Endometrial cancer biomarkers

Abstract

The present invention provides methods for diagnosing and prognosing endometrial cancer in readily accessible isolated gynecological samples, particularly from fluid samples of the female reproductive tract, by detecting the expression levels of one or more proteins. The present invention also provides kits comprising means for detecting said proteins for use in diagnosing and prognosing the disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 21382680, filed July 23, 2021.

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

[0003] Endometrial cancer (EC) is the most frequently observed invasive tumor of the female genital tract and the fourth most common cancer in women in developed countries, with an estimated 66,570 cases diagnosed and 12,940 deaths in the United States in 2021. Its early diagnosis is associated with a 95% 5-year survival rate. However, if diagnosed at an advanced stage, the 5-year survival rate drops dramatically to 69% for local metastasis and 16% for distant metastasis. Currently, there are no screening tools for its early diagnosis, and the diagnostic process begins with the observation of associated symptoms, the most common of which is abnormal vaginal bleeding (AVB). Although 90% of EC patients experience AVB, this symptom is not specific for the disease, and only 9% of tested patients ultimately present with EC. The first diagnostic step is to perform a pathological evaluation of the endometrium with a pipelle biopsy. However, this procedure fails in 22% of cases, and these patients undergo additional invasive procedures, such as hysteroscopy, to be diagnosed. Approximately 7 million women experience AVB annually in Europe, initiating this diagnostic process, resulting in morbidity for the patient and placing a significant burden on the healthcare system. Therefore, improving early diagnosis is a major issue to properly manage EC and reduce the mortality associated with the disease. Differentiation between patients with benign endometrial lesions and those with EC is only achieved 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 uterine aspirate and / or piper biopsy, and is obtained by minimally invasive aspiration of endometrial fluid from within the uterine cavity. As the current diagnostic procedure for uterine aspirate relies on the presence of cellular material, this process unfortunately has a diagnostic failure and associated inadequate collection rate of 8% and 15%, respectively. This increases up to 12% and 22% in postmenopausal women. In these cases, a hysteroscopy-guided biopsy must be performed, an invasive technique that presents an increased risk of complications, including uterine perforation, bleeding and possible harm to other organs.

[0004] US Patent Nos. 5,993,311 and 5,993,323 disclose useful markers that can be determined in uterine aspirates and provide good specificity and sensitivity for the differential diagnosis of EC from other endometrial conditions. US Patent No. 5,993,323 provides a method for the prognosis of EC, making it possible to distinguish between endometrioid endometrial cancer (EEC) and non-endometrioid EC cases (NEEC), two of the subtypes of EC.

[0005] To date, many studies have been conducted to identify EC protein biomarkers, mainly in tissue and serum samples (see, for example, Non-Patent Documents 1 and 2). None of these have been translated into clinical utility.

[0006] Other publications disclosing plasma or serum samples to recover important information on EC yield controversial or contradictory conclusions. For example, in Non-Patent Document 3, midkine (MDK), a secreted heparin-binding growth factor, is proposed as a serum biomarker useful for identifying high-risk patients for EC. In Non-Patent Document 4 (see Figure 2(f)), it was identified that MDK cannot distinguish EC from healthy controls when excluding endometriosis patients from the endometrial cancer subgroup. In fact, Torres et al. conclude that while plasma markers such as TGM2 can accurately diagnose EC, other markers such as MDK may be altered in EC studies by including endometriosis cases, and this needs to be taken into account in future study designs. Finally, MDK has also been identified in tissue biopsies from the cervix, providing information on cervical cancer in the literature of Non-Patent Document 5.

[0007] In any case, it is noteworthy that some of the samples tested (tissue biopsies, plasma and serum) were non-routine gynecologic samples, and all of them (including uterine aspirates) were minimally invasive, precluding their use as readily accessible screening and / or diagnostic tools. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] European Patent No. 3452829 [Patent Document 2] European Patent Application No. 3655778 [Non-patent literature]

[0009] [Non-Patent Document 1] DeSouza LV et al., “Endometrial cancer biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry,” Mol Cell Proteomics MCP, 2007, Vol. 6, pp. 1170–8. [Non-Patent Document 2] Kemik P et al., "Diagnostic and prognostic values ​​of preoperative serum levels of YKL-40, HE-4 and DKK-3 in endometrial cancer," Gynecol Oncol, 2016, vol. 140, pp. 64-9 [Non-Patent Document 3] Tanable et al., "Midkine and its clinical significance in endometrial carcinoma," Cancer Sci, 2008, Vol. 99(6), pp. 1125-1130 [Non-Patent Document 4] Torres et al., "CD44, TGM2 and EpCAM as novel plasma markers in endometrial cancer diagnosis," BMC Cancer, 2019, Vol. 19, p. 401, https: / / doi.org / 10.1186 / s12885-019-5556-x. [Non-Patent Document 5] Moon et al., "Immunohistochemical and quantitative competitive PCR analysis of midkine and pleiotrophin expression in cervical cancer," Gynecologic Oncology, 2003, Vol. 88, pp. 289-297. Summary of the Invention [Problem to be solved by the invention]

[0010] In conclusion, despite efforts, there is still a need for reliable exclusion methods to improve the diagnosis and even prognosis of endometrial cancer and to reduce the current burden on women participating throughout the diagnostic process, as well as highly accurate biomarkers that can be easily evaluated in a clinical setting, obtained from non-invasive samples. [Means for solving the problem]

[0011] The inventors have determined that specific protein markers detectable in isolated samples obtained from methods used in the normal or routine management of gynecology provide valuable diagnostic information in endometrial cancer (EC). Proteins were first analyzed retrospectively from a cohort of 60 patients, including controls, patients with EC and cervical lesions. The set of informative proteins was then retrospectively validated in a larger cohort of 242 patients (106 non-EC, 129 EC and 7 pre-malignant lesions of EC, i.e. hyperplasia).

[0012] Furthermore, the inventors have determined that several proteins detectable in these types of samples are significant prognostic biomarkers for endometrial cancer (EC). These proteins allow for the high sensitivity and specificity of distinguishing ECs with different prognoses, including different histological subtypes and grades and different molecular features, thus minimizing the risk of false positive and false negative classification between these subtypes.

[0013] Thus, in a first aspect, the present invention relates to a method for the diagnosis and / or prognosis of EC comprising determining the presence and / or expression levels of midkine (MDK) in a sample selected from gynecological sampling including or selected from cervical mucus, cytology, Pap smear sample, endometrial biopsy, uterine fluid, uterine washings and combinations thereof from parts of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0014] MDK (MK or MDK), also known as neurite outgrowth-promoting factor 2 (NEGF2), is a protein encoded by the MDK gene in humans. Midkine is a low molecular weight basic heparin-binding growth factor that forms a family with pleiotrophin (NEGF1, 46% homologous to MK). It is a non-glycosylated protein composed of two domains held by disulfide bridges. It is a developmentally important retinoic acid-responsive gene product that is strongly induced during mid-gestation, hence the name midkine. It is mainly restricted to certain tissues in normal adults and is strongly induced during tumorigenesis, inflammation and tissue repair. The canonical amino acid sequence (isoform 1) has a length of 143 amino acids and is identified in the UniProtKB database under accession number P21741 (version of sequence from May 1, 1991 and release 189 of the UniProtKB / Swiss-Prot database from April 7, 2021).

[0015] Another aspect of the invention is the use of MDK as an in vitro marker for the diagnosis and / or prognosis of endometrial cancer in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear-like samples containing cervical mucus, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries.

[0016] In a third aspect, the invention relates to a solid support and a means for detecting the presence and / or determining the expression level of MDK and, optionally, a method for detecting the presence and / or expression level of apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), chaperone containing TCP1 subunit 6A (CCT6A), lipopolysaccharide binding protein precursor (LBP), serum amyloid. A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A) and tissue inhibitor of metalloproteinases 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B dopantase (BDOPA), and phospholipase B receptor activator (GPA) are also involved. Main containing 1 (PLBD1), CD44 antigen, IgG binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3, matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (P PL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1),and means for detecting the presence and / or determining the expression level of one or more proteins selected from the group consisting of lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), proliferation and apoptosis adaptor protein 15 (PEA15), S100 calcium-binding protein A9 (S100A9), Sciellin (SCEL, Sciellin), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG-binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2).

[0017] In other words, this embodiment can also be defined as a new kit comprising a solid support, means for detecting the presence and / or determining the expression level of MDK, and optionally means for detecting the presence and / or determining the expression level of one or more proteins selected from the group listed in Table 1 below, showing the entries (accession numbers) in the UniProtKB / Swiss-Prot database with release numbers accessible on July 23, 2021 at https: / / www.uniprot.org / help / uniprotkb:

[0018] [Table 1] JPEG2024525939000002.jpg255147TIFF2024525939000003.tif255146TIFF2024525939000004.tif236151

[0019] Indeed, also disclosed herein is the use of a kit comprising means for detecting and / or determining the expression of one or more of the above listed proteins in an indicated sample as a tool for the diagnosis and / or prognosis of EC.

[0020] Finally, another aspect of the present invention is a computer-implemented method for carrying out the in vitro method defined in the first aspect, wherein after determining the expression level of one or more of MDK and optionally proteins for diagnosis and / or prognosis of endometrial cancer, said level is given a value and / or score, and optionally calculated by a formula to obtain a calculated value, and depending on said level, score and / or calculated value, a decision is made between the options of being affected or not with EC, and / or between the options of being affected with different ECs that show different prognosis, including different histological subtypes and grades and different molecular features.In other words, a decision is made between the options of being affected or not with EC, and / or between the options of being affected with different EC subtypes, and / or between the options of being affected with different EC grades.

[0021] This aspect arises from an algorithm for carrying out any of the methods defined herein. In the sense of the present invention, the term "algorithm" is also synonymous with the combination of panels or decision diagrams, predictors and data for correctly classifying individual samples.

[0022] Also disclosed herein is a method for the diagnosis and / or prognosis of EC comprising determining the presence and / or expression level of one or more of the following proteins in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries: Midkine (MDK), apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), TCP1 subunit 6A-containing chaperone (CCT6A), lipopolysaccharide-binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A), and Tissue inhibitor of metalloproteinases 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B domain containing 1 (PLBD1), CD44 antigen, IgG binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock Hsp10 protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7),Inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), proliferation and apoptosis cis adaptor protein 15 (PEA15), S100 calcium binding protein A9 (S100A9), scyelin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2). In some examples, the method further comprises determining one or more clinical or characteristic parameters of the subject, particularly selected from the group consisting of blood pressure, blood glucose, age, grading or staging score (i.e., the Federation of Gynecology and Obstetrics (FIGO) grading / staging system), endometrial thickness, CA125 and HE4 molecular markers, AVB, and combinations thereof.

[0023] Also disclosed is a method for the diagnosis and / or prognosis of EC comprising determining the presence and / or expression level of one or more of the proteins from the group listed in Table 1 in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear-like samples, endometrial biopsy, uterine fluid, uterine washings and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0024] Also disclosed herein is a specific prognostic method for the detection of EC histology, comprising determining the presence and / or expression level of one or more of PIGR, PKM, optionally in combination with MDK or any other protein in Table 1, in a sample selected from a gynecological sample collection including cervical mucus, cytology, Pap smear sample, Pap smear-like sample, endometrial biopsy, uterine fluid, uterine washings, and combinations thereof from a portion of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries. In particular, the combination of PIGR and MDK provides an AUC value close to 0.846 for accurate determination (i.e., diagnosis) of EC histology. Notably, the combination of PIGR, RAB2, and MDK also produced an AUC value of 0.89.

[0025] Also disclosed herein is a particular prognostic method for EC grading, comprising determining the presence and / or expression level of one or more of PIGR, HSPE1, optionally in combination with MDK or any other protein in Table 1, in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsy, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries. In particular, the combination of PIGR and MDK provides an AUC value around 0.920 for accurate determination (i.e., diagnosis) of EC grade.

[0026] In a further aspect related to the diagnosis of EC, the present invention provides a method for identifying a subject suspected of having EC, comprising the steps of: a) determining in vitro the presence and / or expression level of midkine (MDK) in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, Pap smear-like samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries; b) comparing the level of MDK from step (a) with a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer and / or with a cut-off value discriminating between endometrial cancer and other gynecological disorders or conditions, wherein the subject is diagnosed with endometrial cancer if the level of MDK is within the value or interval of values ​​from subjects suffering from this cancer and / or if the level of MDK relative to the cut-off value is classified into the endometrial cancer group. The present invention provides a method comprising:

[0027] 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 the steps of: a) determining in vitro the presence and / or expression level of midkine (MDK) in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries; b) comparing the level of MDK from step (a) with a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer and / or with a cut-off value discriminating between endometrial cancer and other gynecological disorders or conditions, wherein the subject is diagnosed with endometrial cancer if the level of MDK is within the value or interval of values ​​from subjects suffering from this cancer and / or if the level of MDK relative to the cut-off value is classified into the endometrial cancer group. The present invention provides a method comprising:

[0028] 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, a follow-up may be performed, taking into account the results of the patient's examination by the physician; A method is provided.

[0029] By determining the level of the marker in a test sample, one skilled in the art can further establish the most appropriate treatment that can be recommended, since the level detected in the sample can reflect the extent (i.e., severity) of the disease. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 shows the results obtained using an ELISA assay for determining MDK, tested on samples from a cohort of subjects and comparing EC and non-EC patients (cervical samples also called cervical mucus or Pap smear-like samples). (A) Dot plot of the distribution of protein concentrations between EC and non-EC patients. (B) Curve of the ROC analysis (Receiver Operating Characteristic analysis) of MDK assessed by ELISA. [Diagram 2] Figure 1 shows the results obtained using (A) mass spectrometry (LC-MS / MS PRM) or (B) ELISA assays for determining MDK, tested on samples from a cohort of subjects and comparing EC and non-EC patients. The dot plots show the distribution of protein concentrations between EC and non-EC patients. Also shown is the ROC analysis (Receiver Operating Characteristic Analysis) of MDK assessed by either LC-MS / MS or ELISA. (C) Correlation between measurements of both techniques. [Diagram 3] Figure 1 shows the results obtained using an ELISA assay for the determination of MDK in uterine aspirates / piper biopsies / uterine fluid, tested on samples from a cohort of subjects and comparing EC and non-EC patients. (A) Dot plot of the distribution of protein concentrations between EC and non-EC patients. (B) ROC analysis (Receiver Operating Characteristic analysis) curve of MDK assessed by ELISA. Detailed Description of the Invention

[0031] (definition) 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 in this application are as set forth below, and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth provides a broader definition.

[0032] The present invention provides new biomarkers for the diagnosis and prognosis of endometrial cancer in samples selected from gynecological sampling including the female reproductive tract (i.e., including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries), cervical mucus, cytology, Pap smear samples, Pap smear-like samples including fluids, endometrial biopsies, uterine fluid, uterine washings and combinations thereof.

[0033] The term "diagnosis" is known to those skilled in the art. As used herein, "diagnosis" is understood as becoming aware of a particular medical condition, complication or risk 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 this process. Diagnosis, in the sense of a diagnostic procedure, can be considered as an attempt to classify an individual's condition into a separate and distinct category that allows medical decisions regarding treatment and prognosis to be made. A diagnostic opinion is then often written in terms of a disease or other condition. However, diagnosis can take many forms. It can be a matter of detecting and naming the presence of a disease, lesion, dysfunction or disorder. It can be a challenge to assign a category for management or prognosis. It can indicate either the degree of abnormality on a continuum or the type of abnormality within a classification. Thus, the term "diagnosis" also encompasses "screening" or "differential diagnosis" of subjects to classify them into several distinct groups, including in particular the classification of asymptomatic subjects, subjects at risk of suffering from EC, subjects already diagnosed as suffering from EC, subjects suffering from EC and exhibiting different prognoses including different histological subtypes (especially EEC vs. NEEC) and grades and different molecular features, etc. Thus, the method of the present invention is a powerful screening tool for the correct classification of all analyzed samples from a subject.

[0034] Generally speaking, the diagnostic markers listed herein are proteins (including characterization of tumors and / or some types of ECs) that are differentially detected at the expression level in isolated samples of control (non-cancer individuals) versus endometrial cancer samples.

[0035] The term "patient" (or subject) as used herein refers to any subject that typically shows one or more signs or symptoms associated with EC. The term "patient" as used herein also refers to any animal that is classified as a female mammal, including but not limited to domestic and farm female animals, primates, and humans. Preferably, the patient is a human female of any age or race.

[0036] The in vitro diagnostic, including screening, method of the first aspect of the present invention may 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.

[0037] The term "reference value" as used herein refers to a predefined standard used as a criterion for evaluating values ​​or data obtained from samples collected from a subject. The reference value or reference level can be an absolute value (i.e., a cutoff value or cutoff discrimination value), a relative value, a value with an upper or lower limit, a range of values ​​(i.e., a range of possible cutoff values), an average value, a median value, a mean value, or a value compared to a particular control or baseline value. The reference value or reference range can be based on the value of an individual sample, such as a value at an earlier time point obtained from a sample from the subject being tested. The reference value or range can be based on multiple samples, such as a population of subjects from an age-matched group over time, or on a pool of samples that includes or excludes the sample being tested. Reference values ​​were determined for the biomarkers of the present invention. The reference values ​​for proteins (i.e., MDK) can be from lower and higher values ​​as disclosed in the following examples. The range of values ​​(protein levels) of each biomarker and the specific combination of values ​​of the different biomarkers provide correct classification of subjects with high sensitivity and specificity.

[0038] When the expression level is determined at the protein level, the "reference expression level" is a predetermined value of the protein amount, and when the expression level is determined at the mRNA level, the "reference expression level" is a predetermined value of the mRNA amount. The sample is taken from a subject or a group of subjects in which the presence or absence, stage, histological subtype or grade or course of the disease has been appropriately performed before. This value is used as a threshold value to distinguish subjects in which the condition being analyzed exists from subjects in which such condition does not exist (i.e., subjects with endometrial cancer from subjects without endometrial cancer) in order to determine, among other things, the histological subtype of the disease, the risk of developing or suffering from endometrial cancer. This reference control level is also useful for determining whether the subject should start a medical regimen and how effective the regimen is. The one or more subjects from which the "reference control level" is derived may include subject(s) in which the condition does not exist, subject(s) in which the condition exists, or both. Those skilled in the art can use their general knowledge to more appropriately select a 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 group of selected 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, "reference control level" is a cut-off value defined by conventional ROC analysis (Receiver Operating Characteristic Analysis). As the skilled artisan will understand, the optimal cut-off value is defined 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] "Prognosis" as used herein refers to the prediction of the likely progression and outcome of disease.This includes neoplasia grading (an attempt to express in replicable terms the level of cell differentiation in neoplasia, since increased anaplasia correlates with the aggressiveness of neoplasia), neoplasia staging (an attempt to express in replicable terms the extent of neoplasia in patients), neoplasia histological subtype, and neoplasia molecular subtype.As used herein, prognosis, in certain embodiments, refers to the distinction between endometrioid and non-endometrioid endometrial cancer, or between low and high histological grade endometrial cancer, or between molecular subtypes of endometrial cancer, or between patients with high or low risk of recurrence.

[0040] As mentioned above, a first aspect of the present invention is a method for the diagnosis and / or prognosis of endometrial cancer comprising determining the presence and / or expression level of MDK in an isolated sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings and combinations thereof from parts of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0041] Samples from the female reproductive tract, from the vulva to the cervix, are obtained from common, routine examinations typically performed once a year by a gynecologist.

[0042] In a specific embodiment of the first aspect, the sample is a Pap smear-like sample, specifically the fluid contained in the Pap smear and / or cervical mucus.In other words, the sample is a cervical sample, specifically the fluid contained in the cervical sample.This cervical mucus can be easily obtained by using a cytobrush, similar to that used for cytological analysis in routine gynecological examination.

[0043] In another specific embodiment, the sample is uterine aspirate (also called piper biopsy or uterine fluid).This is obtained from a common and routine gynecological examination used to determine the presence of carcinoma in the gynecological tract, and is generally known as a sample that is less traumatic and uncomfortable than histoscopy or endometrial curettage biopsy.Importantly, even in this type of sample, MDK can distinguish between EC and non-EC.

[0044] This is the first time that MDK has been detected as informative in this type of routine sample.Thus, the present invention relates to the use of MDK as a diagnostic marker in samples selected from gynecological sampling including cervical mucus, cytology, Pap smear-like samples, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof from female genital tract parts including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries.In particular, the present invention relates to the use of MDK as a diagnostic and / or prognostic marker for EC.

[0045] As described in the Examples section, when MDK is determined, high sensitivity and specificity is achieved at certain levels of expression in any sample from this part of the female genital tract (e.g., AUC=0.910, SE=82.8, SP=87.8). When expression levels of additional markers were also determined in samples isolated from these genital tract structures (i.e., fluids from the cervix in particular), specificity was maintained while sensitivity was increased, allowing classification of most of the true positive patients.

[0046] Thus, in another particular embodiment of the method of the first aspect, the method further comprises determining the presence and / or expression level of one or more of the other proteins of Table 1. In another more particular embodiment, the method further comprises determining the presence and / or expression level of one or more of the following proteins: Apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), TCP1 subunit 6A-containing chaperone (CCT6A), lipopolysaccharide-binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acylate transferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A) and tissue inhibitor of metalloproteinase 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B domain-containing 1 (PLBD1), CD44 antigen, IgG-binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8 L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), proliferation and apoptosis adaptor protein 15 (PEA15),S100 calcium-binding protein A9 (S100A9), Scielin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG-binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2).

[0047] In more particular embodiments, the method comprises determining the presence and / or expression levels of 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the proteins in the panel of proteins that always include MDK. Yet another particular embodiment of the in vitro method comprises determining the presence and / or expression levels of 2, 3 or 4 of the proteins in the panel of proteins that always include MDK.

[0048] In a more particular embodiment of the method, the method comprises determining in the isolated sample the presence and / or expression level of a protein in at least one binary set of the groups listed in any one of Tables 4 and 6.

[0049] In a more particular embodiment of the method, the method comprises determining in the isolated sample the presence and / or expression level of proteins in at least one of the binary sets of the group consisting of MDK, LCN2; MDK, FN1; MDK, PLBD1; MDK, APOB; MDK, CD44; MDK, FCGBP; MDK, EPS8L1; MDK, ANXA3; MDK, C4BPA; MDK, MMP8; MDK, NEDD-8; MDK, CAMP; MDK, TIMP2; MDK, HSPE1; MDK, CALU; MDK, C1QA; MDK, LDHA; MDK, TIMP2; MDK, PIGR; MDK, KRT8; MDK, PPL; MDK, SPRR1A; MDK, STMN1; MDK, CAPS; MDK, CA1; MDK, CCT6A; MDK, VIM; MDK, TCP1; and MDK, AGRN.

[0050] In a more particular embodiment of the method, the method comprises determining in the isolated sample the presence and / or expression level of a protein in at least one set of triplets of the groups listed in any one of Tables 5 and 7.

[0051] In another specific embodiment, the method comprises the steps of: in the isolated sample, MDK, ANXA7, CSE1L; MDK, LCN2, FGFBP1; MDK, LCN2, ANXA7; MDK, FGFBP1, LCN2; MDK, APOF, FCGBP; MDK, IMPA1, FCGBP; MDK, LCN2, CD44; MDK, STX7, FCGBP; MDK, ITIH2, FCGBP; MDK, LCN2, EPS8L1; MDK, FGFBP, EPS8L1; MDK, ANXA7, FCGBP; MDK, LCN2, PLBD1; MDK, FCGBP , CD44;MDK, PLBD1, FCGBP;MDK, ANXA3, FCGBP;MDK, PLBD1, LGALS1;MDK, LCN2, IMPA1;MDK, FCGBP, VIM;MDK, FCGBP, LMNB1;MDK, PLBD1, ATP6V1G1; MDK, APOB, PKM;MDK, ITIH2, LCN2;MDK, PLBD1, CALU;MDK, LCN2, APOF;MDK, PLBD1, EPS8L1;MDK, APOC1, ANXA7;MDK, FCGBP, GYG1;MDK, PLBD1, PKM;M DK, FCGBP, LSP1; MDK, TIMP2, CD44; MDK, ANXA3, PLBD1; MDK, PLBD1, HSPE1; MDK, PLBD1, CA1; MDK, FCGBP, HCLS1; MDK, STX7, IMPA1; MDK, ANXA7, STX 7;MDK, STX7, EPS8L1;MDK, PLBD1, LSP1;MDK, C1R, ANXA7;MDK, STX7, PEA15;MDK, PLBD1, HCLS1;MDK, LCAT, FCGBP;MDK, PLBD1, LDHA;MDK, APOB, S10 0A9;MDK, ANXA7, SCEL;MDK, SERPINA3, FCGBP;MDK, FCGBP, ITGB2;MDK, SCEL, ANXA3;MDK, S100A9, PLBD1;MDK, S100A9, LSP1;MDK, C1R, FCGBP;MDK , FGFBP1, FCGBP; MDK, APOC1, S100A9; MDK, PPL, PLBD1; MDK, TCP1, PEA15; MDK, ANXA3, LSP1; MDK, PPL, IMPA1; MDK, S100A9, MMP8; MDK, FGFBP1, PPL;and determining the presence and / or expression level of protein in at least one triplet set of the group consisting of MDK, S100A9, PPL;

[0052] In a more particular embodiment, the method comprises determining the presence and / or expression level of a set defined by MDK, ANXA7 and CSE1L in the isolated sample. In yet another specific embodiment, the method comprises the steps of: in the isolated sample, MDK, LCN2, PEA15, LDHA; MDK, LCN2, PEA15, PKM; MDK, FGFBP1, FCGBP, EPS8L1; MDK, LCN2, HSPE1, CD44; MDK, FGFBP1, EPS8L1, CTNNB1; MDK, LCN2, IMPA1, LDHA; MDK, IMPA1, FCGBP, PEA15; MDK, IMPA1, FCGBP, PKM; MDK, FCGBP, IMPA1, PEA15; MDK, CCT6A, FGFBP1, EPS8L1; MDK , STX7, FCGBP, PKM; MDK, IMPA1, FCGBP, GYG1; MDK, FGFBP1, SCEL, EPS8L1; MDK, STX7, FCGBP, CA1; MDK, STX7, FCGBP, NAMPT; MDK, FGFBP1, EPS8L1, CAPS; MD K, STX7, FCGBP, FCGBP; MDK, SCEL, STX7, FCGBP; MDK, FGFBP1, ANXA7, CAPS; MDK, FGFBP1, EPS8L1, RAB21; MDK, SCEL, STX7, FCGBP; MDK, STX7, FCGBP, NEDD8 -MDP1;MDK, ITIH2, FCGBP, CAPS;MDK, FGFBP1, EPS8L1, PKM;MDK, IMPA1, FCGBP, HCLS1;MDK, APOF, ANXA3, FCGBP;MDK, FGFBP1, EPS8L1, STMN1;MDK, STX7 ,FCGBP,LGALS1;MDK,FCGBP,LSP1,VIM;MDK,FGFBP1,EPS8L1,LGALS1;MDK,ANXA3,FCGBP,LMNB1;MDK,FCGBP,IMPA1,MMP8;MDK,FGFBP1,EPS8L1,STMN1; MDK, SCEL, IMPA1, PEA15; MDK, FGFBP1, ANXA7, LGALS1; MDK, TCP1, PEA15, LMNB1; MDK, FCGBP, GYG1, CA1; MDK, SCEL, IMPA1, PKM; MDK, FGFBP1, EPS8L1, LDH A;MDK, FCGBP, KRT8, GYG1;MDK, FCGBP, GYG1, LMNB1;MDK, FCGBP, GYG1, LSP1;MDK, FCGBP, KRT8, GYG1;MDK, FCGBP, GYG1, LGALS1;MDK, FCGBP, CAPS, GYG1;determining the presence and / or expression level of protein in at least one set of 4 values ​​from the group consisting of: MDK, FGFBP1, EPS8L1, LDHA; MDK, FCGBP, CAPS, GYG1; MDK, C1R, FCGBP, LGALS1; MDK, FGFBP1, SCEL, PEA15; MDK, FGFBP1, TIMP2, CHMP4B; MDK, ANXA3, STMN1, PKM; MDK, ANXA7, SCEL, CHMP4B; MDK, FCGBP, GYG1, LDHA; MDK, S100A9, MMP8, LGALS1; MDK, C1R, FCGBP, GYG1; MDK, C4BPA, FGFBP1, PPL; MDK, ITIH2, FGFBP1, PPL; and MDK, APOC1, FGFBP1, PPL;

[0053] In another embodiment of the first aspect, optionally in combination with any of the above or below embodiments, the method comprises the steps of: a) determining in vitro in a sample isolated from the female reproductive tract the expression level of MDK, optionally in combination with one or more of the proteins listed in any one of the previous embodiments; b) comparing the level of MDK from step (a) and, if determined, the level of one or more other proteins with a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer and / or with a cut-off value that distinguishes between endometrial cancer and other endometrial disorders or conditions; c) diagnosing the subject with endometrial cancer if the level of at least MDK is within a value or range of values ​​from subjects suffering from this cancer and / or if the level of at least MDK relative to the cut-off value is classified into the endometrial cancer group.

[0054] The term other gynecological disorders or conditions includes, but is not limited to, healthy endometrium, endometriosis, endometrial polyps, endometrial fibroids, atrophic endometrium, physiological damage to the uterus, or any other condition that may cause abnormal vaginal bleeding.

[0055] In another particular embodiment of the method disclosed herein, the method further comprises a step of determining one or more clinical or characteristic parameters of the subject, particularly selected from the group consisting of blood pressure, blood glucose, age, grading or staging score (i.e., Federation of Gynecology and Obstetrics (FIGO) stage), endometrial thickness, CA125 and HE4 molecular markers, AVB, and combinations thereof.

[0056] In another particular embodiment of the method according to the first aspect or any of its embodiments, the expression levels of MDK and the other protein are determined at the protein level.

[0057] In more particular embodiments, the protein level is determined by an assay or technique selected from the group consisting of an immunoassay, a bioluminescence assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, a lateral flow assay, mass spectrometry, and combinations thereof.

[0058] In another specific embodiment, the expression level of the protein is determined using an antibody or a fragment thereof capable of specifically binding to the protein.

[0059] More particularly, the antibody or fragment thereof forms part of a kit.

[0060] Alternatively, the expression level is determined at the mRNA level, and this alternative method is described in more detail below.

[0061] In a second aspect, the present invention relates to the use of MDK as an in vitro marker for the diagnosis and / or prognosis of endometrial cancer in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, Pap smear-like samples, endometrial biopsies, uterine fluid, uterine washings and combinations thereof from parts of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0062] In a particular embodiment of the second aspect, the sample is a Pap smear, in particular the fluid contained in the Pap smear and / or cervical mucus.

[0063] In another particular embodiment of the second aspect, the MDK is also a member of the family of proteins that are involved in the expression of the MDK protein, such as apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), chaperone containing TCP1 subunit 6A (CCT6A), lipopolysaccharide binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalli. lipocalin-2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A) and tissue inhibitor of metalloproteinases 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B domain-containing 1 (PLBD1), CD44 antigen, IgG-binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1),Used as an in vitro marker in a panel of multiple biomarkers including one or more of proliferation and apoptosis adaptor protein 15 (PEA15), S100 calcium binding protein A9 (S100A9), scyelin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2),

[0064] In another particular embodiment of the second aspect, MDK is also used as an in vitro marker in a panel of biomarkers that includes one or more other proteins from the group listed in Table 1.

[0065] These panels of biomarkers, including MDK, in certain embodiments, include from 2 to all of the listed markers. In particular, the panels include 2, 3, 4, 5, 6, 7, 8, 9, and 10 biomarkers, one of which is MDK.

[0066] Also disclosed are panels of biomarkers that include one or more of the proteins in Table 1. In particular examples, panels that include from 2 to all of the listed markers. In particular, panels include 2, 3, 4, 5, 6, 7, 8, 9, and 10 biomarkers.

[0067] Diagnosis of EC due to MDK and / or one or more of the proteins listed above can be performed by the use of a kit comprising means for determining the expression level of these proteins, as indicated. As mentioned above, the third aspect of the invention comprises a solid support, means for detecting the presence and / or determining the expression level of MDK, and optionally one or more of the proteins listed in Table 1, in particular apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), TCP1 subunit 6 (TCP1), and / or apolipoprotein B (APOB). A-containing chaperone (CCT6A), lipopolysaccharide-binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich These include SPRR1A and tissue inhibitor of metalloproteinases 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B domain-containing 1 (PLBD1), CD44 antigen, IgG-binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein phagocytosis (HSC), and phospholipase B domain-containing protein 1 (PHD1). MilliE (Hsp10) member 1 (HSPE1), Calumenin (CALU), Lactate dehydrogenase A (LDHA), Polymeric immunoglobulin receptor (PIGR), Keratin 8 (KRT8), Periplakin (PPL), Stathmin 1 (STMN1), Calcyphosine (CAPS), Carbonic anhydrase 1 (CA1), Vimentin (VIM), T complex 1 (TCP1), Agrin (AGR), Annexin A7 (ANXA7), Inositol monophosphatase 1 (IMPA1), Syntaxin 7 (STX7),Inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), Galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), Pyruvate kinase isozyme M1 / M2 (PKM), Glycogenin 1 (GYG1), Lymphocyte-specific protein 1 (LSP1), Hematopoietic cell-specific Lyn substrate 1 (HCLS1), Proliferation and apoptosis adaptor protein 15 (PEA15), S100 calcium-binding protein A9 (S100A9) and means for detecting the presence and / or determining the expression level of one or more selected from the group consisting of scyellin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2). A "device" or "kit" in the sense of the present invention is an assay or method for determining a biomarker (level of a protein of interest in a sample) or a panel of biomarkers (combination) according to the present invention, which can be used to perform an assay or method for diagnosis and / or prognosis of EC or for selection of patients for medical regimens once diagnosed. Examples are carrier plates, test stripes, biochip arrays, electrochemical sensors, etc., known in the art, which contain reagent means for detecting the presence and level of a protein of interest. A kit, as used herein, refers to a product comprising the different reagents (or reagent means) necessary to perform the method of the present invention, packaged to allow transport and storage. Materials suitable for packaging the components of the kit include quartz, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes. Instructions in different formats for performing the method are also included in the kit in some embodiments. The particular format of the instructions may be selected from an electronic aid capable of storing readable or understandable instructions, such as a leaflet, an electronic storage medium (e.g., magnetic disk, tape), or an optical medium (e.g., CD-ROM, DVD), or audio material.

[0068] Certain embodiments of the kits of the present invention comprise means (i.e., reagent means) for detecting and / or quantifying the expression level of at least one of the binary sets from the group consisting of MDK, LCN2; MDK, FN1; MDK, PLBD1; MDK, APOB; MDK, CD44; MDK, FCGBP; MDK, EPS8L1; MDK, ANXA3; MDK, C4BPA; MDK, MMP8; MDK, NEDD-8; MDK, CAMP; MDK, TIMP2; MDK, HSPE1; MDK, CALU; MDK, C1QA; MDK, LDHA; MDK, TIMP2; MDK, PIGR; MDK, KRT8; MDK, PPL; MDK, SPRR1A; MDK, STMN1; MDK, CAPS; MDK, CA1; MDK, CCT6A; MDK, VIM; MDK, TCP1; and MDK, AGRN.

[0069] In another particular embodiment, the set of binary values ​​is selected from at least one of Table 4 and / or Table 6 below (see Examples).

[0070] In another particular embodiment of the kit of the invention, MDK, ANXA7, CSE1L; MDK, LCN2, FGFBP1; MDK, LCN2, ANXA7; MDK, FGFBP1, LCN2; MDK, APOF, FCGBP; MDK, IMPA1, FCGBP; MDK, LCN2, CD44; MDK, STX7, FCGBP; MDK, ITIH2, FCGBP; MDK, LCN2, EPS8L1; MDK, FGFBP, EPS8L1; MDK, ANXA7, FCGBP; MDK, LCN2, PLBD1; MDK, FCGBP, CD44 ;MDK, PLBD1, FCGBP;MDK, ANXA3, FCGBP;MDK, PLBD1, LGALS1;MDK, LCN2, IMPA1;MDK, FCGBP, VIM;MDK, FCGBP, LMNB1;MDK, PLBD1, ATP6V1G1;MDK, APOB, PKM; MDK, ITIH2, LCN2; MDK, PLBD1, CALU; MDK, LCN2, APOF; MDK, PLBD1, EPS8L1; MDK, APOC1, ANXA7; MDK, FCGBP, GYG1; MDK, PLBD1, PKM; MDK, FCGBP, LSP1;MDK, TIMP2, CD44;MDK, ANXA3, PLBD1;MDK, PLBD1, HSPE1;MDK, PLBD1, CA1;MDK, FCGBP, HCLS1;MDK, STX7, IMPA1;MDK, ANXA7, STX7; MDK, STX7, EPS8L1; MDK, PLBD1, LSP1; MDK, C1R, ANXA7; MDK, STX7, PEA15; MDK, PLBD1, HCLS1; MDK, LCAT, FCGBP; MDK, PLBD1, LDHA; MDK, APOB, S100 A9;MDK, ANXA7, SCEL; MDK, SERPINA3, FCGBP; MDK, FCGBP, ITGB2; MDK, SCEL, ANXA3; MDK, S100A9, PLBD1; MDK, S100A9, LSP1; MDK, C1R, FCGBP; MDK, FGFBP1, FCGBP;MDK, APOC1, S100A9;MDK, PPL, PLBD1;MDK, TCP1, PEA15;MDK, ANXA3, LSP1;MDK, PPL, IMPA1;MDK, S100A9, MMP8;MDK, FGFBP1, PPL;and means (i.e., reagent means) for detecting and / or quantifying the expression level of at least one triplet set of the group consisting of MDK, S100A9, and PPL;

[0071] In another particular embodiment, the set of ternaries is selected from at least one of Table 5 and / or Table 7 below (see Examples).

[0072] In another specific embodiment of the kit of the present invention, MDK, LCN2, PEA15, LDHA; MDK, LCN2, PEA15, PKM; MDK, FGFBP1, FCGBP, EPS8L1; MDK, LCN2, HSPE1, CD44;.MDK, FGFBP1, EPS8L1, CTNNB1; MDK, LCN2, IMPA1, LDHA; MDK, IMPA1, FCGBP, PEA15; MDK, IMPA1, FCGBP, PKM; MDK, FCGBP, IMPA1, PEA15; MDK, CCT6A, FGFBP1, EPS8L1; MDK, STX7, FCGBP, PKM; MDK, IMPA1, FCGBP, GYG1; MDK, FGFBP1, SCEL, EPS8L1; MDK, STX7, FCGBP, CA1; MDK, STX7, FCGBP, NAMPT; MDK, FGFBP1, EPS8L1, CAPS; MDK, STX7, FCGBP, FCGBP; MDK, SCEL, STX7, FCGBP; MDK, FGFBP1, ANXA7, CAPS; MDK, FGFBP1, EPS8L1, RAB21; MDK, SCEL, STX7, FCGBP; MDK, STX7, FCGBP, NEDD8-MDP1; MDK, ITIH2, FCGBP, CAPS; MDK, FGFBP1, EPS8L1, PKM; MDK, IMPA1, FCGBP, HCLS1; MDK, APOF, ANXA3, FCGBP; MDK, FGFBP1, EPS8L1, STMN1; MDK, STX7, FCGBP, LGALS1; MDK, FCGBP, LSP1, VIM; MDK, FGFBP1, EPS8L1, LGALS1; MDK, ANXA3, FCGBP, LMNB1; MDK, FCGBP, IMPA1, MMP8; MDK, FGFBP1, EPS8L1, STMN1; MDK, SCEL, IMPA1, PEA15; MDK, FGFBP1, ANXA7, LGALS1; MDK, TCP1, PEA15, LMNB1; MDK, FCGBP, GYG1, CA1; MDK, SCEL, IMPA1, PKM; MDK, FGFBP1, EPS8L1, LDHA; MDK, FCGBP, KRT8, GYG1; MDK, FCGBP, GYG1, LMNB1; MDK, FCGBP, GYG1, LSP1; MDK, FCGBP, KRT8, GYG1; MDK, FCGBP, GYG1, LGALS1; MDK, FCGBP, CAPS, GYG1;means (i.e., reagent means) for detecting and / or quantifying the expression level of at least one of the set of quaternaries from the group consisting of MDK, FGFBP1, EPS8L1, LDHA; MDK, FCGBP, CAPS, GYG1; MDK, C1R, FCGBP, LGALS1; MDK, FGFBP1, SCEL, PEA15; MDK, FGFBP1, TIMP2, CHMP4B; MDK, ANXA3, STMN1, PKM; MDK, ANXA7, SCEL, CHMP4B; MDK, FCGBP, GYG1, LDHA; MDK, S100A9, MMP8, LGALS1; MDK, C1R, FCGBP, GYG1; MDK, C4BPA, FGFBP1, PPL; MDK, ITIH2, FGFBP1, PPL; and MDK, APOC1, FGFBP1, PPL;

[0073] In another particular embodiment of the first aspect, the method for diagnosis and / or prognosis, in particular in which the level of MDK is determined in a gynecological and / or cervical sample, is a method for diagnosing endometrial cancer recurrence or risk of recurrence, further comprising determining in an isolated female sample the presence and / or expression level of one or more of the proteins selected from the group consisting of MUC1, PRSS8, PNP, APEH, MUC16, C9, SERPINC1, SERPINA1, F2, AMBP, HP, SERPINA3, CFB, ORM2, CAT, GNAI2, A1BG, FN1, C7, ASTRGL1, B4GALT1, CAPS, CBX3, CD163, CDV3, DMBT1, DSG3, EHD1, GOLM1, MUC5AC, NME1, NT5E, PDLIM5, RDX and VASP.

[0074] MDK in combination with one or more of the others listed in the previous paragraph is, in certain embodiments, determined in a cervical sample and / or a uterine aspirate.

[0075] Accordingly, there is provided a method for diagnosing endometrial cancer recurrence or risk of recurrence, comprising the step of detecting the presence or absence and / or levels of the following proteins: MUC1, PRSS8, PNP, APEH, MUC16, C9, SERPINC1, SERPINA1, F2, AMBP, HP, SERPINA3, CFB, ORM2, CAT, GNAI2, A1BG, FN1, C7, ASTRGL1, B4GALT1, CAPS, CBX3, CD163, CD Also disclosed are methods in which one or more of V3, DMBT1, DSG3, EHD1, GOLM1, MUC5AC, NME1, NT5E, PDLIM5, RDX, and VASP are determined in an isolated sample selected from gynecological sampling of portions of the female reproductive tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries, including cervical mucus, cytology, Pap smear samples, Pap smear-like samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof.

[0076] As shown in the Examples below, these markers provided a notable accuracy (AUC>0.75) for distinguishing between recurrent and non-recurrent EC patients. In particular, one or more markers selected from ASTRGL1, B4GALT1, CAPS, CBX3, CD163, CDV3, DMBT1, DSG3, EHD1, GOLM1, MUC16, MUC5AC, NME1, NT5E, PDLIM5, PRSS8, RDX and VASP enabled such a distinction in isolated samples with only a presence / absence statistical analysis.

[0077] For example, all specific embodiments regarding the type of sample or the number of markers determined indicated for the method of the first aspect also apply to any of the methods allowing diagnosis of recurrence or risk of recurrence of endometrial cancer.

[0078] "Recurrence", also known as relapse or recurrent, is the recurrence of a past (typically medical) condition. "Recurrence risk" relates to the probability of such a recurrence.

[0079] Thus, in certain embodiments of a method for diagnosing endometrial cancer recurrence or risk of recurrence, the method comprises the steps of: a) determining the expression level of one or more of the listed proteins in a sample isolated from the female reproductive tract in vitro; b) comparing the levels of one or more of these other proteins of step (a) with a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer resulting from recurrence; c) diagnosing the subject with recurrence of endometrial cancer if the level of at least one of the proteins is within a value or interval of values ​​from subjects suffering from recurrence of endometrial cancer.

[0080] In another particular embodiment of the first aspect, the method for diagnosis and / or prognosis, in particular in which the MDK level is determined in a gynecological and / or cervical sample, is a method for diagnosis of endometrial cancer subtypes and / or endometrial cancer molecular classification, further comprising the step of determining in an isolated female sample the presence and / or expression level of one or more proteins selected from the group consisting of LBP, VWF, GPLD1, SAA4, APOF, C4BPA, SPRR1A, SERPIND1, APOB, SCEL, LCAT, SERPINA3, LMO7, C1R, MUC4, FN1, SPRR1B, C1QA, ITIH2, TIMP2, APOC1, GRN, ANXA3, S100A9, PLBD1, PIGR, SERPINH1, HSPE1.

[0081] MDK in combination with one or more of the others listed in the previous paragraph is, in certain embodiments, determined in a cervical sample and / or a uterine aspirate.

[0082] Therefore, also disclosed herein is a method of diagnosing endometrial cancer subtype and / or endometrial cancer molecular classification, wherein one or more of the following proteins (i.e., presence or absence and / or levels thereof): LBP, VWF, GPLD1, SAA4, APOF, C4BPA, SPRR1A, SERPIND1, APOB, SCEL, LCAT, SERPINA3, LMO7, C1R, MUC4, FN1, SPRR1B, C1QA, ITIH2, TIMP2, APOC1, GRN, ANXA3, S100A9, PLBD1, PIGR, SERPINH1, HSPE1 are determined in an isolated sample selected from gynecological sampling including cervical mucus, cytology, Pap smear sample, endometrial biopsy, uterine fluid, uterine washings and combinations thereof of the female genital tract portions including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0083] For example, all specific embodiments regarding the type of sample or the number of markers determined indicated for the method of the first aspect also apply to any of the methods allowing the diagnosis of types of endometrial cancer in terms of the molecular distinction between them.

[0084] In a particular example of a method for diagnosing endometrial cancer molecular subtypes, the subtype is selected from the group consisting of POLE hypermutation or POLE mutation (POLEMut), MMR deficiency (MMRd) based on MSI: microsatellite instability (MSI) or loss of mismatch repair protein expression; low copy number (low CN) or no specific molecular profile (NSMP); high copy number (high CN) or p53 abnormality based on mutation-like immunostaining (p53 abn).

[0085] The data in the examples show the statistical significance between the EC subtypes obtained with each of these markers analyzed in Pap smear-like samples (cervical samples, also called cervical mucus) of patients.

[0086] In certain embodiments of the diagnostic method for diagnosing endometrial cancer subtype, the method comprises the steps of: a) determining the expression level of one or more of the listed proteins in a sample isolated from the female reproductive tract in vitro; b) comparing the levels of one or more of these other proteins of step (a) to a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects afflicted with a particular endometrial cancer subtype; c) diagnosing the subject with endometrial cancer subtype if the level of at least one of the proteins is within a value or interval of values ​​from subjects afflicted with that endometrial cancer subtype.

[0087] In another particular embodiment of the kit of the present invention, the means for detecting the expression level of the protein is a means for performing an assay or technique selected from the group consisting of an immunoassay, a bioluminescence assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, a lateral flow assay, mass spectrometry, and combinations thereof.

[0088] In an even more particular embodiment, the means for detecting the expression level of a protein in the kit is an antibody or a fragment thereof.

[0089] In yet another more specific embodiment, the kit is for performing an enzyme-linked immunosorbent assay (ELISA).

[0090] In another particular embodiment of the kit of the present invention, it further comprises a panel diagram for classifying individual samples.

[0091] In a preferred embodiment, the reagent means for assaying the levels of different biomarkers (proteins) comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying the biomarkers forming the kit. Thus, in the particular case of a kit comprising reagents for assaying the levels of MDK (and optionally one or more of the proteins listed above), the reagents specific for said biomarkers (i.e. antibodies that specifically bind to the proteins) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the antibodies present in the kit. Thus, these kits are simplified kits that mainly comprise reagent means for detecting the levels of MDK (and optionally one or more of the proteins listed above).

[0092] In another particular embodiment, the kits of the invention are conceived as point-of-care tests, more particularly, they are in the form of lateral flow tests or electrochemical sensors.

[0093] In another particular embodiment, the kit according to the invention comprises a support (particularly a solid support) and one or more sample inlet ports for the deposition of a biological fluid sample, in particular total cervical fluid, and a reaction area comprising a means / reagent for specifically binding a marker protein, in particular an antibody, the sample inlet ports being connected to the reaction area. In another more particular embodiment, the kit comprises as many sample inlet ports as there are markers to be detected (1, 2 or 3 or 4) and corresponding reaction areas connected thereto. In another embodiment, the kit comprises one single inlet port and a capillary track connecting to the same number of reaction areas, said capillary track directing a portion of the sample to each corresponding connected reaction area. A kit comprising more than one reaction area is a multiplex kit.

[0094] In a more particular embodiment, the kit comprises means for detecting the expression levels of between 2 and 80 sets of combinations.

[0095] In the particular case where the kits of the present invention are ELISA kits, they comprise a solid support and antibodies or fragments thereof that specifically bind to the target proteins to be detected, these antibodies being conjugated to a reporter molecule capable of generating a signal.

[0096] "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 be in the form of strips, tubes, beads, disks or microplates, or any other surface suitable for performing immunoassays.

[0097] As used herein, a "reporter molecule" refers to a molecule that, by its chemical nature, provides an analytically identifiable signal that allows for the detection of antigen-bound antibodies. Detection can be either 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 the case of enzyme immunoassays, the enzyme is generally conjugated to the second antibody by glutaraldehyde or periodate. However, as will be readily recognized, a wide variety of different conjugation techniques exist and 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 a particular enzyme are generally selected for the production of 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 above-mentioned chromogenic substrates. 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, followed by the emission of light of a characteristic color that is visually detectable by light microscopy. Both immunofluorescence and EIA techniques are well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent and bioluminescent molecules and / or dyes and other chromogenic substances, can also be used.

[0098] 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.

[0099] The binding assay for measuring biomarker levels can use solid phase or homogeneous format.Suitable assay methods include sandwich or competitive binding assay.Examples of sandwich immunoassays are described in U.S. Patent No. 4,168,146 and U.S. Patent No. 4,366,241, both of which are incorporated herein by reference in their entirety.Examples of competitive immunoassays include those disclosed in U.S. Patent No. 4,235,601, U.S. Patent No. 4,442,204 and U.S. Patent No. 5,208,535, each of which is incorporated herein by reference in its entirety.

[0100] Multiplex assay formats can be used to measure multiple biomarkers, for example, multiplexing through the use of binding reagent arrays, multiplexing using spectral discrimination of labels, for example, multiplexing flow cytometric analysis of binding assays performed on particles using the Luminex® system.

[0101] The assays (methods and kits) of the present invention may be performed by any suitable method. In one embodiment, biomarker levels are measured in a single sample, and the measurements may be performed in a single assay chamber or assay device, including, but not limited to, a single well of an assay plate, a single assay cartridge, a single lateral flow device, a single assay tube, and the like. Biomarker levels may be measured using any of several techniques available to those of skill in the art, such as direct physical measurements (e.g., mass spectrometry) or binding assays (e.g., immunoassays, agglutination assays, and immunochromatography assays). The method may also include measuring signals resulting from chemical reactions, such as changes in optical absorbance, changes in fluorescence, production of chemiluminescence or electrochemiluminescence, changes in reflectance, refractive index, or light scattering, accumulation or release of a detectable label from a surface, oxidation or reduction or redox species, current or potential, changes in magnetic field, and the like. Suitable detection techniques can detect binding events by measuring the engagement of a labeled binding reagent through measurement of the label's photoluminescence (e.g., via measurement of fluorescence, time-resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multiphoton fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic field, enzymatic activity (e.g., by measuring enzymatic activity via an enzymatic reaction that causes a change in optical absorbance or fluorescence, or causes chemiluminescence). Alternatively, detection techniques that do not require the use of labels may be used, for example, techniques based on measurement of the analyte's mass (e.g., surface acoustic wave measurements), refractive index (e.g., surface plasmon resonance measurements), or intrinsic luminescence.

[0102] In another aspect, the present invention relates to the use of the kit of the invention according to the third aspect and / or any one of its embodiments for the diagnosis and / or prognosis of EC.

[0103] Thus, in a particular embodiment, the present invention relates to the use of the kit of the present invention in any of the methods of the present invention.

[0104] In any of the embodiments provided above or below for any of the aspects and embodiments of the invention, the expression levels of MDK and optionally any other proteins are determined at the protein level, in which embodiment, the protein markers include, but are not limited to, all homologs, fragments and precursors of the markers, including native sequence peptides, isoforms, chimeric polypeptides, modified forms of the polypeptides and derivatives thereof.

[0105] In certain embodiments provided above or below, the expression level is determined by immunochemistry, as indicated. The following paragraphs set out this option in more detail.

[0106] The term "immunochemistry" as used herein refers to various techniques for detecting antigens (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 said antigens. Visualization of antibody-antigen interactions 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 reaction. Alternatively, the antibody can be tagged with a fluorophore such as fluorescein or rhodamine. Immunochemistry techniques can be direct or indirect. Direct methods are one-step staining methods and include a labeled antibody (e.g., FITC-conjugated antiserum) that reacts directly with the antigen. This technique utilizes only one antibody, and is therefore simple and rapid, but is less sensitive as there is little signal amplification as in the indirect method, and is less commonly used than the indirect method. Indirect methods include 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 strategies due to signal amplification resulting from the binding of several secondary antibodies to each primary antibody when the secondary antibodies are conjugated to fluorescent or enzymatic reporters.

[0107] Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules and is able to recruit avidin, streptavidin or neutravidin enzyme complexes. Apart from its higher sensitivity, the indirect method also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies need to be produced. In the direct method, it may be necessary to label each primary antibody for each antigen of interest. It should be noted that immunochemical techniques can also be used to detect specific nucleic acid sequences, if a tagged nucleic acid probe (designed to specifically bind to a specific target nucleic acid sequence) can be subsequently detected with a labeled antibody. Thus, detection of a protein can be performed by using a tagged nucleic acid designed to bind to a specific sequence of the target protein RNA, and then detecting said tagged nucleic acid with a labeled antibody that selectively binds to the tag.

[0108] Suitable immunoassay procedures include enzyme-linked immunosorbent assays (ELISAs, e.g., multiplex ELISAs), enzyme immunodot assays, agglutination assays, antibody-antigen-antibody sandwich assays, antigen-antibody-antigen sandwich assays, immunochromatography, or other immunoassay formats well known to those skilled in the art, such as radioimmunoassays, chemiluminescence assays, lateral flow assays or electrochemical assays, as well as protein microarray formats.

[0109] Thus, in some aspects of any of the above or below aspects, the expression level of the protein is measured by immunoassay. In another embodiment, in combination with any of the embodiments provided above or below, the expression level of the protein is determined by ELISA, more particularly by multiplex ELISA.

[0110] Alternatively, the expression level of a protein can be determined by bioluminescence, fluorescence, chemiluminescence, electrochemistry, or mass spectrometry.

[0111] Alternatively, protein expression levels can be determined by measuring the levels of a protein's proteotypic peptides (peptides having amino acid sequences that are uniquely associated with the test protein in a given proteome) by mass spectrometry.

[0112] As mentioned above, in some embodiments of any of the aspects of the invention in combination with any of the embodiments provided above or below, the expression level of the protein is determined using an antibody or a fragment thereof capable of binding to the target protein. These antibodies can be used as a "means" for determining the expression of the target protein in the kit of the invention. The following paragraphs set out this option in more detail.

[0113] 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 that has 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 portion of an antigen that is recognized by the immune system (B cells, T cells or antibodies).

[0114] The antibodies used for specific detection can be polyclonal or monoclonal. Well-known means for preparing and characterizing antibodies exist in the state of the art. Methods for generating polyclonal antibodies are well-known in the prior art. Briefly, polyclonal antibodies are prepared by immunizing an animal with a protein. Serum is then collected from the immunized animal and the antibody is isolated. A wide range of animal species can be used for the production of antisera. Typically, the animals used for the production of antisera can be rabbits, mice, rats, hamsters, guinea pigs or goats.

[0115] Additionally, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure 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. The method for preparing monoclonal antibodies generally begins along the same lines as polyclonal antibody preparation. The immunogen is injected into the animal as an antigen. The antigen may be mixed with an adjuvant, such as complete or incomplete Freund's adjuvant. Immunization is repeated with the same antigen at approximately two-week intervals.

[0116] An antibody or fragment thereof for detecting a target protein (i.e., MDK and optionally one or more of the listed proteins) can be included in the kit. The kit may further include other means (additives, solvents) for visualizing the antibody-protein interaction.

[0117] Alternatively, the expression level of MDK, and, if determined, the level of one or more of the above listed proteins, is determined at the mRNA level. The following paragraphs set out this option in more detail.

[0118] Thus, in another embodiment of the kit or its use, the kit is a microarray.

[0119] In another embodiment, the kit is a microarray containing a defined set of genes encoding protein endometrial cancer markers. All the embodiments provided above for the specific proteins (2 to 11 of the list) analyzed whose expression is significantly altered by endometrial disease are also specific embodiments of the microarray.

[0120] In addition, the kit of the present invention includes a reagent for detecting the protein encoded by a constitutive gene. The availability of such additional reagents allows normalization of measurements made in different samples (e.g., the sample to be analyzed and the control sample) to exclude that differences in expression of biomarkers are due to different amounts of total protein in the samples rather than actual differences in the relative levels of expression. Constitutive genes in the present invention are genes that are always active or constantly transcribed, constitutively expressed, and code for proteins that perform essential cellular functions. Constitutively expressed proteins that can be used in the present invention include, but are not limited to, beta-2-microglobulin (B2 M), ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin, and actin.

[0121] In one embodiment, the amount of mRNA of each one of the markers is detected by polymerase chain reaction, for example, using oligonucleotide primers that hybridize to one or more polynucleotide endometrial cancer markers or the complements of such polynucleotides.In another embodiment, the amount of mRNA is detected using hybridization techniques that use oligonucleotide probes that hybridize to one or more polynucleotide endometrial cancer markers or the complements of such polynucleotides.

[0122] When using mRNA detection, the method of the present invention can be carried out by combining isolated mRNA with a reagent to convert it to cDNA according to standard methods well known in the art, treating the converted cDNA with an amplification reaction reagent (such as a cDNA PCR reaction reagent) in a container 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. In the case of mRNA, the analysis step can be achieved by detecting the presence of polynucleotide endometrial cancer markers in the sample using Northern blot analysis. The analysis step can be further achieved by quantitatively detecting the presence of polynucleotide endometrial cancer markers in the amplification product and comparing the amount of the detected marker to a panel of expected values ​​for the known presence or absence of such markers in normal and malignant tissues derived using similar primers.

[0123] In another embodiment, the invention provides a method for detecting mRNA by (a) isolating mRNA from a sample and converting the mRNA into cDNA by combining with a reagent; (b) treating the converted cDNA with amplification reaction reagents and a nucleic acid primer that hybridizes to one or more polynucleotide endometrial cancer markers to produce an amplification product; (c) analyzing the amplification product to determine the abundance of mRNA encoding the protein endometrial cancer marker; and (d) comparing the determined amount of mRNA to the amount detected for a panel of predictive values ​​for normal and diseased tissues (e.g., malignant tissues) derived using a similar method.

[0124] In certain embodiments of the present invention, RT-PCR can be used to amplify the mRNA of protein endometrial cancer markers for detection and analysis.Other embodiments of the present invention use quantitative RT-PCR to quantitatively determine the amount of mRNA of protein endometrial cancer markers.Further embodiments of the present invention use real-time RT-PCR for quantification and analysis.

[0125] All proteins listed herein, or in the corresponding tables herein, UniprotKB (https: / / www.uniprot.org / help / uniprotkb) database accession numbers correspond to the version accessible on July 23, 2021 (database release).

[0126] AGRIN has the Uniprot database accession number O00468. The protein is a heparin sulfate basement membrane glycoprotein involved in the formation and maintenance of the neuromuscular junction.

[0127] PIGR is also known as the polymeric immunoglobulin receptor and 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.

[0128] CD44, also known as the CD44 antigen, has the Uniprot database accession number P16070, 5 Oct 2010-v3. It mediates cell-cell and cell-matrix interactions through its affinity for HA and possibly other ligands such as osteopontin, collagen, and matrix metalloproteinases (MMPs).

[0129] LDHA, also known as L-lactate dehydrogenase A chain, has the Uniprot database accession number P00338, 23 January 2007-v2. This protein is involved in step 1 of the alternative pathway for the synthesis of (S)-lactate from pyruvate.

[0130] Some of these proteins have been associated with EC grading / diagnosis in tissue samples. However, there is no meaningful data correlating with values ​​in samples selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings and combinations thereof from female genital tract parts including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries. As mentioned above, detection of markers in these types of samples represents an advantage in collecting data from routine sampling in clinical practice of gynecological diseases, avoiding the cost and importantly invasive tissue biopsy.

[0131] A further aspect of the invention was a method for deciding or recommending whether to initiate a medical regimen for a subject suffering from endometrial cancer, in particular depending on the prognosis. In a particular embodiment of this method, a) determining in vitro the presence and / or expression level of midkine (MDK) in a sample selected from gynecological sampling including cervical mucus, cytology, Pap smear samples, endometrial biopsies, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries; b) comparing the levels of MDK from step (a) with corresponding reference values ​​or reference intervals for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer and / or with cut-off values ​​discriminating between endometrial cancer and other gynecological disorders or conditions; c) if the level of MDK is within a value or range of values ​​from subjects suffering from this cancer and / or the level of MDK relative to the cut-off value is classified into the endometrial cancer group; i) if a subject is diagnosed with or suspected of having endometrial cancer, it is recommended that a medical regimen consisting of total hysterectomy and bilateral salpingo-oophorectomy, optionally complemented by pelvic and para-aortic lymphadenectomy and omentectomy, be instituted; ii) if the patient is diagnosed as not having endometrial cancer, then follow-up, if applicable, will be carried out taking into account the results of the doctor's examination of the patient; diagnosing the subject with endometrial cancer; Includes.

[0132] In another particular embodiment method of determining or recommending whether to initiate a medical regimen for a subject suffering from endometrial cancer, usually depending on an established prognosis, an adjuvant treatment selected from radiation therapy, brachytherapy, hormonal therapy, chemotherapy, targeted therapy and combinations thereof is recommended.

[0133] This method of determining or recommending whether to initiate a medical regimen in a subject suffering from endometrial cancer is also applicable to previously disclosed methods for diagnosing endometrial cancer recurrence or risk of recurrence, and / or diagnosing endometrial cancer subtype and / or endometrial cancer molecular classification.

[0134] Therefore, also disclosed herein is a method for the treatment of EC in a subject suffering from this disease, comprising carrying out the method of diagnosis and / or prognosis of the first aspect or any one of its embodiments, and further comprising treating the subject by total hysterectomy and / or bilateral salpingo-oophorectomy, optionally complemented with pelvic and para-aortic lymphadenectomy and / or omentectomy.In a particular embodiment of the proposed treatment method, the subject also undergoes recommended adjuvant treatment selected from radiation therapy, brachytherapy, hormone therapy, chemotherapy, targeted therapy and combinations thereof.

[0135] Also provided is a method for detecting levels of MDK, and / or optionally one or more of the proteins listed in Table 1, in an isolated sample from a subject, comprising: (a) obtaining from a subject a sample selected from a gynecological sampling including cervical mucus, cytology, Pap smear sample, endometrial biopsy, uterine fluid, uterine washings, and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries; (b) detecting the presence and / or expression level of midkine (MDK) in the isolated sample by (i) contacting the sample with a means capable of binding to the corresponding protein and detecting said binding, or (ii) contacting the sample with a means capable of binding to the corresponding RNA that will be translated into the protein and detecting said binding. A method is disclosed that includes:

[0136] Another aspect of the present invention is to provide an algorithm for performing any of the diagnostic and / or prognostic methods defined in the above aspects and embodiments, or for performing any of the above disclosed methods for diagnosing endometrial cancer recurrence or risk of recurrence and / or endometrial cancer subtype and / or endometrial cancer molecular classification.

[0137] As indicated, this fourth aspect relates to a computer implemented method for carrying out the method defined in the first aspect, wherein after the determination of the expression level of one or more of MDK and optionally proteins for the diagnosis and / or prognosis of endometrial cancer, said level is given a value and / or score, optionally calculated in a mathematical formula to obtain a calculated value, and depending on said level, score and / or calculated value a decision is made between the options of being affected or not affected by EC and / or between EC exhibiting different prognoses including different histological subtypes and grades and different molecular features.

[0138] In a particular embodiment, the algorithm is a computer-implemented method for diagnosing and / or prognosing EC, in particular for disease prognosis by determining EC subtype, in particular EEC or NEEC. The algorithm allows to make a decision whether a sample is derived from a subject suffering from EC, and also allows to make a decision whether a sample derived from a subject suffering from EC is suffering from EEC or NEEC. In a more particular embodiment, the algorithm provides a recommended treatment. There is therefore also provided a computer-implemented method for carrying out the method defined above, in which after determining the expression level of one or more of the proteins for diagnosing and / or prognosing EC, said level is given a value and / or a score, optionally calculated in a formula to obtain a calculated value, and depending on said level, score and / or calculated value, a decision is made between the options of suffering from EC or not, and / or between the options of suffering from different EC subtypes.

[0139] In other words, in another particular embodiment of the algorithm of the invention, after determining the expression level of one or more of the proteins for the diagnosis and / or prognosis of EC, said level is given a value and / or score, optionally calculated in a mathematical formula to obtain a calculated value, and depending on said level, score and / or calculated value a decision is made between the options of being affected with EC or not and / or between the options of being affected with different EC subtypes.

[0140] In a particular embodiment, the algorithm is a computer-implemented method for diagnosing and / or prognosing EC, in particular for disease prognosis by determining EC grade classification, in particular low-grade or high-grade EC, according to a known grading or staging system, such as that of FIGO. In another particular embodiment, the algorithm is a computer-implemented method for diagnosing and / or prognosing EC, in particular for disease prognosis by determining EC tissue subtype, such as endometrioid versus non-endometrioid endometrial cancer. The algorithm allows for a determination to be made whether a sample is derived from a subject suffering from EC, and also allows for a determination to be made whether a sample derived from a subject suffering from EC suffers from low-grade or high-grade EC. In a more particular embodiment, the algorithm provides a recommended treatment. There is therefore also provided a computer implemented method for carrying out the method defined above, wherein after determining the expression level of one or more of the proteins for the diagnosis and / or prognosis of EC, said level is given a value and / or score, optionally calculated in a mathematical formula to obtain a calculated value, and depending on said level, score and / or calculated value a decision is made between the options of being affected with EC or not and / or between the options of being affected with different EC grades.

[0141] In other words, in another particular embodiment of the algorithm of the invention, after determining the expression level of one or more of the proteins for the diagnosis and / or prognosis of EC, said level is given a value and / or score, optionally calculated in a mathematical formula to obtain a calculated value, and depending on said level, score and / or calculated value a decision is made between the options of being affected with EC or not and / or between the options of being affected with different EC grades.

[0142] The in vitro method of the invention provides diagnostic and / or prognostic information. In one embodiment, the method of the invention further comprises the steps of (i) collecting the diagnostic and / or prognostic information and (ii) storing the information on a data carrier.

[0143] In the sense of the present invention, a "data carrier" should be understood as any means containing meaningful information data for the diagnosis and / or prognosis of endometrial cancer, such as a piece of paper. The carrier can also be any entity or device capable of holding the prognosis data. For example, the carrier can include a storage medium, such as a ROM, e.g. a CD ROM or a semiconductor ROM, or a magnetic recording medium, e.g. a floppy disk or a hard disk. Furthermore, the carrier can be a transmissible carrier, such as an electric or optical signal, which can be transmitted via an electric or optical cable or by radio or other means. If the diagnosis / prognosis data is embodied in a signal that can be directly transmitted by a cable or other device or means, the carrier can be constituted by such a cable or other device or means. Other carriers concern USB devices and computer archives. Examples of suitable data carriers are paper, CD, USB, a computer archive in a PC, or a voice registration with the same information.

[0144] Throughout the specification and claims, the word "comprises" and variations of this word are not intended to exclude other technical features, additives, ingredients or steps. Furthermore, the word "comprises" encompasses the case of "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 present invention. The following examples are provided by way of illustration and are not intended to limit the present invention. Furthermore, the present invention covers all possible combinations of the specific preferred embodiments described herein. EXAMPLES

[0145] Example 1: Discovery cohort. Validation cohort and informative analysis of markers. Informative data from analysis of MDK levels in cervical mucus samples (routine non-invasive gynecological sampling)

[0146] Materials and Methods: The discovery phase to identify potential biomarkers using fluids contained in Pap smears, i.e. cervical mucus samples, was carried out using a shotgun label-free proteomic approach. The study included 60 patients (20 EC patients, 20 controls suffering from AVB without endometrial cancer or cervical lesions, and 20 controls suffering from cervical lesions without endometrial lesions). Data were analyzed using MaxQuant and R software. The levels of 110 peptides corresponding to 75 proteins identified in the discovery phase were further measured in the validation phase in 242 patients (106 non-EC, 129 EC, 7 pre-malignant tumors with EC) by mass spectrometry (LC-PRM). The analysis was performed using Skyline software, SPSS and R software. The clinicopathological characteristics of the patients included in the validation study are shown in Table 2.

[0147] Results: The discovery test made it possible to determine a total of 2,888 proteins identified with one or more peptides in our samples. Statistical analysis made it possible to identify 75 potential proteins differentially expressed between EC and non-EC patients to be further evaluated and validated. The validation phase revealed a great potential of 60 of these proteins measured in cervical samples to reach a non-invasive diagnosis of EC comparing EC and non-EC patients (adj. p-value < 0.05, fold change > 2, AUC > 0.7) (Table 3). Specifically, 19 proteins achieved an AUC > 0.75 and 7 proteins an AUC > 0.8. Furthermore, reproducing the results obtained by mass spectrometry, the ELISA assay of the best performing proteins was tested in all samples reaching an AUC = 0.91 in this dataset for diagnosing EC (comparing 127 EC patients with 106 non-EC patients) (Figure 1). The validation studies made it possible to identify a set of proteins significant as prognostic biomarkers for differentiating histological subtypes and grades of EC patients. Specifically, when comparing 35 patients with low grade (G1) EC with 37 patients with high grade (G3) EC, a set of 34 protein biomarkers had significant adjusted p-values ​​(Table 3, bold) and 39 proteins had fold changes higher than 1.3 (Table 3, shaded). A comparison was also made between low and intermediate grades, G1 and G2, and high grade (G3). The significant and highly expressed proteins are shown in Table 3. When comparing 102 patients with endometrioid endometrial cancer (EEC) with 25 patients with non-endometrioid endometrial cancer, with regard to histological subtypes, a set of 33 proteins was identified as differentially expressed (fold change higher than 1.3-fold) and 31 proteins as significant (adj. p-values ​​< 0.05). All these results are shown in Table 3. Tables 8 and 9 also show two or three protein panels that, in combination, allow for differentiation of histological subtypes and grades in EC patients.

[0148] Table 3 below shows the AUC, SE and SP of another marker, MDK. Note that MDK provided the best values.

[0149] [Table 2]

[0150] [Table 3] JPEG2024525939000007.jpg255146JPEG2024525939000008.jpg81151

[0151] When MDK was combined with two and three panel protein biomarkers, the AUC values ​​of the combinations increased compared to MDK alone, primarily by increasing the sensitivity for diagnosing EC. The data are shown in Tables 4 and 5 below. Similarly, proteins providing significant information regarding prognosis were combined in Tables 6 and 7. As shown earlier, sensitivity is important considering the symptomatology of the disease, which is common with other disorders of the female reproductive tract.

[0152] [Table 4] JPEG2024525939000010.jpg208155

[0153] [Table 5] JPEG2024525939000012.jpg255149JPEG2024525939000013.jpg187153

[0154] [Table 6]

[0155] [Table 7]

[0156] [Table 8-1] JPEG2024525939000017.jpg128150

[0157] [Table 8-2] TIFF2024525939000019.tif197146

[0158] [Table 9-1]

[0159] [Table 9-2]

[0160] Example 2: Markers indicative of disease recurrence. The discovery study included 20 patients, of whom 9 recurred after 5 years of follow-up. These recurrent women recurred between 10 and 119 months after surgery. Specifically, 4 endometrioid EC (2 G2 and 2 G3) and 5 non-endometrioid EC recurred, whereas 10 EEC (2 G1, 7 G2, 1 G3) and 1 non-EEC did not recur. Thus, we compared the 9 recurring patients with the 11 non-recurring patients.

[0161] Statistical analysis showed that 19 proteins with AUC>0.75 distinguished between recurrent and non-recurrent EC patients (MUC1, PRSS8, PNP, APEH, MUC16, C9, SERPINC1, SERPINA1, F2, AMBP, HP, SERPINA3, CFB, ORM2, CAT, GNAI2, A1BG, FN1, and C7). Furthermore, presence-absence statistical analysis identified 18 differentially expressed proteins between recurrent and non-recurrent EC patients (ASTRGL1, B4GALT1, CAPS, CBX3, CD163, CDV3, DMBT1, DSG3, EHD1, GOLM1, MUC16, MUC5AC, NME1, NT5E, PDLIM5, PRSS8, RDX, and VASP).

[0162] The following Tables 10(A) and (B) show the data.

[0163] [Table 10A]

[0164] [Table 10B]

[0165] The following Table 11 lists the names of the biomarkers used.

[0166] [Table 11] TIFF2024525939000025.tif255152TIFF2024525939000026.tif161154

[0167] Example 3: ELISA-EC diagnosis (MDK) MDK is a good EC diagnostic biomarker when measured by both mass spectrometry (MS) and antibody-based assay (ELISA technique). The levels of MDK in cervical mucus of 241 patients (n=128 EC, 113 non-EC) showed statistical differences for discriminating between EC and non-EC patients, achieving an AUC of 0.91 when measured by MS and an AUC of 0.92 when measured by ELISA assay. A correlation of 0.91 is shown between measurements of both techniques.

[0168] The data are shown in Figure 2, showing (A) mass spectrometry (LC-MS / MS PRM) data or (B) data derived from an ELISA assay to determine MDK. Samples from a cohort of subjects were tested, comparing EC and non-EC patients. In (C), the correlation between the measurements of both techniques can be at a high correlation level, making the marker suitable for implementation at the point of care.

[0169] Example 4: MDK in uterine fluid The level of MDK was also demonstrated to be a potential diagnostic marker for EC in uterine fluid obtained from endometrial biopsies, specifically pipel biopsies. Specifically, MDK levels showed a p-value <0.0001 and AUC=0.69 between EC patients (n=118) and non-EC patients (n=129). Thus, in addition to the information from MDK that can be obtained from sample types during routine gynecological examinations, this marker also provides information from other more invasive sample collections, such as endometrial biopsies. These biopsies may be performed when certain diseases are suspected, so that when this marker is analyzed, the analysis of early EC or the possibility of not being EC can also be detected at the same time.

[0170] The data are shown in Figure 3, where data from an ELISA assay for determining MDK in uterine fluid tested on samples from a cohort of subjects and comparing EC and non-EC patients are shown. (A) Dot plot of the distribution of protein concentrations between EC and non-EC patients. (B) ROC analysis (Receiver Operating Characteristic analysis) curve of MDK assessed by ELISA.

[0171] Example 5: Molecular Classification A total of 28 proteins (LBP, VWF, GPLD1, SAA4, APOF, C4BPA, SPRR1A, SERPIND1, APOB, SCEL, LCAT, SERPINA3, LMO7, C1R, MUC4, FN1, SPRR1B, C1QA, ITIH2, TIMP2, APOC1, GRN, ANXA3, S100A9, PLBD1, PIGR, SERPINH1, HSPE1) were able to significantly distinguish molecular subgroups of EC: presence of POLE exonuclease domain hotspot mutations (POLE) hypermutations or POLE mutations (POLEMut), MSI: MMR-deficient (MMRd) based on microsatellite instability (MSI) or loss of mismatch repair protein expression; low copy number (lowCN) or no specific molecular profile (NSMP); high copy number (highCN) or p53 aberrant (p53 abn) based on mutation-like immunostaining-FC: fold change.

[0172] The data is shown in Table 12: [Table 12]

[0173] Further aspects / embodiments are defined in the following sections.

[0174] Clause 1. A method for the diagnosis and / or prognosis of endometrial cancer, comprising determining the presence and / or expression level of midkine (MDK) in a sample selected from a gynecological sampling including or selected from cervical mucus, cytology, Pap smear sample, endometrial biopsy, uterine fluid, uterine washings and combinations thereof from portions of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes and ovaries.

[0175] Clause 2. The method of clause 1, wherein the sample is a Pap smear, in particular the fluid contained in the Pap smear and / or cervical mucus.

[0176] Clause 3. The method of any of clauses 1 to 2, further comprising determining the presence and / or expression level of one or more of the proteins listed in Table 1, in particular one or more of the following proteins: Apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), TCP1 subunit 6A-containing chaperone (CCT6A), lipopolysaccharide-binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acylate transferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A) and tissue inhibitor of metalloproteinase 2 (TIMP2), lipocalin-2 (LCN2), phospholipase B domain-containing 1 (PLBD1), CD44 antigen, IgG-binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8 L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), proliferation and apoptosis adaptor protein 15 (PEA15),S100 calcium-binding protein A9 (S100A9), Scielin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG-binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2).

[0177] Clause 4. The method of any of clauses 1 to 3, comprising determining the presence and / or expression level of two, three or four of the proteins.

[0178] Clause 5. In the isolated sample, MDK, LCN2; MDK, FN1; MDK, PLBD1; MDK, APOB; MDK, CD44; MDK, FCGBP; MDK, EPS8L1; MDK, ANXA3; MDK, C4BPA; MDK, MMP8; MDK, NEDD-8; MDK, CAMP; MDK, TIMP2; MDK, HSPE1; MDK, CALU; MDK, C1QA; MDK, LDHA; MDK, TIMP2; MDK, PIGR; MDK, The method of clause 4, comprising determining the presence and / or expression level of protein in at least one binary set of the group consisting of: KRT8; MDK, PPL; MDK, SPRR1A; MDK, STMN1; MDK, CAPS; MDK, CA1; MDK, CCT6A; MDK, VIM; MDK, TCP1; and MDK, AGRN; and / or at least one binary set listed in either Table 4 and Table 6.

[0179] Clause 6. In the isolated sample, MDK, ANXA7, CSE1L; MDK, LCN2, FGFBP1; MDK, LCN2, ANXA7; MDK, FGFBP1, LCN2; MDK, APOF, FCGBP; MDK, IMPA1, FCGBP; MDK, LCN2, CD44; MDK, STX7, FCGBP; MDK, ITIH2, FCGBP; MDK, LCN2, EPS8L1; MDK, FGFBP, EPS8L1; MDK, ANXA7, FCGBP; MDK, LCN2, PLBD1; MDK, FCGBP, CD44; MDK, PLBD1, FCGBP P;MDK, ANXA3, FCGBP;MDK, PLBD1, LGALS1;MDK, LCN2, IMPA1;MDK, FCGBP, VIM;MDK, FCGBP, LMNB1;MDK, PLBD1, ATP6V1G1;MDK, APOB, PKM;MDK, ITIH2, LCN2;MDK, PLBD1, CALU;MDK, LCN2, APOF;MDK, PLBD1, EPS8L1;MDK, APOC1, ANXA7;MDK, FCGBP, GYG1;MDK, PLBD1, PKM;MDK, FCGBP, LSP1;MDK, TIMP2, CD 44;MDK, ANXA3, PLBD1;MDK, PLBD1, HSPE1;MDK, PLBD1, CA1;MDK, FCGBP, HCLS1;MDK, STX7, IMPA1;MDK, ANXA7, STX7;MDK, STX7, EPS8L1;MDK, PLBD1, L SP1;MDK, C1R, ANXA7;MDK, STX7, PEA15;MDK, PLBD1, HCLS1;MDK, LCAT, FCGBP;MDK, PLBD1, LDHA;MDK, APOB, S100A9;MDK, ANXA7, SCEL;MDK, SERPINA3, at least one set of triplets from the group consisting of FCGBP;MDK, FCGBP, ITGB2;MDK, SCEL, ANXA3;MDK, S100A9, PLBD1;MDK, S100A9, LSP1;MDK, C1R, FCGBP;MDK, FGFBP1, FCGBP;MDK, APOC1, S100A9;MDK, PPL, PLBD1;MDK, TCP1, PEA15;MDK, ANXA3, LSP1;MDK, PPL, IMPA1;MDK, S100A9, MMP8;MDK, FGFBP1, PPL; and MDK, S100A9, PPL;and / or determining the presence and / or expression level of protein in at least one set of ternaries listed in any of Tables 5 and 7;

[0180] Clause 7. The method of clause 6, comprising determining the presence and / or expression levels of the set of triplets, MDK, ANXA7 and CSE1L, in the isolated sample.

[0181] Article 8. a) determining in vitro in a sample isolated from the female reproductive tract the expression level of MDK, optionally in combination with one or more of the proteins listed in any one of claims 3 to 7; b) comparing the level of MDK from step (a), and, if determined, one or more other proteins, with a corresponding reference value or reference interval for each protein selected from values ​​or intervals of values ​​from subjects suffering from endometrial cancer, and / or with a cut-off value discriminating between endometrial cancer and other endometrial disorders or (i.e. healthy) endometrial conditions, wherein the subject is diagnosed with endometrial cancer if the level of at least MDK is within the value or interval of values ​​from subjects suffering from this cancer and / or if the level of at least MDK relative to the cut-off value is classified into the endometrial cancer group. 8. The method according to any one of clauses 1 to 7, comprising:

[0182] Clause 9. The method of any one of clauses 1 to 8, wherein the expression level is determined at the protein level.

[0183] Clause 10. The method of any of clauses 1 to 9, wherein the protein level is determined by an assay or technique selected from the group consisting of an immunoassay, a bioluminescence assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0184] Clause 11. The method of any one of clauses 9 to 10, wherein the expression level of the protein is determined using an antibody or a fragment thereof capable of binding to the protein.

[0185] Clause 12. The method of clause 11, wherein the antibody or fragment thereof forms part of a kit.

[0186] Clause 13. Use of MDK as an in vitro marker for the diagnosis and / or prognosis of endometrial cancer in a sample selected from a gynecological sample collection including one or more of cervical mucus, cytology, Pap smear sample, endometrial biopsy, uterine fluid, uterine washings from any portion of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries.

[0187] Clause 14. A solid support and a means for detecting the presence and / or determining the expression level of MDK, and optionally one or more proteins listed in Table 1, in particular apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), chaperone containing TCP1 subunit 6A (CCT6A), lipopolysaccharide binding protein precursor (LPC1), lipopolysaccharide binding protein precursor (LPC2), lipopolysaccharide binding protein precursor (LPC3), lipopolysaccharide binding protein precursor (LPC4), lipopolysaccharide binding protein precursor (LPC5), lipopolysaccharide binding protein precursor (LPC6), lipopolysaccharide binding protein precursor (LPC7), lipopolysaccharide binding protein precursor (LPC8), lipopolysaccharide binding protein precursor (LPC9), lipopolysaccharide binding protein precursor (LPC10), lipopolysaccharide binding protein precursor (LPC11), lipopolysaccharide binding protein precursor (LPC12), lipopolysaccharide binding protein precursor (LPC13), lipopolysaccharide binding protein precursor (LPC14), lipopolysaccharide binding protein precursor (LPC15), lipopolysaccharide binding protein precursor (LPC16), lipopolysaccharide binding protein precursor (LPC17), lipopolysaccharide binding protein precursor (LPC18), lipopolysaccharide binding protein precursor (LPC19), lipopolysaccharide binding protein precursor (LPC19), lipopolysaccharide binding protein precursor (LPC19), lipopolysaccharide binding protein precursor (LPC19), lipopolysaccharide binding protein precursor (LPC11), lipopolysaccharide binding protein precursor (LPC12), lipopolysaccharide binding protein precursor (LPC13), lipopolysaccharide binding protein precursor (LPC14), lipopolysaccharide binding protein precursor (LPC15), lipopolysaccharide binding protein precursor (LPC16), lipopolysaccharide binding protein precursor (LPC17), lipopolysaccharide binding protein precursor (LPC BP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A) and tissue inhibitor of metalloproteinases 2 (TIMP2), lipocalin-2 (LCN2). , phospholipase B domain containing 1 (PLBD1), CD44 antigen, IgG binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T complex 1 (TCP1), agrin (AGR), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transport V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM),and a means for detecting the presence and / or determining the expression level of one or more proteins selected from the group consisting of glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), proliferation and apoptosis adaptor protein 15 (PEA15), S100 calcium-binding protein A9 (S100A9), scyellin (SCEL), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), and exportin-2 (XPO2).

[0188] Clause 15. A computer implemented method for carrying out the method defined in any one of clauses 1 to 3, wherein after the determination of the expression level of one or more of MDK and optionally proteins for the diagnosis and / or prognosis of endometrial cancer, said level is given a value and / or score, optionally calculated with a mathematical formula to obtain a calculated value, and depending on said level, score and / or calculated value a decision is made between the options of being affected or not affected by EC and / or between different ECs exhibiting different prognoses including different histological subtypes and grades and different molecular features.

[0189] References list ●DeSouza LV, et al, "Endometrial cancer biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry", Mol Cell Proteomics MCP- 2007, vol. no.6, pp.:1170-8. ●EP3452829B1 ●EP3655778A1 ●Tanable et al., "Midkine and its clinical significance in endometrial carcinoma", cancer Sci-2008, vol. no. 99(6), pp.: 1125-1130 ●Kemik P, et al. "Diagnostic and prognostic values of preoperative serum levels of YKL-40, HE-4 and DKK-3 in endometrial cancer", Gynecol Oncol- 2016; vol. no.140, pp.:64-9. ●Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values"

Claims

**Claim 1** A method for diagnosing and / or predicting the prognosis of endometrial cancer, comprising the step of determining the presence and / or expression level of a protein in a sample selected from gynecological sampling including or selected from endocervical mucus, cytology, Papanicolaou smear samples, endometrial biopsy, uterine fluid, uterine lavage fluid, and combinations thereof, from a part of the female genital tract including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries, The protein contains metalloproteinase tissue inhibitor 2 (TIMP2), agrin (AGRIN), midkine (MDK), apolipoprotein B (APOB), complement C1q subcomponent subunit A (C1QA), fibronectin 1 (FN1), serpin family D member 1 (SERPIND1), apolipoprotein F precursor (APOF), apolipoprotein C1 (APOC1), chaperonin containing TCP1 subunit 6A (CCT6A), lipopolysaccharide-binding protein precursor (LBP), serum amyloid A4 (SAA4), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), lipocalin 2 (LCN2), lecithin:cholesterol acyltransferase (LCAT), C4b-binding protein alpha chain (C4BPA), complement C1r (C1R), fibroblast growth factor-binding protein 1 (FGFBP1), small proline-rich protein 1B (SPRR1B), small proline-rich protein 1A (SPRR1A), lipocalin-2 (LCN2), phospholipase B domain-containing 1 (PLBD1), CD44 antigen, IgG-binding protein Fc fragment (FCGBP), epidermal growth factor receptor kinase substrate 8-like protein 1 (EPS8L1), annexin A3 (ANXA3), matrix metalloproteinase-8 (MMP8), NEDD-8 protein, cathelicidin antimicrobial peptide (CAMP), heat shock protein family E (Hsp10) member 1 (HSPE1), calumenin (CALU), lactate dehydrogenase A (LDHA), polymeric immunoglobulin receptor (PIGR), keratin 8 (KRT8), periplakin (PPL), stathmin 1 (STMN1), calcyphosine (CAPS), carbonic anhydrase 1 (CA1), vimentin (VIM), T-complex 1 (TCP1), annexin A7 (ANXA7), inositol monophosphatase 1 (IMPA1), syntaxin 7 (STX7), inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), galectin 1 (LGALS1), ATPase H+ transporting V1 subunit G1 (ATP6V1G1), pyruvate kinase isozyme M1 / M2 (PKM), glycogenin 1 (GYG1), lymphocyte-specific protein 1 (LSP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1).A method that is one or more proteins selected from proliferation and apoptosis adaptor protein 15 (PEA15), S100 calcium-binding protein A9 (S100A9), serum amyloid A (SAA), serpin family A member 3 (SERPINA3), integrin subunit beta 2 (ITGB2), Fc fragment of IgG-binding protein (FCGBP), NEDD8-MDP1 protein (NEDD8-MDP1), charged multivesicular body protein 4B (CHMP4B), exportin-2 (XPO2), alpha-2-macroglobulin-like protein 1 (A2ML1), amyloid-beta precursor protein (APP), biotinidase (BTD), CD44 antigen (CD44), collagen alpha-1(XII) chain (COL12A1), collagen alpha-1(I) chain (COL1A1), collagen alpha-1(III) chain (COL3A1), catenin beta-1 (CTNNB1), FAM107B protein (FAM107B), glycoprotein phospholipase D (GPLD1), progranulin (GRN), insulin-like growth factor-binding protein complex acid-labile subunit (IGFALS), lamin-B1 (LMNB1), LIM domain only protein 7 (LMO7), mucin-4 (MUC4), nicotinamide phosphoribosyltransferase (NAMPT), Ras-related protein (RAB21), serpin H1 (SERPINH1), SPP1 (osteopontin), transmembrane protease serine 11E (TMPRSS11E), and von Willebrand factor (VWF). **Claim 2** The method according to claim 1, wherein the sample is a Papanicolaou smear, specifically the fluid contained in the Papanicolaou smear and / or endocervical mucus. **Claim 3** The method according to claim 1, comprising the step of determining the presence and / or expression level of two, three, or four of said proteins. **Claim 4** The method according to claim 3, comprising the step of determining the presence and / or expression level of a protein in at least one binary set listed in either Table 4 or Table 6 in the isolated sample. **Claim 5** The method according to claim 3, comprising the step of determining the presence and / or expression level of a protein in at least one ternary set listed in either Table 5 or Table 7 in the isolated sample. **Claim 6** The method according to claim 1, comprising the step of determining the presence and / or expression level of TIMP2 in the isolated sample. **Claim 7** a) determining the expression levels of one or more proteins selected from TIMP2, AGRIN, MDK, APOB, C1QA, FN1, SERPIND1, APOF, APOC1, CCT6A, LBP, SAA4, ITIH2, LCN2, C4BPA, C1R, FGFBP1, SPRR1B, SPRR1A, LCN2, PLBD1, CD44 antigen, FCGBP, EPS8L1, ANXA3, MMP8, NEDD-8 protein, CAMP, HSPE1, CALU, LDHA, PIGR, KRT8, PPL, STMN1, CAPS, CA1, VIM, TCP1, ANXA7, IMPA1, STX7, ITIH2, LGALS1, ATP6V1G1, PKM, GYG1, LSP1, HCLS1, PEAR15, S100A9, SCEL, SERPINA3, ITGB2, FCGBP, NEDD8-MDP1, CHMP4B, XPO2, A2ML1, APP, BTD, CD44, COL12A1, COL1A1, COL3A1, CTNNB1, FAM107B, GPLD1, GRN, IGFALS, LMNB1, LMO7, MUC4, NAMPT, RAB21, SERPINH1, SPP1, TMPRSS11E, and VWF in a sample isolated from the female reproductive tract, in vitro; b) comparing the levels of said one or more proteins of step (a) and, if determined, the levels of one or more other such proteins to corresponding reference values or reference intervals for each protein selected from values or value intervals from subjects suffering from endometrial cancer, and / or comparing to a cut-off value for discriminating endometrial cancer from other endometrial disorders or (i.e., healthy) endometrial states, wherein if at least the levels of said one or more proteins of step (a) are within the corresponding values or value intervals from subjects suffering from this cancer, and / or if at least the levels of said one or more proteins of step (a) relative to the corresponding cut-off value are classified into the endometrial cancer group, diagnosing the subject with endometrial cancer; The method according to claim 1, comprising: Claim 8 A method for diagnosing recurrence or risk of recurrence of endometrial cancer, and further comprising the step of determining the presence and / or expression level of one or more of the proteins selected from the group consisting of MUC1, PRSS8, PNP, APEH, MUC16, C9, SERPINC1, SERPINA1, F2, AMBP, HP, SERPINA3, CFB, ORM2, CAT, GNAI2, A1BG, FN1, C7, ASTRGL1, B4GALT1, CAPS, CBX3, CD163, CDV3, DMBT1, DSG3, EHD1, GOLM1, MUC5AC, NME1, NT5E, PDLIM5, RDX and VASP in the isolated female sample, the method according to claim 1.

9. A method for diagnosing endometrial cancer subtypes, and further comprising the step of determining the presence and / or expression level of one or more of the proteins selected from the group consisting of LBP, VWF, GPLD1, SAA4, APOF, C4BPA, SPRR1A, SERPIND1, APOB, SCEL, LCAT, SERPINA3, LMO7, C1R, MUC4, FN1, SPRR1B, C1QA, ITIH2, TIMP2, APOC1, GRN, ANXA3, S100A9, PLBD1, PIGR, SERPINH1, HSP E1 in the isolated female sample, the method according to claim 1.

10. The method according to claim 1, wherein the expression level is determined at the protein level.

11. The method according to claim 10, wherein the protein level is determined by an assay or technique selected from the group consisting of immunoassay, bioluminescence assay, fluorescence assay, chemiluminescence assay, electrochemical assay, mass spectrometry, and combinations thereof.

12. The method according to claim 11, wherein the expression level of the protein is determined using an antibody or a fragment thereof capable of binding to the protein.

13. The method according to claim 12, wherein the antibody or a fragment thereof forms part of a kit.

14. Use of one or more proteins selected from TIMP2, AGRIN, MDK, APOB, C1QA, FN1, SERPIND1, APOF, APOC1, CCT6A, LBP, SAA4, ITIH2, LCN2, C4BPA, C1R, FGFBP1, SPRR1B, SPRR1A, LCN2, PLBD1, CD44 antigen, FCGBP, EPS8L1, ANXA3, MMP8, NEDD-8 protein, CAMP, HSPE1, CALU, LDHA, PIGR, KRT8, PPL, STMN1, CAPS, CA1, VIM, TCP1, ANXA7, IMPA1, STX7, ITIH2, LGALS1, ATP6V1G1, PKM, GYG1, LSP1, HCLS1, PEA15, S100A9, SCEL, SERPINA3, ITGB2, FCGBP, NEDD8-MDP1, CHMP4B, XPO2, A2ML1, APP, BTD, CD44, COL12A1, COLIA1, COL3A1, CTNNB1, FAM107B, GPLD1, GRN, IGFALS, LMNB1, LMO7, MUC4, NAMPT, RAB21, SERPINH1, SPP1, TMPRS11E, and VWF as in vitro markers for the diagnosis and / or prognosis determination of endometrial cancer in a sample selected from gynecological sample collection including one or more of cervical mucus, cytology, Papanicolaou smear sample, endometrial biopsy, uterine fluid, uterine lavage fluid from a female reproductive tract portion including one or more of the vulva, vagina, cervix, uterus, fallopian tubes, and ovaries.

15. Use of a kit for the diagnosis and / or prognosis determination of endometrial cancer as defined in claim 1, wherein the kit comprises a solid support and means for detecting the presence of and / or determining the expression level of the protein. Use of the kit, wherein the protein is one or more proteins selected from TIMP2, Agrin, MDK, ApoB, C1QA, FN1, SERPIND1, ApoF, ApoC1, CCT6A, LBP, SAA4, ITIH2, LCN2, C4BPA, C1R, FGFBP1, SPRR1B, SPRR1A, LCN2, PLBD1, CD44 antigen, FCGBP, EPS8L1, ANXA3, MMP8, NEDD-8 protein, CAMP, HSPE1, CALU, LDHA, PIGR, KRT8, PPL, STMN1, CAPS, CA1, VIM, TCP1, ANXA7, IMPA1, STX7, ITIH, LGMALS1, ATP6V1G1, PKM, GYG1, LSP1, HCLS1, PEA15, S100A9, SCEL, SERPINA3, ITGB2, FCGBP, NEDD8-MDP1, CHMP4B, XPO2, A2ML1, APP, BTD, CD44, COL12A1, COL1A1, COL3A1, CTNNB1, FAM107B, GPLD1, GRN, IGFALS, LMN1, LMO7, MUC4, NAMPT, RAB21, SERPINH1, SPP1, TMPRSS11E, and VWF.

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