KPYM as a marker for endometrial cancer

Abstract

To provide non-invasive methods of diagnosing and prognosing endometrial cancer.SOLUTION: A method disclosed herein comprises determining the level of expression of MMP9 in a uterine aspirate fluid sample collected from the female genital tract. The present invention further provides kits for diagnosing the disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application Publication No. 16168328.9, filed May 4, 2016.

[0002] The present invention relates to the diagnosis and prognosis of endometrial carcinoma. [Background technology]

[0003] Endometrial cancer (EC) is the most frequent and aggressive tumor of the female reproductive tract and the fourth most common cancer in women in developed countries, accounting for an estimated 54,870 cases and 10,170 deaths in the United States in 2015. Today, 70% of EC cases are diagnosed early, when the tumor is still localized within the endometrium and is associated with an overall 5-year survival rate of 96%. However, 30% of EC patients are diagnosed only at an advanced stage of the disease, which is associated with a significantly reduced 5-year survival rate. This rate drops to 67% when myometrial invasion and / or lymph node involvement are already present, and to 18% when distant metastasis is present. Therefore, improving early diagnosis is a major challenge for managing EC appropriately and reducing disease-related mortality.

[0004] Early detection of EC patients is supported by the presence of symptoms such as abnormal vaginal bleeding, which are present in 93% of women diagnosed with EC. However, many other benign diseases cause similar symptoms. Distinguishing between patients with benign endometrial lesions and those with EC is achieved only after a lengthy diagnostic process consisting of a pelvic examination and transvaginal ultrasound, followed by a confirmatory histopathological examination of an endometrial biopsy. The preferred biopsy used in this procedure is called a uterine aspirate and / or pipelle biopsy, which is obtained by minimally invasive aspiration of endometrial fluid from the uterine cavity. Currently, diagnostic procedures in uterine aspirates rely on the presence of cellular material, and this process unfortunately has diagnostic failures and associated inadequate sampling rates of 8% and 15%, respectively. This increases to 12% and 22% in postmenopausal women. In these cases, a hysteroscopically guided biopsy is necessary. However, this invasive technique carries an increased risk of complications, including uterine perforation, bleeding, and potential harm to other organs.

[0005] Despite numerous studies on EC tumor tissues and normal endometrium, the development of proteome-based diagnostic assays remains challenging. The lack of translation of results from these studies into the clinic is due to two key factors: i) the lack of biofluid studies to develop EC biomarkers. The majority of studies have been based on tissue and / or serum or plasma. However, the search for plasma or serum biomarkers is extremely challenging due to the low concentrations of available biomarkers and the wide dynamic range of protein abundance. ii) the lack of validation studies to bridge the gap between the discovery and validation stages of the biomarker pipeline. Biomarker discovery experiments are prone to false discoveries due to biological variability and the small number of samples involved.

[0006] Therefore, despite the efforts made, there remains a need for biomarkers that allow for early stage endometrial cancer diagnosis with high sensitivity and specificity. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have demonstrated that uterine fluid samples can be used to diagnose endometrial cancer with high sensitivity and specificity. We found that the genomic DNA contained several robust markers that made it an appropriate sample for this purpose. [Means for solving the problem]

[0008] As shown below, the present inventors were able to identify for the first time 27 proteins that are differentially expressed in uterine fluid samples from patients with endometrial cancer. Surprisingly, all 27 proteins exhibit very high sensitivity and specificity (see Table 1 below), minimizing the risk of false-positive or false-negative diagnoses.

[0009] These findings open the door to using uterine fluid samples as biosamples for disease diagnosis instead of blood / serum or tissue biopsies. A useful diagnostic biomarker should not only improve the discrimination between patients suffering from the disease and benign cases, but also be economically beneficial and advantageous in clinical scenarios. For a diagnostic biomarker for EC, reducing the number of invasive biopsies and diagnostic costs is of great value. Therefore, the identification of biomarkers, the object of this invention, in easily accessible biofluids such as uterine fluid samples obtained by minimally invasive procedures already performed in current diagnostic processes represents a significant advance in the early diagnosis of disease.

[0010] Therefore, the present invention represents a major advance in the early diagnosis of endometrial cancer.

[0011] Thus, in a first aspect, the present invention provides a method for diagnosing or prognosing endometrial cancer, the method comprising determining the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1 in an isolated liquid sample obtained from the female reproductive tract.

[0012] It is noteworthy that the biomarkers of the first aspect of the invention have been concluded to be associated with cancer individually and collectively, and maintain strong associations with commonly altered molecular processes in cancer, such as cell motility, cell death, and survival in particular.

[0013] Thus, in a second aspect, the present invention provides the use of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, or GTR1 as an in vitro marker for the diagnosis or prognosis of endometrial cancer in isolated fluids obtained from the female reproductive tract. This embodiment may also be formulated as a method for detecting one or more endometrial cancer markers in a subject, comprising: (a) obtaining a fluid sample from the female reproductive tract; and (b) detecting in the sample an amount of at least one endometrial cancer marker selected from PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1.

[0014] In a third aspect, the present invention provides a method for diagnosing or prognosing endometrial cancer in a method of the first aspect of the invention, comprising measuring the expression of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1. The use of a means for determining the level is provided.

[0015] Importantly, the protein biomarkers that are the subject of this invention can be assessed by simple and low-cost methods such as immunochemistry or ELISA, platforms that are widely available in hospitals. As a result, these protein biomarkers can be easily implemented as routine clinical diagnostic kits, reducing costs for the health system. Furthermore, biomarker-based diagnostic kit tests provided by this invention could improve the current process of diagnosis and provide useful diagnostic or prognostic information for diseases in uterine aspirates.

[0016] Thus, in a fourth aspect, the present invention provides a kit comprising a solid support and means for detecting the expression level of two or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1.

[0017] In a further aspect, the present invention provides a method for identifying a subject suspected of having endometrial cancer, the method comprising: a) determining in vitro the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1 in a fluid sample obtained from the female reproductive tract of a subject; and b) comparing the level of step (a) with a reference control level, wherein if the level determined in step (a) is higher than the reference control level, it indicates that the subject is suspected of being afflicted with endometrial cancer; Includes:

[0018] In a further aspect, the present invention provides a method for determining or recommending whether to initiate a medical regimen in a subject suspected of having endometrial cancer, comprising: a) determining in vitro the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1 in a fluid sample obtained from the female reproductive tract of a subject; and b) diagnosing endometrial cancer or determining whether the subject is suspected of having endometrial cancer if the protein level in the test sample is higher than the reference control level; Including, i) if a subject is diagnosed with or suspected of having endometrial cancer, initiation of a medical regimen is recommended; ii) If the patient is diagnosed as not having endometrial cancer, optional follow-up will be performed taking into account the results of the patient's examination by the physician. A method is provided.

[0019] By determining the marker levels in a test sample, one skilled in the art can further establish what is the most appropriate therapy that can be recommended, as the levels detected in the sample may reflect the progression (i.e., severity) of the disease.

[0020] Furthermore, if it is determined that a subject has or is suspected of having endometrial cancer and therefore must begin a medical regimen, the markers of the present invention can be used to monitor how effective the regimen is; a decrease or return of the marker to normal levels (i.e., to the levels of a cancer-free control subject) can indicate that the patient is responding favorably to the medical regimen and therefore that the regimen is effective, whereas if the level of the marker does not change significantly or increases, this can indicate that the regimen is not effective. Finally, the levels of the marker can be measured after completion of treatment to control recurrence.

[0021] Thus, in a further aspect, the present invention provides a method for determining the efficacy of a medical regimen in a patient already diagnosed with endometrial cancer, comprising: (a) measuring in vitro the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1 in a fluid sample obtained from the female genital tract of the subject prior to administration of the medical regimen; (b) upon initiation of administration of the medical regimen, measuring in vitro the level of the marker in a fluid sample obtained from the female genital tract of the subject; and (c) comparing the levels measured in steps (a) and (b), such that if the level measured in step (b) is lower than the level measured in step (a), it indicates that the medical regimen is effective in treating endometrial cancer. or (i) measuring in vitro the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1 in a fluid sample obtained from the female genital tract of the subject upon initiation of administration of the medical regimen; and (ii) comparing the level measured in step (i) with a reference control level of the marker; Including, If the level measured in step (i) is not as high as the reference control level, it provides a method to indicate that the medical regimen is effective in treating endometrial cancer.

[0022] This method of the invention can be used to determine treatment outcomes (evaluations made to assess the results of management and procedures used in the fight against disease in order to determine the effectiveness, efficacy, safety, practicality, etc. of these interventions in individual cases or sequences).

[0023] As shown below, the inventors also surprisingly found that MMP-9 was significantly more highly expressed in uterine aspirate samples (also known as "pipelle biopsies," or fluid contained in endometrial biopsies) obtained from subjects with endometrial cancer compared to healthy controls.

[0024] Thus, in a further aspect, we provide

[0025] 1. A method for diagnosing or prognosing endometrial cancer, comprising determining the expression level of MMP9 in an isolated uterine aspirate sample;

[0026] 2. Use of MMP-9 as an in vitro marker for diagnosing or prognosing endometrial cancer in uterine aspirate samples;

[0027] 3. A method for identifying a subject suspected of having endometrial cancer, comprising: a) determining the expression level of MMP9 in uterine aspirate samples in vitro; and b) comparing the level of step (a) with a reference control level, wherein if the level determined in step (a) is higher than the reference control level, it indicates that the subject is suspected of having endometrial cancer;

[0028] 4. A method for determining or recommending whether to initiate a medical regimen in a subject suspected of having endometrial cancer, comprising: a) determining in vitro the expression level of MMP9 in a uterine aspirate sample; and b) diagnosing endometrial cancer or determining whether the subject is suspected of having endometrial cancer if the protein level in the test sample is higher than the reference control level; Including, i) if a subject is diagnosed with or suspected of having endometrial cancer, initiation of a medical regimen is recommended; ii) If the patient is diagnosed as not having endometrial cancer, optional follow-up will be performed taking into account the results of the patient's examination by the physician. Methods; and

[0029] 5. A method for determining the effectiveness of a medical regimen in a patient already diagnosed with endometrial cancer, comprising: (a) determining in vitro the expression level of MMP9 in a uterine aspirate sample isolated from the female genital tract of a subject prior to administration of a medical regimen; (b) upon initiation of administration of the medical regimen, measuring in vitro the level of the marker in an isolated uterine aspirate sample obtained from the subject's female reproductive tract; and (c) comparing the levels measured in steps (a) and (b), such that if the level measured in step (b) is lower than the level measured in step (a), it indicates that the medical regimen is effective in treating endometrial cancer. or (i) determining in vitro the expression level of MMP9 in an isolated uterine aspirate sample obtained from the female genital tract of the subject once administration of the medical regimen has begun; and (ii) comparing the level measured in step (i) with a reference control level of the marker. Including, If the level measured in step (i) is not as high as the reference control level, it indicates that the medical regimen is effective in treating endometrial cancer. is provided.

[0030] In a final aspect, the present invention provides a workflow for validating potential protein markers for endometrial cancer by performing mass spectrometry in targeted capture mode. From a list of potential endometrial cancer protein markers, at least one surrogate peptide for each protein was selected according to the criteria of detectability by mass spectrometry and uniqueness of the amino acid sequence. Variants of these peptides containing amino acids labeled with stable heavy isotopes of carbon and nitrogen were synthesized. Fluids obtained from the female reproductive tract were individually proteolyzed with trypsin and supplemented with an equal amount of a stable isotope-labeled synthetic peptide mixture. Samples were analyzed by high-performance liquid chromatography hyphenated with a hybrid high-resolution mass spectrometer, which (a) determined the detected peptides, which correlate with their elution time under defined chromatographic separation conditions. This method involves (a) generating an acquisition strategy containing a list of peptide ions to be identified; (b) performing mass analysis by repeatedly isolating the listed peptide ions during their elution time window using a quadrupole analyzer; (c) performing collision-induced fragmentation of the isolated peptide ions; and (d) analyzing the resulting peptide fragment ions using a high-resolution analyzer. For each peptide, the signals of the fragment ions of interest were extracted to construct an elution profile of the peptide that could be integrated. Normalization of the area attributed to endogenous peptides was performed with the area of ​​each stable-isotope-labeled peptide. The method also includes a step of confirming the identity of the peptides by spectral matching. DETAILED DESCRIPTION OF THE INVENTION

[0031] All terms used herein in this application are to be understood in their ordinary meaning as known in the art, unless otherwise specified. Other, more specific definitions of certain terms used herein are set forth below and are intended to apply equally throughout the specification and claims, unless another expressly defined definition confers a broader definition.

[0032] The present invention provides novel biomarkers for the diagnosis or prognosis of endometrial cancer in female genital fluids.

[0033] The term "diagnosis" is known to those skilled in the art. As used herein, "diagnosis" is understood as recognizing the complications or risk of a particular medical condition in a subject, determining the nature of a disease or condition, or distinguishing one disease or condition from another. It refers to both the process of attempting to determine or identify a possible disease or disorder and the opinion reached by that process. Diagnosis, in the sense of a diagnostic procedure, can be considered as an attempt to classify an individual's condition into a distinct category that allows medical decisions regarding treatment and prognosis to be made. The diagnostic opinion is then often described in terms of a disease or other condition. However, diagnosis can take many forms. It can detect the presence and identify a disease, pathology, dysfunction, or disability. It can also be an attempt to assign a category for management or prognosis. It can also indicate either a continuum of abnormality degrees or a categorical abnormality type.

[0034] The in vitro diagnostic method of the first aspect of the present invention can be performed using samples from (a) asymptomatic subjects, (b) subjects already identified as suspected of having endometrial cancer, (c) subjects already diagnosed with endometrial cancer in a complementary confirmatory diagnostic assay, or (d) subjects at high risk of having the disease.

[0035] As used herein, "prognosis" refers to a prediction of the likely progression and outcome of a disease. It includes grading a neoplasm (an attempt to describe in repeatable terms the level of cellular differentiation in a neoplasm, when increasing anaplasia correlates with neoplastic aggressiveness) and staging a neoplasm (an attempt to describe in repeatable terms the extent of a neoplasm in a patient).

[0036] The term "fluid sample obtained from the female reproductive tract" refers to a fluid produced by the uterine tract, which forms part of the female reproductive tract, and collected by aspiration, such as by vacuum aspiration (i.e., an "aspiration sample"). In accordance with the present invention, the aspiration of the fluid is performed without a prior step of injecting saline; that is, the term "aspiration" does not encompass a sample produced by washing the uterus.

[0037] In another embodiment of the method of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises (a) detecting in vitro in a test sample any of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD 44, CLIC1, TPIS, GSTP1, and GTR1, and (b) comparing the expression level of each of the tested proteins to a reference control value. In another embodiment of the method of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the method comprises (a) measuring in vitro in a test sample the expression level of one or more proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1, and (b) comparing the expression level of each of the tested proteins to a reference control value, wherein overexpression of a protein is indicative of endometrial cancer or a poor prognosis.

[0038] In the present invention, the term "reference control level" referred to in the methods of the first and second aspects of the present invention should be understood as a predetermined value of a given molecular marker, and in this case, any protein is mentioned in the first or second aspect and in certain embodiments, which is derived from the level of that molecular marker in a sample or group of samples. When the expression level is determined at the protein level, the "reference expression level" is a predetermined value of the amount of protein, while when the expression level is determined at the mRNA level, the "reference expression level" is a predetermined value of the amount of mRNA. The sample is taken from a subject or group of subjects in which the presence, absence, stage, or progression of the disease is appropriately preceded. This value is used as a threshold value to distinguish between subjects with the condition to be analyzed and those without the condition (i.e., subjects with endometrial cancer and subjects without endometrial cancer), particularly to determine the stage of the disease, the risk of developing or suffering from endometrial cancer. This reference control level is also useful for determining whether a subject should start a medical regimen and how effective the regimen is. The one or more subjects from which the "reference control level" is obtained may include subjects who do not have the condition, subjects who have the condition, or both. Those skilled in the art can use their general knowledge to select a more appropriate subject or group of subjects to obtain a reference control level for each of the methods of the present invention. Methods for obtaining reference values ​​from a selected group of subjects are well known in the state of the art (Burtis CA et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values"). In certain cases, the "reference control level" is a cut-off value determined by conventional ROC analysis (receiver operating characteristic analysis). As those skilled in the art will recognize, the optimal cut-off value is determined according to the specific application of the diagnostic or prognostic method, i.e., the purpose, the target population for diagnosis or prognosis, the balance between specificity and sensitivity, etc.

[0039] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method further comprises determining the expression level of one or more proteins selected from the group consisting of ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0040] PERM, also known as myeloperoxidase or MPO, has the Uniprot database accession number P05164, 19 Feb 2014-v4. MPO is a protein released by leukocytes that plays an important role in inflammation and oxidative stress at the cellular level.

[0041] CADH1, also known as cadherin-1 or E-cadherin, has the Uniprot database accession number P12830, July 1, 1993-v3. This protein is involved in mechanisms regulating cell-cell adhesion, epithelial cell motility, and proliferation. It acts as an effective invasive suppressor.

[0042] SPIT1, also known as Kunitz-type protease inhibitor 1, has the Uniprot database accession number O43278, March 15, 2005-v2. This protein is an inhibitor of HGF activators. It also acts as an inhibitor of matriptase (ST14).

[0043] ENO, also known as alpha-enolase, has the Uniprot database accession number P06733, January 23, 2007-v2. It is a multifunctional enzyme that plays a role not only in glycolysis but also in various processes such as growth control, hypoxia tolerance, and allergic responses. It can also function in the intravascular and pericellular fibrinolytic system due to its ability to function as a receptor and activator of plasminogen on the cell surface of several cell types, including leukocytes and neurons. It also stimulates immunoglobulin production.

[0044] MMP9, also known as matrix metalloproteinase-9, has Uniprot accession number P14780, 2009-11-24-v3. This protein may play an essential role in localized proteolysis of extracellular matrix and leukocyte migration. It may play a role in osteoclast resorption of bone. It cleaves KiSS1 at Gly-|-Leu bonds. It cleaves type IV and V collagens into large C-terminal 3 / 4 fragments and shorter N-terminal 1 / 4 fragments. It degrades fibronectin but not laminin or Pz-peptide.

[0045] NAMPT, also known as nicotinamide phosphoribosyltransferase, has the Uniprot database accession number P43490, November 1, 1995-v1. This enzyme is the rate-limiting component in the mammalian NAD biosynthetic pathway, catalyzing the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to produce nicotinamide mononucleotide, an intermediate in the biosynthesis of NAD.

[0046] LDHA, also known as L-lactate dehydrogenase A chain, has the Uniprot database accession number P00338, January 23, 2007-v2. This protein is involved in step 1 of the subpathway that synthesizes (S)-lactate from pyruvate.

[0047] CASP3, also known as caspase-3, has the Uniprot database accession number P42574, 11 Oct 2005-v2. It is involved in the activation cascade of caspases responsible for the execution of apoptosis.

[0048] KPYM, also known as pyruvate kinase PKM, has the Uniprot database accession number P14618, January 23, 2007-v4. It is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP and playing a general role in caspase-independent cell death of tumor cells.

[0049] PRDX1, also known as peroxiredoxin-1, has the Uniprot database accession number Q06830, June 1, 1994-v1. It is involved in cellular redox regulation.

[0050] OSTP, also known as osteopontin, has Uniprot database accession number P10451, July 1, 1989-v1. It acts as a cytokine involved in enhancing the production of interferon-gamma and interleukin-12 and reducing the production of interleukin-10, and is essential in the pathway leading to type I immunity.

[0051] PDIA1, also known as protein disulfide-isomerase, has the Uniprot database accession number P07237, November 1, 1997-v3. It catalyzes the formation, breaking, and rearrangement of disulfide bonds.

[0052] MIF, also known as macrophage migration inhibitory factor, has the Uniprot database accession number P14174, 2007-01-23-v4. It is involved in the innate immune response to bacterial pathogens.

[0053] CTNB1, also known as catenin beta-1, has the Uniprot database accession number P35222, February 1, 1994-v1. It acts as a negative regulator of centrosome cohesion and prevents anoikis of malignant renal and intestinal epithelial cells.

[0054] K2C8, also known as keratin, type II cytoskeleton 8, has Uniprot database accession number P05787, 2007-01-23-v7. Together with KRT19, it helps link the contractile apparatus and dystrophin in striated muscle costomere.

[0055] ANXA2, also known as annexin-2, has the Uniprot database accession number P07355, January 23, 2007-v2. It is a calcium-regulated membrane-binding protein whose affinity for calcium is greatly enhanced by anionic phospholipids. It binds calcium ions with high affinity and may be involved in heat stress responses.

[0056] CAPG, also known as macrophage capping protein, has the Uniprot database accession number P40121, 2010-11-30-v2. It is a calcium-sensitive protein that reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. It may play an important role in macrophage function.

[0057] FABP5, also known as fatty acid binding protein (epidermis), has the Uniprot database accession number Q01469, January 23, 2007-v3. It shows high specificity for fatty acids and may be involved in keratinocyte differentiation.

[0058] MUC1, also known as mucin-1, has the Uniprot database accession number P15941, May 18, 2010-v3. The alpha subunit has cell adhesion properties. It can act as both an adhesive and anti-adhesive protein, providing a protective layer to epithelial cells against bacterial and enzymatic attack.

[0059] CAYP1, also known as calcyphosine, has the Uniprot database accession number Q13938, November 1, 1997-v1. It is a calcium-binding protein that may play a role in cellular signaling events.

[0060] XPO2, also known as exportin-2, has the Uniprot database accession number P55060, March 29, 2005-v3. Among other things, this protein has been disclosed as an export receptor for importin-alpha, mediating the re-export of importin-alpha from the nucleus to the cytoplasm after import substrates (cargo), and as binding cooperatively to importin-alpha and the GTPase Ran in its active GTP-bound form.

[0061] NGAL, also known as neutrophil gelatinase-binding lipocalin, is a It has database accession number P80188, November 1, 1995-v2. It is involved in apoptosis caused by interleukin 3 (IL3) deficiency and innate immunity.

[0062] SG2A1, also known as mammaglobin-B, has the Uniprot database accession number O75556, November 1, 1998-v1. It can bind androgens and other steroids.

[0063] ANXA1, also known as annexin-1, has the Uniprot database accession number P04083, v2, January 23, 2007. It has been disclosed to play an important role in the innate immune response, modulate inflammatory processes, have anti-inflammatory activity, and promote inflammation resolution and wound healing, among other things.

[0064] HSPB1, also known as heat shock protein beta-1, has the Uniprot database accession number P04792, 26 Sep. 2001-v2. It is involved in stress resistance and actin organization.

[0065] PIGR, also known as the polymeric immunoglobulin receptor, has the Uniprot database accession number P01833, 26 Jun 2007-v4. This receptor binds polymeric IgA and IgM at the basolateral surface of epithelial cells.

[0066] CH10, also known as the 10 kDa heat shock protein, mitochondrial, has the Uniprot database accession number P61604, January 23, 2007-v2. It, along with CPN60, is essential for mitochondrial protein biogenesis. It binds to CPN60 in the presence of Mg-ATP, inhibiting the latter's ATPase activity.

[0067] CD44, also known as the CD44 antigen, has the Uniprot database accession number P16070, October 5, 2010-v3. It mediates cell-cell and cell-matrix interactions through its affinity for HA and possibly also for other ligands such as osteopontin, collagen, and matrix metalloproteinases (MMPs).

[0068] CLIC1, also known as chloride intracellular channel protein 1, has the Uniprot database accession number O00299, January 23, 2007-v4. It inserts into membranes to form chloride ion channels. Channel activity is pH dependent. Membrane insertion appears to be redox regulated and can only occur under oxidizing conditions. It is involved in cell cycle regulation.

[0069] TPIS, also known as triosephosphate isomerase, has the Uniprot database accession number P60174, 2011-10-19-v3. This protein is involved in the pathway gluconeogenesis, which is part of carbohydrate biosynthesis.

[0070] GSTP1, also known as glutathione S-transferase P, has the Uniprot database accession number P09211, January 23, 2007-v2. It negatively regulates CDK5 activity via p25 / p35 translocation to prevent neurodegeneration.

[0071] GTR1 has the Uniprot database accession number P11166, October 3, 2006-v2. It is a facilitative glucose transporter. This isoform may be responsible for constitutive or basal glucose uptake. It has very broad substrate specificity. It is capable of transporting a wide range of aldoses, including both pentoses and hexoses.

[0072] In another embodiment of the method of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the method comprises: (a) measuring in vitro, in an isolated liquid sample obtained from the female reproductive tract, the expression level of one or more proteins selected from a first group of proteins: PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1, and the expression level of one or more proteins selected from a second group consisting of ENOA, KPYM, PDIA1, ANXA2, and FABP5; and (b) comparing the expression level of each of the proteins to be tested with a reference value, wherein overexpression of the protein is indicative of endometrial cancer or a poor prognosis.

[0073] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of two proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0074] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of three proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0075] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of four proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0076] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of five proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0077] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises the step of detecting one or more of: PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, The method includes determining the expression levels of six proteins selected from the group consisting of LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0078] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of seven proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0079] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of eight proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0080] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of nine proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0081] In one embodiment of the first aspect of the present invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of 10 proteins selected from the group consisting of PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0082] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression level of two or more of the following markers: MMP9, LDHA, KPYM, PERM, SPIT1, NAMPT, and CADH1.

[0083] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression level of one of the following markers: MMP9, LDHA; MMP9, KPYM; MMP9, PERM; MMP9, SPIT1; MMP9, NAMPT; LDHA, KPYM; LDHA, PERM; LDHA, SPIT1; LDHA, NAMPT; KPYM, PERM; KPYM, SPIT1; KPYM, NAMPT; PERM, SPIT1; PERM, NAMPT; and SPIT1, NAMPT.

[0084] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression level of one of the following group of markers: MMP9, LDHA, KPYM; MMP9, LDHA, PERM; MMP9, LDHA, SPIT1; MMP9, LDHA, NAMPT; MMP9, KPYM, PERM; MMP9, KPYM, SPIT1; MMP9, KPYM, NAMPT; MMP9, PER M, SPIT1;MMP9, PERM, NAMPT;MMP9, SPIT1, NAMPT;LDHA, KPYM, PERM;LDHA, KPYM, SPIT1;LDHA, KPYM, NAMPT;LDHA, PERM, SPIT 1; LDHA, PERM, NAMPT; LDHA, SPIT1, NAMPT; KPYM, PERM, SPIT1; KPYM, PERM, NAMPT; KPYM, SPIT1, NAMPT; and PERM, SPIT1, NAMPT.

[0085] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression level of one of the following group of markers: MMP9, LDHA, KPYM, PERM; MMP9, LDHA, KPYM, SPIT1; MMP9, LDHA, KPYM, NAMPT; MMP9, LDHA, PERM, SPIT1; MMP9, LDHA, PERM, NAMPT; MMP9, LDHA, SPI T1, NAMPT;MMP9, KPYM, PERM, SPIT1;MMP9, KPYM, PERM, NAMPT;MMP9, KPYM, SPIT1, NAMPT;MMP9, PERM, SPIT1, NAMPT;LDHA, KP YM, PERM, SPIT1; LDHA, KPYM, PERM, NAMPT; LDHA, KPYM, SPIT1, NAMPT; LDHA, PERM, SPIT1, NAMPT; and KPYM, PERM, SPIT1, NAMPT.

[0086] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression level of one of the following groups of markers: MMP9, LDHA, KPYM, PERM, SPIT1; MMP9, LDHA, KPYM, PERM, NAMPT; MMP9, LDHA, KPYM, SPIT1, NAMPT; MMP9, LDHA, PERM, SPIT1, NAMPT; MMP9, KPYM, PERM, SPIT1, NAMPT; and LDHA, KPYM, PERM, SPIT1, NAMPT.

[0087] In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the expression levels of MMP9, LDHA, KPYM, PERM, SPIT1, NAMPT, and CADH1.

[0088] As reported below, when the expression level of MMP9 is determined in uterine aspirates, accurate diagnostic / prognostic information (AUC values ​​of approximately 0.89-0.90) is achieved.

[0089] In an attempt to improve the robustness of MMP9 as an EC biomarker, the inventors surprisingly found that when MMP9 detection was combined with detection of one or more of the following proteins: KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2, a substantial improvement in sensitivity was achieved, reaching AUC values ​​of up to about 0.96. This finding was surprising because when MMP9 was combined with other proteins, the AUC resulting from the combination was either unaffected or worsened compared to that provided by MMP9 alone.

[0090] Accordingly, in one embodiment of any of the methods and uses provided by the present invention above or below In this state, the amount of MMP9 and one or more proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0091] In another embodiment of any of the methods and uses provided by the present invention above or below, the amount of MMP9 associated with one protein selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0092] In another embodiment of any of the methods and uses provided by the present invention above or below, the amount of MMP9 associated with two proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0093] In another embodiment of any of the methods provided by the invention, above or below, the amount of MMP9 associated with three proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0094] In another embodiment of any of the methods provided by the invention, above or below, the amount of MMP9 associated with four proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0095] In another embodiment of any of the methods provided by the invention, above or below, the amount of MMP9 associated with five proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2 is determined.

[0096] In any of the embodiments provided above or below, for any of the aspects of the invention, expression levels are determined at the protein level. In this embodiment, protein markers include, but are not limited to, native sequence polypeptides, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of the marker, including modified forms of the polypeptide and derivatives thereof.

[0097] In any of the embodiments provided above or below, the expression level is determined by immunochemistry.

[0098] As used herein, the term "immunochemistry" refers to a variety of techniques for detecting antigens of interest (usually proteins and peptides, in this case any of the proteins listed above, alone or in combination) in a sample by utilizing the principle of antibodies specifically binding to the antigen. Visualizing the antibody-antigen interaction can be achieved in several ways. In the most common example, the antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction. Alternatively, the antibody can be conjugated to an enzyme such as fluorescein or rhodamine. Antigens can also be tagged with fluorophores such as fluorescein. Immunochemistry techniques can be direct or indirect. Direct methods are single-step staining methods that involve a labeled antibody (e.g., FITC-conjugated antiserum) that reacts directly with the antigen. While this technique uses only one antibody and is therefore simple and rapid, it is less sensitive due to the lack of signal amplification found in indirect methods and is therefore less commonly used. Indirect methods involve an unlabeled primary antibody (first layer) that binds to the target antigen in the sample and a labeled secondary antibody (second layer) that reacts with the primary antibody. This method is more sensitive than direct detection methods when the secondary antibody is conjugated to a fluorescent or enzyme reporter, due to the signal amplification resulting from the binding of several secondary antibodies to each primary antibody.

[0099] Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules that can recruit avidin, streptavidin, or neutravidin enzyme complexes. In addition to their high sensitivity, indirect methods also have the advantage of requiring the production of relatively few standard conjugated (labeled) secondary antibodies. Direct methods require labeling each primary antibody for every antigen of interest. It should be noted that specific nucleic acid sequences can also be detected using immunochemical techniques, provided that tagged nucleic acid probes (designed to specifically bind to specific target nucleic acid sequences) can subsequently be detected with labeled antibodies. Thus, protein detection can be achieved by using tagged nucleic acids designed to bind to specific sequences in the target protein RNA, followed by detection of the tagged nucleic acid with a labeled antibody that selectively binds to the tag.

[0100] Suitable immunoassay procedures include enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography, or other immunoassay formats known to those skilled in the art.

[0101] In one embodiment, in combination with any of the embodiments provided above or below, the expression level of the protein is determined by immunoassay.

[0102] In another embodiment, in combination with any of the embodiments provided above or below, the expression level of the protein is determined by ELISA.

[0103] Alternatively, the expression level of a protein can be determined by bioluminescence, fluorescence, chemiluminescence, electrochemistry, or mass spectrometry.

[0104] In another embodiment, in combination with any of the embodiments provided above or below, the expression level of the protein is determined using an antibody or fragment thereof capable of binding to the target protein.

[0105] The term "antibody or fragment thereof capable of binding to a target protein" should be understood as any immunoglobulin or fragment thereof capable of selectively binding to a target protein. This includes monoclonal and polyclonal antibodies. The term "fragment thereof" encompasses any portion of an antibody having a size and conformation suitable for binding to an epitope of the target protein. Suitable fragments include F(ab), F(ab'), and Fv. An "epitope" is the part of an antigen that is recognized by the immune system (B cells, T cells, or antibodies).

[0106] The antibodies used for specific detection can be polyclonal or monoclonal. There are well-known means in the state of the art for preparing and characterizing antibodies. Methods for generating polyclonal antibodies are well known in the prior art. Briefly, an animal is immunized for a protein. Polyclonal antibodies are prepared by immunizing an animal with the antigen, followed by collection of serum from the immunized animal and isolation of the antibodies. A wide range of animal species can be used for the production of antisera. Typically, animals used for the production of antisera can be rabbits, mice, rats, hamsters, guinea pigs, or goats.

[0107] Additionally, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, this involves immunizing a suitable animal with a protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody-producing cells. Methods for preparing monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies. An immunogen is injected into an animal as an antigen. The antigen can be mixed with an adjuvant, such as complete or incomplete Freund's adjuvant. Immunizations are repeated with the same antigen at approximately two-week intervals.

[0108] In another particular embodiment of the third aspect, the means for carrying out the invention form part of a kit. An antibody or fragment thereof for detecting the target protein can be included in the kit. The kit can further comprise means (additives, solvents) for visualizing antibody-protein interactions.

[0109] These antibodies can be used as a "means" for determining the expression of the target protein in the fifth aspect of the present invention.

[0110] Also, all of the embodiments provided above in the first aspect of the invention with respect to the proteins to be analyzed (sets of proteins comprising 2 to 10 of the list and 2, 3, 4, 5, or 6 specific markers) are specific embodiments of the use of the third aspect of the invention.

[0111] Alternatively, the expression level is determined at the mRNA level.

[0112] In one embodiment, the amount of mRNA of each one of the markers is detected, for example, by polymerase chain reaction using oligonucleotide primers that hybridize to one or more polynucleotide endometrial cancer markers or complements of such polynucleotides. In other embodiments, the amount of mRNA is detected using hybridization techniques using oligonucleotide probes that hybridize to one or more polynucleotide endometrial cancer markers or complements of such polynucleotides.

[0113] When mRNA detection is used, the method can be carried out by combining isolated mRNA with reagents to convert it to cDNA according to standard methods known in the art, treating the converted cDNA in a container with an amplification reaction reagent (such as a cDNA PCR reaction reagent) along with an appropriate mixture of nucleic acid primers, reacting the contents of the container to produce an amplification product, and analyzing the amplification product to detect the presence of one or more polynucleotide endometrial cancer markers in the sample. For mRNA, the analyzing step can be carried out using Northern blot analysis to detect the presence of polynucleotide endometrial cancer markers in the sample. The analyzing step can be further accomplished by quantitatively detecting the presence of polynucleotide endometrial cancer markers in the amplification product and comparing the amount of detected marker to a panel of expected values ​​for the known presence or absence of such markers in normal and malignant tissues obtained using similar primers.

[0114] In another embodiment, the present invention provides a method for detecting endometrial cancer by (a) isolating mRNA from a sample and converting the mRNA into cDNA in combination with a reagent; (b) treating the converted cDNA with an amplification reaction reagent and a polynucleotide endometrial cancer marker, a nucleic acid primer that hybridizes to one or more of the endometrial cancer markers to produce an amplification product; and (c) detecting a protein endometrial cancer marker. (d) analyzing the amplified product to determine the amount of mRNA present encoding the marker; and (d) comparing the amount of mRNA determined to the amount detected for a panel of expected values ​​for normal and diseased tissues (e.g., malignant tissues) obtained using similar methods.

[0115] In certain embodiments of the invention, RT-PCR can be used to amplify the mRNA of protein endometrial cancer markers for detection and analysis. Other embodiments of the invention use quantitative RT-PCR to quantitatively determine the amount of mRNA of protein endometrial cancer markers. Further embodiments of the invention use real-time RT-PCR for quantification and analysis.

[0116] In a fourth aspect, the present invention provides a kit.

[0117] In one embodiment of the fourth aspect of the present invention, the means for measuring the expression level is an antibody or a fragment thereof that specifically binds to the target protein.

[0118] The number of specific antibodies or fragments thereof included in the kit depends on the number of proteins to be detected. In this regard, the previous embodiments of the method of the first aspect of the present invention provide several sets of proteins to be determined for proper diagnosis or prognosis of endometrial cancer, and these protein sets include 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins. Starting from this information, a person skilled in the art can select one of the aforementioned sets for the detection of each protein and select a more suitable antibody or fragment thereof from those already available. Incorporation of the selected antibody on a suitable solid support can be carried out using routine methods.

[0119] In one embodiment of the fourth aspect, optionally in combination with any of the embodiments provided above or below, the kit comprises means for detecting the expression level of two or more proteins selected from MMP9, LDHA, KPYM, PERM, SPIT1, NAMPT, and CADH1.

[0120] In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises means for determining the expression level of one of the following markers: MMP9, LDHA; MMP9, KPYM; MMP9, PERM; MMP9, SPIT1; MMP9, NAMPT; LDHA, KPYM; LDHA, PERM; LDHA, SPIT1; LDHA, NAMPT; KPYM, PERM; KPYM, SPIT1; KPYM, NAMPT; PERM, SPIT1; PERM, NAMPT; SPIT1, NAMPT; MMP9, GSTP1; MMP9, HSPB1; and MMP9, CH10.

[0121] In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises means for determining the expression level of one of the following markers: MMP9, LDHA, KPYM; MMP9, LDHA, PERM; MMP9, LDHA, SPIT1; MMP9, LDHA, NAMPT; MMP9, KPYM, PERM; MMP9, KPYM, SPIT1; MMP9, KPYM, NAMPT; MMP9, PE RM, SPIT1;MMP9, PERM, NAMPT;MMP9, SPIT1, NAMPT;LDHA, KPYM, PERM;LDHA, KPYM, SPIT1;LDHA, KPYM, NAMPT;LDHA, PERM, SPIT 1; LDHA, PERM, NAMPT; LDHA, SPIT1, NAMPT; KPYM, PERM, SPIT1; KPYM, PERM, NAMPT; KPYM, SPIT1, NAMPT; and PERM, SPIT1, NAMPT.

[0122] In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises means for determining the expression level of one of the following markers: MMP9, LDHA, KPYM, PERM; MMP9, LDHA, KPYM, SPIT1; MMP9, LDHA, KPYM, NAMPT; MMP9, LDHA, PERM, SPIT1; MMP9, LDHA, PERM, NAMPT; MMP9, LDHA, SP IT1, NAMPT;MMP9, KPYM, PERM, SPIT1;MMP9, KPYM, PERM, NAMPT;MMP9, KPYM, SPIT1, NAMPT;MMP9, PERM, SPIT1, NAMPT;LDHA, KP YM, PERM, SPIT1; LDHA, KPYM, PERM, NAMPT; LDHA, KPYM, SPIT1, NAMPT; LDHA, PERM, SPIT1, NAMPT; and KPYM, PERM, SPIT1, NAMPT.

[0123] In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprises means for determining the expression level of one of the following markers: MMP9, LDHA, KPYM, PERM, SPIT1; MMP9, LDHA, KPYM, PERM, NAMPT; MMP9, LDHA, KPYM, SPIT1, NAMPT; MMP9, LDHA, PERM, SPIT1, NAMPT; MMP9, KPYM, PERM, SPIT1, NAMPT; and LDHA, KPYM, PERM, SPIT1, NAMPT.

[0124] In another embodiment of the fourth aspect, optionally in combination with any of the embodiments provided above or below, the kit comprises means for detecting expression levels of MMP9, LDHA, KPYM, PERM, SPIT1, NAMPT, and CADH1.

[0125] In any of the embodiments of the fourth aspect of the invention provided above, the kit may optionally comprise means for detecting the expression level of one or more proteins selected from ENOA, KPYM, PDIA1, ANXA2, and FABP5.

[0126] In another embodiment, the present invention provides a kit comprising a solid support and a means for detecting the expression level of MMP9 and one or more proteins selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2.

[0127] In another embodiment, the present invention provides a kit comprising means for determining the expression level of MMP9 and one protein selected from the group consisting of KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2.

[0128] In another embodiment of the fourth aspect of the present invention, the kit is an ELISA kit. In this embodiment, the kit comprises a solid support and a means for determining the expression level of any of the group of proteins provided above. In another embodiment, the kit comprises a solid support and antibodies or fragments thereof that specifically bind to the target protein to be detected, wherein these antibodies are conjugated to a reporter molecule capable of generating a signal.

[0129] "Solid supports" include nitrocellulose membranes, glass, or polymers. The most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Solid supports can also be strips, tubes, beads, or other similar structures. The surface may be in the form of a wafer, a disc, or a microplate, or any other surface suitable for performing an immunoassay.

[0130] As used herein, a "reporter molecule" refers to a molecule whose chemical nature provides an analytically identifiable signal that allows for the detection of antigen-bound antibodies. Detection can be qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores, or radionuclide-containing molecules (i.e., radioisotopes). In enzyme immunoassays, enzymes are generally conjugated to the second antibody using glutaraldehyde or periodate. However, as will be readily recognized, a variety of different conjugation techniques are readily available to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase, among others. Substrates used with particular enzymes are generally selected to produce a detectable color change upon hydrolysis by the corresponding enzyme. For example, 5-bromo-4-chloro-3-indolylphosphate / nitroblue tetrazolium is suitable for use with alkaline phosphatase conjugates. For peroxidase conjugates, 1,2-phenylenediamine, 5-aminosalicylic acid, 3,3:5,5:tetramethylbenzidine, or tolidine are commonly used. It is also possible to use fluorogenic substrates that yield fluorescent products rather than the chromogenic substrates mentioned above. Examples of fluorogenic substrates are fluorescein and rhodamine. When activated by irradiation with light of a specific wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing an excited state in the molecule, which then emits light of a characteristic color that is visually detectable with a light microscope. Immunofluorescence and EIA techniques are both well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent, bioluminescent molecules, and / or dyes and other chromogenic substances, can also be used.

[0131] The choice of a particular reporter molecule-conjugated antibody will be determined in large part by the intended use and user of the test kit of the invention.

[0132] In another embodiment, the kit is a microarray.

[0133] In another embodiment, the kit is a microarray containing a defined group of genes encoding protein endometrial cancer markers. All of the embodiments provided above for specific sets of proteins having 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins whose expression is significantly altered by endometrial disease are also specific embodiments of microarrays.

[0134] The in vitro methods of the invention provide diagnostic and prognostic information. In one embodiment, the methods of the invention further comprise the steps of (i) collecting the diagnostic or prognostic information and (ii) storing the information on a data carrier.

[0135] In the sense of the present invention, a "data carrier" should be understood as any means containing data of meaningful information for the diagnosis or prognosis of endometrial cancer, such as paper. The carrier may also be any entity or device capable of transferring the prognostic data. For example, the carrier may comprise a storage medium, such as a CD-ROM or a semiconductor ROM, or a magnetic recording medium, such as a floppy disk or a hard disk. Furthermore, the carrier may be a transmissible carrier, such as an electrical or optical signal, which may be transmitted via an electrical or optical cable or by wireless or other means. If the prognostic data is embodied in a signal that can be directly transmitted by a cable or other device or means, the carrier may be constituted by such a cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carriers are paper, a CD, a USB, a computer archive in a PC, or an audio recording containing the same information.

[0136] Throughout the specification and claims, the word "comprise" and variations of that word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" and variations thereof encompass the term "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided for illustrative purposes and are not intended to limit the invention. Furthermore, the present invention encompasses all possible combinations of the specific preferred embodiments described herein. [Example]

[0137] Example 1

[0138] reagent Albumin and IgG Depletion SpinTrap columns were purchased from GE Healthcare (catalog number 28-9480-20). Lys C endoproteinase MS grade was purchased from Thermo Scientific (catalog number 90051). Solid-phase extraction cartridges, Sep, were purchased from Waters. Pak tC18, 50 mg, was obtained (catalog number WAT054960). All other reagents were obtained from Sigma-Aldrich.

[0139] Patients and specimen collection A total of 38 patients (20 women suffering from EC and 18 non-EC controls, i.e., women with EC symptoms but not diagnosed with EC) participated in this prospective study at the Vall d'Hebron University Hospital (Barcelona, ​​Spain) between 2012 and 2015. All patients signed an informed consent form approved by the Vall d'Hebron Ethics Committee (approval number: PR_AMI_50-2012).

[0140] Uterine fluid samples were collected by aspiration using a Cornier Pipette (Eurogine Ref. 03040200) in the clinician's office or operating room prior to surgery and transferred to a 1.5 ml microtube. Phosphate-buffered saline was added at a 1:1 (v / v) ratio, and the soluble fraction (supernatant) was separated from the solid fraction (pellet) by centrifugation at 2500 rcf for 20 minutes. The separated fractions were kept at -80°C until use.

[0141] Preparation of samples for validation studies Uterine aspirate supernatants from 20 EC patients and 18 non-EC controls were sonicated to disrupt cryptic microvesicles, protein aggregates, and / or mucus for 5 cycles at 100% amplitude for 5 seconds (Labsonic M). Albumin and immunoglobulin G were then depleted from 50 μl supernatant samples using an Albumin & IgG Depletion Spin Trap kit according to the manufacturer's instructions. Total protein concentration was measured by Bradford assay, performed in triplicate. Each of the 38 samples was then separated into two 25 μg aliquots to generate replicates for the entire process, except for one sample with insufficient material for replication. Samples were diluted to a final volume of 120 μl in 50 mM ammonium bicarbonate and denatured by adding 185 μl of 10 M urea suspended in 50 mM ammonium bicarbonate. The samples were incubated at 22°C for 20 minutes with agitation, followed by 10 minutes of incubation in a Branson 5510 sonicator. The samples were then reduced with 7.8 μl of 200 mM dithiothreitol for 60 min at 37°C and alkylated with 12.2 μl of 400 mM iodoacetamide for 30 min in the dark at 22°C. The samples were digested with Lys C (protease / total protein ratio 1 / 150; w / w) for 4 hours at 37°C. The urea concentration was then diluted to 1 M with 50 mM ammonium bicarbonate buffer, and the samples were incubated overnight at 37°C with trypsin (protease / total protein ratio 1 / 50; w / w). Trypsin activity was quenched by adding 1 μl of anhydrous formic acid per 100 μl of solution. The samples were spiked with a mixture of synthetic peptides and then desalted onto solid-phase extraction cartridges (Sep Pak tC18, 50 mg, Waters). The eluate was then evaporated to dryness in a vacuum centrifuge and resuspended in 0.1% formic acid before LC-PCR analysis.

[0142] LC-MS / MS PRM configuration The LC-MS setup consisted of a Dionex Ultimate 3000 RS LC chromatography system configured for high-pressure binary gradient and operated in column-switching mode. Mobile phase A consisted of 0.1% formic acid in water, phase B consisted of 0.1% formic acid in acetonitrile, and the loading phase consisted of 0.05% trifluoroacetic acid in water and 1% acetonitrile. The equivalent of 250 ng of each digested sample was injected and loaded onto a trap column (75 μm x 2 cm, C18 PepMap 100, 3 μm) at 5 μL / min. The analytical column (75 μm x 15 cm, C18 PepMap 100, 2 μm) was further eluted at 300 nL / min with a linear gradient starting from 2% A to 35% B over 48 min. MS analysis was performed on a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) operated in PRM mode. The MS cycle began with a full MS1 scan performed at a resolving power of 70,000 (200 m / z) followed by a time-scheduled targeted PRM scan acquired at a resolving power of 35,000 (200 m / z) with a standardized collision energy of 20. The quadrupole isolation window for PRM events was set to 1 m / z unit, and the duration of the scheduled time window for each pair of endogenous and isotopically labeled peptides was set to 2 min.

[0143] statistical analysis All analyses were performed using SPSS version 20.0 (IBM, USA) and Graph Pad Prism v.6.0 (GraphPad Software, CA, USA). The averaged mild / severe area ratio was calculated between replicates. Linear correlations between peptides of the same protein signature were calculated using the Pearson correlation coefficient. Due to non-normally distributed datasets, as assessed by the Kolmogorov-Smirnova test and the Shapiro-Wilk test, comparisons of the expression of monitored peptides between tumor and control samples were evaluated by the nonparametric Mann-Whitney U test. A P value below 0.05 with a fold change of more than 3 was considered statistically significant. Receiver operating characteristic (ROC) curves were used to calculate the relationship between sensitivity and specificity for EC versus non-EC control groups, thereby assessing the diagnostic ability of each biomarker candidate.

[0144] result We compared the expression of each candidate biomarker between 20 EC patients and 18 non-EC controls. Importantly, both patients and controls were postmenopausal women with abnormal vaginal bleeding, and although 93% of patients with EC exhibited these clinical features, only 15% of them would ultimately be diagnosed with EC.

[0145] Based on the Bradford assay, 250 ng of total protein concentration after albumin and IgG depletion was injected for each sample. Integration of the total ion chromatograms of MS1 scans further confirmed the consistent amount of protein injected between samples. After MS data curation, the relative levels (light / heavy ratio) of 98 monitored peptides in MS2 were analyzed using the Mann-Whitney test for comparison between tumor and control samples. Fifty-eight peptides corresponding to 32 proteins showed significant differences between the two groups with p values ​​<0.05 and fold changes >1.5: PERM, CADH1, SPIT1, ENOA, MMP9, LDHA, CASP3, KPYM, PRDX1, OSTP, PDIA1, NAMPT, MIF, CTNB1, K2C8, ANXA2, CAPG, FABP5, MUC1, CAYP1, XPO2, NGAL, SG2A1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, and GTR1. All of these proteins were overexpressed in tumor samples compared with control samples.

[0146] To further evaluate their utility as biomarkers for diagnosing EC, receiver operating characteristic (ROC) analyses were performed to determine the sensitivity and specificity of each biomarker. Interestingly, all proteins achieved excellent area under the curve (AUC) values ​​for defining an increased likelihood of EC in minimally invasive uterine aspirates, ranging from 0.71 to 0.95. The 10 best-performing individual proteins were PERM, CADH1, SPIT1, ENOA, MMP9, LDHA, CASP3, KPYM isoform M1-M2, PRDX1, and OSTP isoform A, all with AUC values ​​greater than 0.9.

[0147] [Table 1]

[0148] These results allow us to conclude that these proteins exhibit very high sensitivity and specificity in isolated fluid samples obtained from the female reproductive tract.

[0149] Furthermore, we will further elucidate the origin of these proteins in relation to cancer and their subcellular location. To better understand these biomarkers, we also conducted a bioinformatics analysis using Ingenuity Pathway Analysis (IPA). As expected, data integration led to the identification of cancer, inflammatory diseases, organismal injuries and abnormalities, and diseases of the reproductive system as major diseases associated with these biomarkers. Five major molecular and cellular functions associated with these proteins include cell motility, cell death and survival, cell development, cell proliferation, and cell-cell signaling and interactions, all of which are important processes altered in cancer. These proteins were found primarily in the cytoplasm, plasma membrane, and extracellular space, indicating that they originate from the secretions of endometrial epithelial and inflammatory cells or from cell necrosis in proximal tissues. These characteristics were important for facilitating the use of biomarkers to diagnose EC in proximal fluids associated with the female reproductive tract.

[0150] Example 2

[0151] The diagnostic potential of several proteins was evaluated in uterine aspirate samples from 116 women by liquid chromatography with mass spectrometry detection using parallel reaction monitoring acquisition (LC-PRM) as previously described.

[0152] Uterine aspirate processing included collection by aspiration using specialized equipment and dilution into tubes with PBS1x saline solution at a 1:1 (v / v) ratio. The liquid fraction was then separated from the cellular fraction by centrifugation at 2,500 x g for 20 minutes. Protein biomarkers were assessed using the supernatant of the uterine aspirate.

[0153] Of the 116 women, 69 were diagnosed with EC, including 49 with endometrioid EC (EEC) and 20 with non-endometrioid serous EC (SEC). The remaining 47 women were non-EC women diagnosed with conventional endometriosis or benign disease.

[0154] To obtain significant EC biomarkers, the expression of each marker, measured as the light / heavy area ratio obtained in the LC-PRM study (the configuration of the LC-PRM was the same as in Example 1), was compared between the tumor group (n=69) and the non-EC group (n=47) using the nonparametric Mann-Whitney U test. An adjusted p-value of less than 0.05 was considered statistically significant. Receiver operating characteristic (ROC) analysis was used to evaluate the specificity and sensitivity of the biomarkers, and the area under the ROC curve (AUC) was evaluated for each protein. The results are summarized in Table 2 below.

[0155] [Table 2]

[0156] From a more robust biomarker, we turned our attention to improving the diagnostic information provided by MMP9. To this end, logistic regression models were fitted to the data to evaluate the power of different combinations of proteins to classify samples in two clinical categories (cancer and control). ROC curves were generated for each of these regression models. AUC, sensitivity, and specificity at the "optimal" cutoff point for discrimination between groups were obtained. The optimal cutoff corresponded to the threshold that maximized the distance to identity (diagonal line). The criterion for optimality was maximum (sensitivity + specificity). 95% confidence intervals (CI) of the AUC were calculated using the Delong method (20). 95% CIs of the sensitivity and specificity values ​​were calculated using the averaging method and bootstrap resampling described by Fawcett (Fawcett T., "An Introduction to ROC Analysis"). Analysis”, Pattern Recognition Lett. 2006, v. 27, pages 861-874. All ROC analyses were performed using the R “pROC” package (Robin X. et al., “pROC: an open-source "Package for R and S+ to analyze and compare ROC curves," BMC Bioinformatics, 2011, v.12, page 77. To assess the robustness of each protein panel, a "leave-one-out" cross-validation procedure was performed by applying a logistic regression model adjusted for the remaining samples in the dataset to each sample in the dataset, thereby obtaining new ROC curves, after which conventional ROC analysis was performed. In a similar manner, the discriminatory ability of the diagnostic protein panel was further validated by applying a logistic regression model adjusted for the initial set (Cohort 1: the cohort in Example 2) to each sample in an independent set of samples (Cohort 2: the cohort in Example 1), thereby obtaining new ROC curves, after which conventional ROC analysis was performed.

[0157] Thus, MMP9 biomarker values ​​were found to be significantly improved when assessed in combination with KPYM, ENOA, PRDX1, MIF, GSTP1, CAPG, CADH1, HSPB1, PDIA1, LDHA, CLIC1, CASP3, FABP5, TPIS, LDHA, CTNB1, CH10, NAMPT, and ANXA2, in contrast to other proteins that, when combined with MMP9, negatively impact the EC biomarker values ​​of MMP9 alone.

[0158] A specific example of a positive combination is the combination of MMP9 and KPYM, which showed an AUC value of 0.96. This finding was surprising because the individual AUC values ​​for MMP9 and KPYM were 0.89 and 0.90, respectively, resulting in an increased AUC value for MMP9 when combined.

[0159] In contrast, the combination of MMP9 and PERM did not improve the detection accuracy of EC. In Examples 1 and 2, the individual AUC values ​​obtained for MMP9 were 0.91 and 0.89, respectively. The AUC values ​​for PERM were 0.96 and 0.86, respectively. However, the combination did not report any improvement in AUC value. The combination of MMP9 and PERM had an AUC value of 0.89.

[0160] Example 3

[0161] Detection of MMP9 and KPYM by ELISA technique. ELISA kits (R&D Systems and USCN Life Science and Technology Company, respectively) were used according to the manufacturer's protocol. For MMP9, 105 uterine aspirate samples were analyzed using dilutions of 1:10, 1:100, or 1:1000. For KPYM, due to a lack of sample material, only 39 uterine aspirate samples could be analyzed using dilutions of 1:2, 1:4, or 1:10. All samples were measured in duplicate, and the mean values ​​were reported as ng / mL. Linear correlation between the results of the LC-PRM and ELISA assays was calculated using the Pearson correlation coefficient. ELISA results were found to be highly correlated with those observed by mass spectrometry. Therefore, MMP9 and KPYM can be used in antibody-based techniques to diagnose EC.

Claims

1. A method for diagnosing endometrial cancer, comprising determining the expression level of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in a body fluid sample isolated from the female reproductive tract.

2. 1. A method for identifying a subject suspected of having endometrial cancer, said method comprising: (a) determining in vitro the expression level of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in a body fluid sample isolated from the female reproductive tract; and (b) comparing the expression level of step (a) with a reference control level; Including, If the expression level determined in step a) is higher than the reference control level, it indicates that the subject is suspected of suffering from endometrial cancer.

3. 1. A method for determining or recommending whether to initiate a medical regimen in a subject suspected of having endometrial cancer, the method comprising: determining in vitro the expression level of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in a body fluid sample isolated from the female reproductive tract; Including, A method in which, if the expression level of the protein in the sample is higher than the reference control level, endometrial cancer is diagnosed or the subject is suspected of suffering from endometrial cancer.

4. 1. A method for determining the effectiveness of a medical regimen for a subject already diagnosed with endometrial cancer, the method comprising: a) measuring in vitro the expression levels of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in a body fluid sample isolated from the female reproductive tract prior to administering the medical regimen; and b) upon initiating a medical regimen, measuring in vitro the expression level of said marker in a body fluid sample isolated from the female reproductive tract of said subject; and c) comparing the expression levels measured in step (a) and step (b), and if the expression level measured in step (b) is lower than the expression level measured in step (a), this indicates that the medical regimen is effective in treating endometrial cancer; Including, Alternatively, the method comprises: (i) upon initiating the medical regimen, measuring in vitro the expression levels of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in a body fluid sample isolated from the female genital tract of the subject; and (ii) comparing the expression level measured in step (i) with a reference control level, wherein if the expression level measured in step (i) is not higher than the reference control level, this indicates that the medical regimen is effective in treating endometrial cancer; A method comprising:

5. The method of any one of claims 1 to 4, wherein the expression level is determined at the protein level.

6. 6. The method of claim 5, wherein the expression level of a protein is determined using an antibody or a fragment thereof capable of binding to the protein.

7. The method of claim 6 , wherein the antibody or fragment thereof forms part of a kit.

8. The method of any one of claims 1 to 7, wherein the isolated sample is a uterine aspirate.

9. The method of any one of claims 1 to 8, further comprising: (i) collecting diagnostic information; and (ii) storing said data on a data medium.

10. Use of KPYM; or KPYM and one or more markers selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 as an in vitro marker for diagnosing endometrial cancer in a body fluid sample isolated from the female reproductive tract.

11. Use of a means for detecting the expression level of KPYM; or KPYM and one or more markers selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1 in the method of any one of claims 1 to 9.

12. A kit for diagnosing endometrial cancer, comprising a solid support and means for detecting the expression level of KPYM; or KPYM and one or more proteins selected from the group consisting of ANXA1, ANXA2, CAPG, CAYP1, CASP3, CD44, CADH1, XPO2, CTNB1, ENOA, FABP5, GSTP1, HSPB1, CH10, K2C8, NGAL, LDHA, MIF, MMP9, MUC1, NAMPT, PIGR, PRDX1, SG2A1, GTR1, SPIT1, OSTP, TPIS, and PDIA1.

13. The kit of claim 12, wherein the means for detecting the expression level of the protein is an antibody or a fragment thereof.

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