cancer, is thought to arise from the fallopian tubes[3,16]. The 5-year survival rate for nonmetastatic ovarian cancer is approximately 90%, decreasing to about 60% for localized disease and to about 20% for metastatic disease[9]. The lack of early-stage screening methods with high specificity, specificity, or both contributes significantly to the high mortality rates observed. Furthermore, early-stage ovarian cancer presents with nonspecific symptoms, and thus diagnosis is often made after the malignancy has spread outside the ovaries

[18] .

[0105] Ovarian cancer cells are characterized by an enhanced oxidative stress environment, which is further amplified in chemoresistant ovarian cancer [8]. The inventor first reported that the key oxidative enzyme myeloperoxidase (MPO) is expressed by ovarian cancer cells and tissues

[23] . This finding was surprising, as MPO, an abundant heme protein known to be present only in neutrophils and monocytes, plays an essential role in immune surveillance and host defense mechanisms

[19] . The inventor reported crosstalk between MPO and the key pro-oxidant enzyme inducible nitric oxide synthase (iNOS) as a key mechanism of apoptosis suppression in ovarian cancer cells

[23] . Further findings from the inventor's laboratory strongly indicated the potential benefit of the combination of serum MPO and free iron as biomarkers in the early detection and prognosis of ovarian cancer [9].

[0106] Mature MPO is a symmetrically glycosylated homodimer of approximately 150 kDa. Encoded by a single gene on chromosome 17, the MPO monomer is composed of a 59 kDa heavy (α) and a 13.5 kDa light (β) subunit covalently linked by disulfide bonds

[11] . The formation of mature glycosylated heme-containing dimeric MPO from monomeric pro-MPO is a complex process involving numerous proteolytic reactions and post-translational modifications

[31] . Furthermore, the expression level of MPO depends on allelic polymorphisms in the promoter region. In particular, a G to A substitution at position 463 (G-463A) reduces MPO transcription 25-fold

[22] . Indeed, this single nucleotide polymorphism (SNP) in MPO has been reported to increase the risk of breast and ovarian cancer [4,5,32]. The main function of mature MPO, an oxidant-generating enzyme, is to catalyze the formation of hypochlorous acid (HOCl), a potent antimicrobial substance. Mature MPO is then degraded by HOCl to a monomer (MPO-Cl). Monomeric MPO has been reported to be detected in the plasma of patients with acute inflammation

[12] . The impact of MPO dimerization on the structural and functional properties of MPO is not fully understood.

[0107] In this study, the inventors showed that ovarian cancer cells express only the monomeric form of MPO, which distinguishes between early and late stages of the disease. This novel finding strongly indicates that MPO is a critical biomarker for the early detection of this disease.

[0108] B. Method 1. Patient population: Serum (n=15) was collected from patients with suspected EOC attending the Gynecologic Oncology Department at Karmanos Cancer Institute who were invited to participate in a prospective study. Patients provided informed consent (Wayne State University Human Subject Committee protocol number 027201MP2E) and agreed to provide blood samples before treatment (chemotherapy or surgery). Cases included early to late stage diagnoses and a range of histologies. Stage I was defined as early stage compared with the remaining stages II to IV (II-IV).

[0109] Benign controls: Sera (n=14) from patients with benign gynecologic conditions were obtained through the Cooperative Human Tissue Network (CHTN). These included women diagnosed with ovarian cysts, peritonitis (inflammation), or uterine fibroids.

[0110] Healthy control sera (n=8) were sourced from women recruited through local community organizations. These women were healthy and had no history of cancer. Basic information, such as age, race, and evidence of benign gynecologic conditions, if present, was obtained at the time of informed consent. The age and racial makeup of this group overlapped with, but was not specifically matched to, patients with ovarian cancer and benign conditions.

[0111] 2. Cell lines and cell cultures: Human cell lines: MDAH-2774, OvCar-3, OV-21, OV-90, TOV112D (kindly provided by Gen Sheng Wu, Wayne State University, Detroit, Michigan), SKOV-3, A2780, CRL-1671, BXPC-3, COLO-3, DLCL-2, and HTB-4 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cell lines were grown in 75 cm cultures containing their respective medium according to the manufacturer's protocol. 2The cells were cultured in cell culture flasks (Corning Incorporated, Corning, NY). The medium was supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin containing 10% heat-inactivated FBS at 37°C under 5% CO2. The culture medium was changed every 2 days. For each experiment, the cells were plated in 60 mm x 15 mm cell culture dishes at approximately 2X10 cells / dish. 6 Cells were plated at 100% cell density and cultured for an additional 24 h before RNA and protein collection. All experiments were performed in triplicate.

[0112] 3. Myeloperoxidase enzyme immunoassay assay: Prior to performing the ELISA, monomeric MPO was purified from all samples by gel filtration on Sephacryl S-200 HR, which removes traces of dimeric MPO. Myeloperoxidase Enzyme Immunometric Assay Kit (Assay Designs Catalog No. 900-115). We used Assay Designs' human Myeloperoxidase Enzyme Immunometric Assay (EIA) kit, an established assay in our laboratory, following the manufacturer's protocol. Briefly, the kit uses a monoclonal antibody against MPO immobilized on a microtiter plate that binds to MPO in the standard or sample. A native MPO standard is provided with the kit. A rabbit polyclonal antibody against MPO is added to bind to the MPO captured on the plate, followed by the addition of goat anti-rabbit IgG conjugated to horseradish peroxidase, which binds to the polyclonal MPO antibody. The enzyme reaction is stopped and the resulting color is read at 450 nm. The measured optical density, either in the standards or in the samples, is directly proportional to the concentration of MPO. The sensitivity of the assay, defined as the concentration of human MPO, was determined to be 0.019 ng / mL.

[0113] 4. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) for MPO: Total RNA was isolated from ovarian cancer cell lines using a monophasic solution of phenol and GITC / Trizol as previously described. [24–27] Quantification of RNA samples was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts).

[0114] Complementary DNA (cDNA) was prepared as follows: a 20 μl reaction volume containing 1 μg total RNA, 1 μg oligo(dT, 500 μg / ml; Invitrogen, Waltham, MA) and 1 μl 10 mM dNTP mix (Invitrogen, Waltham, MA) was heated to 65°C for 5 min and then immediately cooled on ice. A master mix containing 4 μl 5x First Strand Buffer, 2 μl 0.1 M DTT and 1 μl RNaseOut Recombinant Ribonuclease Inhibitor (40 units / μl; Invitrogen, Waltham, MA) was added and incubated at 42°C for 2 min. 1 μl (200 units) SuperScript II (Invitrogen, Waltham, MA) was added to each reaction and incubated at 42°C for 50 min. Finally, the enzyme was inactivated by heating at 70°C for 15 min.

[0115] Quantitative RT-PCR was performed using the Express SYBR Green RT-PCR kit (Life Technologies, Grand Island, New York) and the Cepheid 1.2f Detection System (Cepheid, Sunnyvale, CA). The total reaction volume of 25 μl contained 12.5 μl of 2x QuantiTect SYBR Green RT-PCR master mix, 3 μl of cDNA template, and 0.2 μM of each target-specific primer designed to amplify a portion of each gene. Optimal oligonucleotide primers for real-time RT-PCR amplification of reverse transcribed cDNA were selected with the aid of the software program Oligo 4.0 (National Bioscience Inc., Plymouth, MN). The sequences utilized for MPO (NM_000250) were as follows: sense (5'-3') TIFF2024515726000004.tif5128 and antisense (3'-5') 79 bp standard with TIFF2024515726000005.tif4128. For β-actin (NM_001101): Sense (5'-3') TIFF2024515726000006.tif4128 and antisense (3'-5') 79 bp standard with TIFF2024515726000007.tif4128. PCR reaction conditions were programmed as follows: an initial cycle at 95°C for 60 seconds, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 63 seconds (for MPO) and 58°C for 10 seconds (β-actin). This was followed by a final cycle at 72°C for 30 seconds to complete product synthesis.

[0116] To quantify each target transcript, a standard curve was generated using serial dilutions of a standard plasmid (Invitrogen, Waltham, MA). After PCR, melting curve analysis was performed to verify the specificity of the PCR product as a single peak. In all experiments, controls containing all reaction components except the template were included. The amount of mRNA was then normalized to the abundance of the housekeeping gene, β-actin.

[0117] To evaluate the validity of using β-actin as an internal standard and the change in the amount of β-actin, mRNA was tested as an external standard. The normalized values ​​of mRNA were then divided by those in the control. Unpaired Student's t-test was used for group comparison.

[0118] 5. Receiver Operating Characteristics (ROC): We estimated diagnostic performance using receiver operating characteristic (ROC) curves and confidence bands.[10,21] ROC curves allow the assessment of sensitivity and specificity of continuous biomarkers over the full range of potential cutoffs.

[0119] 6. Western Blot: Total protein was isolated from various human ovarian cancer cells (SKOV-3, A2780, OvCar-3, MDAH-2774, and TOV112D), serum from ovarian cancer patients, and healthy volunteers. Total protein (50 ug) was subjected to non-reducing Western blots and MPO was detected using a monoclonal MPO antibody (Santa Cruz Biotechnology, Dallas, TX) as previously described [7,15,20,28].

[0120] 7. Statistical analysis: Data were analyzed using SPSS 19.0 for Windows (SPSS for Windows, Chicago, IL). Data were analyzed using one-way ANOVA with Student-Newman-Keuls post-hoc comparisons. In all analyses, a significance value of p<0.05 was considered statistically significant.

[0121] C. Results In this study, we demonstrated the presence of only monomeric forms of MPO in cells derived from ovarian cancer (Figure 2). Both monomeric and dimeric forms of MPO were present in serum from ovarian cancer patients, with monomeric MPO levels being significantly higher (Figure 2). However, only dimeric MPO forms were detected in serum from healthy controls (Figure 2). Furthermore, MPO expression was unique to ovarian cancer cells and was not detected in various other types of cancer cell lines (Figure 3). More importantly, we demonstrated the ability of serum monomeric MPO to differentiate between early stage ovarian cancer, healthy controls, and inflammatory benign gynecological disorders such as peritonitis and fibroids (Figures 4, 5, p<0.05). Furthermore, serum monomeric MPO differentiated early stage ovarian cancer from late stage ovarian cancer (Figures 4, 5, p<0.05).

[0122] D. Discussion The inventor's laboratory has been investigating the role of oxidative stress and inflammation in the pathogenesis of ovarian cancer for over 20 years. Indeed, the inventor was the first to show that myeloperoxidase is present in all ovarian cancer cell lines and tissues, with minimal or no detectable presence in normal ovarian tissues

[23] . Furthermore, crosstalk between MPO and inducible nitric oxide synthase (iNOS), a key pro-oxidant enzyme, was shown to be an important mechanism of apoptosis suppression in EOC cells

[23] . In this mechanism, MPO increases S-nitrosylation of caspase-3, inhibiting its activity, thereby oxidizing the nitrosonium cation (NOS), a labile nitrosated species that suppresses apoptosis, a hallmark of ovarian cancer. + It uses nitric oxide (NO) produced by iNOS as a one-electron substrate to generate acetylcholinesterase (ACE) [2].

[0123] We previously demonstrated the potential benefit of a combination of serum MPO and free iron as a biomarker in the early detection and prognosis of ovarian cancer in a small number of patients [9]. To date, MPO has not been validated as a biomarker for the early detection and prognosis of ovarian cancer. In this study, we showed that MPO was present in its monomeric form in all ovarian cancer cells tested (Figure 2). The reason for this phenomenon remains unclear and is currently under investigation. A possible explanation is due to the enhanced inherent oxidative environment that characterizes ovarian cancer cells [13, 29]. The fact that monomeric form of MPO is detected in the blood of ovarian cancer patients but not in healthy individuals strongly indicates its potential use as a biomarker for this disease. Indeed, these results clearly demonstrated that monomeric MPO significantly differentiates between healthy and benign inflammatory conditions, early stage, and late stage ovarian cancer (Figures 4, 5).

[0124] Due to the low prevalence of ovarian cancer in US women, an ovarian cancer diagnostic or screening test must have a minimum specificity of 99.6% before it can be routinely used in the general population of postmenopausal women [6,17]. Such a test could offset the potential morbidity and mortality that may be associated with surgical complications for patients with false positive ovarian cancer screening tests [6,17]. Ovarian cancer screening tests should also have high sensitivity and adequate positive predictive value (PPV) [17,18]. Routine screening for ovarian cancer in the general population is not recommended because traditional screening methods are not sufficiently sensitive or specific [1]. Thus, the development of sensitive and specific methods for early detection has been a priority as a means to improve the diagnosis and treatment of this disease.

[0125] In conclusion, the technical idea of ​​using monomeric serum MPO levels as a means to infer the source of MPO and thus to distinguish between ovarian cancer-associated MPO and inflammation-associated MPO is novel. If this finding, determined in a small population of ovarian tumors, is confirmed in a larger population, a valuable biomarker will be identified. The results from this study strongly indicate that MPO is a critically needed biomarker for the early detection of this disease.

[0126] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and in the steps or in the sequence of steps of the methods without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0127] References The following references, and the references cited throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are individually incorporated by reference herein. TIFF2024515726000008.tif143160TIFF2024515726000009.tif237160TIFF2024515726000010.tif237160

Claims

1. A compound for use in a method for treating a subject having endometriosis, wherein the compound comprises hormonal therapy, the method comprising administering the compound for endometriosis to a subject in whom the level of monomeric MPO has been evaluated in a biological sample derived from the subject, the subject having or having been determined to have a level of monomeric MPO in the biological sample that is higher than the level of monomeric MPO in a control sample, and the control comprising a level of monomeric MPO representative of the level of monomeric MPO in a subject without endometriosis, said compound.

2. (i) the biological sample comprises serum, plasma, or a tissue sample; and / or (ii) the subject is female, optionally a female in puberty, perimenopause, or menopause; and / or (iii) the hormonal therapy comprises contraception or hormone replacement therapy; and / or (iv) the level of monomeric MPO in the biological sample derived from the subject has been quantified, optionally the level of monomeric MPO is standardized; A compound for use according to Claim 1.

3. the subject has been evaluated for iron levels; optionally, (i) the subject has been evaluated for iron levels by having one or more of a serum iron test, a free iron test, a total iron binding capacity (TIBC) test, or a ferritin test in a biological sample derived from the subject; and / or (ii) the subject has been determined to have abnormal iron levels, optionally the subject has been determined to have a TIBC lower than normal or a ferritin higher than normal, or the amount of free iron in a biological sample derived from the subject has been determined to be higher than normal; A compound for use according to Claim 1 or 2.

4. (a) a step of evaluating monomeric MPO in a biological sample derived from a subject, (b) a step of comparing the measured level with a control level or a control sample, and (c) a step of detecting a subject having endometriosis based on the measured level of monomeric MPO comprising a method for detecting a subject having endometriosis, wherein the step of evaluating the monomeric MPO comprises quantifying the level of the monomeric MPO in the biological sample; the monomeric MPO is (i) when not detected in the biological sample derived from the subject, (ii) if not significantly different from a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, or (iii) if less than a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis, it is indicated that the subject does not have endometriosis; or the monomeric MPO is (i) if more than a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, or (ii) if not significantly different from a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis, it is indicated that the subject has endometriosis; the method.

5. (i) the biological sample comprises serum, plasma, or a tissue sample, and optionally, the biological sample comprises a serum or plasma sample; and / or (ii) the method further comprises the step of evaluating the iron level in the biological sample from the subject, and optionally, the step of detecting the iron level in the biological sample from the subject comprises using one or more of a serum iron test, a free iron test, a total iron binding capacity (TIBC) test, or a ferritin test; and / or (iii) the method comprises size fractionation of the biological sample, the size fractionation providing a fraction or biological sample that contains molecules of 75 kDa and does not contain molecules of 150 kDa or larger, and optionally, the method comprises the step of evaluating monomeric MPO in the fraction containing the 75 kDa molecules; The method according to claim 4.

6. (i) the step of evaluating the monomeric MPO comprises using one or more antibodies that specifically bind to monomeric MPO; and / or (ii) the step of evaluating the monomeric MPO comprises an ELISA assay; and / or (iii) the step of evaluating the monomeric MPO comprises contacting the biological sample or the fraction containing the 75 kDa molecules with an anti-MPO antibody or an MPO-binding molecule under conditions that permit binding of MPO to the anti-MPO antibody, and optionally, the anti-MPO antibody or the binding molecule is linked to a solid support, and the method further comprises the step of washing the solid support to remove unbound molecules; and / or (iv)the method further comprises contacting the biological sample or the fraction with a capture antibody or an antigen-binding molecule; The method according to claim 4 or 5.

7. （I） (A)when the monomeric MPO is (i)not detected in a biological sample from the subject, (ii)not significantly different from a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, or (iii)less than a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis; and (B)when the iron level is normal, it is indicated that the subject does not have endometriosis; or (II) (A)when the monomeric MPO is (i)more than a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, or (ii)not significantly different from a control comprising the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis; and (B)when the iron level is abnormal, optionally, the TIBC is lower than normal and / or ferritin is more than normal in a biological sample from the subject, and / or the amount of free iron is more than normal in a biological sample from the subject, it is indicated that the subject has endometriosis; The method according to claim 4.

8. (i)the subject is a subject having one or more symptoms of endometriosis; and / or (ii)the subject is a female, optionally, the female is a female in puberty, perimenopause, or menopause; and / or (iii)the subject is undergoing hormone therapy, optionally, the hormone therapy includes contraception or hormone replacement therapy; and / or (iv)the level of the monomeric MPO is standardized; The method according to claim 4.

9. (a)evaluating monomeric MPO in a biological sample from a subject, (b)comparing the measured level with a control level or a control sample, and (c)determining the efficacy of a therapeutic agent based on the measured level of monomeric MPO A method for monitoring a subject being treated for endometriosis using a therapeutic agent, comprising: the step of evaluating the monomeric MPO includes quantifying the level of monomeric MPO in the biological sample; When the monomeric MPO is (i) not detected in a biological sample from the subject, (ii) not significantly different from a control including the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, (iii) less than a control including the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis, or (iv) decreased as compared to the level of monomeric MPO before treatment of the subject with a therapeutic agent, the therapeutic agent is determined to be effective; or When the monomeric MPO is (i) detected in a biological sample from the subject, (ii) increased as compared to a control including the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject without endometriosis, (iii) not significantly different from or more than a control including the level of monomeric MPO representing the level of monomeric MPO in a biological sample from a subject with endometriosis, or (iv) not significantly different from or increased as compared to the level of monomeric MPO before treatment of the subject with a therapeutic agent, the therapeutic agent is determined to be ineffective; the method.

10. (i) the biological sample includes serum, plasma, or a tissue sample, preferably the biological sample includes a serum or plasma sample; (ii) the method further includes a step of evaluating the iron level in the biological sample from the subject, optionally, the step of detecting the iron level in the biological sample from the subject includes using one or more of a serum iron test, a free iron test, a total iron binding capacity (TIBC) test, or a ferritin test; and / or (iii) the method includes a size fractionation of the biological sample, the size fractionation provides a fraction or biological sample that includes molecules of 75 kDa and does not include molecules of 150 kDa or larger, optionally, the method includes a step of evaluating monomeric MPO in the fraction including the 75 kDa molecules; The method according to claim 9.

11. (i) the step of evaluating the monomeric MPO includes using one or more antibodies that specifically bind to the monomeric MPO; and / or (ii) the step of evaluating the monomeric MPO includes an ELISA assay; and / or (iii) the step of evaluating the monomeric MPO comprises contacting the biological sample or the fraction containing the 75 kDa molecule with an anti-MPO antibody or an MPO-binding molecule under conditions that allow binding of MPO to the anti-MPO antibody, and optionally, the anti-MPO antibody or the binding molecule is linked to a solid support, and the method further comprises a step of washing the solid support to remove unbound molecules; and / or (iv) the method further comprises a step of contacting the biological sample or the fraction with a capture antibody or an antigen-binding molecule; The method according to claim 9 or 10.

12. (i) the step of evaluating the monomeric MPO comprises quantitatively detecting the level of monomeric MPO in the biological sample; and / or (ii) the subject is a subject having one or more symptoms of endometriosis; and / or (iii) the subject has been diagnosed with endometriosis; and / or (iv) the method further comprises a step of evaluating the level of monomeric MPO in a biological sample from the subject obtained prior to treatment; and / or (v) the method further comprises a step of evaluating the level of monomeric MPO in a biological sample from the subject obtained after one or more treatments; The method according to claim 9.

13. (i) the subject is female, and optionally, the female is a female in puberty, perimenopause, or menopause; and / or (ii) the subject is undergoing hormone therapy, and optionally, the hormone therapy includes contraception or hormone replacement therapy; and / or (iii) the level of the monomeric MPO is normalized; The method according to claim 9.

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