BR122024027006A2 - METHOD FOR DIAGNOSING ENDOMETRIAL CANCER IN A SAMPLE OF UTERINE FLUID, KIT AND COMPUTER-IMPLEMENTED METHOD FOR PERFORMING SAID DIAGNOSTIC METHOD, AND METHOD FOR DECIDING OR RECOMMENDING A MEDICAL REGIMEN - Google Patents

Abstract

The present invention provides a method for diagnosis and prognosis of endometrial cancer in readily available isolated samples by detecting the level of expression of one or more proteins. In particular from uterine fluid samples. The invention also provides kits comprising means for detecting said proteins for use in the diagnosis and prognosis of the disease. A pharmaceutical composition and a therapeutic method, each of which is distinguished by the fact that an antibody-drug conjugate and an immune checkpoint inhibitor are administered in combination, wherein the antibody-drug conjugate is formed by attaching a drug ligand represented by formula (1) (wherein A represents an antibody-binding position) to an antibody via a thioether bond; and a pharmaceutical composition and a therapeutic method, each of which is distinguished by the fact that the antibody-drug conjugate is contained and is also distinguished by being used for the treatment of a disease that can be attenuated through an anti-tumor immunity activating effect.

Description

[001] Divided from BR 11 2020 000926 1, filed on 07/20/2018.

[002] This application claims the benefit of European Patent Application EP17382483.0 filed on July 21, 2017.

FIELD OF TECHNIQUE

[003] The present invention relates to the diagnosis and prognosis of endometrial cancer.

TECHNIQUE FUNDAMENTALS

[004] 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, considering 61,380 diagnosed cases and 10,920 estimated deaths in 2017 in the United States. Currently, 70% of EC cases are diagnosed in the early stages of the disease where the tumor is still located in the endometrium and is associated with an overall 5-year survival rate of 96%. However, 30% of patients with EC are diagnosed only at an advanced stage of the disease associated with a drastic reduction in the 5-year survival rate, which is reduced to 67% when myometrial invasion and/or lymph node involvement is already present and to 18% in cases of distant metastasis. Therefore, improving early diagnosis is a key issue to appropriately manage EC and reduce mortality associated with the disease.

[005] Early detection of patients with EC is favored by the presence of symptoms such as abnormal vaginal bleeding present in 93% of women diagnosed with EC. However, many other benign disorders generate similar symptoms. Discrimination of patients with benign endometrial pathologies and EC is achieved only after a tedious diagnostic process consisting of 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 pipelle biopsy and is obtained by a minimally invasive aspiration of endometrial fluid from within the uterine cavity. Because current diagnostic procedures in uterine aspirates rely on the presence of cellular material, this process unfortunately has a diagnostic failure and an associated inadequate sampling rate of 8% and 15%, respectively. This is increased in postmenopausal women by up to 12% and 22%. In these cases, a hysteroscopy-guided biopsy needs to be performed, as this invasive technique carries an increased risk of complications, including uterine perforation, hemorrhage, and possible injury to other organs.

[006] To date, many studies have been conducted to identify EC protein biomarkers, mainly in tissue and serum samples (see, for example, 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, or 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). None of them have translated into clinical utility.

[007] Although biopsies should provide information on tumor histology and tumor grade to aid in risk stratification of patients with EC and guide surgical testing procedures, unfortunately, the limited number of cells available for examination and high interobserver variability in pathologic interpretation result in 40-50% discordance in histotype and grade of EC between biopsies and final hysterectomy specimens. Therefore, identification of sensitive, specific, and reproducible biomarkers that improve diagnosis, prognosis, and preoperative assessment of histologic type and grade of EC tumors is critical to appropriately manage patients with EC and reduce the mortality and morbidity associated with this disease.

[008] Among the subtypes or variety of manifestations of EC, endometroid endometrial cancer (EEC) is the most common histology in EC and has a good prognosis when compared to cases of non-endometroid EC (NEEC). NEEC represents about 20% of all EC cases, but accounts for more than 50% of recurrences and deaths resulting from EC. Among NEEC, serous EC (SEC) is the most common subtype. There are no tests available to discriminate between these two histologies with different results.

[009] In conclusion, despite the efforts applied, there is still a need for biomarkers that allow the diagnosis, and even prognosis of endometrial cancer with high sensitivity and specificity, in particular, the need for biomarkers for clinical practice. SUMMARY OF THE INVENTION

[010] The inventors have determined that certain protein markers detectable in exosomes isolated from uterine fluid provide valuable diagnostic information in endometrial cancer (EC). Many of these proteins have been validated in the uterine fluid itself without isolation of the exosome fraction. Therefore, the proteins are relevant diagnostic biomarkers that allow discrimination with high sensitivity and specificity between EC and non-cancer controls when analyzed in uterine fluid.

[011] Furthermore, the inventors have determined that some proteins that can be detected in said uterine fluid, and also in the exosome fraction of said uterine fluid, are relevant prognostic biomarkers of endometrial cancer (EC). These proteins allow discrimination between endometrial cancer subtypes with high sensitivity and high specificity, and therefore allow minimizing the risk of false positive and false negative classification among these subtypes.

[012] Furthermore, since proteins are detectable in samples that are obtained without invasive practices, such as in uterine fluid samples, which are, successively, common sample types in the diagnosis of EC, this represents a real advantage and an improvement in the clinical practice of classifying and managing patients with EC.

[013] In previous studies, the inventors have demonstrated that the fluid fraction of uterine aspirates has an important value for screening EC protein biomarkers (see, for example, Martínez-Garcia E, et al. “Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples”, Oncotarget -2016, vol. no. 7(33), pp.:53102-53114). This paper shows a study conducted with a sequential workflow based on LC-PRM, illustrating the importance of a biomarker verification phase to bridge the gap between clinical discovery and practice. This study highlighted the benefit of 26 proteins for diagnosing EC patients from non-EC patients (controls).

[014] In relation to novel diagnostic biomarkers for EC identified in uterine fluid exosomes, one aspect of the invention is a method for diagnosing EC, the method comprising determining the expression level of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 in a sample of uterine fluid from the female genital tract.

[015] Another aspect of the invention is the use of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 as an in vitro marker for diagnosing EC in a sample of uterine fluid from the female genital tract. This aspect can also be formulated as an in vitro method for detecting one or more markers of endometrial cancer in an individual, comprising: (a) obtaining a sample of fluid from the female genital tract; and (b) detecting in the sample an amount of at least one endometrial cancer marker selected from AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 in a sample of uterine fluid from the female genital tract, with this marker providing information on the diagnosis of EC. In general terms, it covers the use of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 as an in vitro marker for diagnosing endometrial cancer in a uterine fluid sample isolated from the female genital tract.

[016] The diagnosis of EC with one or more proteins listed above can be performed by using kits that comprise means for determining the level of expression of these proteins. Therefore, it is also an aspect of the invention a kit comprising a solid support and means for detecting the expression level of one or more of the following proteins AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166.

[017] Another aspect of the invention is the use of said kits, which comprise means for determining one or more of the proteins defined above, for the diagnosis of EC.

[018] An additional aspect related to the diagnosis of EC provides a method for identifying an individual suspected of suffering from EC, the method comprising: a) determining, in vitro, the level of expression of one or more proteins selected from the group consisting of: AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, CLIC1, BCAM and KPYM, in a sample of female uterine fluid; b) optionally determining, in vitro, the expression level of one or more proteins selected from the group consisting of: AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RS16, RSSA, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 in a fraction containing exosome isolated from uterine fluid; and c) compare the level of step (a) and optionally that of step (b) with a reference control level, whereby if the level determined in step (a) and optionally that of step (b) is greater than the reference control level, it is indicative that the individual is suspected of suffering from endometrial carcinoma.

[019] In a further aspect, the present invention provides a method for deciding or recommending whether to initiate a medical regimen of an individual suspected of suffering from endometrial carcinoma, said method comprising the steps of: a) determining, in vitro, the expression level of one or more proteins selected from the group consisting of: AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9 and KPYM in a female uterine fluid sample; b) optionally determining, in vitro, the expression level of one or more proteins selected from the group consisting of: AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RS16, RSSA, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 in a fraction containing exosome isolated from uterine fluid; and c) comparing the level of step (a) and optionally that of step (b) with a reference control level, wherein if the level determined in step (a) and optionally that of step (b) is higher than the reference control level, it is indicative that the individual is suspected of suffering from endometrial carcinoma. wherein: i) if the individual is diagnosed as suffering from endometrial carcinoma, or is suspected of suffering from endometrial carcinoma, then initiation of the medical regimen is recommended, and ii) if the patient is diagnosed as not suffering from endometrial carcinoma, follow-up is optionally carried out taking into account the result of an examination of the patient by a physician.

[020] By determining the level of marker in a test sample, the skilled person can further establish which will be the most appropriate therapy that can be recommended, because the level detected in the sample can reflect the extent (i.e., the severity) of the disease.

[021] Regarding the new prognostic biomarkers for EC in uterine fluid, a first aspect of the invention consists of a method for differential diagnosis of endometrial cancer, the method comprising determining the level of expression of CTNB1 in a sample of uterine fluid from the female genital tract.

[022] Another aspect consists of a method for prognosis of endometrial cancer, the method comprising determining the level of expression of one or more of the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 in a sample of uterine fluid from the female genital tract.

[023] The methods provide important diagnostic and prognostic information, since an accurate prognosis can be made because two different histological manifestations of EC, i.e. endometrioid endometrial cancer (EEC) and non-endometrioid endometrial cancer (NEEC), can be differentiated, with EEC being likely to have a better outcome (i.e. better prognosis). In other words, the determination of proteins in the uterine fluid sample allows to diagnose EEC and NEEC differently.

[024] As will be described in the examples below, the above-mentioned proteins allowed the diagnosis of EC or an accurate prognosis of EC, in terms of which they were differentially expressed in samples isolated from patients suffering from EEC and from patients suffering from NEEC. Proteins with diagnostic value could be differentially detected due to differential expression in uterine fluid samples from individuals with EC relative to non-EC samples (healthy controls). Proteins with prognostic value were significantly increased in EEC subtype compared to levels in NEEC subtype, with the exception of CAPG which was increased in NEEC tumors. Thus, they allowed the classification of tumors of the most prevalent histological subtypes. Therefore, the proteins are usable to improve diagnosis, prognosis and/or preoperative risk assessment, and to aid in the prediction of optimal surgical treatment. Similarly, as indicated below, some protein panels (protein combinations) have been developed with these and other proteins that allow accurate discrimination of EC vs. non-EC samples, and also for accurate discrimination of histological types of EC (AUC of 0.99). In combination with histopathological examination, these panels are expected to eliminate the need for more invasive diagnostic sampling and help predict optimal surgical treatment for patients with EC.

[025] Therefore, in general terms, one aspect of the invention consists of a method of prognosis of endometrial cancer (EC), the method comprising determining the level of expression of one or more of the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 in a sample of uterine fluid from the female genital tract.

[026] A second aspect of the invention is the use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 as an in vitro marker for prognosis of EC in a sample of uterine fluid from the female genital tract. This aspect can also be formulated as an in vitro method for detecting one or more markers of endometrial cancer in a subject, comprising: (a) obtaining a sample of fluid from the female genital tract; and (b) detecting in the sample an amount of at least one endometrial cancer marker selected from PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, said marker providing differential diagnostic information for EEC and NEEC. In general terms, it encompasses the use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3 and MX1 as an in vitro marker for endometrial cancer prognosis in a uterine fluid sample isolated from the female genital tract.

[027] In a third aspect, the invention provides the use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 for the prognosis of endometrial cancer in a method of the first aspect. That is, the proteins are used to differentially diagnose EEC and NEEC.

[028] Above all, the protein biomarkers of the present invention can be assessed by easy and low-cost methods, such as immunochemistry, chemiluminescent assay, or ELISA, platforms that are widely available in hospitals. Consequently, these protein biomarkers can be easily implemented as routine clinical diagnostic and/or prognostic kits with reduced costs to the healthcare system. Furthermore, a biomarker-based diagnostic kit test provided by the present invention can improve the current diagnostic and prognostic process by conferring on uterine aspirates the ability to provide valuable diagnostic and prognostic information of the disease.

[029] Therefore, in a fourth aspect the present invention provides the use of a kit for the prognosis of EC, the kit comprising a solid support and means for detecting the expression level of one or more of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, and, optionally, means for detecting the expression level of one or more of the following proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[030] This aspect may also be formulated as a kit comprising a solid support and means for detecting the expression level of one or more of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1, and optionally means for detecting the expression level of one or more of the following proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA for use in the prognosis of EC.

[031] New kits have been developed that facilitate the implementation of the methods and uses of the invention. Thus, in a fifth aspect, the invention also provides a kit comprising a solid support and means for detecting the expression level of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[032] An additional aspect consists of a kit comprising a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[033] In a further aspect, the present invention provides the use of means for determining the expression level of: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1 for the prognosis of endometrial cancer in the method of the first aspect of the invention. Thus, these are means for the differential diagnosis of EEC and NEEC. Also provided is the use of means for determining the level of expression of: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, and optionally means for determining the level of expression of XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA, for the prognosis of endometrial cancer in the method of the first aspect of the invention.

[034] In a further aspect, the present invention provides a method for identifying an individual suspected of suffering from endometrial cancer and for further identifying the EC subtype, differentially diagnosing EEC from NEEC, the method comprising: a) determining, in vitro, the expression level of one or more proteins selected from the group consisting of: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1; and optionally the expression level of one or more of XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA proteins in a uterine fluid sample from the individual's female genital tract; and b) comparing the level from step (a) with a reference control level, wherein if the level determined in step (a) is greater than the reference control level for PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1 and less than the reference control level for CAPG, this is indicative that the individual is suspected of suffering from NEEC and suffers from EEC.

[035] In this aspect of the invention, the reference control level is indistinctly chosen from non-EC and NEEC samples. As set out above, the expression levels in EEC are higher than the expression levels in NEEC, except for CAPG. In addition, the levels of CADH1, CAPG, CTNB1 and CD44 are also higher in non-EC (non-cancer) control samples.

[036] In a further aspect, the present invention provides a method for deciding or recommending whether to initiate a medical regimen of a subject suffering from endometrial cancer based on prognosis, the method comprising the steps of: a) determining, in vitro, the expression level of one or more proteins selected from the group consisting of: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, and, optionally, the expression level of one or more proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA in a sample of uterine fluid from the female genital tract of the subject; individual; and b) establishing the prognosis of said EC by distinguishing EEC and NEEC, if the protein level in the test sample is greater than a reference control level for PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, and less than the reference control level for CAPG; wherein: i) if the individual is diagnosed as suffering from endometrial cancer, or is suspected of suffering from endometrial cancer, and the individual is differentially diagnosed as suffering from EEC, then initiation of the medical regimen for EEC is recommended; ii) if the individual is diagnosed as suffering from endometrial cancer, or is suspected of suffering from endometrial cancer, and the individual is differentially diagnosed as suffering from NEEC, then initiation of the medical regimen for NEEC is recommended; and by determining the marker level in a test sample, the skilled individual can additionally establish which is the most appropriate therapy that can be recommended, because the level detected in the sample can reflect the aggressiveness of the disease.

[037] Finally, another aspect of the present invention consists in providing an algorithm for performing any of the diagnostic and/or prognostic methods as defined in the previous aspects. In the sense of the invention, the term “algorithm” is also synonymous with panel or decision diagrams, predictors and combinatorial analyses of data to correctly categorize an individual sample.

[038] According to aspects and embodiments of the invention, the diagnosis and prognosis of EC can be performed using a mathematical algorithm that evaluates a detectable level of biomolecules, proteins, antibodies, and/or mRNA, comprising one or more of the previously described EC diagnostic and prognostic biomarkers, either in conjunction with, or independently of, other clinical parameters, to correctly categorize an individual sample as originating from a healthy patient, a patient with a non-malignant endometrial disease, a patient with a pre-malignant endometrial disease, a patient with endometrial cancer, or, as previously described, further categorize an individual sample as originating from an individual with a specific histological subtype of endometrial cancer, an individual having a specific histological grade or stage of the disease, or an individual with a specific molecular subtype of EC.

[039] The classification algorithm may be as simple as determining whether or not the amount of a specific biomarker or subset of medical biomarkers is above or below a particular cut-off number. Where multiple biomarkers are used, the classification algorithm may be a linear regression formula. Alternatively, the classification algorithm may be the product of any of a number of learning algorithms. In the case of complex classification algorithms, it may be necessary to perform the algorithm on the data, thereby determining the classification, using a computer, e.g., a programmable digital computer. In either case, the status may then be recorded on a tangible medium, e.g., in a computer-readable format such as a memory unit or disk or simply printed on paper. The result could also be reported on a computer screen. This algorithm is used as a diagnostic and/or prognostic method, and in particular forms part of the kits for performing the methods disclosed in the first aspects.

[040] After determining the expression level of one or more of the proteins for the diagnosis and/or prognosis of EC based on said computed levels, scores and/or values, a decision is made between the options of suffering from premalignant lesions or not, and/or EC, and/or between the options of suffering from different subtypes of EC.

BRIEF DESCRIPTION OF THE DRAWINGS

[041] Figure 1 shows a ROC curve for markers that distinguish EEC from NEEC, specifically cases of NEEC are serous endometrial cancer (SEC). The sensitivity and specificity of CTNB1, XPO2 and CAPG individually; and of the combination (or protein panel) of the three are shown.

DETAILED DESCRIPTION OF THE INVENTION

[042] All terms as used in this application, except where otherwise stated, are to be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in this application are as set forth below and are intended to apply uniformly throughout the specification and claims unless a definition expressly set forth to the contrary provides a more comprehensive definition.

[043] The present invention provides novel biomarkers for the diagnosis and prognosis of endometrial cancer in female genital tract fluid.

[044] The term “diagnosis” is familiar to those skilled in the art. As used herein, “diagnosis” is understood to mean becoming aware of a complication or risk of a particular medical condition in an individual; determining the nature of the disease or condition; or distinguishing one disease or condition from another. It refers both to the process of attempting to determine or identify the possible disease or disorder, and to the judgment reached by that process. A diagnosis, in the sense of a diagnostic procedure, may be thought of as an attempt to classify an individual condition into separate and distinct categories allowing medical decisions about treatment and prognosis to be made. Accordingly, a diagnostic judgment is generally stated in terms of a disease or other condition. However, a diagnosis may take many forms. It may be a matter of detecting the presence and naming the disease, injury, dysfunction, or disability. It may be an exercise in assigning a category for management or for prognosis. It may indicate a degree of abnormality on a continuum or type of abnormality in a classification. In general terms, diagnostic markers listed in this description are those proteins differentially detected in expression level in samples isolated from controls (individuals without cancer) versus endometrial cancer samples (including various types of EC).

[045] The in vitro method of the first aspect of the invention may be performed with a sample from: (a) an asymptomatic individual, (b) an individual who has already been identified as being suspected of suffering from endometrial cancer, (c) an individual already diagnosed with endometrial cancer, as a complementary confirmatory diagnostic assay or (d) an individual at high risk of suffering from the disease.

[046] In the present invention, the term “reference control level” referred to in the methods of any of the aspects of the invention should be understood as a predefined value of a given molecular marker or a combination of the given molecular markers, in the present case any of the proteins listed in the first or second aspects as well as in particular embodiments, which is derived from the levels of said marker or molecular markers in a sample or group of samples. If the expression level is determined at the protein level, then the “reference expression level” is a predefined value of protein quantity, while if the expression level is determined at the mRNA level, then the “reference expression level” is a predefined value of mRNA quantity. The samples are taken from an individual or group of individuals in which the presence, absence, stage, subtype or histological grade, or course of the disease has been appropriately performed beforehand. This value is used as a threshold to discriminate individuals in whom the condition to be analyzed is present from those in whom the condition is absent (i.e., an individual with endometrial cancer from individuals without endometrial cancer), to determine the histological subtype of the disease, the risk of developing or suffering from endometrial carcinoma, among others. This reference control level is also useful in determining whether the individual needs to start a medical regimen and how effective the regimen is. The individual or individuals from which the “reference control level” is derived may include individuals in whom the condition is absent, individuals in whom the condition is present, or both. Individuals skilled in the art, using general knowledge, are able to choose the most appropriate individual or group of individuals to obtain the reference control level for each of the methods of the present invention. Methods for obtaining the reference value from the group of selected individuals are well known in the state of the art (Burtis C. A. et al., 2008, chapter 14, section “Statistical Treatment of Reference Values”). In a particular case, “reference control level” is a cut-off value defined by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). As those skilled in the art will appreciate, an optimal cut-off value will be defined according to the particular applications of the diagnostic or prognostic method: purpose, target population for diagnosis or prognosis, balance between specificity and sensitivity, etc.

[047] “Prognosis” as used herein refers to the prediction of the likely progression and outcome of a disease. It includes: neoplasm classification (an attempt to express in replicable terms the level of cellular differentiation in neoplasms as increasing anaplasia correlates with the aggressiveness of the neoplasm), neoplasm testing (an attempt to express in replicable terms the extent of the neoplasm in the patient), histologic subtype of neoplasm, and molecular subtype of neoplasm. As used herein, prognosis means, in particular embodiments, the differentiation between endometrioid endometrial cancer and non-endometrioid endometrial cancers.

[048] The term “female genital tract fluid sample” refers to a fluid produced by the uterine organ that forms part of the female genital tract and that has been collected by aspiration, such as vacuum aspiration (i.e., “uterine aspirate sample”), or by a Cornier Pipelle, and/or any other method that recovers fluid from the uterine cavity. It therefore includes uterine lavages. According to the present invention, the aspiration of the fluid is, in particular, carried out without a prior saline infusion step. That is, the term “aspirate” does not cover those samples resulting from uterine lavages.

[049] In the present invention, “exosomes” referred to interchangeably as “extracellular vesicles”, “microvesicles”, “exosome-like vesicles” or “uterosomes” are cell-derived vesicles that are present in many eukaryotic fluids, including blood, urine, and culture medium of cell cultures. The reported diameter of exosomes is between 30 and 100 nm, which is larger than low-density lipoproteins (LDL) but much smaller than, for example, red blood cells. Exosomes are released from the cell when multivesicular bodies fuse with the plasma membrane or released directly from the plasma membrane. Exosomes can potentially be used for diagnosis, prognosis, therapy, and as biomarkers for health and disease. For “exosome-containing fraction isolated from UA” should be understood as any purified fraction of uterine aspirate that primarily comprises exosomes. Non-limiting examples of methods for isolating exosomes from uterine aspirates will be detailed below.

[050] In a particular embodiment of the aspect relating to novel diagnostic biomarkers for EC identified in uterine fluid exosomes, optionally in combination with any prior or subsequent embodiments, an aspect of the invention consists of a method of diagnosing EC in which the sample is a uterine fluid aspirate (UA).

[051] In another particular embodiment of the EC diagnostic method, optionally in combination with any prior or subsequent embodiments, the sample is an exosome-containing fraction isolated from uterine aspirate (uterine fluid sample), and the method comprises determining the expression level of one or more proteins selected from the group consisting of: AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RSSA, RS16, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166.

[052] In another embodiment, the method of diagnosing EC comprises determining the expression level of one or more proteins selected from AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RS16, RSSA, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3, and CD166 in a exosome-containing fraction isolated from UA; and the expression level of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, and KPYM in a sample of uterine fluid from the female genital tract, the latter sample without isolation or purification of exosomes.

[053] In a particular embodiment, the method of diagnosing EC comprises determining the expression level of two or more proteins, more particularly, three or more proteins, and even more than four proteins.

[054] In another particular embodiment, the method of diagnosing EC further comprises determining the expression level of one or more proteins selected from the group consisting of: PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, PDIA1, KPYM, ANXA2, and FABP5.

[055] In another particular embodiment, optionally in combination with any prior or subsequent embodiment, the method of diagnosing EC comprises determining the expression level of at least one set of proteins (i.e., biomarkers) selected from the group consisting of: AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; and AGRIN, MMP9, in an exosome-containing fraction isolated from UA; and/or determining the expression level of at least one set of proteins (i.e., biomarkers) selected from the group consisting of: MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BCAM, MMP9; MMP9, AGRIN in a uterine aspirate.

[056] In yet another particular embodiment, optionally in combination with any prior or subsequent embodiment, the method of diagnosing EC further comprises determining the expression level of at least one set of proteins (i.e., biomarkers) selected from the group consisting of the list in Table D (illustrated at the end of this description).

[057] Another aspect of the invention is the use of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 as an in vitro marker for diagnosing EC in a female genital tract fluid sample. This aspect can also be formulated as an in vitro method for detecting one or more endometrial cancer markers in an individual, comprising: (a) obtaining a female genital tract fluid sample; and (b) detecting in the sample an amount of at least one endometrial cancer marker selected from AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 in a sample of uterine fluid from the female genital tract, with this marker providing information on the diagnosis of EC. In general terms, it covers the use of one or more proteins selected from the group consisting of AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 as an in vitro marker for diagnosing endometrial cancer in a uterine fluid sample isolated from the female genital tract.

[058] The diagnosis of EC with one or more proteins listed above can be performed by using kits that comprise means for determining the level of expression of these proteins. In a particular embodiment of the diagnostic kit, the same comprises means for detecting the expression level of one or more of the following proteins AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RSSA, RS16, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB3 and CD166 from a fraction containing exosome isolated from UA.

[059] In another particular embodiment, optionally in combination with any of the preceding or subsequent embodiments, the kits further comprise means for detecting the level of expression of one or more of the following proteins PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, PDIA1, KPYM, ANXA2, and FABP5.

[060] Likewise, in another more particular embodiment, the kits comprise means for detecting the expression level of one or more proteins that provide particular prognostic information (i.e., prognostic biomarkers) and selected from PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, LDHA, AGR2, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3 and MX1.

[061] Another aspect of the invention is the use of said kits, which comprise means for determining one or more proteins defined above, for the diagnosis of EC.

[062] In another particular embodiment of the use of the kits, the means for detecting the level of expression of the proteins are means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescent assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[063] The Uniprot database accession numbers of all proteins listed in this document correspond to versions accessible on July 7, 2017. In addition, they are indicated in the Examples Tables.

[064] AGRIN has Uniprot database accession number O00468. This protein is a heparin sulfate basal lamina glycoprotein involved in the formation and maintenance of the neuromuscular junction.

[065] MVP, also known as major vault protein, has Uniprot database accession number Q14764. It is involved in signal transduction.

[066] VAMP8, also known as vesicle-associated membrane protein 8, has Uniprot database accession number Q9BV40. It is a soluble N-ethylmaleimide-sensitive attachment factor protein receptor (SNARE) involved in autophagy through direct control of autophagosome membrane fusion with the lysosome membrane.

[067] SYIC, also known as isoleucine tRNA ligase, has Uniprot database accession number P41252.

[068] RAB8A, also known as Ras-related protein Rab-8A, has Uniprot database accession number P61006. It is a Rab small GTPase involved in intracellular membrane diapedesis.

[069] MX1, also known as interferon-induced GTP protein Mx1, has Uniprot database accession number P20591. It has antiviral activity against a broad range of RNA viruses and some DNA viruses.

[070] IMB1, also known as importin subunit beta-1, has Uniprot database accession number Q14974. It is involved in nuclear protein import.

[071] SMD3, also known as small nuclear ribonucleoprotein Sm D3, has Uniprot database accession number P62318. It is a component of the spliceosome.

[072] ILF2, also known as Interleukin 2 enhancer binding factor, has Uniprot database accession number Q12905. It is involved in the regulation of the IL2 gene.

[073] TERA, also known as transitional endoplasmic reticulum ATPase, has Uniprot database accession number P55072. This protein is involved in the formation of the transitional endoplasmic reticulum.

[074] RL11, also known as 60S ribosomal protein L11, has Uniprot database accession number P62913. This protein is a component of the ribosome and is therefore involved in protein synthesis in the cell.

[075] BCAM, also known as basal cell adhesion molecule, has Uniprot database accession number P50895. This protein is a Laminin alpha-5 receptor.

[076] ANXA2, also known as Annexin A2, has Uniprot database accession number P07355. This protein is a calcium-regulated membrane-binding protein that inhibits PSCK9-enhanced LOLR degradation.

[077] LAT1, also known as neutral amino acid transporter small subunit 1, has Uniprot database accession number Q01650. This protein is involved in cellular amino acid uptake.

[078] RUVB1, also known as Pontin 52 or INO80 complex subunit H, has Uniprot database accession number Q9Y265. This protein is a component of the histone acetyltransferase NuA4 complex, which is involved in the transcriptional activation of selected genes primarily by acetylation of nucleosomal histones H4 and H2A.

[079] SH3L3, also known as SH3 domain-binding protein glutamic acid-rich type 3 or SH3 domain-binding protein 1, has Uniprot database accession number Q9H299. This protein may be involved in the modulation of glutaredoxin activity.

[080] RPL13A, also known as 60S ribosomal protein, has Uniprot database accession number P40429. It is involved in selective translational inhibition of interferon gamma-induced transcription in inflammatory processes.

[081] RS16, also known as 40S ribosomal protein S16, has Uniprot database accession number P62249.

[082] RSSA, also known as 40S ribosomal protein SA or Laminin receptor 1, has Uniprot database accession number P08865. It requires assembly and/or stability of the 40S ribosomal subunit.

[083] RL12, also known as 60S ribosomal protein L12, has Uniprot database accession number P30050.

[084] RL29, also known as 60S ribosomal protein L29, has Uniprot database accession number P47914. This protein is a component of the large ribosomal subunit.

[085] PPIA, also known as peptidyl-prolyl cis-trans isomerase A, cyclophilin A or rotamase A, has Uniprot database accession number P62937. This protein is involved in protein folding.

[086] AGR2, also known as anterior gradient protein 2 homolog or HPC8, has Uniprot database accession number O95994. This protein is involved in MUC2 post-transcriptional synthesis and secretion.

[087] PODXL, also known as Podocalyxin or GCTM-2 antigen, has Uniprot database accession number O00592. It is involved in the regulation of adhesion, cell morphology and cancer progression.

[088] CD59, also known as CD59 glycoprotein or MAC-inhibitory protein, has Uniprot database accession number P13987. It is involved in inhibiting the action of the complement membrane attack complex (MAC).

[089] TACD2, also known as tumor-associated calcium signal transducer 2 or Trop-2 cell surface glycoprotein, has Uniprot database accession number P09758. This protein may function as a growth factor receptor.

[090] FAS, also known as fatty acid synthase, has Uniprot database accession number P49327. It is involved in the formation of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH.

[091] SORT, also known as Sortilin or Neurotensin receptor 3, has Uniprot database accession number Q99523. This protein functions as a sorting receptor in the Golgi compartment and as a clearance receptor on the cell surface.

[092] ADA10, also known as Disintegrin and metalloproteinase domain-containing protein 10, has Uniprot database accession number O14672. It is involved in the proteolytic release of several cell surface proteins, such as the membrane-binding precursor of TNF-alpha.

[093] PGBM, also known as basement membrane-specific heparin sulfate proteglycan core protein or Perlecan, has Uniprot database accession number P98160. It is involved in repair of electrostatic negative membrane charge and vascularization.

[094] ITA3, also known as Integrin alpha-3 or VLA-alpha subunit 3, has Uniprot database accession number P26006. It is a receptor for fibronectin, laminin, collagen, epiligrin, thrombospondin, and CSPG4.

[095] RUXE, also known as small nuclear ribonucleoprotein E, has Uniprot database accession number P62304. This protein is involved in histone 3' end processing. It may indirectly play a role in hair development.

[096] PSMD2, also known as 26S proteasome non-ATPase regulatory subunit 2, has Uniprot database accession number Q13200. It is involved in the ATP-dependent degradation of ubiquitinated proteins.

[097] VPS35, also known as Vacuolar protein sorting-associated protein 35, has Uniprot database accession number Q96QK1. It is involved in preventing missorting of selected transmembrane cargo proteins into the lysosomal degradation pathway.

[098] ANXA4, also known as Annexin A4 or Lipocortin IV, has Uniprot database accession number P09525. It is involved in processes related to membrane fusion and exocytosis.

[099] PLD3, also known as Phospholipase D3 or choline phosphatase 3, has Uniprot database accession number Q8IV08. This protein may be involved in APP processing.

[0100] S10AC, also known as S100-A12 Protein or Calgranulin-C, has Uniprot database accession number P80511. This protein is involved in the regulation of inflammatory processes and immune response.

[0101] CD14, also known as monocyte differentiation antigen CD14, has Uniprot database accession number P08571. This protein is a coreceptor for bacterial lipopolysaccharide (LPS) involved in the innate immune response to bacterial LPS.

[0102] LAMP2, also known as lysosome-associated membrane glycoprotein 2, has Uniprot database accession number P13473. It is involved in chaperone-mediated autophagy.

[0103] CLD6, also known as Claudin-6 or Skulin, has Uniprot database accession number P56747. It is involved in tight junction-specific obliteration of the intercellular space.

[0104] IF2B3, also known as insulin-like growth factor 2 mRNA binding protein 3 or VICKZ family member 3, has Uniprot database accession number O00425. It is involved in the recruitment of target transcripts into cytoplasmic protein-RNA complexes.

[0105] MLEC, also known as Malectin, has Uniprot database accession number Q14165. This protein is involved in the early steps of N protein glycosylation.

[0106] H10, also known as Histone H1.0 or Histone H1', has Uniprot database accession number P07305. This protein is involved in the condensation of nucleosome chains into higher-order structures.

[0107] CD166, also known as CD166 antigen, has Uniprot database accession number Q13740. It is involved in heterotypic and homotypic cell-cell contacts.

[0108] CD81, also known as CD81 antigen or Tetraspanin-28, has Uniprot database accession number P60033. This protein may be involved in regulating lymphoma cell growth.

[0109] AR6P1, also known as ADP-ribosylation factor-like protein 6 interacting protein 1, has Uniprot database accession number Q15041. This protein is involved in SLC1A1/EAAC1-mediated glutamate transport.

[0110] VAC14, also known as VAC14 homologue protein or Tax1-binding protein 2, has Uniprot database accession number Q08AM6. This protein is involved in the biogenesis of endosome-bearing vesicles (ECV)/multivesicular body (MVB) transport intermediates of early endosomes.

[0111] ITB3, also known as Integrin beta-3, has Uniprot database accession number P05106. This protein is involved in cell signaling transduction since it forms cellular receptors for various ligands, such as fibronectin, laminin or osteopontin among others.

[0112] PIGR, also known as polymeric immunoglobulin receptor, has Uniprot database accession number P01833, 26 June 2007 - v4. This receptor binds polymeric IgA and IgM on the basolateral surface of epithelial cells.

[0113] VIME, also known as Vimentins, consists of class III intermediate filaments found in various non-epithelial cells, especially mesenchymal cells. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. It has Uniprot database accession number P08670, 23 January 2007 - v4.

[0114] CTNB1, also known as beta-1 catenin, has Uniprot database accession number P35222, 1 February 1994 - v1. It acts as a negative regulator of centrosome cohesion and blocks anoikis of malignant epithelial cells of the kidney and intestine.

[0115] CAYP1, also known as calcifosin, has Uniprot database accession number Q13938, 1 November 1997 - v1. It is a calcium-binding protein that may play a role in cell signaling events.

[0116] WFDC2, WAP quadruple disulfide core domain protein 2, is a broad-range protease inhibitor, also known as Epididymal secretory protein E4. It has Uniprot database accession number Q14508 23 January 2002 - v2.

[0117] CADH1, also known as cadherin-1 or E-cadherin, has Uniprot database accession number P12830, 1 July 1993 - v3. This protein is involved in mechanisms that regulate cell-cell adhesions, motility and proliferation of epithelial cells. It has a powerful invasive suppressor role.

[0118] CD44, also known as CD44 antigen, has Uniprot database accession number P16070, 5 October 2010 - v3. It mediates cell-cell and cell-matrix interactions through its affinity for HA, and possibly also through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs).

[0119] XPO2, also known as exportin-2, has Uniprot database accession number P55060, 29 March 2005 - v3. Among others, this protein has been revealed as an export receptor for importin-alpha, mediating importin-alpha re-export from the nucleus to the cytoplasm after import substrates (cargoes) and cooperatively binding to importin-alpha and the GTPase Ran in its active GTP-bound form.

[0120] SG2A1, also known as mammaglobin-B, has Uniprot database accession number O75556, 1 November 1998 - v1. It can bind androgens and other steroids.

[0121] ENOA, also known as alpha-enolase, has Uniprot database accession number P06733, 23 January 2007 - v2. It is a multifunctional enzyme that, in addition to its role in glycolysis, plays a part in several processes such as growth control, hypoxia tolerance and allergic responses. It may also function in the intravascular and pericellular fibrinolytic system due to its ability to serve as a receptor and plasminogen activator on the cell surface of several cell types such as leukocytes and neurons. It stimulates the production of immunoglobulin.

[0122] LEG3, known as galectin-3 and also referred to as Galectin-3, is a galactose-specific lectin that binds IgE. May mediate with alpha-3, beta-1 integrins the CSPG4 stimulation of endothelial cell migration. Together with DMBT1, required for terminal differentiation of columnar epithelial cells during early embryogenesis (by similarity). In the nucleus: acts as a pre-mRNA splicing factor. Involved in acute inflammatory responses including neutrophil activation and adhesion, monocyte-macrophage chemoattraction, opsonization of apoptotic neutrophils, and mast cell activation. Has Uniprot database accession number P17931, 25 November 2008 - v5.

[0123] LEG1 is also known as LEG1 protein homologue, involved in early liver development. It has Uniprot database accession number P09382, 29 March 2005 - v2.

[0124] CAPG, also known as macrophage capping protein, has Uniprot database accession number P40121, 30 November 2010 - v2. It is a calcium-sensitive protein that reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. It may play an important role in macrophage function.

[0125] PRDX1, also known as peroxiredoxin-1, has Uniprot database accession number Q06830, 1 June 1994 - v1. It is involved in the redox regulation of the cell.

[0126] CLIC1, also known as chloride intracellular channel protein 1, has Uniprot database accession number O00299, 23 January 2007 - v4. Inserts into membranes and forms chloride ion channels. Channel activity is pH dependent. Membrane insertion appears to be redox regulated and can occur only under oxidizing conditions. Involved in cell cycle regulation.

[0127] PDIA1, also known as protein disulfide isomerase, has Uniprot database accession number P07237, 1 November 1997 - v3. It catalyzes the formation, rupture, and rearrangement of disulfide bonds.

[0128] KPYM, also known as pyruvate kinase PKM, has Uniprot database accession number P14618, 23 January 2007 - v4. It is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP, and plays a general role in caspase-independent cell death of tumor cells.

[0129] GSTP1, also known as glutathione S-transferase P, has Uniprot database accession number P09211, 23 January 2007 - v2. It regulates CDK5 activity negatively via p25/p35 translocation to prevent neurodegeneration

[0130] GTR1 has Uniprot database accession number P11166, 3 October 2006 - v2. It is a facilitative glucose transporter. This isoform may be responsible for constitutive or basal glucose uptake. It has a very broad substrate specificity; it can transport a wide range of aldoses including pentoses and hexoses.

[0131] CH10, also known as mitochondrial heat shock protein 10 kDa, has Uniprot database accession number P61604, 23 January 2007 - v2. It is essential for mitochondrial protein biogenesis, together with CPN60. It binds to CPN60 in the presence of Mg-ATP and suppresses the latter's ATPase activity.

[0132] MIF, also known as macrophage migration inhibitory factor, has Uniprot database accession number P14174, 23 January 2007 - v4. It is involved in the innate immune response to bacterial pathogens.

[0133] PEBP1, phosphatidylethanolamine-binding protein 1, binds ATP, opioids, and phosphatidylethanolamine. It is a serine protease inhibitor that inhibits thrombin, neuropsin, and cymotrypsin, but not trypsin, tissue-type plasminogen activator, and elastase (by similarity). It inhibits the kinase activity of RAF1 by inhibiting its activation and dissociating the RAF1/MEK complex and acting as a competitive inhibitor of MEK phosphorylation. It has Uniprot database accession number P30086, 23 January 2007 - v3.

[0134] TPIS, also known as triosephosphate isomerase, has Uniprot database accession number P60174, 19 October 2011 - v3. This protein is involved in pathway gluconeogenesis, which is part of carbohydrate biosynthesis.

[0135] NGAL, also known as neutrophil gelatinase-associated lipocalin, has Uniprot database accession number P80188, 1 November 1995 - v2. It is involved in apoptosis due to interleukin 3 (IL3) deprivation and in innate immunity.

[0136] LDHA, also known as L-lactate dehydrogenase chain A, has Uniprot database accession number P00338, 23 January 2007 - v2. This protein is involved in step 1 of the subpathway that synthesizes (S)-lactate from pyruvate.

[0137] Some of these proteins have been related to EC grading in tissue samples. However, no significant data are correlated with values in uterine fluid samples from the female genital tract. As explained above, the detection of markers in uterine fluids (aspirates or washes) implies the advantage of collecting data from routine sampling in the clinical practice of gynecological diseases, avoiding costs and, above all, tissue biopsies.

[0138] As indicated previously, the invention also aims as a first aspect at a method of differential diagnosis of endometrial cancer, the method comprising determining the level of expression of CTNB1 in a sample of uterine fluid from the female genital tract.

[0139] In a particular embodiment of the method of differential diagnosis of endometrial cancer, the uterine fluid sample is a uterine aspirate fluid sample from the female genital tract.

[0140] In another particular embodiment, the method further comprises determining the expression level of one or more of the following proteins: MMP9, AGRIN, CAPG, HSPB1 and XPO2.

[0141] In yet another particular embodiment, further comprising determining the level of expression of one or more of the following proteins: PIGR, VIME, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, CD59, CLD6, IF2B3, PLD3, and MX1.

[0142] In another particular embodiment of the method for differential diagnosis of endometrial cancer, it further comprises determining the expression level of one or more of the following proteins: PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, CAYP1, SG2A1, LDHA, PERM, OSTP, SPIT1, NAMPT, CASP3, K2C8, MUC1, ANXA1, ANXA2, FABP5 and WFDC2.

[0143] In another particular embodiment, the method further comprises determining the expression level of a protein selected from the group consisting of: XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[0144] In another particular embodiment, the method further comprises determining the expression level of one or more proteins selected from the group consisting of CLD6, BCAM, IF2B3, PLD3, and MX1 in an exosome-containing fraction isolated from a uterine aspirate.

[0145] In yet another particular embodiment, the method further comprises determining the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0146] In another particular embodiment of the method, the differential diagnosis of endometrial cancer is determined by distinguishing endometrioid endometrial cancer from non-endometroid endometrial cancer and non-cancer.

[0147] In another particular embodiment of the method, the expression level is determined at the protein level.

[0148] In a more particular embodiment, the protein level is determined by an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, a mass spectrometry, and combinations thereof.

[0149] In another particular embodiment, the level of protein expression is determined using an antibody or a fragment thereof capable of binding to the protein.

[0150] More particularly, said antibody or fragment thereof forms part of a kit.

[0151] As indicated, another aspect of the invention consists of the use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, as an in vitro marker for the prognosis of endometrial cancer in a sample of uterine fluid from the female genital tract. More particularly, the use of at least the expression level of CTNB1.

[0152] The invention also relates to the use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 for the prognosis of endometrial cancer, in the method of any of the aspects and embodiments.

[0153] Another aspect also consists of the use of a kit for the prognosis of endometrial cancer, and for the differential diagnosis of endometrial cancer, the kit comprising a solid support and means for detecting the level of expression of one or more of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, and, optionally, means for detecting the level of expression of one or more of the following proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[0154] In a particular embodiment of use of the kit, the kit comprises a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0155] In a particular embodiment of use of the kit, the means for detecting the level of expression of the proteins are means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0156] More particularly, the means for detecting the level of expression of proteins are antibodies or fragments thereof.

[0157] Another aspect of the invention is a kit comprising a solid support and means for detecting the expression level of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA. In particular, it comprises means for detecting at least the expression level of CTNB1.

[0158] Another aspect is a kit comprising a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0159] In a particular embodiment of the kits, they further comprise means for detecting the expression level of at least one set of proteins selected from the group consisting of MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BCAM, MMP9; MMP9, AGRIN; AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9, and those sets of proteins listed in Table D.

[0160] In a more particular embodiment of the kits, the means for detecting the level of expression of the proteins are means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0161] More particularly, the kits comprise means for detecting the level of expression of proteins, antibodies or fragments thereof.

[0162] In another particular embodiment of the kits, they are kits for performing an enzyme-linked immunosorbent assay.

[0163] In another particular embodiment, the kits further comprise a panel diagram for categorizing an individual sample.

[0164] The invention also relates to a computer-implemented method for performing the differential diagnosis method as disclosed above and as defined in any of the embodiments, wherein after determining the level of expression of one or more of the proteins for the diagnosis and/or for the prognosis of EC, said levels are given a value and/or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein as a function of said levels, scores and computed values, a decision is made between the options of suffering from EC or not and/or between the options of suffering from among different subtypes of EC.

[0165] As indicated previously, the invention also aims as another aspect a method for the prognosis of EC. In a particular embodiment of the first aspect of the invention, the method comprises determining the level of expression of one or more of PIGR, VIME, LEG1 and CAPG in a sample of uterine fluid from the female genital tract.

[0166] In another particular embodiment of this aspect of the invention, optionally in combination with any preceding or succeeding embodiment, the uterine fluid sample is a uterine aspirate fluid sample from the female genital tract.

[0167] It is highly advantageous that proteins could be differentially detected in uterine aspirate, since minimal manipulation is required and clinically significant information can be obtained.

[0168] In another particular embodiment of this aspect, the method comprises determining the level of expression of one or more of the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, and CADH1. As will be illustrated in the examples, determining the levels of one of the seven proteins allowed a highly sensitive and specific diagnosis of the EC subtype with an area under the ROC curve (AUC) of 0.74 to 0.85 (with a 95% confidence interval).

[0169] The inventors also determined that detecting the level of expression of other proteins in said uterine fluid, in particular in uterine aspirate, allowed improving the robustness (sensitivity and specificity) of EC subtype classification, and also of disease diagnosis. Therefore, in another particular embodiment of the first aspect and other aspects, optionally in combination with any previous or subsequent embodiment, the method further comprises determining the level of expression of one or more of the following proteins: XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, CAPG, CD44, LEG1, LEG3 and LDHA.

[0170] More particularly, it further comprises determining the expression level of a protein selected from the group consisting of: XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, CAPG, CD44, LEG1, LEG3 and LDHA.

[0171] In another particular embodiment of the first aspect and other aspects of the invention, optionally in combination with any preceding or succeeding embodiment, the method comprises determining the expression level of two proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA, wherein at least one of the proteins is selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1 and CAPG.

[0172] In another particular embodiment, the method comprises determining the expression level of two proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA.

[0173] In another particular embodiment of the first aspect of the method and additional method aspects, the method comprises determining the expression level of two proteins selected from the group consisting of AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1.

[0174] In another particular embodiment of the first aspect of the method and additional method aspects of the invention, optionally in combination with any preceding or succeeding embodiment, the method comprises determining the expression level of three proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA, wherein at least one of the proteins is selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1 and CAPG.

[0175] In another particular embodiment of the first method aspect and additional method aspects of the invention, optionally in combination with any prior or later embodiment, the method comprises determining the expression level of three proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA.

[0176] In another particular embodiment of the first method aspect and additional method aspects of the invention, the method comprises determining the expression level of three proteins selected from the group consisting of AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1.

[0177] In another particular embodiment of the method for prognosis of EC, the method further comprises determining the expression level of one or more proteins selected from the group consisting of CLD6, BCAM, IF2B3, PLD3 and MX1 in an exosome-containing fraction isolated from uterine aspirate.

[0178] The inventors have detected in a fraction of the uterine aspirate, said fraction comprising in a more purified form the exosomes that certain markers (proteins) were differentially expressed in EEC and NEEC. Therefore, the invention encompasses the optional step of determining in a processed uterine aspirate (the exosome fraction) particular markers or verifying the prognosis determined in the uterine fluid, or to increase the sensitivity of the method in case uterine fluid without isolation of exosomes does not provide relevant data.

[0179] In a more particular embodiment, the method for the prognosis or differential diagnosis of EC comprises determining the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0180] Particular combinations selected from LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR (in Table B below); as well as those listed in Table C, (illustrated at the end of this description) were determined to have high prognostic value when protein expression levels were detected in a uterine aspirate. Table B below shows AUC values and 95% confidence interval (CI). The data are derived from the analysis of EEC and NEEC samples. In Table C, the AUC values are also indicated.

[0181] On the other hand, Table A shows AUC and 95% CI values of the following combinations: CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3, providing important prognostic information when protein expression levels are detected in an exosome-containing fraction isolated from uterine aspirate. The data are derived from the analysis of EEC and NEEC samples. TABLE A TABLE B

[0182] In a more particular embodiment, the method comprises determining the expression level of CTNB1, XPO2 and CAPG. As will be illustrated and described below (Figure 1) this combination allows for a highly sensitive and specific differential diagnosis of EEC versus NEEC (serous EC; SEC).

[0183] In another particular embodiment of the first aspect and additional method aspects of the invention, optionally in combination with any preceding or succeeding embodiment, the method comprises determining the expression level of four, five, six, seven, eight, nine, ten or eleven proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA, wherein at least one of the proteins is selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1 and CAPG.

[0184] In another particular embodiment of the first aspect and additional method aspects of the invention, optionally in combination with any preceding or succeeding embodiment, the method comprises determining the level of expression of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, and CD59.

[0185] In any of the embodiments provided above or below, for any of the aspects of the invention, the expression level is determined at the protein level. In this embodiment, the protein markers include, but are not limited to, native sequence peptides, isoforms, chimeric polypeptides, all homologues, fragments, and precursors of the markers, including modified forms of the polypeptides and derivatives thereof. In a more particular embodiment, the protein level is determined by an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0186] In particular embodiments provided above or below, the level of expression is determined by immunochemistry.

[0187] The term “immunochemistry” as used herein refers to a variety of techniques for detecting antigens (usually proteins and peptides, and in the present case any of the proteins listed above alone or in combination) in a sample by exploiting the principle of antibodies specifically binding to said antigens. Visualization of an antibody-antigen interaction can be accomplished in a number of ways. In the most common case, an antibody is conjugated to an enzyme, such as peroxidase, which can catalyze a color-producing reaction. Alternatively, the antibody can also be labeled with a fluorophore, such as fluorescein or rhodamine. The immunochemistry technique can be direct or indirect. The direct method is a one-step staining method and involves a labeled antibody (e.g., FITC-conjugated antiserum) reacting directly with the antigen. Although the technique uses only one antibody and is therefore simple and rapid, sensitivity is lower due to a small signal amplification, as with indirect methods, and they are less commonly used than indirect methods. The indirect method involves 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 because of signal amplification due to the binding of multiple secondary antibodies if the secondary antibody is conjugated to the fluorescent or enzyme reporter.

[0188] Additional amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit avidin, streptavidin or Neutravidin enzyme complexes. The indirect method, in addition to its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies need to be generated. With the direct method, it would be necessary to label each primary antibody for each antigen of interest. It should be borne in mind that immunochemical techniques can also be used to detect certain nucleic acid sequences if a labeled nucleic acid probe (designed to specifically bind to a certain target nucleic acid sequence) can subsequently be detected with a labeled antibody. Thus, protein detection can be accomplished by using a labeled nucleic acid designed to bind to a specific sequence of the target protein RNA, and then detecting said labeled nucleic acid with a labeled antibody that selectively binds to the label.

[0189] Suitable immunoassay procedures include enzyme-linked immunosorbent assays (ELISA, such as a multiplexing ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography, or other immunoassay formats well known to those of ordinary skill in the art, such as radioimmunoassay, as well as protein microarray formats.

[0190] In one embodiment, in combination with any of the embodiments provided above or below, the level of protein expression is determined by an immunoassay.

[0191] In another embodiment, in combination with any of the embodiments provided above or below, the level of protein expression is determined by ELISA; more particularly, multiplexing ELISA.

[0192] Alternatively, the level of protein expression can be determined by bioluminescence, fluorescence, chemiluminescence, electrochemistry, or mass spectrometry.

[0193] Alternatively, the level of protein expression can be determined by measuring the levels of the protein's proteolytic peptides (peptides with an amino acid sequence uniquely associated with the protein studied in a given proteome) by mass spectrometry.

[0194] In another embodiment, in combination with any of the embodiments provided above or below, the level of protein expression is determined using an antibody or a fragment thereof capable of binding to the target proteins.

[0195] The term “antibody or a fragment thereof capable of binding to target proteins” should be understood as any immunoglobulin or fragment thereof capable of selectively binding to the target protein. It includes both monoclonal and polyclonal antibodies. The term “fragment thereof” encompasses any part of an antibody having the appropriate size and conformation to bind an epitope of the target protein. Suitable fragments include F(ab), F(ab') and Fv. An “epitope” is the part of the antigen being recognized by the immune system (B cells, T cells or antibodies).

[0196] Antibodies used for specific detection may be polyclonal or monoclonal. There are well-known means in the art for preparing and characterizing antibodies. Methods for generating polyclonal antibodies are well known in the art. Briefly, polyclonal antibodies are prepared by immunizing an animal with the protein; then, serum from the immunized animal is collected and the antibodies isolated. A wide range of animal species can be used for the production of antisera. Typically, the animal used for the production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat.

[0197] Furthermore, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the disease-associated protein. The immunizing composition can be administered in an amount effective to stimulate antibody-producing cells. Methods for preparing monoclonal antibodies are generally initiated along the same lines as for polyclonal antibody preparation. The immunogen is injected into animals as antigen. The antigen can be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At approximately two-week intervals, immunization is repeated with the same antigen.

[0198] In another particular embodiment of the third aspect, the means for carrying out the invention form part of a kit. The antibody or fragment thereof for detecting the target proteins may be included in a kit. The kit may further comprise means (additives, solvents) for visualizing antibody-protein interactions.

[0199] These antibodies can be used as “means” for determining the expression of the target proteins in the fifth aspect of the invention.

[0200] Thus, in a particular embodiment of the first method aspect and additional method aspects of the invention, the expression level of a protein is determined using an antibody or a fragment thereof capable of binding to the protein.

[0201] In another particular embodiment, said antibody or fragment thereof forms part of a kit.

[0202] All embodiments provided above under the first method aspect and additional method aspects of the invention, considering the proteins to be analyzed (two to eleven from the list), are also particular embodiments of the use of the second, third and fourth aspects of the invention.

[0203] Alternatively, the expression level is determined at the mRNA level.

[0204] In one embodiment, the amounts of mRNA for each of the markers are detected via a polymerase chain reaction using, for example, oligonucleotide primers that hybridize to one or more polynucleotide endometrial cancer markers or complements of such polynucleotides. In other embodiments, the amount of mRNA is detected using a hybridization technique, which employs oligonucleotide probes that hybridize to one or more polynucleotide endometrial cancer markers or complements of such polynucleotides.

[0205] When using mRNA detection, the method can be performed by combining isolated mRNA with reagents to convert to cDNA according to standard methods well known in the art, treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a vessel along with an appropriate mixture of nucleic acid primers; reacting the contents of the vessel to produce amplification products; and analyzing the amplification products to detect the presence of one or more polynucleotide endometrial cancer markers in the sample. For mRNA, the step of analysis can be performed using Northern Blot analysis to detect the presence of polynucleotide endometrial cancer markers in the sample. The step of analysis can be further performed by quantitatively detecting the presence of polynucleotide endometrial cancer markers in the amplification product, and comparing the amount of markers detected against a panel of expected values for the known presence or absence of such markers in normal and malignant tissue derived using similar primers.

[0206] In another embodiment, the invention provides a method wherein mRNA is detected by: (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more of the polynucleotide endometrial cancer markers in the endometrial cancer marker to produce amplification products; (c) analyzing the amplification products to determine the amount of mRNA present that encodes the protein endometrial cancer marker; and (d) comparing the determined amount of mRNA in a detected amount against a panel of expected values for normal and diseased tissue (e.g., malignant tissue) derived using similar methods.

[0207] In particular embodiments of the invention, RT-PCR can be used to amplify mRNA for protein endometrial cancer markers for detection and analysis. Other embodiments of the invention use quantitative RT-PCR to quantitatively determine the amount of mRNA for protein endometrial cancer markers. Additional embodiments of the invention use real-time RT-PCR for quantification and analysis.

[0208] Considering the device or kits of the invention, said kits are in particular embodiments of the invention provided for the analysis of patient samples. Such devices or kits will include reagents that specifically identify one or more proteins, where at least a subset of proteins is selected from proteins listed above, as well as from Tables A to F, listed below. Devices of interest include arrays, where reagents are spatially separated on a substrate such as a slide, gel, multi-well plate, etc. Alternatively, reagents may be provided as a kit comprising reagents in a suspension or suspendable form, for example reagents bound to microspheres. Reagents of interest include reagents specific for autoantibody markers. Such reagents may include antigenic proteins or peptides, and the like. Such devices or kits may further comprise cytokine-specific antibodies or fragments thereof; and the like.

[0209] As indicated above, one aspect of the invention is the use of kits comprising a solid support and means for detecting the level of expression of one or more of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, and, optionally, means for detecting the level of expression of one or more of the following proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA, said kits for the prognosis of endometrial cancer, distinguishing EEC from NEEC.

[0210] In a particular embodiment of the use of the kits, the kits are those comprising a solid support and means for detecting the expression level of two, three, four, five, six, seven, eight, nine, ten and eleven proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA. More particularly, the kits comprising a solid support and means for detecting the expression level of three of these proteins.

[0211] In a particular embodiment of use of the kits, they are kits comprising a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0212] In a more particular embodiment of the use of the kits, the kits are those that comprise a solid support and means for detecting the level of expression of CTNB1, XPO2 and CAPG.

[0213] In another particular embodiment of the use of the kits, the means for detecting the level of expression of the proteins are antibodies or fragments thereof that specifically bind to target proteins.

[0214] According to the fifth aspect, the invention relates to kits comprising a solid support and means for detecting the expression level of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[0215] In a particular embodiment of the fifth aspect, the kits comprise a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0216] In a more particular embodiment, the kits comprise means for detecting the expression level of a combination of 2 to 500 sets, and more particularly, 2 to 400.

[0217] In another particular embodiment, optionally in combination with any of the preceding or subsequent embodiments, the kits further comprise means for detecting the expression level of at least one set of proteins selected from the group consisting of MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BCAM, MMP9; MMP9, AGRIN; AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9, and those protein sets listed in Table D.

[0218] Particular combinations selected from Table E below; as well as those listed in Table D, (illustrated at the end of this description) have been found to have high diagnostic value when protein expression levels were detected in a uterine aspirate. Table E below shows AUC and 95% confidence interval (CI) values. The data are derived from the analysis of non-EC and EC (including EEC and NEEC) samples. In Table D, AUC are also indicated. TABLE E. Combinations of diagnostic value in uterine aspirate TABLE F. Combinations of diagnostic value in an exosome-containing fraction isolated from uterine aspirate

[0219] On the other hand, Table F shows AUC and 95% CI values of the following combinations: AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9, providing important diagnostic information when protein expression levels were detected in an exosome-containing fraction isolated from uterine aspirate. The data are derived from the analysis of non-EC and EC (including EEC and NEEC) samples.

[0220] As indicated for the fourth aspect, in a particular embodiment of this fifth aspect, the means for detecting the level of expression of the proteins are antibodies or fragments thereof that specifically bind to the target proteins.

[0221] In another particular embodiment of the fourth and fifth aspects of the invention, the kits are ELISA kits. In this embodiment, the kit comprises a solid support and means for determining the level of expression of any of the proteins and combinations of proteins provided above. In another embodiment, the kit comprises a solid support and antibodies or fragments thereof that specifically bind to the target proteins to be detected, wherein these antibodies are conjugated to a reporter molecule capable of producing a signal.

[0222] The “solid support” includes a membrane of nitrocellulose, glass, or a polymer. The most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Solid supports may be in the form of strips, tubes, microspheres, microplate discs, or any other surface suitable for conducting an immunoassay.

[0223] The “reporter molecule” as used herein means a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of antibody bound to antigen. Detection may be qualitative or quantitative. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores, or radionuclide containing molecules (i.e., radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate. As will be readily recognized, however, there are a wide variety of different conjugation techniques that are readily available to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase, among others. The substrates to be used with the particular enzymes are generally chosen to produce, upon hydrolysis by the corresponding enzyme, a detectable color change. For example, 5-bromo-4-chloro-3-indolyl phosphate/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. Likewise, it is possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted previously. Examples of fluorogenic substrates are fluorescein and rhodamine. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing an excitable state in the molecule, followed by the emission of light in a characteristic color visually detectable with a light microscope. Immunofluorescence and EIA techniques are well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent and bioluminescent molecules and/or dyes, and other chromogenic substances, can also be used.

[0224] The choice of a particular reporter molecule-conjugated antibody will, for the most part, be determined by the intended use and user of the test kit of the present invention.

[0225] Binding assays for measuring biomarker levels may use solid phase or homogeneous formats. Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Pat. Nos. 4,168,146 and 4,366,241, both of which are incorporated herein in their entireties by reference. Examples of competitive immunoassays include those disclosed in U.S. Pat. Nos. 4,235,601 , 4,442,204 , and 5,208,535 , each of which is incorporated herein in their entireties by reference.

[0226] Multiple biomarkers can be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral discrimination of markers, multiplexing by flow cytometric analysis of binding assays performed on particles, e.g., using the Luminex® system.

[0227] The assays of the present invention may be conducted by any suitable method. In one embodiment, biomarker levels are measured in a single sample, and those measured may be conducted in a single assay chamber or assay device, including, without limitation, a single well of an assay plate, a single assay cartridge, a single lateral flow device, a single test tube, etc. Biomarker levels may be measured using any of a number of techniques available to those of skill in the art, for example, direct physical measurements (e.g., mass spectrometry) or binding assays (e.g., immunoassays, agglutination assays, and immunochromatographic assays). The method may also comprise measuring a signal that results from chemical reactions, for example, a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemiluminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable markers from the surface, oxidation or reduction or redox species, an electric current or potential, changes in magnetic fields, etc. Suitable detection techniques can detect binding events by measuring the participation of labeled binding reagents by measuring the labels through their photoluminescence (e.g., by measuring fluorescence, time-resolved fluorescence, evanescent wave fluorescence, upconversion phosphor, multiphoton fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic fields, enzymatic activity (e.g., by measuring enzymatic activity through enzymatic reactions that cause changes in optical absorbance or fluorescence or cause the emission of chemiluminescence). Alternatively, detection techniques that do not require the use of labels can be used, for example, techniques based on mass measurement (e.g., surface acoustic wave measurements), refractive index (e.g., surface plasmon resonance measurements), or the inherent luminescence of an analyte.

[0228] In another embodiment, the kit is a microarray.

[0229] In another embodiment, the kit is a microarray that includes a defined set of genes encoding protein endometrial cancer markers. All embodiments provided above for particular proteins to be analyzed (two through eleven from the list) whose expression is significantly altered by endometrial disease are also particular microarray embodiments.

[0230] In another particular embodiment, the kits of the invention further comprise a panel diagram for categorizing an individual sample.

[0231] A further aspect of the present invention was a method of deciding or recommending whether to initiate a medical regimen of a subject suffering from endometrial cancer based on prognosis. In a particular embodiment of this method, it comprises a) determining, in vitro, the expression level of one or more proteins selected from the group consisting of: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, and, optionally, the expression level of one or more proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA in a sample of uterine fluid from the female genital tract of the subject; and b) establishing the prognosis of said EC by distinguishing EEC and NEEC, if the protein level in the test sample is greater than a reference control level for PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, and less than the reference control level for CAPG; wherein: i) if the individual is diagnosed as suffering from endometrial cancer, or is suspected of suffering from endometrial cancer, and the individual is differentially diagnosed as suffering from EEC, then initiation of the medical regimen for EEC is recommended, which consists of total hysterectomy and bilateral salpingo-oophorectomy, optionally supplemented with lymphadenectomy; ii) If the individual is diagnosed as suffering from endometrial cancer, or is suspected of suffering from endometrial cancer, and the individual is differentially diagnosed as suffering from NEEC, then initiation of medical regimen for NEEC is recommended, which consists of total hysterectomy and bilateral salpingo-oophorectomy, pelvic and para-aortic lymphadenectomy, omentectomy and peritoneal biopsies.

[0232] In another particular embodiment, the method of deciding or recommending whether to initiate a medical regimen of an individual suffering from endometrial cancer based on the established prognosis, an adjuvant treatment is recommended selected from radiotherapy, chemotherapy and combinations thereof if the individual is diagnosed as suffering from EEC. In yet another particular embodiment, and if the individual is diagnosed as suffering from NEEC, chemotherapy is recommended as adjuvant treatment.

[0233] Another aspect of the present invention is to provide an algorithm for performing any of the diagnostic and/or prognostic methods as defined in the previous aspects and modalities.

[0234] In a particular embodiment, the algorithm is a computer-implemented method for diagnosing EC and/or for prognosing EC, in particular for the prognosis of the disease by determining an EC subtype, in particular EEC or NEEC. This algorithm allows to make a decision whether a sample is from an individual suffering from EC or not; and also whether a sample is from an individual suffering from EC suffering from EEC or NEEC. In a more particular embodiment, the algorithm provides a recommended treatment. Therefore, there is also provided a computer-implemented method for carrying out the method as defined above, wherein after determining the level of expression of one or more of the proteins for the diagnosis and/or for the prognosis of EC, said levels are given a value and/or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein as a function of said computed levels, scores and/or values, a decision is made between the options of suffering from EC or not and/or between the options of suffering from different subtypes of EC.

[0235] In other words, in another particular embodiment of the algorithm of the invention, after determining the level of expression of one or more of the proteins for the diagnosis and/or prognosis of EC, said levels are given as a value and/or score, and, optionally, are computed in a mathematical formula to obtain a computed value; in which, based on said computed levels, scores and/or values, a decision is made between the options of suffering from EC or not and/or between the options of suffering from different subtypes of EC.

[0236] The invention also encompasses a method for prognosis of endometrial cancer by distinguishing endometrioid endometrial cancer (EEC) from non-endometrioid endometrial cancer (NEEC), the method comprising determining the level of expression of one or more of the following PIGR, and VIME proteins in a sample isolated from the female genital tract.

[0237] The inventors have discovered for the first time that these two proteins are useful in distinguishing EC subtypes by diverging from a sample of the female genital tract. The sample may in particular be selected from a tissue biopsy of the uterine organ and a fluid of the genital tract (i.e. uterine aspirate). This method further comprises detecting the expression level of one or more proteins selected from the group consisting of CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA. The expression level is in particular determined at the protein level, in particular by means of antibodies or fragments of said antibodies that specifically bind to the proteins. Kits of parts and means that are used to carry out the method are also part of the invention.

[0238] With the assay performed by the inventors, a protein was identified that provides fundamental information about EC. This is the case of VIME, which was differentially expressed in EEC samples and NEEC (SEC) samples. Therefore, these proteins not only allow the classification of the EC subtype, but also confirm the diagnosis. The invention encompasses a method for diagnosing and/or prognosing EC, the method comprising determining the level of expression of VIME in a sample isolated from a female, in particular, from the female genital tract.

[0239] Furthermore, the samples tested allow determining the differential expression of some proteins in the fluid sample from the female genital tract between EC and endometrial hyperplasia. Endometrial hyperplasia is a thickening of the endometrium caused by excess estrogen stimuli. It is a benign disease. However, it is considered a precursor lesion of EC, and must be distinctively diagnosed. In an assay with EC samples (of any subtype; EEC and NEEC) and with non-EC sample controls including cases of hyperplasia, 16 proteins were found differentially abundant with adjusted p-values <0.05 and successive changes greater than 2 between hyperplasias and the other non-EC controls (women with normal endometrium or benign diseases other than hyperplasias, such as fibroids or polyps). Four of them, NAMPT, ENOA, CATD and GSTP1 showed AUC greater than 0.85. After appropriate validation, a diagnosis of EC hyperplasias is made. Therefore, the invention also relates to a method for diagnosing and/or prognosing endometrial hyperplasia in an individual, the method comprising determining the level of expression of one or more of the following proteins: NAMPT, ENOA, CATD and GSTP1, in a sample isolated from a female, in particular from the female genital tract. Early detection of hyperplasia, a precursor lesion of EC, allows for increased follow-up of patients in order to detect any malignancy as early as possible. Related to this aspect, the invention also provides a method for monitoring a medical regimen for hyperplasia using one or more of the NAMPT, ENOA, CATD and GSTP1 markers of the invention: a reduction or return to a normal level of the marker (i.e. to the level of a control individual without hyperplasia) may indicate that the patient has responded favorably to the medical regimen and, therefore, said regimen is effective; If the marker level does not change significantly or increases, this may indicate that the regimen is not effective and/or there is a high risk of hyperplasia progressing to EC. In such cases, surgical treatment should be performed.

[0240] The in vitro methods of the invention provide diagnostic and/or prognostic information. In one embodiment, the methods of the invention further comprise the steps of (i) collecting the diagnostic and/or prognostic information, and (ii) saving the information to a data carrier.

[0241] Within the scope of the invention a “data carrier” should be understood as any medium that contains information data significant for the diagnosis and/or prognosis of endometrial carcinoma, such as paper. The carrier may also be any entity or device capable of carrying the prognostic data. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disk or a hard disk. Furthermore, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be transported via an electrical or optical cable or by radio or other means. When the diagnostic/prognostic data is embodied in a signal that can be transported directly by a pipe or other device or medium, the carrier may consist of such a cable or other device or medium. Other carriers refer to USB devices and computer files. Examples of suitable data carriers are paper, CDs, USB, computer files on PCs, or sound recordings with the same information.

[0242] Throughout the description and claims, the word “comprise” and its variations are not intended to exclude other technical features, additives, components or steps. Additionally, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration and are not intended to limit the present invention. Additionally, the present invention encompasses all possible combinations of particular and preferred embodiments described herein.

EXAMPLES EXAMPLE 1. Diagnostic and prognostic biomarkers of EC in exosomes derived from uterine fluid, and in uterine fluid. 1. Samples, reagents and methods 1.1 Description of patients and sample

[0243] Patients were recruited from three different institutions: HUVH (Hospital Universitari Vall d’Hebron, Barcelona, Spain), HUAV (Hospital Universitari Arnau de Vilanova, Lleida, Spain) and UMCF (University Medical Center of Freiburg, Freiburg, Germany). Each participating institution obtained ethical approval and samples were obtained after participants had given written informed consent.

[0244] Uterine aspirates (UAs) were obtained by aspiration with a Cornier Pipelle (Gynetics Medical Products). Samples were placed in 1.5 mL tubes and kept on ice throughout processing that included addition of phosphate buffered saline (PBS) at a 1:1 (v/v) ratio, gently pipetting the sample, and centrifugation at 2500 g (4 °C) in an F45-30-11 rotor (Eppendorf Microcentrifuge 5417R) for 20 min to remove the cellular fraction. The remaining supernatant (SN) fraction was then aliquoted and frozen at −80 °C until needed.

[0245] For exosome isolation (abbreviated ELVs), ELVs were obtained from UA SNs by differential centrifugation, following a modification of an ELV isolation protocol previously described by Thery et al. http://www.ncbi.nlm.nih.gov/pubmed/18228490. Briefly, SNs were thawed and diluted in PBS to a final volume of 25 mL. A centrifugation step at 10,000xg (4 °C) for 30 min was performed in a Thermo Scientific Heraeus MultifugeX3R centrifuge (FiberLite F15-8x-50c rotor) to remove cellular debris, macroparticles, and apoptotic bodies. The resulting MV-enriched pellet was resuspended in 50 μL PBS and frozen at -80 °C. Then, the supernatant was transferred to ultracentrifuge tubes (Beckman Coulter) and loaded with PBS to perform a first ultracentrifugation step at 100,000xg (4 °C) for 2 h in a Thermo Scientific Sorvall WX UltraSeries centrifuge with an AH-629 rotor. The supernatant from this second centrifugation was the soluble fraction and was frozen at −80 °C. This first pellet was resuspended in PBS and centrifuged again at 100,000xg (4 °C) for 1 h. The final pellet enriched in ELVs (possibly along microvesicles (MVs) and some remaining apoptotic bodies) was resuspended in 50 μL of PBS. Five microliters of MVs and ELVs pellets were reserved at -80 °C for particle size distribution and quantification by NTA while the rest of the sample was frozen at -80 °C for protein extraction.

1.2 Protein extraction and identification by liquid chromatography-parallel reaction monitoring (LC-PRM) analysis

[0246] For the ascertainment phase, ELVs were resuspended in a lysis buffer composed of 40 mM Tris pH 8, 300 mM NaCl, 10 mM EDTA, 2% Triton X-100 and protease inhibitors 1:100 (Sigma-Aldrich) in a 1:1 (v/v) ratio. Then, the samples were frozen at -20°C for at least 8 hours and then thawed on ice and sonicated five cycles of 5 seconds at 100% amplitude (Labsonic M, Sartorius Stedim Biotech) to ensure membrane disruption. The extracted proteins were stored at -20°C until needed.

[0247] The validation phase was performed on ELVs and uterine aspirate (UA) without the need to isolate ELVs. To extract proteins from ELVs for the validation phase, the detergent contained in the lysis buffer was changed to <1% NP-40, to produce protein extraction already suitable for direct solution digestion and LC-MS/MS. The rest of the procedure remained the same. To extract proteins from the fluid fractions of UAs; they were sonicated 5 cycles and 5 seconds each at 100% amplitude (Labsonic M, Sartorius Stedim Biotech) to disrupt microvesicles, protein aggregates and/or mucus present in the sample. Then, the Albumin and IgG Depletion Spin Trapping Kit (GE Healthcare) was used following the manufacturer's instructions to remove albumin and immunoglobulin G from the samples. All extracted proteins were stored at -20°C until needed. Diagnostic and prognostic value data were also obtained with the uterine fluid sample (ELVs or UA without ELVs isolation) where no albumin and immunoglobulin G depletion was performed.

[0248] For the discovery phase, peptides dissolved in 0.1% formic acid were first loaded onto a 150 μm ID x 20 cm nano-LC on a house-packed column (Jupiter C18, 3 μm, 300 A, Phenomenex, Torrance, CA) and separated with an EASY nanoLC system (Thermo Scientific). For elution of the peptides, a 1-h linear gradient of 5-40% ACN (0.2% FA) at a constant flow rate of 600 nL/min was used. The LC system was coupled to a QExactive plus Orbitrap tandem mass spectrometer (Thermo Fisher Scientific), and RAW files were acquired with XCalibur software (Thermo Fisher Scientific). Tandem mass spectra were performed with a Top-12 method with a precursor isolation window of m/z 2.0. The resolution was 70,000 at m/z 400 for the survey scan (with AGC 1e6, maximum injection time 200 ms, and a scan range of m/z 300–2000) and 17,500 for MS/MS spectra (AGC at 5e5, maximum injection time 50 ms, and scan range m/z 200–2000). The normalized collision energy (NCE) was set to 25, and the exclusion time was set to 10 s.

[0249] Considering protein identification, for SILAC quantification, RAW files were analyzed using MaxQuant (version 1.3.0.3). Default parameters were adopted: MS/MS fragment error tolerance of 20 ppm, Carbamidomethylamine (C) fixed and Oxidation (M) as well as Acetyl (Protein N-term) as variable modification. Arginine (Arg10) and lysine (Lys8) were selected for heavy labeling and applied the “match between cycles” option. RAW files were searched in the HUMAN SwissProt database. For Presence/Absence analysis, a label-free analysis was performed with PEAKS 7.0 (http://www.bioinfor.com/). For database searching, RAW files were searched in the Human Uniprot database containing 37254 entries. For the search parameters, a precursor error tolerance was set at 10 ppm and for MS2 fragments the tolerance was set at 0.01 Da. A maximum of 2 erroneous cleavages for Trypsin were allowed. Carbamidomethylation (C), Deamidation (NQ) and Oxidations (M) were variable modifications. Only peptides with false discovery rates (FDR) lower than 1% were considered. For validation of potential biomarkers, PEAKS results were uploaded to Scaffold Proteome software (http://www.proteomesoftware.com/).

[0250] Statistical analysis of the ascertainment phase was performed to obtain two different results: (1) Qualitative data consisting of proteins that were present or absent in the different patient subgroups; and (2) quantitative data consisting of expression measures obtained from SILAC ratios representing abundance of each protein vs. internal standards.

[0251] For qualitative data, a Fisher's exact test was applied to each protein to compare presence/absence between groups (p-value < 0.001). For quantitative data, a Student's t-test was performed between each pair of groups in order to select differentially expressed proteins. The test was performed only for those proteins that were present in more than 4 individuals per group. In order to solve the problem of multiple comparisons derived from performing many tests (one per protein), p-values were adjusted to obtain a strong control over FDR using the Benjamini and Hockberg methods (adj.-value < 0.25 and FC > 1.3).

[0252] All analyses were performed using the statistical program “R” (R version 3.2, Copyright (C) 2015 The R Foundation for Statistical Computing).

[0253] A total of 54 proteins were selected for the targeted reaction monitoring (SRM) experiment based on the results of the discovery phase (49), along with 5 candidates selected from previous results from our group. Two unique peptides per protein were selected as SRM monitors based on their detectability in previous mass spectrometric experiments. For each selected peptide, an isotopically labeled version (15N2,13C6-Lysine, 15N4,13C6-Arginine) was purchased and spiked into each sample to be used as an internal standard. The internal standards were spiked at a concentration within the linear response range, which was established for each individual peptide based on experimental dilution curves (data not shown). In parallel, isotropically labeled peptides were mixed and used to generate an MS2 spectral library and a retention time knowledge database.

[0254] A total of 85 endogenous peptides and their corresponding internal standards were measured by SRM on Lys-C- and trypsin-digested samples from an independent cohort of 107 patients. The five most intense transitions per peptide were monitored on a triple quadrupole mass spectrometer (5500 Q-Trap, AB Sciex Instruments, Foster, CA, USA) equipped with a 25 cm reversed-phase chromatography column with an inner diameter of 75 μM, packed with 1.9 μM C18 particles (NikkyoTechnos Co., Ltd. Japan) and a 2 cm pre-column (Acclaim PepMap 100, C18, 15 μM, 100A). Loading buffer: H2O with 0.1% formic acid; elution buffer: ACN, 0.1% formic acid. The flow rate was 250 nL/min and a chromatographic gradient ranging from 5 to 40% elution buffer in 40 min was used. Empty cycles were performed between SRM measurements of biological samples to avoid sample carryovers. Measurements were performed in a programmed SRM mode using a 300 s window and a target cycle time of 1.5 s.

[0255] For the data analysis of the verification phase, transition clusters corresponding to the targeted peptides were evaluated with Skyline v2.5 based on the co-elution of transition traces associated with a targeted peptide, in its light and heavy form; the correlation between the relative SRM intensities of the light and heavy counterpart. All peptide peaks were visually inspected. The areas of all transitions were used as an input to a linear mixed-effects model implemented in the MSstats Skyline plugin (v3.3) to calculate successive protein fold changes and adjusted p-values across the different sample groups.

[0256] For predictor development, MSstats was used to estimate the amount of proteins present in all samples based on log2-transformed transition areas, which were then used as input variables to a logistic regression model between defined groups. The classification ability of each protein was evaluated within 2/3 of the database using the area under the curve (AUC) as a performance indicator. The most discriminative protein was selected as the first classifier. The most discriminative proteins were iteratively added while increasing AUC values (deltaAUC > 0.02). The classification evaluation procedure was repeated 500 times using a different subset of patients in each iteration. Sample subsets were generated by randomly selecting patients from the original cohort without replacement and balanced for each sample subgroup. A final consensus model was composed of the combination of proteins, which were mostly selected in 500 repetitions. The final model was fitted to the full database and predictive accuracy was quantified using the area under the ROC curve, sensitivity, specificity and precision. The pROC package for the statistical program “R” was used to extract ROCs, to calculate AUCs and other performance values (i.e. sensitivity, specificity and precision); these were obtained considering the “optimal cutoff” as the point where the sum of sensitivities and specificities reached the maximum value.

2. Results 2.1 Verification phase

[0257] For the observation phase of this project, uterine aspirates from 10 patients with EEC (also abbreviated EC1), 10 NEEC (also abbreviated EC2, and who were particularly SEC) and 10 control patients (N=30) were used to isolate ELVs.

[0258] Once the purity of the isolated vesicles was ensured, the same amount of Super-SILAC mix (internal standard) was spiked into each of the 30 samples (EEC n=10, NEEC n=10 and CTRL n=10) at a Heavy/Light ratio of 2:1, just prior to separation by 1D-SDS-PAGE. Each gel lane was sliced into 10 bands, and each band was subjected to trypsin digestion to extract peptides, resulting in 300 individual LC-MS/MS cycles. Protein database searching of MS/MS data resulted in the overall identification of 2138 proteins, considering only those proteins identified with at least 1 unique peptide, and those peptides with a false discovery rate (FDR) less than 1%. The MS/MS spectra were further processed with MaxQuant software leading to the quantification of 920 proteins, which is an overall quantification rate of 43% of the identified proteomes.

[0259] A total of 152 proteins were differentially expressed with an adjusted p-value < 0.25 and successive fold changes lower than -1.3 or higher than 1.3. From this, 147 proteins were potential diagnostic biomarkers (differential CTRL vs. EC comparison), and 28 proteins were potential prognostic biomarkers (differential EEC vs. NEEC comparison). In parallel to the quantitative analysis, a presence/absence study was conducted in order to take into account proteins that failed to be quantified because they are devoid of the heavy counterpart, but are still relevant to be present in a given group. For this reason, RAW files were reanalyzed with PEAKS software and a Fisher test was performed to select those proteins significantly present in a group (p-value < 0.001). A total of 30 candidates were included following this analysis, corresponding to 29 potential diagnostic biomarkers and 9 potential prognostic biomarkers.

[0260] Above all, a final list of 54 candidates was generated by combining (i) the relative quantification analysis, (ii) the presence/absence study, and (iii) biological criteria such as the exclusion of proteins whose family was overrepresented in the candidate list, or proteins downregulated in cancer; without respecting the statistical constraints stated above. From these 54 candidate biomarkers, 50 corresponded to diagnostic biomarkers and 15 to prognostic biomarkers, from which 37 had only diagnostic potential, 2 had only prognostic potential and 13 had both diagnostic and prognostic potential.

2.2 Verification of protein markers in UAs ELVs

[0261] Once the list of 54 candidates described in the previous section was obtained, the aim was to verify the potential of these candidates using LC-SRM in a new and larger cohort of patients. As in the verification phase study, the ELVs fraction of UAs was the selected analysis sample. In addition to evaluating the individual potential of each marker to diagnose EC and differentiate between histological subtypes, it was also imagined to generate diagnostic and prognostic models combining different candidates. Furthermore, the classification power of each candidate in the total fluid fraction of UAs was evaluated in an independent cohort.

[0262] A total of 107 patients were recruited for the verification phase (cohort B), divided into three groups: EEC or EC1 (n=45), NEEC or EC2 (n=21) and CTRL (n=41). Specifically, NEEC corresponded to patients diagnosed with serous endometrial cancer (SEC). From these, UA ELVs were isolated and characterized as seen in the previous sections (data not shown). The list of 54 protein candidate biomarkers generated in the verification phase was verified by LC-SRM. In this way, two unique tryptic peptides were selected per protein, from which a total of 85 endogenous peptides were finally monitored by programmed SEM. However, only 69 of the 85 peptides corresponding to 51 proteins were detected; three of the candidates (TNR6, CLH1 and PSB3) were not detected in any of the samples.

[0263] As a result of this SRM experiment, it was observed that 43 of the 48 (89.6%) potential diagnostic biomarkers (i.e., significant differences in abundance were observed between CTRL and EC) were also significant in this verification phase (adj. p-value < 0.01), thus confirming their potential as individual diagnostic biomarkers. A total of 29 of the 45 quantified proteins showed high accuracy to individually discriminate between EC and CTRL cases (AUC values greater than 0.75, highlighted in bold in Table 6). The 5 most significant individual biomarkers were AGRIN (AUC= 0.90, CI95: 0.85-0.96), TACD2 (AUC= 0.87, CI95: 0.81-0.94), SORT (AUC= 0.86, CI95: 0.79-0.93), MVP (AUC= 0.86, CI95: 0.78-0.93) and FAS (AUC= 0.85, CI95: 0.78-0.92). Table 2.1 below shows the list of biomarkers selected for diagnosis of CE in ELVs.

[0264] Considering the potential prognostic biomarkers (i.e., comparison of EC1 (EEC) vs. EC2 (NEEC)) only 5 proteins out of the 15 candidates were found to be differentially expressed in the verification phase (adj. p-value < 0.01). The verified biomarkers were CLD6, IF2B3, BCAM, PLD3 and MX1. A total of 4 out of 45 quantified proteins showed high accuracy to individually discriminate between EC1 and EC2 cases with AUC values greater than 0.75: CLD6 (AUC= 0.88, CI95: 0.76-1.00), BCAM (AUC= 0.87, CI95: 0.760.97), IF2B3 (AUC= 0.80, CI95: 0.68-0.93) and PLD3 (AUC= 0.79, CI95: 0.66-0.93). Table 2.2 below shows the list of biomarkers selected for EC prognosis in ELVs.

2.3 Candidate verification by targeted proteomics in whole fluid from UAs

[0265] In order to understand whether it would be feasible to transfer the use of these biomarkers to a sample that is easier to access and does not require the isolation of extracellular vesicles, we aimed to study the biomarkers identified in exosomes in the total fluid fraction of UAs. For this, a total of 67 patients (cohort C) were selected and divided into three groups: EEC (n=22), NEEC (n=20) and CTRL (n=25).

[0266] For this part of the study, a total of 51 proteins were analyzed. Two unique tryptic peptides were selected per protein, from which a total of 75 endogenous peptides were finally monitored by programmed SEM. A total of 37 proteins were only detected and subsequently quantified for AUC estimation.

[0267] The results obtained in this verification study were evaluated against those previously performed in order to understand the feasibility of translating ELV-based biomarkers into whole fluid from UAs. First, it was observed that the detectability of ELV biomarkers in whole fluid from UAs was very limited compared to the detectability when ELVs were analyzed; of the 51 biomarkers in ELVs, only 34 (66.7%) were detected in UAs.

[0268] In Table 1.1 and Table 1.2 below, the list of significant biomarkers for diagnosis of EC in total fluid fraction of UAs, and for prognosis of EC (comparison between EEC and NEEC) in said total fluid fraction of UAs. TABLE 1.1. Diagnostic biomarkers in uterine aspirates TABLE 1.2. Prognostic biomarkers in uterine aspirates TABLE 2.1. Diagnostic biomarkers in exosome-containing fraction (ELV) isolated from UA: TABLE 2.2. Biomarkers for prognosis of CE in ELVs.

EXAMPLE 2. Prognosis of EC distinguishing cases of EEC (EC1) from NEEC (EC2) in a cohort of 116 patients, in uterine aspirates. 1. Samples, reagents and methods 1.1. Description of patients and sample

[0269] A total of 116 women were recruited at Vall Hebron University Hospital (Barcelona, Spain), Hospital Universitari Arnau de Vilanova (Lleida, Spain) and University Medical Center Freiburg (Freiburg, Germany) from 2012 to 2015. Informed consent forms, approved by the Ethics Committees of each Hospital, were signed by all patients. All women registered for the EC diagnostic process due to suspected EC, i.e., presentation of abnormal uterine bleeding (AUB) and/or an endometrial thickness greater than 4 mm for postmenopausal women and 8 mm for premenopausal women based on transvaginal ultrasound results. Of the 116 women, 69 were diagnosed with EC, including 49 endometrioid EC (EEC) and 20 non-endometrioid serous EC (SEC or NEEC). The remaining 47 women were non-EC women with normal endometrium or diagnosed with benign disorders (including endometrial hyperplasias). Clinicopathological features of the patients are described in the following Patient Table. TABLE 3. Clinical features of women enrolled in the study. * One case with indeterminate grade

[0270] Uterine aspirates were collected by aspiration with a Cornier Pipelle (Eurogine Ref. 03040200) and transferred to 1.5 ml microtubes. Phosphate buffer saline was added in a 1:1 (v/v) ratio and centrifuged at 2,500 x g for 20 min in order to separate the fluid fraction from the cellular fraction. Uterine aspirate fluids, varying volumes from 100 μl to 1 ml, were kept at -80°C until used.

1.2. Sample preparation for liquid chromatography-parallel reaction monitoring (LC-PRM) analysis

[0271] Sample preparation for LC-PRM analysis was performed as previously described by Martinez-Garcia E, et al. “Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples”, Oncotarget -2016, vol. no. 7(33), pp.:53102-53114 (supra). Briefly, fluid fractions of uterine aspirates were sonicated and 50 μL of each sample was depleted of albumin and immunoglobulin G proteins using the Albumin & IgG Depletion Spin Trapping Kit (GE Healthcare). Total protein concentration was estimated by a Bradford assay. Then, all 116 samples were divided into two equal aliquots of 12.5 μg to perform subsequent steps and analyses with technical duplicates. Samples were denatured, reduced, and alkylated prior to a two-step sequential proteolysis using first MS grade endoproteinase Lys-C (Thermo Scientific) at a 1/150 (w/w) protease/total protein ratio for 4 h at 37°C, and second, trypsin (Promega) at a 1/50 (w/w) protease/total protein ratio at 37°C overnight. A mixture of stable isotope-labeled synthetic peptides (Thermo Fisher, crude quality) was spiked into each sample (C-terminal arginine 13C6, 15N4, Δm = 10 Da, C-terminal lysine 13C6, 15N2, Δm = 8 Da or when not applicable a heavy leucine 13C6, 15N1, Δm = 7 Da or phenylalanine 13C9, 15N1, Δm = 10 Da). Finally, samples were purified by solid-phase extraction (Sep Pak tC18, 50 mg, Waters), vacuum-dried and resuspended in 0.1% formic acid prior to LC-PRM analysis. As in example 1, prognostic value data were also obtained with uterine fluid (UA) sample where no albumin and immunoglobulin G depletion was performed.

1.3. LC-PRM adjustment

[0272] Peptide separation was performed on a Dionex Ultimate 3000 RSLC chromatography system operated in column switching mode. The equivalent of 250 ng of digested sample was injected and loaded onto a trapping column (75 μm x 2 cm, C18 pepmap 100, 3μm) using a mobile phase of 0.05% trifluoroacetic acid and 1% acetonitrile in water at a flow rate of 5 μl/min. Peptides were further eluted onto the analytical column (75 μm x 15 cm, C18 pepmap 100, 2μm) at 300 nl/min by a linear gradient starting from 2% solvent A to 35% solvent B in 48 min. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile. PRM analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Scientific). The MS cycle consisted of a full MS1 scan performed at a resolving power of 70,000 (at 200 m/z) followed by time-programmed targeted MS2 scans with a normalized collision energy of 20 acquired at a resolving power of 35,000 (at 200 m/z). The quadrupole isolation window of precursor ions was set to 1 m/z unit for the MS2 events, and the duration of the time-programmed windows for each pair of endogenous and isotopically labeled peptides was set to 2 min.

1.4. PRM data processing

[0273] PRM data were processed as described in Martinez et al (supra). Briefly, the extracted ion chromatogram (XIC) areas of the five most intense fragment ions of each precursor (i.e., PRM transition) were extracted using the Skyline (v3.1) program (McCoss Lab, University of Washington, USA). Peptide identity was confirmed using a spectral matching approach based on the cosine of the spectral contrast angle (cos θ) calculated between the peak areas of the five transitions of the reference (a PRM acquisition of the synthetic peptide mixture without the biological matrix) and the areas of the corresponding transitions for the endogenous and heavy peptides in the clinical samples. Peptide detection and identification were accepted if both the cos θ of the endogenous and the isotope-labeled version of a peptide were greater than 0.98. MS measurements reduce the generated quantification score limits below 0.98, and in these cases area values were replaced by an estimate of the background.

1.5. Statistical analysis

[0274] The light/heavy area ratio of each peptide was extracted from Skyline and the mean across duplicates was calculated. Statistical analysis was performed in SPSS (v20.0) (IBM, Armonk, NY, USA) and Graph Pad Prism (v.6.0) (GraphPad Software, La Jolla, CA, USA). Comparison of monitored peptide levels between patient groups was performed using the nonparametric Mann-Whitney U test, as the data did not follow a normal distribution. p-values were adjusted for multiple comparisons using the Benjamini-Hochberg FDR method. Adjusted p-values less than 0.05 were considered statistically significant. Receiver operating characteristic (ROC) analysis was used to assess the specificity and sensitivity of the biomarkers and the area under the ROC curve (AUC) was estimated for each individual protein.

1.6. Development of classifiers

[0275] A logistic regression model was fitted to the data in order to assess the power of the different protein combinations to classify samples into two clinical categories. ROC curves were generated for each of these regression models; the AUC, and the sensitivity and specificity at the “ideal” cutoff point for discrimination between groups were obtained. The ideal cutoff corresponded to the threshold that maximized the distance to the identity (diagonal) line. The ideal criterion was: max (sensitivities + specificities). AUCs with 95% confidence intervals (CI) were computed with the Delong method. The 95% CIs of the sensitivity and specificity values were computed with bootstrap resampling and the averaging methods described by Fawcett. All ROC analyses were performed using the R package “pROC” (Robin et al., “pROC: and open-source package for R and S+ to analyze and compare ROC curves”, BMC Bioinformatics-2011, vol. no. 12:77). To assess the robustness of each protein panel, the “leave-one-out” cross-validation procedure was performed by applying to each sample in the dataset the logistic regression model fitted to the remaining samples in the dataset, thus deriving a new ROC curve and subsequently performing the usual ROC analysis.

2. Results

[0276] EEC is the most common histology in EC and has a good prognosis when compared with non-endometrioid EC (NEEC) cases. NEEC represents about 20% of all EC cases, but accounts for more than 50% of recurrences and deaths resulting from EC. Among NEEC, serous EC (SEC) is the most common subtype. After investigating the abundance of the 51 proteins in the cohort of 49 EEC cases and 20 SEC cases, the levels of eleven proteins were significantly increased in uterine aspirate samples from EEC patients (adjusted p-value <0.05), as described in Table 4. Among these, six proteins had successive fold changes greater than 2 and presented the highest individual AUC values: PIGR with 0.85 (95% CI, 0.734-0.958), CAYP1 with 0.83 (95% CI, 0.725-0.942), CTNB1 with 0.78 (95% CI, 0.670-0.895), SG2A1 with 0.77 (95% CI, 0.661-0.880), VIME with 0.76 (95% CI, 0.645–0.881), and WFDC2 with 0.74 (95% CI, 0.624–0.855). Table 4. Proteins differentially diagnosing EEC vs SEC (NEEC) *p-value <0.05

[0277] The inventors found that classification robustness was improved if in addition to determining the expression level of one or more of the proteins in Table 4, the levels of other proteins were analyzed. In particular, the levels of one or more of XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA.

[0278] Following the same procedure as described previously, all possible combinations of two to twelve proteins, and in particular two and three proteins, were evaluated among the diagnostic and predictive biomarkers to identify protein panels that would improve the outcome of individual biomarkers. A three-protein combination consisting of CTNB1, XPO2, and CAPG was the best performing panel for discriminating between EEC and SEC in uterine aspirate fluid samples with an AUC of 0.99 (95% CI, 0.90-1). This panel achieved 95% (95% CI, 85%-100%) sensitivity and 95.9% (95% CI, 89.8%-100%) specificity (Figure 1). After completion of leave-one-out cross-validation, the values were 95% sensitivity and 89.8% specificity.

[0279] As can be deduced from Figure 1, both CTNB1 and CAPG markers provided interesting sensitivity and specificity values to differentially diagnose EEC from SEC, but, surprisingly, the combination (panel) of CTNB1, CAPG and also XPO2 provided a highly sensitive and differentially specific diagnosis. Therefore, the panel should be understood as a diagnostic panel, but also as a reliable prognostic panel, allowing the classification of the EC subtype. The earlier the correct classification of the disease, the better it will be translated into an increased survival rate basically due to the correct medical regimen applied as early as possible.

[0280] In this study, a method is disclosed to reliably distinguish EEC and SEC histologies using a liquid biopsy obtained from the female genital tract (i.e., uterine aspirate fluid). This represents a real improvement, since the current diagnostic procedure relies on the histological examination of the limited cellular content of this sample and is associated with important drawbacks: an average of 22% of undiagnosed patients, and up to 50% of incorrect histotyping and/or grading of EC cases.

[0281] A clinical strength of this investigation is that the women enrolled in the study covered the wide range of women who enroll in the EC diagnostic process. Regardless of the EC patients, the two most common histologies, EEC and SEC cases, were included. Non-EC patients covered all women with suspected EC mainly due to AUB or endometrial thickening. This included women suffering from benign pathological conditions (mainly polyps, fibroids and endometrial hyperplasia), and women with normal endometrium. Furthermore, this study included both premenopausal women with functional endometrium and postmenopausal women presenting predominantly with an atrophic endometrium.

[0282] Considering the role of the studied proteins to improve the assignment of the risk group of EC patients, current leading risk stratification systems, such as the European Society for Medical Oncology (ESMO) classification, focus on histology, grade, myometrial invasion and lymphovascular invasion of tumors. Since most of the information is not available preoperatively, the histological subtype and grade become key factors for assignment of the risk group and for determining the extent of the surgical testing procedure. The combination of three proteins (CTNB1, XPO2 and CPG) derived from the present invention allowed an accurate discrimination between histological EC types EEC and NEEC (SEC) with a sensitivity of 95.0% and specificity of 95.9%.

[0283] These three proteins have been previously implicated in EC. Beta-catenin (CTNB1) plays an important role in epithelial cell-cell adhesion and in the transcription of essential genes responsible for cell proliferation and differentiation in the Wnt signaling pathway. Consistent with the observations in the current trial, CTNB1 has been described in EEC tissue specimens but not in NEEC tumors. Capping macrophage protein (CAPG) modulates cell motility by remodeling actin filaments. It is involved in cell migration and invasiveness in several types of cancers. Unlike CTNB1, higher levels of CAPG were reported in more aggressive SEC tumors compared to EEC cases at the tissue level, consistent with the results in uterine aspirates reported in the present paper. Finally, exportin-2 (XPO2), also known as cellular apoptosis susceptibility protein, plays a role in the mitotic spindle checkpoint. XPO2 depletion leads to cell cycle arrest and has consequently been associated with tumor proliferation; although it has also been associated with tumor invasion and metastasis. Increased XPO2 levels have been observed in many cancer types, including EC, and have been positively associated with higher cancer grade and worse patient outcome. Although no significant differences in XPO2 levels were observed between EEC and SEC cases in this study, its inclusion in the CTNB1 and CAPG panel significantly improved its performance.

[0284] Notably, this study addresses an important clinical need in EC, as the uterine aspirate-based proteomic approach described here will form the basis for identifying proteomic signatures that classify EC tumors into more clinically relevant risk groups to aid in predicting surgical treatment. Therefore, a first step has been achieved with a signature to accurately classify the most common histologies. Overall, the development of uterine aspirate-based biomarker signatures is expected to improve the management of EC patients and save considerable healthcare costs.

[0285] As indicated in the description, particular protein sets from Tables C and D are listed below: TABLE C. Protein sets for EC prognosis (EEC vs. NEEC) in uterine fluid samples TABLE D. Protein sets for diagnosis of EC in uterine fluid samples

[0286] The invention also relates to the contents of the following clauses:

[0287] Clause 1. Method of prognosis of endometrial cancer, the method comprising determining the level of expression of one or more of the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 in a sample of uterine fluid from the female genital tract.

[0288] Clause 2. The method of clause 1, wherein the uterine fluid sample is a uterine aspirate fluid sample from the female genital tract.

[0289] Clause 3. The method of any one of clauses 1 to 2 comprising determining the expression level of one or more of the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, and WFDC2

[0290] Clause 4. The method of any one of clauses 1 to 3, further comprising determining the expression level of one or more of the following proteins: XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA

[0291] Clause 5. The method of any one of clauses 1 to 4, further comprising determining the expression level of a protein selected from the group consisting of: XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA.

[0292] Clause 6. The method of any one of clauses 1 to 5, the method further comprising determining the expression level of one or more proteins selected from the group consisting of CLD6, BCAM, IF2B3, PLD3 and MX1 in an exosome-containing fraction isolated from a uterine aspirate.

[0293] Clause 7. The method of any one of clauses 1 to 6, comprising determining the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0294] Clause 8. The method of any one of clauses 1 to 7, wherein the prognosis of endometrial cancer is determined by distinguishing endometrioid endometrial cancer from non-endometrioid endometrial cancer.

[0295] Clause 9. The method of any one of clauses 1 to 8, wherein the expression level is determined at the protein level.

[0296] Clause 10. The method of any one of clauses 1 to 9, wherein the protein level is determined by an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0297] Clause 11. The method of any one of clauses 9 to 10, wherein the protein expression level is determined using an antibody or a fragment thereof capable of binding to the protein.

[0298] Clause 12. The method of clause 11, wherein said antibody or fragment thereof forms part of a kit.

[0299] Clause 13. Use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, as an in vitro marker for the prognosis of endometrial cancer in a sample of uterine fluid from the female genital tract.

[0300] Clause 14. Use of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 for the prognosis of endometrial cancer, in the method as defined in any of the preceding clauses 1 to 13.

[0301] Clause 15. Use of a kit for the prognosis of endometrial cancer, the kit comprising a solid support and means for detecting the expression level of one or more of the following proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1, and, optionally, means for detecting the expression level of one or more of the following proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA.

[0302] Clause 16. Use of a kit according to clause 15, the kit comprising a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0303] Clause 17. Use of a kit according to any one of clauses 15 to 16, wherein the means for detecting the level of expression of the proteins is a means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0304] Clause 18. Use according to any of clauses 15 to 17, wherein the means for detecting the level of expression of the proteins are antibodies or fragments thereof.

[0305] Clause 19. A kit comprising a solid support and means for detecting the expression level of one or more proteins selected from the group consisting of PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA.

[0306] Clause 20. A kit comprising a solid support and means for detecting the expression level of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM;BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C.

[0307] Clause 20. The kit of clause 19 further comprising means for detecting the expression level of at least one set of proteins selected from the group consisting of MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BCAM, MMP9; MMP9, AGRIN; AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9, and those sets of proteins listed in Table D.

[0308] Clause 21. The kit of any one of clauses 19 to 20, wherein the means for detecting the level of expression of the proteins is a means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof.

[0309] Clause 22. Kit according to any one of clauses 19 to 21, wherein the means for detecting the level of expression of the proteins are antibodies or fragments thereof.

[0310] Clause 23. A kit according to any one of clauses 19 to 22 which is a kit for performing an enzyme-linked immunosorbent assay.

[0311] Clause 24. A kit according to any one of clauses 19 to 23, further comprising a panel diagram for categorizing an individual sample.

[0312] Clause 25. A computer-implemented method for performing the method as defined in any one of clauses 1 to 12, wherein after determining the level of expression of one or more of the proteins for the diagnosis and/or prognosis of EC, said levels are given as a value and/or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein as a function of said computed levels, scores and/or values, a decision is made between the options of suffering from EC or not and/or between the options of suffering from different subtypes of EC. CITATION 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. - 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. - Martínez-Garcia E, et al. “Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate”, Oncotarget -2016, vol. node. 7(33), pp.:53102-53114 - Robin et al., “pROC: and open-source package for R and S+ to analyze and compare ROC curves”, BMC Bioinformatics-2011, vol. node. 12:77.

Claims (20)

1. Method for the diagnosis of endometrial cancer, CHARACTERIZED by the fact that the method comprises determining the level of expression of AGRIN in a sample of uterine fluid from the female genital tract. 2. The method of claim 1, wherein the uterine fluid sample is a uterine aspirate fluid sample from the female genital tract. 3. The method of claim 1 or 2, wherein the uterine fluid sample is an exosome-containing fraction isolated from a uterine aspirate. 4. Method according to claim 3, CHARACTERIZED by the fact that it comprises determining, in a fraction containing exosome isolated from a uterine aspirate, the expression level of at least one set of proteins selected from the group consisting of: AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9; and AGRIN, BCAM. 5. Method according to claim 2, CHARACTERIZED by the fact that it comprises determining, in a uterine aspirate, the expression level of at least one set of proteins selected from the group consisting of: AGRIN, MMP9, PODXL; AGRIN, MMP9; and AGRIN, MMP9, PIGR. 6. The method of any one of claims 1 to 5, further comprising determining the expression level of one or more proteins selected from PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENOA, PDIA1, KPYM, ANXA2, and FABP5. 7. Method according to any one of claims 1 to 6, CHARACTERIZED by the fact that it comprises determining the expression level of at least one set of proteins selected from the group consisting of: AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; and AGRIN, MMP9, in a fraction containing exosome isolated from UA; and determining the expression level of at least one set of proteins selected from the group consisting of: MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BCAM, MMP9; MMP9, AGRIN, in a uterine aspirate. 8. Method according to any one of claims 1 to 7, CHARACTERIZED by the fact that the expression level is determined at the protein level. 9. The method of any one of claims 1 to 8, wherein the protein level is determined by an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof. 10. The method of claim 8 or 9, wherein the protein expression level is determined using an antibody or fragment thereof capable of binding to the protein. 11. The method of claim 10, wherein said antibody or fragment thereof forms part of a kit. 12. Method according to any one of claims 1 to 11, CHARACTERIZED by the fact that it is carried out by means of a kit comprising a solid support and means for detecting the level of expression of AGRIN. 13. Method according to any one of claims 1 to 12, CHARACTERIZED by the fact that it comprises: a) determining, in vitro, the level of expression of AGRIN, in a sample of female uterine fluid; b) optionally, determining, in vitro, the level of expression of AGRIN, in an exosome-containing fraction isolated from the uterine fluid; and c) comparing the level of step (a) and, optionally, that of step (b) with a reference control level, in which if the level determined in step (a) and, optionally, that of step (b) is greater than the reference control level it is indicative that the individual is suspected of suffering from endometrial carcinoma. 14. Kit, CHARACTERIZED by the fact that it comprises a solid support and means for detecting the level of expression of AGRIN and, optionally, means for detecting the level of expression of the proteins of at least one set of proteins selected from the group consisting of AGRIN, CD81, TERA; AGRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9; AGRIN, BCAM; AGRIN, MMP9, PODXL; AGRIN, MMP9; and AGRIN, MMP9, PIGR. 15. The kit of claim 14 wherein the means for detecting the level of protein expression are means for performing an assay or technology selected from the group consisting of an immunoassay, a bioluminescent assay, a fluorescence assay, a chemiluminescence assay, an electrochemical assay, mass spectrometry, and combinations thereof. 16. The kit of claim 14 or 15, wherein the means for detecting the level of expression of the proteins are antibodies or fragments thereof. 17. The kit of any one of claims 14 to 16, characterized in that it is a kit for performing an enzyme-linked immunosorbent assay. 18. The kit of any one of claims 14 to 17, further comprising a panel diagram for categorizing an individual sample. 19. A method for deciding or recommending whether to initiate a medical regimen for an individual suspected of suffering from endometrial carcinoma, the method CHARACTERIZED by the fact that it comprises the steps of: a) determining, in vitro, the level of expression of AGRIN in a sample of female uterine fluid; b) optionally, determining, in vitro, the level of expression of AGRIN in an exosome-containing fraction isolated from the uterine fluid; and c) comparing the level of step (a) and, optionally, that of step (b) with a reference control level, wherein if the level determined in step (a) and, optionally, that of step (b) is higher than the reference control level it is indicative that the individual is suspected of suffering from endometrial carcinoma, wherein: i) if the individual is diagnosed as suffering from endometrial carcinoma or is suspected of suffering from endometrial carcinoma, then initiation of the medical regimen is recommended, and j) if the patient is diagnosed as not suffering from endometrial carcinoma, a follow-up is optionally carried out taking into account the result of an examination of the patient by a physician. 20. A computer-implemented method for performing the method as defined in any one of claims 1 to 12, wherein after determining the expression level of AGRIN and the proteins of the protein sets for diagnosing EC, said levels are given a value and/or a score, and optionally computed in a mathematical formula to obtain a computed value; wherein as a function of said computed levels, scores and/or values, a decision is made between the options of suffering from EC or not.