CTNB1 as a marker for endometrial cancer

Abstract

To provide a method that diagnoses and prognoses endometrial cancer in a readily available isolated sample, in particular, a uterine fluid sample by detecting the expression level of one or more proteins, and provide a kit comprising means of detecting the protein for use in disease diagnosis and prognosis.SOLUTION: A method for prognosis of endometrial cancer (EC) comprises determining 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, in a uterine fluid sample from a female genital tract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is based on European Patent Application EP17382483.0 filed on July 21, 2017. They assert their interests.

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

[0003] Endometrial cancer (EC) is the most common cancer of the female reproductive tract. It is a frequently observed invasive tumor, the fourth most common cancer among women in developed countries, and in the United States, it is the 20th most common cancer. In 2017, there were 61,380 diagnosed cases and an estimated 10,920 deaths. Currently, E 70% of C cases are diagnosed in the early stages of the disease, and in the early stages, the tumor is still localized within the endometrium. This is associated with a 96% overall 5-year survival rate. However, 30% of EC patients have a 5-year survival rate. Diagnosed only in the advanced stages of the disease, associated with a significant decline in the myometrium, the 5-year survival rate is higher. If invasion and / or lymph node involvement is already present, the rate drops to 67%, and in the case of distant metastasis... It decreases to 18%. Therefore, improving early diagnosis is important for properly managing EC and the disease. This is a major challenge in reducing mortality rates associated with [the condition].

[0004] Early detection of EC patients is crucial, as abnormal vaginal bleeding, which is present in 93% of women diagnosed with EC, is important. While the presence of symptoms such as these is advantageous, many other benign diseases can cause similar symptoms. It is caused by pelvic examination and transvaginal ultrasound. The distinction between patients with benign endometrial lesions and patients with EC is made by pelvic examination and transvaginal ultrasound. After a lengthy diagnostic process consisting of examinations, a histopathological examination was performed to confirm the endometrial biopsy. This is achieved only by [specific method]. The preferred biopsy used in this procedure is the uterine aspirate and / or pip. This is called an elle biopsy and is obtained by minimally invasive aspiration of endometrial fluid from the uterine cavity. The diagnostic procedure for uterine aspirates relies on the presence of cellular material, so unfortunately this process is difficult to perform. The failures in the selection process resulted in associated inappropriate sampling rates of 8% and 15%, respectively. The appropriate sampling rate increases to 12% and 22% in postmenopausal women. In this example, a hysteroscopy-guided biopsy is required, and this invasive technique can lead to the child The risk of complications, including perforation of the cervix, bleeding, and potential harm to other organs, increases.

[0005] To date, we have primarily focused on identifying EC protein biomarkers in tissue and serum samples. Many studies have been conducted on this (see, for example, Non-Patent Document 1 or 2). It does not contribute to any practical use as flooring.

[0006] Biopsies provide information on tumor histology and tumor grading, and are used for risk stratification of EC patients. It should be helpful and guide surgical staging procedures, but unfortunately, the cells available for examination are not sufficient. Due to the limited number of samples and the large inter-observer variability in pathological interpretation, biopsy and final uterine There is a 40-50% discrepancy in the histological type and grade of EC between excised specimens. Therefore, E A highly sensitive, specific, and reproducible method for improving the diagnosis of histological type and malignancy grade of tumors, prognosis, and preoperative evaluation. Identifying biomarkers will help in the proper management of EC patients and in the reduction of mortality and morbidity associated with this disease. It is extremely important in order to reduce the rate.

[0007] EC subtype or manifestation variety Among these, endometrioid endometrial carcinoma (EEC) is the most common histological structure in EC, and non-endometrioid EC The prognosis is favorable compared to that of NEEC cases. NEEC accounts for approximately 20% of all EC cas es, but accounts for more than 50% of EC recurrences and EC-related deaths. Among NEECs, serous EC ( SEC) is the most common subtype. There are no currently available tests that can distinguish between these two histological structures with different outcomes.

PRIOR ART DOCUMENT

NON-PATENT LITERATURE

[0008]

NON-PATENT LITERATURE 1

NON-PATENT LITERATURE 2

SUMMARY OF THE INVENTION

PROBLEM TO BE SOLVED BY THE INVENTION

[0009] In conclusion, despite the considerable efforts that have been made, there still remains a need for biomarkers that enable diagnosis and even prognosis of endometrial cancer with high sensitivity and specificity, particular ly biomarkers for clinical practice. [Means for Solving the Problem]

[0010] The inventors found that certain detectable protein markers in exosomes isolated from uterine fluid provide valuable diagnostic information for endometrial cancer (EC). Many of these proteins have been verified in uterine fluid itself, without isolating the exosome fraction . Therefore, when analyzed in uterine fluid, this protein is a meaningful diagnostic biomarker that enables distinguishing EC from non-cancer controls with high sensitivity and specificity.

[0011] Furthermore, the present inventors found that several proteins detectable in uterine fluid and the exosome fraction of uterine fluid are meaningful prognostic biomarkers for endometrial cancer (EC) . These proteins enable distinguishing endometrial cancer subtypes with high sensitivity and high specificity , thereby minimizing the risk of false positive and false negative classification between these subtypes.

[0012] Furthermore, since the protein can be detected in samples obtained without invasive medical procedures, for example in uterine fluid samples, which are a conventional sample type for EC diagnosis, it is expected to bring true advantages and improvements in the clinical practice of classification and management of EC patients.

[0013] In previous studies, the inventors have shown that the fluid fraction of uterine aspirate has important value for screening EC protein biomarkers (see, for example, Martinez-Garcia E, et al. “Development of a sequential wo rkflow based on LC-PRM for the verificat ion of endometrial cancer protein biomar kers in uterine aspirate samples”,Oncota Refer to rget-2016, vol.no.7(33), pp.:53102-53114. (Referring to illumination). This document describes the process implemented using a sequential workflow based on LC-PRM. This research highlights the importance of the biomarker validation phase in bridging the gap between discovery and clinical practice. This study shows that 26 factors were used to diagnose EC patients from non-EC patients (controls). The advantages of protein were emphasized.

[0014] Regarding novel diagnostic biomarkers of EC identified in uterine fluid exosomes, this invention One aspect of this method is a diagnostic method for EC, which involves a uterine fluid sample taken from the female reproductive tract. AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC 1, SMD3, ILF2, TERA, RL11, BCAM, ANXA2, LAT1, RU VB1, SH3L3, RPL13A, RS16, RSSA, RL12, PDIA1, RL 29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LA T1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD 3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14, ITB Determine the expression level of one or more proteins selected from the group consisting of 3 and CD166. This includes the following.

[0015] Another aspect of the present invention is an instrument for diagnosing EC in uterine fluid samples from the female reproductive tract. As intromarkers, AGRIN, MVP, VAMP8, SYIC, RAB8A, MX 1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, AN XA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL 12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS3 5, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3 , CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P 1. One or more proteins selected from the group consisting of VAC14, ITB3, and CD166. This involves the use of an in vitro that detects one or more endometrial cancer markers in a subject. It can also be constructed as a method, and the method is (a) to obtain a liquid sample from the female reproductive tract. (b) AGRIN, MVP, VAMP in uterine fluid samples from the female reproductive tract 8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TE RA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL1 3A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, M MP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, P ODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD1 Selected from 4, H10, CD81, AR6P1, VAC14, ITB3 and CD166 The amount of at least one endometrial cancer marker in the sample provides information for diagnosing EC. Including detection. Generally, endometrial cancer in uterine fluid samples isolated from the female reproductive tract. AGRIN, MVP, VAMP8, SYI as in vitro markers for diagnosing C, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL 11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS 16, RSSA, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, K PYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10 Selected from the group consisting of CD81, AR6P1, VAC14, ITB3 and CD166. It includes the use of one or more proteins.

[0016] The diagnosis of EC using one or more of the proteins listed above involves assessing the expression levels of these proteins. This can be done using a kit that includes means for making a determination. For this reason, a solid carrier and 1 or more The following proteins are listed above: AGRIN, MVP, VAMP8, SYIC, RAB8A, MX1 , IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, ANX A2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL1 2, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, F AS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS35 , ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P1 This also includes a kit that includes means for detecting the expression levels of VAC14, ITB3, and CD166. This is the state of affairs in the Ming Dynasty.

[0017] Another aspect of the present invention involves determining one or more of the proteins defined above for the diagnosis of EC. This involves using a kit that includes the means to do so.

[0018] Further aspects related to the diagnosis of EC include methods for identifying individuals suspected of having EC. There is, a) AGRIN, MVP, VAMP8, SYIC, RAB8 in female uterine fluid samples A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCA M, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSS A, RL12, PDIA1, RL29, PPIA, AGR2, MMP9, CLIC1, B The expression level of one or more proteins selected from the group consisting of CAM and KPYM is controlled by an invisible vein. The step of judging by the toro, b) AGRIN, MVP, and TACD2 in the exosome-containing fraction isolated from uterine fluid , FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RS1 6, RSSA, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA 1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, M LEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14 The expression level of one or more proteins selected from the group consisting of ITB3 and CD166 A step of making an arbitrary determination in vitro, and c) The level of step (a) and optionally the level of step (b) are compared to the reference level. Includes a comparison step, the level determined in step (a) and optionally step (b) If the level determined is higher than the reference control level, the subject is diagnosed with endometrial cancer. To provide a method to indicate that there is a suspicion of [something].

[0019] In a further embodiment, the present invention relates to a medical register for a person suspected of having endometrial cancer. A method for deciding or recommending whether or not to initiate mendation, a) AGRIN, MVP, VAMP8, SYIC, RAB8 in female uterine fluid samples A, MX1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCA M, ANXA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSS From A, RL12, PDIA1, RL29, PPIA, AGR2, MMP9 and KPYM A step to determine the expression level of one or more proteins selected from a group in vitro. , b) AGRIN, MVP, and TACD2 in the exosome-containing fraction isolated from uterine fluid , FAS, VAMP8, SYIC, SORT, LAT1, TERA, RUVB1, RS1 6, RSSA, SMD3, ADA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VPS35, ILF2, PDIA 1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6, IF2B3, CD59, M LEC, PGBM, S10AC, CD14, H10, CD81, AR6P1, VAC14 The expression level of one or more proteins selected from the group consisting of ITB3 and CD166 A step of making an arbitrary determination in vitro, and c) The level of step (a) and optionally the level of step (b) are compared to the reference level. Includes a comparison step, the level determined in step (a) and optionally step (b) If the level determined is higher than the reference control level, the subject is diagnosed with endometrial cancer. It indicates that there is a suspicion that... i) The subject has endometrial cancer or is suspected of having endometrial cancer. If rejected, initiation of a medical regimen is recommended, and ii) If the patient is diagnosed as not having endometrial cancer, the doctor's examination results Considering the results, follow-up will be conducted at the discretion of the individual. Provide a method.

[0020] By determining the marker level of the test sample, the level detected in the sample is quickly Since it can reflect the progression of the disease (i.e., the severity), those skilled in the art can further determine which is the most appropriate to recommend. It is possible to determine whether it is a treatment method.

[0021] In relation to a new prognostic biomarker for EC in uterine fluid, a first aspect of the present invention relates to intrauterine A method for the differential diagnosis of uterine cancer, comprising the expression of CTNB1 in uterine fluid samples from the female reproductive tract. This method includes determining the level.

[0022] Another aspect is a method for determining the prognosis of endometrial cancer, wherein a uterine fluid sample from the female reproductive tract is used. PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9 The expression level of one or more proteins including CD59, CLD6, IF2B3, PLD3, and MX1 This method includes determining the bell.

[0023] This method provides important diagnostic and prognostic information because it addresses the two different histological manifestations of EC. In other words, endometrioid endometrial carcinoma (EEC) and non-endometrioid endometrial carcinoma (NEEC) can be differentiated. Therefore, it allows for accurate prognosis, and EEC has a better, more likely outcome. (That is, a better prognosis). In other words, the determination of proteins in uterine fluid samples This allows for a differential diagnosis between EEC and NEEC.

[0024] As shown in the following example, the aforementioned protein affects patients with EEC and NEEC In isolated samples from patients suffering from the disease, the protein was expressed differentially, which is characteristic of E. Diagnosis of C or accurate prognosis of EC became possible. The proteins of diagnostic value are In relation to non-EC samples (healthy controls), the difference in uterine fluid samples from EC subjects. Due to its subsequent expression, it can be detected differentially. CAPG was increased in NEEC tumors. With the exception of the NEEC subtype, the EEC subtype has a prognostic value compared to the NEEC subtype. Protein levels increased significantly. Therefore, this protein is the most common histological This has made it possible to classify tumor subtypes. Therefore, the protein is important for diagnosis, prognosis, and / Or to improve preoperative risk assessment and to support the prediction of optimal surgical treatment. It is a useful tool. As will also be shown below, it accurately distinguishes between EC samples and non-EC samples. It is possible to distinguish them, and the EC organizational type (AUC of 0.99) also distinguishes them accurately, these and other types Several protein panels (combinations of proteins) containing proteins have been developed. Combining these panels with histopathological examination necessitates more invasive diagnostic sampling. This is expected to eliminate the need for surgical intervention and help predict the optimal surgical treatment for EC patients.

[0025] Therefore, generally speaking, one aspect of the present invention is a method for prognosis of endometrial cancer (EC). PIGR, VIME, CTNB1, CAYP1 in uterine fluid samples from the female reproductive tract ,SG2A1,WFDC2,CADH1,CD44,LEG3,LEG1,CAPG,A GR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 The method includes determining the expression level of one or more proteins in MX1.

[0026] A second aspect of the present invention is for determining the prognosis of EC in uterine fluid samples from the female reproductive tract. PIGR, VIME, CTNB1, CAYP1, SG2A as in vitro markers 1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, B CAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1 This involves the use of one or more proteins selected from the group consisting of the following. An in vitro method for detecting one or more endometrial cancer markers, wherein (a) the female reproductive tract or (b) the step of obtaining a liquid sample, and (b) PIGR, VIME, C in the sample TNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, L EG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, The amount of at least one endometrial cancer marker selected from IF2B3, PLD3, and MX1 A step to detect the marker, wherein the marker provides information for the differential diagnosis of EEC and NEEC. It can also be constructed as a method that includes steps. Generally, isolated from the female reproductive tract PIG as an in vitro marker for determining the prognosis of endometrial cancer in uterine fluid samples R, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD4 4, LEG3, LEG1, CAPG, AGR2, PODXL, MMP9, CD59, CL A protein selected from the group consisting of D6, BCAM, IF2B3, PLD3, and MX1. This includes the use of one or more of the following.

[0027] In a third aspect, the present invention relates to the method of the first aspect for the prognosis of endometrial cancer , PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1 , CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP A selection from the group consisting of 9, CD59, CLD6, IF2B3, PLD3 and MX1. It provides the use of one or more proteins. That is, proteins are used to differentiate between EEC and NEEC. It is used for that purpose.

[0028] Importantly, the objective of the protein biomarkers of this invention is to provide immunologically active materials widely available in hospitals. It can be evaluated using simple and low-cost methods such as epidemiological chemistry, chemiluminescence assays, or ELISA. As a result, these protein biomarkers are used in routine clinical diagnosis and / or prognosis. It can be easily implemented as a set, reducing the cost of the healthcare system. In addition, the present invention provides Diagnostic kits based on biomarkers will improve current methods of diagnosis and prognosis. This enables the uterine aspirate to provide valuable diagnostic and prognostic information about the disease.

[0029] Therefore, in a fourth aspect, the present invention provides the use of a kit for the prognosis of EC. The kit includes a solid carrier and the following proteins: PIGR, VIME, CTNB1, C AYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CA PG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, A means for detecting one or more expression levels of PLD3 and MX1, and optionally the following proteins Quality XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA (one or more of these) Includes means for detecting the expression level.

[0030] This embodiment provides a solid carrier and the following protein PIGR for use in the prognosis of EC. VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59 means for detecting one or more expression levels of CLD6, IF2B3, PLD3, and MX1, and In addition, optionally include the following proteins: XPO2, PRDX1, CLIC1, PDIA1, KPYM , ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGA It can also be constructed as a kit that includes means for detecting one or more expression levels of L and LDHA. can.

[0031] A new kit has been developed to facilitate the implementation of the method and use of the present invention. Therefore, In a fifth aspect, the present invention relates to a solid carrier and PIGR, VIME, CTNB1, CA YP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAP G, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, P LD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LD A means for detecting the expression level of one or more proteins selected from the group consisting of HA. We also offer a set.

[0032] Additional embodiments include solid carriers and LAMP, MMP9, PIGR;AGRIN, MMP9 , PIGR;AGR2, PIGR, PLD3;AGR2, BCAM, PODXL;BCA M, PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; C LD6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA ;AGRIN, BCAM;ANXA, BCAM;BCAM, RAB8A;BCAM, SY At least one of the groups consisting of IC;CLD6, IFB3; and the sets listed in Table C is selected. It is a kit that includes a means for detecting the expression level of one set of proteins.

[0033] In an additional aspect, the present invention relates to the prognosis of endometrial cancer in the method of the first aspect of the present invention. For PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, C ADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL Determine the expression levels of MMP9, CD59, CLD6, IF2B3, PLD3, and MX1. This provides a means for the differential diagnosis of EEC and NEEC. Yes. PIGR, VIM for the prognosis of endometrial cancer in the method of the first aspect of the present invention. E, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG 3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CL A means for determining the expression levels of D6, IF2B3, PLD3, and MX1, and optionally XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, C Determine the expression levels of H10, MIF, PEBP1, TPIS, NGAL, and LDHA. The use of the means is also provided.

[0034] In a further aspect, the present invention relates to the differential diagnosis of NEEC and EEC, thereby enabling the uterus To identify individuals suspected of having endometrial cancer, and further to identify EC subtypes. It is a law, a) PIGR, VIME, CTNB1, and CAY in uterine fluid samples from the female reproductive tract of the subject. P1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG , AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PL One or more protein expression levels selected from the group consisting of D3 and MX1, and optionally, Proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GST P1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL and LDHA The above steps involve determining the expression levels of the proteins in vitro, and b) A step of comparing the level of step (a) with a reference control level, (a) The level determined is PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, POD Reference reference levels for XL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1. If the level is higher than the CAPG reference control level, the subject has NEEC. The present invention provides a method including the step of indicating that the person is suspected of having EEC.

[0035] In this embodiment of the present invention, the reference control level is unknown from non-EC and NEEC samples. Clearly selected. As revealed above, with the exception of CAPG, the expression levels of EEC are The expression levels are higher than those of NEEC. Furthermore, CADH1, CAPG, CTNB1 and CD4 Level 4 is also higher than in the non-EC (cancer-free) control sample.

[0036] In a further embodiment, the present invention relates to the prognostic function of subjects suffering from endometrial cancer. A method for determining or recommending whether or not to initiate a medical regimen, a) PIGR, VIME, CTNB1, in uterine fluid samples from the female reproductive tract of the subject CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, C APG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3 The expression level of one or more proteins selected from the group consisting of PLD3 and MX1, and optional Proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, G STP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA A step of determining the expression level of 1 or more in vitro, and b) Protein levels in the test sample are PIGR, VIME, CTNB1, CAYP 1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1 If it is higher than the baseline control level and lower than the CAPG baseline control level, then EEC and Steps to establish the prognosis of EC by distinguishing NEEC, Includes, i) The subject has endometrial cancer or is suspected of having endometrial cancer. If a diagnosis is made and the subject is differentially diagnosed as having EEC, the medical regimen for EEC will be developed. It is recommended to start; ii) If the subject has endometrial cancer or is suspected of having endometrial cancer If diagnosed and differential diagnosis indicates that the subject has NEEC, the medical regimen for NEEC The start of the program is recommended, and By determining the marker level of the test sample, the level detected in the sample is Because it may reflect the aggressiveness of the disease, those skilled in the art can further establish the most suitable treatment methods that can be recommended. To provide a method.

[0037] Finally, another aspect of the present invention relates to the diagnostic and / or prognostic methods defined in the above aspects. The objective is to provide an algorithm for performing the deviation. In the sense of the present invention, The term "algorithm" refers to a panel or decision for correctly classifying individual samples. It is also synonymous with diagrams, predictors, and combinations of data.

[0038] According to the aspects and embodiments of the present invention, the diagnosis and prognosis of EC are as described above. Detection of biomolecules, proteins, antibodies, and / or mRNA containing one or more of the biomarkers is possible. A mathematical algorithm to evaluate the level of competence, in conjunction with other clinical parameters, or with other clinical parameters. Use independently of bed parameters, in healthy patients, patients with non-malignant endometrial disease, Individual samples derived from patients with premalignant endometrial disease and endometrial cancer. It is possible to correctly classify, or, as described above, specific histological subtypes of endometrial cancer. Subjects with a specific histological grade or stage, or specific EC characteristics Individual samples are further classified as originating from subjects with specific molecular subtypes. It is possible.

[0039] The classification algorithm determines the quantity of a specific biomarker or a subset of biomarkers measured. This can be as easy as determining whether it is above or below a specific cutoff number. When using omarkers, the classification algorithm can be a linear regression equation. Alternatively, the classification algorithm can be a linear regression equation. The rhythm may be the product of any of several learning algorithms. In the case of Gorhythm, a computer, such as a programmable digital computer, is used. Therefore, it is necessary to run an algorithm on the data and determine the classification based on that. It is possible. In either case, then, for example, a memory drive or a disk, Recording the status on a tangible medium, either in a computer-readable format or simply by printing it on paper. This can be done. The results can also be displayed on a computer screen. This algorithm is used for diagnosis. Used as a diagnostic and / or prognostic method, and in particular as a kit for carrying out the method disclosed in the preceding aspect It is part of the T.

[0040] For the diagnosis and / or prognosis of EC in a function of level, score, and / or calculated value After determining the expression level of a protein of 1 or higher, the presence or absence of premalignant lesions and / or EC is assessed. A decision is made between the options, and / or the options for disease incidence between different EC subtypes. [Brief explanation of the drawing]

[0041] [Figure 1] Figure 1 shows the ROC curves of markers that distinguish NEEC from EEC, specifically NEEC cases being serous endometrial carcinoma (SEC). The sensitivity and specificity of CTNB1, XPO2, and CAPG, as well as combinations of the three (or panels of proteins), are shown. [Modes for carrying out the invention]

[0042] Detailed description of the invention

[0043] All terms used herein, unless otherwise specified, refer to terms already established in the art. It shall be understood in the ordinary sense, such as knowledge. The specific definition is as described below, and the explicitly defined definition provides a broader definition. Unless otherwise specified, this applies uniformly to the entire specification and claims.

[0044] This invention provides a novel biomarker for the diagnosis and prognosis of endometrial cancer in female reproductive fluid. provide.

[0045] The term “diagnosis” is known to those skilled in the art. When used herein, “diagnosis” means To become aware of complications or risks of a specific medical condition in the subject, disease Or determining the nature of a condition, or distinguishing one disease or condition from another. This is understood to mean that this is for those who are trying to determine or identify a possible disease or disorder. This refers to both the method and the opinion reached through it. Diagnosis, in the sense of the diagnostic procedure, refers to treatment. And classifying an individual's condition into separate, distinct categories that enable medical decisions regarding prognosis. This can be considered an attempt. Subsequently, the diagnostic opinion is explained from the perspective of the disease or other condition. It is often revealed. However, diagnosis can take many forms. Diagnosis detects the presence. It may be possible to name a disease, lesion, dysfunction, or disorder. Diagnosis is a classification of a certain category. This may involve categorizing for management or prognosis purposes. Diagnosis is the degree of abnormality in the continuum. Or it may indicate either of the types of classification abnormalities. Generally, the diagnostics described herein The marker is a control isolation sample for endometrial cancer samples (including multiple EC types). It is a protein that is detected differentially at the hierarchical level in non-cancer individuals.

[0046] An in vitro method according to a first aspect of the present invention involves (a) asymptomatic subjects and (b) subjects suffering from endometrial cancer. (c) Subjects already identified as suspected of having the disease, during a complementary definitive diagnostic assay. (d) Subjects already diagnosed with endometrial cancer or subjects at high risk of developing the disease This can be done using pull commands.

[0047] In the present invention, the term "reference control level" as used in any method of the present invention is The term refers to a given molecular marker or a given combination of molecular markers, in this case, the first or second The form and any of the proteins listed in the detailed embodiment are understood to be a predetermined value. This value should be the molecular marker or multiple markers in the sample or sample group. It is induced from the level of [specific expression level]. When the expression level is determined at the protein level, it is called "reference expression." While "level" refers to a predetermined value for the amount of protein, the expression level is determined at the mRNA level. In this case, the "reference expression level" is a predetermined value for the amount of mRNA. The sample is used to determine the presence of the disease. , absence, stage, histological subtype or grade, or course that has been previously properly investigated This value is taken from the subject or group of subjects. This value is particularly important for the histological subtype of the disease, and for children. In order to determine the risk of developing or contracting endometrial cancer, subjects in whom the condition to be analyzed exists are selected. From subjects where such a condition does not exist (i.e., from subjects with endometrial cancer to subjects without endometrial cancer) It is used as a threshold to distinguish (from) the subject. This reference control level is the medical level of the subject. It is also useful for determining the necessity of starting a regimen and the effectiveness of the regimen. The number of targets that can obtain "Ru" is one or more targets for which the state does not exist, and one or more targets for which the state exists. It may include one or more subjects or both. A person skilled in the art with general knowledge will understand each method of the present invention. A more appropriate target or group of targets can be selected to obtain a reference control level. The method for obtaining reference values ​​from a group of subjects is well known in the latest technology (Burtis CAet al.,2008,Chapter14,section “Statistics” tical Treatment of Reference Values”).Details In such cases, the "reference control level" is determined by conventional ROC analysis (Receiver Operating Characteristics Analysis, Re receiver operating characteristic analysis This is the cutoff value defined by ). As recognized by those skilled in the art, the optimal cutoff value is The set-off value is the specific use of the diagnostic or prognostic method: purpose, target population for diagnosis or prognosis, specificity. It is defined according to factors such as the balance of sensitivity.

[0048] As used herein, "prognosis" refers to the predicted progression and outcome of the disease. The prognosis depends on the classification of the neoplasm (when increasing cataplasia correlates with the aggressiveness of the neoplasm). (An attempt to express the level of differentiation of cells in repeatable terms), neoplasm staging (An attempt to express the degree of neoplasm in a patient using repeatable terminology), neoplasm tissue This includes scientific subtypes and molecular subtypes of neoplasms. Prognosis, as used herein, In detailed embodiments, this means distinguishing between endometrioid endometrial carcinoma and non-endometrioid endometrial carcinoma. .

[0049] The term "fluid sample from the female reproductive tract" refers to the uterine organs, which form part of the female reproductive tract. Generated by a pipe, by suction, for example by vacuum suction or by Cornier pipel le and / or any other method of collecting fluid from the uterine cavity (i.e.) This shows a "uterine aspirate sample." Therefore, the sample contains uterine lavage fluid. According to the present invention... Furthermore, the aspiration of the fluid is performed without the previous saline injection step. That is, "aspirated material The term "uterine lavage fluid" does not include samples derived from uterine lavage fluid.

[0050] In this invention, "exosome" is defined as "extracellular vesicle," "microvesicle," or "exosome." They are interchangeably called "euterosomes" or "vesicles," and are found in blood, urine, and These are cell-derived vesicles present in many eukaryotic cell saturates, including the culture media for cell cultures. The reported diameter of exosomes is 30-100 nm, and they are low-density lipoproteins (L They are larger than DLs, but much smaller than, for example, red blood cells. Exosomes are part of the multimicroendoplasmic reticulum. It is released from the cell when it fuses with the cell membrane, or it is released directly from the cell membrane. Sosomes, in some cases, are used as biomarkers for diagnosis, prognosis, treatment, and health and disease. It can be used. "Exosome-containing fraction isolated from UA" is mainly exosome It should be understood as any purified fraction from uterine aspirate containing somes. A non-restrictive example of a method for isolating exosomes is detailed below.

[0051] Details of a novel diagnostic biomarker for ectoconstrictor identified in uterine fluid exosomes. In a detailed embodiment, the present invention can be optionally combined with any of the embodiments described above or below. This is a diagnostic method for eczema in which the sample is uterine fluid aspirate (UA).

[0052] Another detailed implementation of the EC diagnostic method, optionally combined with any of the embodiments described above or below. In this state, the sample contains exosomes isolated from uterine aspirate (uterine fluid sample). This method is fractional and includes AGRIN, MVP, TACD2, FAS, VAMP8, and SY. IC, SORT, LAT1, TERA, RUVB1, RSSA, RS16, SMD3, A DA10, RPL13A, RL11, IMB1, AGR2, ITA3, RUXE, RL1 2, PSMD2, MX1, VPS35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL29, PLD3, PPIA, ANXA2, SSRA, LA MP2, PODXL, CLD6, IF2B3, CD59, MLEC, PGBM, S10A C, CD14, H10, CD81, AR6P1, VAC14, ITB3, and CD166 This includes determining the expression level of one or more proteins selected from the group consisting of the following:

[0053] In another embodiment, the diagnostic method for EC involves using an exosome-containing fraction isolated from UA. AGRIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT 1, 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, C LD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, One or more selected from CD81, AR6P1, VAC14, ITB3, and CD166 Protein expression levels, as well as AGRIN and MV in uterine fluid samples from the female reproductive tract. P, VAMP8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, I LF2, TERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L 3, RPL13A, RS16, RSSA, RL12, PDIA1, RL29, PPIA, Expression of one or more proteins selected from the group consisting of AGR2, MMP9, and KPYM. This includes determining the level, and the latter sample is subjected to exosome isolation or purification. No.

[0054] In a detailed embodiment, the diagnostic method for EC involves two or more proteins, more specifically three or more proteins. This includes determining the expression levels of the above proteins, and even four or more other proteins.

[0055] In another detailed embodiment, the EC diagnostic method includes PERM, OSTP, CTNB1, CAYP1, XPO2, NGAL, SG2A1, CADH1, SPIT1, MMP9, N AMPT, LDHA, CASP3, PRDX1, MIF, K2C8, CAPG, MUC1 , ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, G STP1, GTR1, ENOA, PDIA1, KPYM, ANXA2, and FABP5 This further includes determining the expression level of one or more proteins selected from the group.

[0056] In other detailed embodiments, optionally combined with any of the embodiments described above or below, EC The diagnostic method involves detecting AGRIN, CD81, and T in the exosome-containing fraction isolated from UA. ERA;AGRIN, CD59, MVP;AGR2, AGRIN, CD81;AGRIN , CD166, MVP;AGRIN, CD81;AGRIN, CD166;AGRIN, CD59; and at least one set selected from the group consisting of AGRIN and MMP9. To determine the expression level of protein (i.e., biomarker), and / or in uterine aspirate MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP 9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BC At least one set selected from the group consisting of AM, MMP9;MMP9, and AGRIN This includes determining the expression level of a protein (i.e., a biomarker).

[0057] In yet another detailed embodiment, optionally combined with any of the embodiments described above or below, The diagnostic method for EC is selected from a small group consisting of the list in Table D (shown at the end of this specification). At the very least, it is necessary to determine the expression level of one set of proteins (i.e., biomarkers). include.

[0058] Another aspect of the present invention is an instrument for diagnosing EC in uterine fluid samples from the female reproductive tract. As intromarkers, AGRIN, MVP, VAMP8, SYIC, RAB8A, MX 1, IMB1, CLIC1, SMD3, ILF2, TERA, RL11, BCAM, AN XA2, LAT1, RUVB1, SH3L3, RPL13A, RS16, RSSA, RL 12, PDIA1, RL29, PPIA, AGR2, MMP9, KPYM, TACD2, FAS, SORT, LAT1, ADA10, ITA3, RUXE, PSMD2, VPS3 5, ANXA4, PLD3, SSRA, LAMP2, PODXL, CLD6, IF2B3 , CD59, MLEC, PGBM, S10AC, CD14, H10, CD81, AR6P 1. One of the proteins selected from the group consisting of VAC14, ITB3, and CD166. The above is the use. This embodiment involves an indication for detecting one or more endometrial cancer markers in a subject. It can also be constructed as the Toro method, and the method is as follows: (a) obtain a liquid sample from the female reproductive tract. (b) AGRIN, MVP, VAM in uterine fluid samples from the female reproductive tract P8, SYIC, RAB8A, MX1, IMB1, CLIC1, SMD3, ILF2, T ERA, RL11, BCAM, ANXA2, LAT1, RUVB1, SH3L3, RPL 13A, 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, CD Choose from 14, H10, CD81, AR6P1, VAC14, ITB3, and CD166. The amount of at least one endometrial cancer marker sampled provides diagnostic information for EC. This includes detection within the uterus. Generally, intrauterine fluid samples isolated from the female reproductive tract. AGRIN, MVP, VAMP8, S as in vitro markers for diagnosing membrane cancer YIC, 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, RUX E, PSMD2, VPS35, ANXA4, PLD3, SSRA, LAMP2, PODX L, CLD6, IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H Select from the group consisting of 10, CD81, AR6P1, VAC14, ITB3, and CD166. This involves the use of one or more selected proteins.

[0059] The diagnosis of EC using one or more of the proteins listed above involves assessing the expression levels of these proteins. This can be done using a kit that includes means for making a determination. Detailed embodiments of the diagnostic kit are shown below. The kit contains the following proteins AG from the exosome-containing fraction isolated from UA. RIN, MVP, TACD2, FAS, VAMP8, SYIC, SORT, LAT1, T ERA, RUVB1, RSSA, RS16, SMD3, ADA10, RPL13A, RL 11, IMB1, AGR2, ITA3, RUXE, RL12, PSMD2, MX1, VP S35, ILF2, PDIA1, ANXA4, MMP9, RAB8A, SH3L3, RL 29, PLD3, PPIA, ANXA2, SSRA, LAMP2, PODXL, CLD6 , IF2B3, CD59, MLEC, PGBM, S10AC, CD14, H10, CD8 1. Detect expression levels of 1 or higher for AR6P1, VAC14, ITB3, and CD166. This includes means to do so.

[0060] In another detailed embodiment, optionally combined with any of the embodiments described above or below, the kit The proteins included are PERM, OSTP, CTNB1, CAYP1, XPO2, and NG. AL, SG2A1, CADH1, SPIT1, MMP9, NAMPT, LDHA, CAS P3, PRDX1, MIF, K2C8, CAPG, MUC1, ANXA1, HSPB1, PIGR, CH10, CD44, CLIC1, TPIS, GSTP1, GTR1, ENO Further testing of the expression levels of A, PDIA1, KPYM, ANXA2, and FABP5 at level 1 or higher. Includes means of outputting.

[0061] In yet another, more detailed embodiment, the kit provides detailed prognostic information (i.e., prognostic information). Iomarker), PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2 , CADH1, CD44, LEG3, LEG1, CAPG, XPO2, PRDX1, CL IC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, LDHA, AGR2, PODXL, MMP9, CD5 9, CLD6, BCAM, IF2B3, PLD3, and MX1 - select one or more of these. The method includes means for detecting protein expression levels.

[0062] Another aspect of the present invention involves determining one or more of the proteins defined above for the diagnosis of EC. This involves using a kit that includes the means to do so.

[0063] In another detailed embodiment of the use of the kit, means for detecting the expression level of a protein This includes immunoassays, bioluminescence assays, fluorescence assays, chemiluminescence assays, and electrochemical assays. Perform an assay or technique selected from the group consisting of assay, mass spectrometry, and combinations thereof. It is a means to that end.

[0064] The Uniprot database accession numbers for all the proteins listed here are This corresponds to the version accessible on July 7, 2017. The accession number is actual Further examples are shown in the table.

[0065] The Uniprot database accession number for AGRIN is O00468. This protein is a heparin sulfate basement membrane glycoprotein involved in the formation and maintenance of neuromuscular junctions. It's about quality.

[0066] MVP is also known as a major vault protein, and in the Uniprot database... - The accession number is Q14764. MVP is involved in signal transduction.

[0067] VAMP8 is also known as vesicle-associated membrane protein 8, and in the Uniprot database... - The accession number is Q9BV40. VAMP8 is an auto-accession to the lysosomal membrane. Soluble N-ethyl maleate involved in autophagy through direct control of phagosome membrane fusion. It is a Mido-sensitive factor-adhering protein receptor (SNARE).

[0068] SYIC is also known as an isoleucine-tRNA ligase, and Uniprot The database accession number is P41252.

[0069] RAB8A is also known as the Ras-related protein Rab-8A, and Unipro The database accession number is P61006. RAB8A is involved in intracellular membrane transport. It is a small GTPase rab involved in [the process].

[0070] MX1 is also known as the interferon-induced GTP-binding protein Mx1, U The niprot database accession number is P20591. MX1 is wide It has antiviral activity against RNA viruses and some DNA viruses.

[0071] IMB1 is also known as importin subunit beta 1, and Uniprot The database accession number is Q14974. IMB1 is involved in nuclear protein transport. They are involved.

[0072] SMD3 is also known as the small nuclear ribonucleoprotein Sm D3, and Uniprot The database accession number is P62318. SMD3 is a spliceosome. It is an ingredient.

[0073] ILF2 is also known as interleukin enhancer binding factor 2, and Unipr The ot database accession number is Q12905. ILF2 is the IL2 gene. It is involved in regulation.

[0074] TERA is also known as a transitional endoplasmic reticulum ATPase, and in the Uniprot database... - The accession number is P55072. This protein is involved in the formation of the transitional endoplasmic reticulum. They are involved.

[0075] RL11 is also known as 60S ribosomal protein L11, and Uniprot The database accession number is P62913. This protein is a ribosome. As a component, it is involved in the synthesis of proteins within cells.

[0076] BCAM is also known as a basal cell adhesion molecule, and is listed in the Uniprot database. The session number is P50895. This protein is a laminin α-5 receptor. .

[0077] ANXA2 is also known as Annexin A2 and is active in the Uniprot database. The session number is P07355. This protein is PSCK9-enhanced LDLR degradation. It is a calcium-regulating membrane-bound protein that inhibits [a specific process].

[0078] LAT1 is also known as the large neutral amino acid transporter small subunit 1. The Uniprot database accession number is Q01650. This protein Quality is involved in the uptake of amino acids by cells.

[0079] RUVB1 is also known as Pontin 52 or INO80 complex subunit H. The Uniprot database accession number is Q9Y265. This protein The process primarily involves the transcription of selected genes through acetylation of nucleosome histones H4 and H2A. It is a component of the NuA4 histone acetyltransferase complex, which is involved in activation.

[0080] SH3L3 is SH3 domain-binding glutamate-rich protein 3 or SH3 It is also known as domain-binding protein 1, and access to the Uniprot database The protein number is Q9H299. This protein is involved in regulating glutarredoxin activity. It is possible.

[0081] RPL13A is also known as the 60S ribosomal protein, and Uniprot The database accession number is P40429. RPL13A is involved in the inflammatory process. It is involved in interferon-gamma-induced selective transcriptional inhibition.

[0082] RS16 is also known as 40S ribosomal protein S16, and Uniprot The database accession number is P62249.

[0083] RSSA is also known as 40S ribosomal protein SA or laminin receptor 1. The Uniprot database accession number is P08865. RSSA is It is necessary for the assembly and / or stability of the 40S ribosome subunit.

[0084] RL12 is also known as 60S ribosomal protein L12, and Uniprot The database accession number is P30050.

[0085] RL29 is also known as 60S ribosomal protein L29, and Uniprot The database accession number is P47914. This protein is a large riboso It is a component of the subunit.

[0086] PPIA stands for peptidyl-prolyl cis-trans isomerase A and cyclophylline A It is also known as rotamase A, and has a Uniprot database accession number. This is P62937. This protein is involved in protein folding.

[0087] AGR2 is an anterior gradient protein 2 homolog. It is also known as HPC8, and the Uniprot database accession number is O The protein is 95994. This protein is involved in the post-transcriptional synthesis and secretion of MUC2.

[0088] PODXL is also known as podocalixin or GCTM-2 antigen, Unipr The ot database accession number is O00592. PODXL is adhesion, cell morphology. It is involved in regulating the state and progression of cancer.

[0089] CD59 is also known as CD59 glycoprotein or MAC inhibitor protein, U The niprot database accession number is P13987. CD59 is the complement. It is involved in inhibiting membrane attack complex (MAC) activity.

[0090] TACD2 is a tumor-associated calcium signaling molecule 2 or cell surface glycoprotein Tr Also known as op-2, its Uniprot database accession number is P09. The value is 758. This protein can function as a growth factor receptor.

[0091] FAS is also known as fatty acid synthase, and access the Uniprot database. The instruction number is P49327. FAS is acetyl-CoA, malonyl-CoA, and NA It is involved in the formation of long-chain fatty acids from DPH.

[0092] SORT is also known as soltirin or neurotensin receptor 3, and Unip The ROT database accession number is Q99523. This protein is Gol It functions as a sorting receptor in the di-isomer region and as a clearance receptor on the cell surface. .

[0093] ADA10 is a protein containing disintegrin and metalloproteinase domains. It is also known as 10, and the Uniprot database accession number is O1467. 2. ADA10 is a membrane-bound precursor of TNF-α and is involved in the metabolism of multiple cell surface proteins. It is involved in the breakdown and release of proteins.

[0094] PGBM is a basement membrane-specific heparan sulfate proteoglycan core protein or perlka It is also known as [a specific database name], and its Uniprot database accession number is P98160. Therefore, PGBMs are involved in the fixation of negative electrostatic charge and angiogenesis.

[0095] ITA3 is also known as integrin α-3 or VLA-3 subunit α. The Uniprot database accession number is P26006. ITA3 is Bronectin, laminin, collagen, epiligrin, thrombospongin, and CSPG It is receptor 4.

[0096] RUXE is also known as small nuclear ribonucleoprotein E, and in the Uniprot database... - The accession number is P62304. This protein is a histone 3' terminal protein. It is involved in loss. It may indirectly be involved in hair growth.

[0097] PSMD2 is also known as the non-ATPase regulatory subunit 2 of the 26S proteasome. This is knowledge, and its Uniprot database accession number is Q13200. PS MD2 is involved in the ATP-dependent degradation of ubiquitinated proteins.

[0098] VPS35 is also known as vacuolar protein sorting-related protein 35, and Unip The ROT database accession number is Q96QK1. VPS35 is selected. It is involved in preventing the misselection of transmembrane cargo proteins into the lysosomal degradation pathway.

[0099] ANXA4 is also known as Annexin A4 or Lipocortin IV, Unipr The ot database accession number is P09525. ANXA4 is related to membrane fusion. It is involved in sphincter and exocytosis.

[0100] PLD3 is also known as phospholipase D3 or choline phosphatase 3, U The niprot database accession number is Q8IV08. This protein is It can be involved in APP processing.

[0101] S10AC is also known as protein S100-A12 or cargranulin-C. Yes, the Uniprot database accession number is P80511. Proteins are involved in regulating inflammatory processes and immune responses.

[0102] CD14 is also known as monocyte differentiation antigen CD14, and is listed in the Uniprot database. The accession number is P08571. This protein is bacterial lipopolysaccharide (LPS). It is a co-receptor for bacterial LPS, which is involved in the innate immune response to ).

[0103] LAMP2 is also known as lysosome-associated membrane glycoprotein 2, and Uniprot The database accession number is P13473. LAMP2 is chaperone-mediated. It is involved in autophagy.

[0104] CLD6 is also known as claudin-6 or Skullin, Unipro t database accession number is P56747. CLD6 is involved in tight junction-specific occlusion of intercellular spaces.

[0105] IF2B3 is also known as insulin-like growth factor 2 mRNA binding protein 3 or VICKZ fa mily member 3, and the Uniprot database accession number is O00425. IF2B3 is involved in the recruitment of target transcripts to cytoplasmic protein-RNA complexes.

[0106] MLEC is also known as malectin, and the Uniprot database accessio n number is Q14165. This protein is involved in the early stage of protein N-glycosylation.

[0107] H10 is also known as histone H1.0 or histone H1', Uniprot database accession number is P07305. This protein is involved in the condensation of nucleosome chains into higher-order structures.

[0108] CD166 is also known as CD166 antigen, and the Uniprot database acce ssion number is Q13740. It is involved in both heterotypic and homotypic cell-cell inter actions.

[0109] CD81 is also known as CD81 antigen or tetraspanin-28, Unipr ot database accession number is P60033. This protein is involved in lymph It may be involved in regulating tumor cell proliferation.

[0110] AR6P1 is also known as ADP-ribosylation factor-like protein 6-interacting protein 1. It is known, and the Uniprot database accession number is Q15041. This protein is involved in SLC1A1 / EAAC1-mediated glutamate transport.

[0111] VAC14 is also known as the protein VAC14 homolog or Tax1-binding protein 2. It is known, and the Uniprot database accession number is Q08AM6. The protein is an endosomal carrier vesicle (ECV) / multivesicle from the early endosome. It is involved in the biosynthesis of MVB transport intermediates.

[0112] ITB3 is also known as integrin β-3, and is listed in the Uniprot database. The query number is P05106. This protein is particularly fibronectin, For several ligands such as minin or osteopontin It is involved in cell signaling because it forms cell receptors.

[0113] PIGR is also known as a high molecular weight immunoglobulin receptor, according to Uniprot data. The base accession number is P01833, June 26, 2007 - v4. The substance binds to high molecular weight IgA and IgM on the basal outer surface of epithelial cells.

[0114] VIME, also known as vimentin, is found in a variety of non-epithelial cells, particularly mesenchymal cells. It is a class III intermediate filament. Vimentin is a nucleus, endoplasmic reticulum, and mitochondrial filament. It binds to the rear either laterally or at the end. Uniprot database accession number for vimentin is P08670, version 4 updated on January 23, 2007.

[0115] CTNB1 is also known as catenin β-1, and its Uniprot database acces sion number is P35222, version 1 updated on February 1, 1994. CTNB1 is a negative regulator of centrosome aggregation, and blocks anoikis in malignant kidney and intestinal epithelial cells.

[0116] CAYP1 is also known as calciphosine, and its Uniprot database acces sion number is Q13938, version 1 updated on November 1, 1997. CAYP1 is a calcium -binding protein that may play a role in cellular signal transduction events.

[0117] WFDC2, namely WAP 4-disulfide core domain protein 2, is also known as epididymal secreted protein E4, and is a broad-spectrum protease inhibitor. The Uniprot database accession number of WFDC2 is Q14508, updated on January 23, 2002 version 2.

[0118] CADH1 is also known as cadherin-1 or E-cadherin, and its Unipro t database accession number is P12830, version 3 updated on July 1, 1993. This protein is involved in the mechanism regulating intercellular adhesion, mobility and proliferation of epithelial cells . It acts as a potent invasion suppressor.

[0119] CD44 is also known as CD44 antigen, and its Uniprot database acces sion number is P16070, version 3 updated on October 5, 2010. It has affinity for HA Therefore, as well as possibly osteopontin, collagen, matrix metalloprotein Affinity for other ligands such as enzymes (MMPs) influences intercellular interactions and cell metabolism. It intervenes in the interaction between trix molecules.

[0120] XPO2 is also known as exportin-2 and is listed in the Uniprot database. The query number is P55060, dated March 29, 2005 - v3. This protein In particular, it transports the importin-α receptor and, after substrate (cargo) transport, moves from the nucleus to the cytoplasm. It intervenes in the re-exportation of importin-α to and, in its active GTP-bound form, importin-α and It is disclosed that it cooperatively binds to the GTPase Ran.

[0121] SG2A1 is also known as mammoglobin B, and is listed in the Uniprot database. The accession number is O75556, November 1, 1998 - v1. SG2A1 is It can bind to endogens and other steroids.

[0122] ENOA is also known as α-enolase, and access the Uniprot database. The production number is P06733, dated January 23, 2007 - v2. ENOA is involved in glycolysis. Similar to its role in growth regulation, hypoxia tolerance, and allergic reactions, it plays a role in a variety of processes. It is a multifunctional enzyme that plays a role on the cell surface of multiple cell types, such as white blood cells and neurons. Due to its ability to function as a rasminogen receptor and activator, it is used in blood vessels and cells. It can also function in the surrounding fibrinolytic system, stimulating immunoglobulin production.

[0123] LEG3 is known as galectin-3, and galectin Also known as -3 (Galectin-3), it is a galactose-specific lectin that binds to IgE. This is because endothelial cell migration is stimulated by CSPG4, and α-3,β-1 integrins are involved. It can intervene. Along with DMBT1, (due to similarity) the most columnar epithelial cells during early embryonic development. It is required for definitive differentiation. In the nucleus, it acts as a pre-mRNA splicing factor. Neutrophil activation and adhesion, chemoattraction of monocytes and macrophages, and opioid neutrophils Involved in acute inflammatory responses, including sonication and mast cell activation. Uniprot Day The database accession number is P17931, dated November 25, 2008 - v5.

[0124] LEG1 is also known as a protein homolog of LEG1 and is involved in early liver development. The Uniprot database accession number is P09382, March 2005. It's the 29th - v2.

[0125] CAPG is also known as a macrophage capping protein, Unipr The ot database accession number is P40121, November 30, 2010 - v2 CAPG reversibly blocks the anti-arrowhead end of actin filaments, but the shape is formed beforehand. The resulting actin filaments are not cleaved; it is a calcium-sensitive protein. PG may play an important role in macrophage function.

[0126] PRDX1 is also known as peroxiredoxin-1, according to Uniprot data. The base accession number is Q06830, dated June 1, 1994 - v1. PRDX 1 is involved in the oxidation-reduction regulation of cells.

[0127] CLIC1 is also known as chloride intracellular channel protein 1, and CLIC1 The Uniprot database accession number is O00299, dated January 23, 2007. -v4. CLIC1 is inserted into the membrane and forms a chloride ion channel. The activity of the substance is pH-dependent. Membrane insertion appears to be oxidation-reduction regulated, and under oxidative conditions... It can occur. It is involved in regulating the cell cycle.

[0128] PDIA1 is also known as a protein disulfide isomerase, and Unipr The ot database accession number is P07237, dated November 1, 1997 - v3. PDIA1 catalyzes the formation, breakdown, and rearrangement of disulfide bonds.

[0129] KPYM is also known as pyruvate kinase PKM, and in the Uniprot database... - Accession number is P14618, January 23, 2007 - v4. KPYM This catalyzes the transfer of the phosphoryl group from phosphoenolpyruvic acid (PEP) to ADP. Solutions that generate ATP and play a common role in caspase-independent cell death of tumor cells. It is a sugar-based enzyme.

[0130] GSTP1 is also known as glutathione S-transferase P, and Unip The ROT database accession number is P09211, dated January 23, 2007 - v2. Yes. GSTP1 negatively regulates CDK5 activity via p25 / p35 translocation, leading to neurodegeneration. To prevent this.

[0131] The Uniprot database accession number for GTR1 is P11166, 2006. October 3rd, 2018 - v2. GTR1 is a facilitating glucose transporter. This isoform GTR1 can be responsible for constitutive or basal glucose uptake. GTR1 has very high substrate specificity. It can transport a wide range of aldoses, including both pentoses and hexoses.

[0132] CH10 is also known as a 10kDa heat shock protein in mitochondria. The Uniprot database accession number is P61604, dated January 23, 2007. -v2. CH10, along with CPN60, is essential for mitochondrial protein biosynthesis. It is essential. It binds to CPN60 in the presence of Mg-ATP and acts as an ATPase of Mg-ATP. It suppresses activity.

[0133] MIF is also known as a macrophage migration inhibitor, according to the Uniprot database. - The accession number is P14174, January 23, 2007 - v4. MIF is It is involved in the innate immune response to bacterial pathogens.

[0134] PEBP1, or phosphatidylethanolamine-binding protein 1, is involved in ATP, opioid It binds to phosphatidylethanolamine, an iodide. PEBP1 binds to thrombin, nucleopropyl alcohol. It inhibits chymotrypsin and chymotrypsin, but (due to similarity) it inhibits trypsin and tissue-type plasma. It is a serine protease inhibitor that does not inhibit suminogen activator or elastase. The kinase activity of RAF1 is inhibited by inhibiting its activation, and RAF1 / MEK By dissociating the complex, it acts as a competitive inhibitor of MEK phosphorylation, thereby inhibiting it. The Uniprot database accession number for AF1 is P30086, dated 2007. This is v3, from the 23rd of the month.

[0135] TPIS is also known as triose phosphate isomerase, and Uniprot Data The database accession number is P60174, dated October 19, 2011 - v3. These proteins are involved in the gluconeogenesis pathway, which is part of carbohydrate biosynthesis.

[0136] NGAL is also known as neutrophil gelatinase-associated lipocalin, and Uniprot The database accession number is P80188, dated November 1, 1995 - v2. NGAL is involved in apoptosis and innate immunity due to interleukin-3 (IL-3) deficiency. It is.

[0137] LDHA is also known as L-lactate dehydrogenase A chain, and Uniprot days The database accession number is P00338, dated January 23, 2007 - v2. The protein is involved in step 1 of the subpathway that synthesizes (S) lactate from pyruvate. Yes, they are.

[0138] Some of these proteins are associated with the EC stage in tissue samples. However, there is no significant data to correlate with the values ​​of uterine fluid samples from the female reproductive tract. As has become clear, the detection of markers in uterine fluid (aspirated or cleansing fluid) can be used in gynecology. The advantages of collecting data from routine sampling in the clinical practice of diseases have been demonstrated, and cost And, importantly, tissue biopsy is avoided.

[0139] As described above, the present invention, in its first aspect, is a method for the differential diagnosis of endometrial cancer, A method including determining the expression level of CTNB1 in uterine fluid samples from the female reproductive tract. Law is also an objective.

[0140] In a detailed embodiment of the differential diagnostic method for endometrial cancer, the uterine fluid sample is from the female reproductive tract. This is a sample of uterine aspirate fluid.

[0141] In another detailed embodiment, this method involves the following proteins: MMP9, AGRIN, This further includes determining the expression levels of one or more CAPG, HSPB1, and XPO2.

[0142] In yet another detailed embodiment, the following proteins: PIGR, VIME, CAYP 1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, CD59, CLD6, IF2B3, PLD3, and MX1 (1 or more) This includes further determining the expression levels mentioned above.

[0143] In another detailed embodiment of the differential diagnostic method for endometrial cancer, the following protein: PRD X1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10 , MIF, PEBP1 , TPIS, NGAL, CAYP1, SG2A1, LDHA, P ERM, OSTP, SPIT1, NAMPT, CASP3, K2C8, MUC1, ANX Determine the expression level of one or more of A1, ANXA2, FABP5, and WFDC2. It also includes.

[0144] Furthermore, in another detailed embodiment, the method involves XPO2, PRDX1, CLIC1, P DIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1 Expression of one protein selected from the group consisting of TPIS, NGAL, and LDHA. This further includes determining the level.

[0145] In another detailed embodiment, the method involves extracting exosome-containing fractions isolated from uterine aspirates. Select from the group consisting of CLD6, BCAM, IF2B3, PLD3, and MX1 in minutes. This further includes determining the expression level of one or more proteins.

[0146] Furthermore, in another detailed embodiment, the method is LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIG R,PLD3;AGR2,BCAM,PODXL;BCAM,PODXL;PIGR,P LD3;BCAM, PIGR;CLD6, RAB8A;CLD6, PODXL;BCAM ,RL29;BCAM,PODXL;CLD6,PPIA,AGRIN,BCAM;AN XA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and and at least one set of protein expression selected from the group consisting of the sets listed in Table C. This further includes determining the level.

[0147] In another detailed embodiment of this method, the differential diagnosis of endometrial cancer is endometrioid endometrial cancer. It is determined by distinguishing it from non-endometrioid endometrial cancer and non-cancerous conditions.

[0148] In another detailed embodiment of the method, the expression level is determined at the protein level.

[0149] In a more detailed embodiment, protein levels are measured in immunoassays and bioluminescence assays. (i) Fluorescence assay, chemiluminescence assay, electrochemical assay, mass spectrometry, and combinations thereof. The determination is made by an assay or technique selected from a group of these.

[0150] In another detailed embodiment, the expression level of a protein is determined by its binding to that protein. The determination is made using an antibody or fragment thereof that can perform the necessary actions.

[0151] More specifically, the antibody or its fragments form part of the kit.

[0152] As noted, another aspect of the present invention relates to endometrium in uterine fluid samples from the female reproductive tract. PIGR, VIME, CTNB1 as in vitro markers for determining cancer prognosis. CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, C APG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3 The use of one or more proteins selected from the group consisting of PLD3 and MX1. More specifically, it involves the use of at least a certain level of CTNB1 expression.

[0153] The present invention relates to a method in any one of the embodiments for the prognosis of endometrial cancer. , PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH 1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MM Selected from the group consisting of P9, CD59, CLD6, IF2B3, PLD3, and MX1. This also relates to the use of one or more proteins.

[0154] Another aspect involves the use of a kit for prognostic and differential diagnosis of endometrial cancer. The kit also includes a solid carrier and the following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, C APG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3 A means for detecting one or more expression levels of PLD3 and MX1, and optionally the following Protein XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP 1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA This includes means for detecting the expression levels of the above.

[0155] In a detailed embodiment of the kit's use, the kit includes a solid carrier and LAMP, MMP 9, PIGR;AGRIN, MMP9, PIGR;AGR2, PIGR, PLD3;AG R2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, PIGR;CLD6, RAB8A;CLD6, PODXL;BCAM, RL29;BCA M, PODXL; CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM; B CAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C A method for detecting the expression levels of at least one set of proteins selected from the group consisting of T Includes steps.

[0156] In a detailed embodiment of the use of the kit, the means for detecting the protein expression level is: Immunoassay, bioluminescence assay, fluorescence assay, chemiluminescence assay, electrochemical assay (i) Performing an assay or technique selected from the group consisting of mass spectrometry and combinations thereof. It is a step.

[0157] More specifically, the means for detecting the protein expression level is an antibody or its fragment That is the case.

[0158] Another aspect of the present invention relates to a solid carrier and PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AG R2, BCAM, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENO A, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and Includes means for detecting the expression level of one or more proteins selected from the group consisting of LDHA. In particular, it is a kit that includes means for detecting at least the expression level of CTNB1.

[0159] Another embodiment includes a solid carrier and LAMP, MMP9, PIGR;AGRIN, MMP9, PIGR;AGR2, PIGR, PLD3;AGR2, BCAM, PODXL;BCAM , PODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CL D6, PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYI C; At least selected from the group consisting of CLD6, IFB3, and the sets listed in Table C. This kit includes a means for detecting the expression level of a set of proteins.

[0160] In a detailed embodiment of the kit, the kit is: MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AGRIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1; MMP 9, RSSA; MMP9, MVP; MMP9, RAB8A; MMP9, VAMP8; BC AM, MMP9;MMP9, AGRIN; AGRIN, CD81, TERA; AGRIN , CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD166, MV P;AGRIN, CD81;AGRIN, CD166;AGRIN, CD59;AGRI At least one of the group consisting of N, MMP9, and the set of proteins listed in Table D The method further includes means for detecting the expression level of a set of proteins.

[0161] In a more detailed embodiment of the kit, the means for detecting the protein expression level is: Munoassay, bioluminescence assay, fluorescence assay, chemiluminescence assay, electrochemical assay , by means of performing an assay or technique selected from the group consisting of mass spectrometry and combinations thereof. be.

[0162] More specifically, the kit uses an antibody or other means to detect the expression level of a protein. Includes fragments of.

[0163] In another detailed embodiment of the kit, the kit performs an enzyme-linked immunosorbent assay. This is a kit for that purpose.

[0164] In another detailed embodiment, the kit includes a panel for classifying individual samples. Includes eargrams as well.

[0165] The present invention carries out a differential diagnosis method as disclosed above and defined in any of the embodiments. A computer implementation method for the diagnosis and / or prognosis of EC, which involves protein After determining an expression level of 1 or higher, a value and / or score are assigned to the level, and a calculated value is obtained. To do so, it is arbitrarily calculated using a formula, and in the functions of level, score and / or calculated value, EC Between the options of being infected or not being infected, and / or different EC subtypes It also concerns the method by which decisions are made among the available options.

[0166] As described above, in another aspect, the present invention also aims to provide a method for the prognosis of EC. In a detailed embodiment of the first aspect of the invention, the method involves collecting a uterine fluid sample from the female reproductive tract. This determines the expression levels of PIGR, VIME, LEG1, and CAPG in the substance. This includes.

[0167] Another detailed embodiment of this aspect of the present invention, optionally combined with any of the embodiments described above or below. Morphologically, a uterine fluid sample is a sample of uterine fluid aspirated from the female reproductive tract.

[0168] The necessary procedures are minimal, and clinically meaningful information can be obtained using uterine aspirates. The ability to detect proteins differentially is a significant advantage.

[0169] In another detailed embodiment of this aspect, the method involves the following proteins: PIGR, VI ME, CTNB1, CAYP1, SG2A1, WFDC2, and CADH1, one or more This includes determining the current level. As described in the examples, one of these seven proteins By determining the level, the area under the ROC curve (AUC) is 0.74~0.85 (trust). This enables highly sensitive and specific diagnosis of EC subtypes within a 95% detection range.

[0170] The inventors have developed a method for detecting the expression levels of other proteins in uterine fluid, particularly in uterine aspirates. This improves the robustness (sensitivity and specificity) of EC subtype classification and disease diagnosis. It was determined that it was possible. For this reason, the first and In other detailed embodiments of the same model, the method involves the following proteins:XPO2,PR DX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH1 0, MIF, PEBP1, TPIS, NGAL, CAPG, CD44, LEG1, LEG 3. Further includes determining the expression level of LDHA at 1 or higher.

[0171] More specifically, the methods include XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL 1 selected from the group consisting of CAPG, CD44, LEG1, LEG3, and LDHA This further includes determining the expression level of one protein.

[0172] Further details of the first and other embodiments of the present invention, optionally combined with any of the embodiments described above or below. In a detailed embodiment, the method involves PIGR, VIME, CTNB1, CAYP1, and SG2. A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1 ,XPO2,PRDX1,CLIC1,PDIA1,KPYM,ENOA,GSTP1, It consists of GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA. This involves determining the expression levels of two proteins selected from the group, and the low expression levels of the proteins. At the very least, one of the following is required: PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC 2. Selected from the group consisting of CADH1, CD44, LEG3, LEG1, and CAPG. ru.

[0173] In another detailed embodiment, the method involves PIGR, VIME, CTNB1, CAYP1 ,SG2A1,WFDC2,CADH1,CD44,LEG3,LEG1,CAPG,A GR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 , MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GS TP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA This involves detecting the expression levels of two proteins selected from a group consisting of the following:

[0174] In another detailed embodiment of the first and further aspects of the method, the method is AGR2, BCA M, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 This includes determining the expression levels of two proteins selected from a given group.

[0175] The first and further method embodiments of the present invention, which can be optionally combined with any of the embodiments described above or below. In another detailed embodiment, the method involves PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AG R2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GST P1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA This includes determining the expression levels of three proteins selected from a group of proteins. At least one of the quality is PIGR, VIME, CTNB1, CAYP1, SG2A1, W Select from the group consisting of FDC2, CADH1, CD44, LEG3, LEG1, and CAPG. It will be selected.

[0176] The first and further method embodiments of the present invention, which can be optionally combined with any of the embodiments described above or below. In another detailed embodiment, the method involves PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AG R2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GST P1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA This includes determining the expression levels of three proteins selected from a given group.

[0177] In another detailed embodiment of the first and further aspects of the present invention, the method is AGR2 , BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and This includes determining the expression levels of three proteins selected from the group consisting of MX1.

[0178] In another detailed embodiment of the method for the prognosis of EC, the method involves isolating from uterine aspirate CLD6, BCAM, IF2B3, PLD3 and M in the exosome-containing fraction This further includes determining the expression level of one or more proteins selected from the group consisting of X1. nothing.

[0179] The inventors found that in a fraction of uterine aspirate containing exosomes in a more purified form, a certain mer We found that the protein was differentially expressed in EEC and NEEC. Therefore, the present invention relates to verifying the prognosis determined by uterine fluid, or to isolating exosomes. Processing to increase the sensitivity of the method when relevant data cannot be obtained from uterine fluid without the use of uterine fluid. The procedure includes an optional step of determining specific markers in the completed uterine aspirate (exosome fraction). .

[0180] In a more detailed embodiment, the method for prognosis or differential diagnosis of EC is LAMP, MMP9 ,PIGR;AGRIN,MMP9,PIGR;AGR2,PIGR,PLD3;AGR 2,BCAM,PODXL;BCAM,PODXL;PIGR,PLD3;BCAM,P IGR;CLD6, RAB8A;CLD6, PODXL;BCAM, RL29;BCAM , PODXL;CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM; BC AM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C To determine the expression level of at least one set of proteins selected from the group consisting of the following: Includes.

[0181] LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIG R,PLD3;AGR2,BCAM,PODXL;BCAM,PODXL;PIGR,P LD3; BCAM, PIGR (Table B below); and those listed in Table C (shown at the end of the specification) A specific combination selected from (s) allows for the detection of protein expression levels in uterine aspirate. At that time, it was judged with a high prognosis value. Table B below shows the AUC and the confidence interval (CI) of the 95th percentile. This is shown. The data comes from the analysis of samples from EEC and NEEC. Table C shows the AUC. The value is also shown.

[0182] On the other hand, Table A shows the following combinations: CLD6, RAB8A; CLD6, PODXL; BCAM ,RL29;BCAM,PODXL;CLD6,PPIA,AGRIN,BCAM;AN XA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3 A It showed a UC value and a 95% CI, and the exosome-containing fraction isolated from uterine aspirate was found to be tan. This provides important prognostic information when the expression level of the protein is detected. The data are from EEC and N This is derived from the analysis of samples from the EEC.

[0183]

number

[0184] In a more detailed embodiment, the method involves reducing the expression levels of CTNB1, XPO2, and CAPG. This includes determining the bell. As explained and illustrated below (Figure 1), this combination This enables highly sensitive and specific differential diagnosis between EEC and NEEC (serous EC; SEC). ru.

[0185] The first and further method embodiments of the present invention, which can be optionally combined with any of the embodiments described above or below. In another detailed embodiment, the method involves PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AG R2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GST P1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA Expression of 4, 5, 6, 7, 8, 9, 10, or 11 proteins selected from the following group. This includes determining the level, and at least one of the proteins being PIGR, VIME, C TNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, L The group is selected from EG1 and CAPG.

[0186] The first and further method embodiments of the present invention, which can be optionally combined with any of the embodiments described above or below. In another detailed embodiment, the method involves the following proteins: PIGR, VIME, CTNB 1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1 The expression levels of CAPG, AGR2, BCAM, PODXL, MMP9, and CD59 were measured. This includes making a judgment.

[0187] In any of the embodiments given above or below, in any aspect of the present invention The expression level is determined at the protein level. In this embodiment, the protein marker While not limited to these, natural sequence peptides, isoforms, and textures are also important. All homologs, fragments, and precursors of polypeptides and markers are included. This includes modified forms of butylates and their derivatives. In a more detailed embodiment, protein Bell offers immunoassays, bioluminescence assays, fluorescence assays, chemiluminescence assays, and electrolysis By an assay or technique selected from the group consisting of biometric assays, mass spectrometry, and combinations thereof. It will be determined accordingly.

[0188] In the detailed embodiments described above or below, the level of expression is determined by immunochemistry. It will be done.

[0189] When used herein, the term "immunochemistry" refers to the antigen in a sample (usually a tan). Proteins and peptides, in this case, any of the above proteins, either individually or in combination, This presentation showcases diverse detection techniques that utilize the principle of antibodies that specifically bind to antigens. The visualization of antibody-antigen interactions can be achieved in several ways. In the most common example, antibody The body binds to enzymes such as peroxidase that can catalyze the color reaction. Alternatively, antibodies can Fluorophores such as ruorethen or rhodamine can also be tagged. Immunization Scientific techniques can be direct or indirect. Direct methods are one-step staining methods. This technique uses labeled antibodies (e.g., FITC-conjugated antiserum) that react directly with the antigen. Because it uses only one, it is simple and quick, but signal amplification is only slightly Therefore, it has lower sensitivity and is not as commonly used as the indirect method. The indirect method involves sample The first layer contains an unlabeled primary antibody that binds to the target antigen, and the second layer contains a labeled secondary antibody that reacts with the primary antibody. (The second layer is included.) This method is used when the secondary antibody is bound to a fluorescent or enzyme reporter. Due to signal amplification caused by the binding of multiple secondary antibodies to each primary antibody, direct detection methods are less effective. It will also become more sensitive.

[0190] When a secondary antibody is bound to multiple biotin molecules, further amplification becomes possible, and Avidin Supplement with a complex of neutraavidin-enzyme, streptavidin-enzyme, or neutraavidin-enzyme. It is possible. The indirect method, apart from having higher sensitivity, requires relatively less generation. Another advantage is that it only requires a small number of standard conjugated (labeled) secondary antibodies. In the direct method, the target and It is necessary to label each of the antigens with a primary antibody. (A certain target nucleus) Tagged nucleic acid probes (designed to specifically bind to acid sequences) are labeled with antibodies. If detectable later, it is also possible to detect certain nucleic acid sequences using immunochemical techniques. It is necessary to keep this in mind. For this reason, protein detection is required for the specific target protein RNA. It uses tagged nucleic acids designed to bind to a sequence, and then selectively binds to the tag. This can be done by detecting tagged nucleic acids using labeled antibodies.

[0191] Suitable immunoassay treatments include enzyme-linked immunosorbent assays (ELISA, for example). Multiplex ELISA, enzyme immunodot assay, agglutination assay, antibody-antigen - Antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography Immunochromography or other immunographs well known to those skilled in the art Examples of assay formats include radioimmunoassays and protein microarray formats. It is possible.

[0192] In one embodiment, in combination with any of the embodiments described above or below, protein The expression level of quality is determined by immunoassay.

[0193] In another embodiment, combined with any of the embodiments described above or below, a protein The expression level is determined by ELISA, particularly multiplex ELISA.

[0194] Alternatively, protein expression levels can be measured by bioluminescence, fluorescence, chemiluminescence, electrochemistry, or mass spectrometry. This can be determined by [the method described].

[0195] Alternatively, the protein expression level is determined by the protein's proteotype peptide (given pro It has an amino acid sequence that is uniquely associated with the protein tested in the teome. The level of peptides can be determined by measuring them using mass spectrometry. In another embodiment, in combination with any of the embodiments described above or below, tamper The expression level of the substance is determined by the antibody or its fragment that can bind to the target protein. It is determined using [this method].

[0196] The term "antibody or fragment thereof that can bind to a target protein" means, An immunoglobulin or fragment thereof that can selectively bind to a target protein and This should be understood as follows: This term includes monoclonal antibodies and polyclonal antibodies. This includes the "fragment" of the target protein's epitope. It comprises any portion of an antibody having a suitable size and structure. Suitable fragments include: Examples include F(ab), F(ab'), and Fv. "Epitope" refers to the immune system (B cells). It is a part of the antigen recognized by T cells or antibodies.

[0197] Antibodies used for specific detection are polyclonal or monoclonal. In the latest technologies for production and characterization, there are well-known methods. Polyclonal Methods for generating antibodies are well known in prior art. In short, it involves using proteins to make animals immune. Polyclonal antibodies are prepared by immunization, then serum is collected from immunized animals, and the antibodies are then processed. Isolate. A wide range of animal species can be used for antiserum preparation. Typically used for antiserum preparation. The animals used may be rabbits, mice, rats, hamsters, guinea pigs, or goats.

[0198] Furthermore, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, this procedure involves immunizing a suitable animal with a disease-related protein. The composition can be administered in an amount effective for stimulating antibody-producing cells. Monoclonal antibody The preparation method is generally initiated following the same guidelines as the preparation of polyclonal antibodies. The substance is injected into the animal as an antigen. The antigen is a complete or incomplete Freund's adjuvant. It can be mixed with any adjuvant. Repeat immunization with the same antigen at intervals of approximately two weeks. do.

[0199] In another detailed embodiment of the third aspect, means of carrying out the present invention are a part of the kit Form. Include an antibody or fragment thereof for detecting the target protein in the kit. The kit provides means (additives, solvents) for depicting antibody-protein interactions. It may further include the following.

[0200] These antibodies are used to determine the expression of a target protein in a fifth embodiment of the present invention. It can be used as a "means."

[0201] Therefore, in detailed embodiments of the first and further methods of the present invention, protein The expression level can be measured using an antibody or fragment thereof that can bind to the protein. It will be determined accordingly.

[0202] In another detailed embodiment, the antibody or a fragment thereof forms part of the kit.

[0203] Under the first and further aspects of the present invention, the proteins to be analyzed (listed 2-11) All of the above embodiments relating to the second, third, and fourth aspects of the present invention also relate to the details of the use of the second, third, and fourth aspects of the present invention. This is an embodiment of the law.

[0204] Alternatively, the expression level can be determined at the mRNA level.

[0205] In one embodiment, the amount of mRNA in each marker is, for example, 1 or more polynucleos Endometrial cancer markers or polynucleotides that hybridize to complement It is detected by polymerase chain reaction using gonucleotide primers. In the application form, the amount of mRNA is one or more polynucleotides, endometrial cancer markers or similar Using oligonucleotide probes that hybridize to the complement of polynucleotides, It is detected using hybridization techniques.

[0206] When using mRNA detection, the method follows standard methods well known in the art, and the isolation is performed. The converted mRNA is combined with a reagent to convert it to cDNA, and the converted cDNA is then processed using a nucleic acid primer. - With the appropriate mixture, the amplification reaction reagent (e.g., cDNA PCR reaction reagent) in the container The contents of the container are reacted to produce an amplified product, and the amplified product is analyzed in the sample. This can be done by detecting the presence of one or more polynucleotide endometrial cancer markers. Regarding mRNA, the presence of polynucleotide endometrial cancer markers in the sample was detected. The analysis step can be achieved using Northern blot analysis. The analysis step is increased The presence of polynucleotide endometrial cancer markers in the vasoconstrictor product was quantitatively detected, and the detected markers were... The amount of such markers obtained in normal and malignant tissue using similar primers Further achieved by comparing with a panel of expected values ​​regarding the known existence or non-existence of obtain.

[0207] In another embodiment, the present invention provides a method for (a) isolating mRNA from a sample (b) The conversion of mRNA to cDNA by combining mRNA with a reagent, (b) the converted cDNA NA is hybridized with an amplification reaction reagent and one or more polynucleotide endometrial cancer markers. (c) Treatment with nucleic acid primers to produce amplification products, and analysis of the amplification products. Then, measure the amount of mRNA encoding the protein endometrial cancer marker, and (d) The amount of mRNA measured in normal tissue and diseased tissue obtained using the same method (for example) The amount detected is compared to a panel of predicted values ​​for malignant tissue. To provide the law.

[0208] In a detailed embodiment of the present invention, RT-PCR is used for detection and analysis. The mRNA of protein endometrial cancer markers can be amplified. Other embodiments of the present invention include Quantitative RT-PCR was used to quantitatively measure the amount of mRNA of the protein endometrial cancer marker. Measured in real time RT-P. Further embodiments of the present invention use real time RT-P for quantification and analysis. Use CR.

[0209] With respect to the device or kit of the present invention, the kit, in a detailed embodiment of the present invention, Provided for the analysis of patient samples. Such devices or kits include at least A subset of proteins is selected from the proteins listed above and from Tables A to F listed below. It includes reagents that specifically identify one or more proteins. Target devices include: Arrays in which reagents are spatially separated on substrates such as slides, gels, and multiwell plates. Examples include reagents in suspension or suspended form, such as reagents bound to beads. It may be provided as a kit containing the drug. The reagents in question are reagents specific to the autoantibody marker. Drugs are one example. Examples of such reagents include antigenic proteins or peptides. Such devices or kits may contain cytokine-specific antibodies or their fragments. It may also include things like [this].

[0210] As described above, one aspect of the present invention relates to a solid carrier and the following proteins PIGR, VIM E, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG 3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CL A means for detecting one or more expression levels of D6, IF2B3, PLD3, and MX1, and The following proteins are particularly important: XPO2, PRDX1, CLIC1, PDIA1, KPYM, EN OA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and A kit comprising means for detecting an expression level of 1 or higher of LDHA, and NEEC's EE This involves using a kit to assess the prognosis of endometrial cancer by differentiating between C.

[0211] In a detailed embodiment of the use of the kit, the kit includes a solid carrier and PIGR, VIME , CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3 , LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD 6, IF2B3, PLD3 and MX1, XPO2, PRDX1, CLIC1, PDIA1 , KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPI 2, 3, 4, 5, 6, 7, 8, 9 selected from the group consisting of S, NGAL, and LDHA. The method includes means for detecting the expression levels of 10 and 11 proteins. More specifically, the kit The system includes a solid carrier and means for detecting three expression levels of these proteins.

[0212] In a detailed embodiment of the kit's use, the kit includes a solid carrier and LAMP, MMP 9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; A GR2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM , PIGR;CLD6, RAB8A;CLD6, PODXL;BCAM, RL29;BC AM, PODXL; CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM; BCAM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the following listed in Table C Detects the expression levels of at least one set of proteins selected from a group consisting of sets. It is a kit that includes the means to use.

[0213] In a more detailed embodiment of the use of the kit, the kit includes a solid carrier and CTNB1, This kit includes means for detecting the expression levels of XPO2 and CAPG.

[0214] In another detailed embodiment of the use of the kit, means for detecting the expression level of a protein This refers to an antibody or a fragment thereof that specifically binds to a target protein.

[0215] In a fifth aspect, the present invention relates to a solid carrier and PIGR, VIME, CTNB1, CA YP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAP G, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, BCAM, IF 2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM , ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGA The expression level of one or more proteins selected from the group consisting of L and LDHA is detected. Regarding kits that include the means.

[0216] In a detailed embodiment of the fifth aspect, the kit includes a solid carrier and LAMP, MMP9 , PIGR;AGRIN, MMP9, PIGR;AGR2, PIGR, PLD3;AGR 2, BCAM, PODXL; BCAM, PODXL; PIGR, PLD3; BCAM, P IGR;CLD6, RAB8A;CLD6, PODXL;BCAM, RL29;BCAM , PODXL;CLD6, PPIA; AGRIN, BCAM; ANXA, BCAM; BC AM, RAB8A; BCAM, SYIC; CLD6, IFB3; and the sets listed in Table C Means for detecting the expression levels of at least one set of proteins selected from the group consisting of the following: Includes.

[0217] In a more detailed embodiment, the kit contains 2 to 500 sets, and more specifically, 2 to 400 Includes means for detecting the expression level of a combination of sets.

[0218] In another detailed embodiment, optionally combined with any of the embodiments described above or below, The kit is compatible with MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AG RIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1 ;MMP9, RSSA;MMP9, MVP;MMP9, RAB8A;MMP9, VAMP 8;BCAM, MMP9;MMP9, AGRIN;AGRIN, CD81, TERA;A GRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD16 6, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; Selected from a group consisting of AGRIN, MMP9, and the set of proteins listed in Table D. The method further includes means for detecting the expression levels of at least one set of proteins.

[0219] Specific combinations selected from Table E and those listed in Table D (shown at the end of this specification) ) is determined to be a high diagnostic value when the expression level of protein in the uterine aspirate is detected. The following table E shows the AUC and the 95% confidence interval (CI). The data is for non-EC and E. This is derived from the analysis of samples from C (including EEC and NEEC). Table D also shows the AUC. .

[0220]

number

[0221] On the other hand, Table F shows the following combinations: AGRIN, CD81, TERA; AGRIN, CD5 9, MVP;AGR2, AGRIN, CD81;AGRIN, CD166, MVP;AG RIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MM The P9 AUC value and 95% CI are shown, and the exosome-containing fraction isolated from uterine aspirate is shown. This provides important prognostic information when protein expression levels are detected within minutes. The data is non-E This is derived from the analysis of C and EC (including EEC and NEEC) samples.

[0222] As shown in the fourth aspect, in a detailed embodiment of this fifth aspect, tamper The means for detecting the expression level of a protein is an antibody or its filament that specifically binds to the target protein. It's a lag issue.

[0223] In another detailed embodiment of the fourth and fifth aspects of the present invention, the kit is an ELISA kit In this embodiment, the kit includes a solid carrier and the above-mentioned protein and protein The method includes means for determining the expression level of any combination of components. In another embodiment, The kit consists of a solid carrier and an antibody or its flag that specifically bind to the detected target protein. These antibodies contain ments and are bound to reporter molecules that can generate signals. Examples of "solid carriers" include nitrocellulose membranes, glass, or polymers. Commonly used polymers include cellulose, polyacrylamide, nylon, and polystyrene. The solid carrier is made of polyvinyl chloride or polypropylene. The solid carrier is a strip, tube, bead In the form of a disc or microplate, or other suitable for performing immunoassays. It may be the surface.

[0224] The "reporter molecule" used herein, due to its chemical properties, is used for the detection of antigen-binding antibodies. This refers to molecules that provide an analytically identifiable signal, enabling detection. It can be either quantitative or quantitative. The most commonly used reporter molecule in this type of assay is yeast It is either an element, a fluorophore, or a molecule containing a radionuclide (i.e., a radioisotope). In enzyme immunoassays, the enzyme is generally glutaraldehyde or periodate. It is bound to the secondary antibody. However, in order to be immediately recognized, a wide variety of different Combination techniques exist and are readily available to those skilled in the art. Commonly used enzymes include: In particular, horseradish peroxidase, glucose oxidase, β-galactosidase Examples include enzymes and alkaline phosphatases. Substrates used with specific enzymes are: Generally, during hydrolysis by the corresponding enzyme, it is selected for the generation of a detectable color change. For example, 5-bromo-4-chloro-3-indolylphosphate / nitrobluteto Lazolium is suitable for use with alkaline phosphatase conjugates and peroxidases. The conjugates include 1,2-phenylenediamine, 5-aminosalicylic acid, and 3,3:5,5:te Tramethylbenzidine or tolidine are commonly used. Instead of the chromogenic substrates mentioned above, fluorescent It is also possible to use a fluorescence-generating substrate that produces the product. An example of a fluorescence-generating substrate is fluorescein. These are cein and rhodamine. When activated by irradiation with light of a specific wavelength, they become fluorescent dyes. Labeled antibodies absorb light energy, inducing an excited state within the molecule, which is then observed using an optical microscope. The light emitted is a characteristic color that is visually detectable. Both immunofluorescence and EIA techniques This method is well-established in this field and is particularly favorable for this method. However, other reporters Substances, such as radioactive isotopes, chemiluminescent molecules and bioluminescent molecules, and / or dyes and other colorants. Sexual substances may also be used.

[0225] The selection of a specific reporter molecule-binding antibody is largely determined by the intended use of the test kit of this invention. It is determined by the purpose and the user.

[0226] In binding assays for measuring the level of biomarkers, solid-phase or homogeneous forms are used. Suitable assay methods include sandwich or competitive binding assays. Examples of Ndwich immunoassays include U.S. Patent No. 4,168,146 and U.S. Patent No. 4, These are described in publications 366 and 241, both of which are incorporated herein by reference in their entirety. It is being noted. Examples of competing immunoassays include U.S. Patent No. 4,235,601, U.S. Disclosed in Japanese Patent No. 4,442,204 and U.S. Patent No. 5,208,535 These include, and each of them is incorporated herein by reference in its entirety. . Multiple biomarkers can be used in multiplex assays, for example, by using a conjugation reagent array. Multiplexing by means of labeling, multiplexing using spectral distinction of labels, for example, Luminex (registered Flow cytometry of binding assays performed on particles using the (trademark) system. It can be measured using multiplexing of the analysis.

[0227] The assay of the present invention can be carried out by any preferred method. In one embodiment, Iomarker levels are measured in a single sample, and these measurements are not limited to this. However, a single well on an assay plate, a single assay cartridge, a single side A single assay chamber or assembly including a flow device, a single assay tube, etc. This can be done with a device. The biomarker level can be achieved using several techniques available to those skilled in the art. For example, direct physical measurement (e.g., mass spectrometry) or binding assay (e.g., immunoassay, coagulation assay) It can be measured using either a collection assay or an immunochromatography assay. The method involves a chemical reaction, such as a change in optical absorbance, a change in fluorescence, chemiluminescence, or electrochemiluminescence. The generation of, changes in reflectance, refractive index or light scattering, accumulation or emission of detectable labels from the surface, Measure signals generated from oxidation or reduction, redox species, electric current or electric potential, magnetic field changes, etc. This may also include the detection of the photoluminescence of the label (e.g., fluorescence, time-resolved). By measuring fluorescence, evanescent wave fluorescence, upconvertrin photon, and multiphoton fluorescence, ), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic field, enzyme activity (for example) Enzymes that cause changes in optical absorbance or fluorescence, or cause the emission of chemiluminescence. By measuring the enzyme activity through the reaction, the labeling of the labeling reagent can be determined. By measuring involvement, coupled events can be detected. Alternatively, the use of labeling may be required. No detection techniques are available, such as mass (e.g., surface acoustic wave measurement), refractive index (e.g., surface plasmon measurement). Techniques based on (luminescence measurement) or measurement of the intrinsic luminescence of the specimen may be used.

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

[0229] In another embodiment, the kit is defined as encoding a protein endometrial cancer marker. This is a microarray containing a set of genes whose expression is significantly altered by endometrial disease. The embodiments given above for the specific proteins to be analyzed (list 2-11) All of these are detailed embodiments of microarrays.

[0230] In another detailed embodiment, the kit of the present invention is used for classifying individual samples. Further includes a channel diagram.

[0231] A further aspect of the present invention relates to the medical treatment of patients with endometrial cancer in terms of prognosis. This was a method for determining or recommending whether or not to initiate the process. Detailed embodiments of this method are described below. And the method a) PIGR, VIME, CTNB1, in uterine fluid samples from the female reproductive tract of the subject CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, C APG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3 The expression level of one or more proteins selected from the group consisting of PLD3 and MX1, and optional Proteins XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, G STP1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDH A step of determining the expression level of A at 1 or higher in vitro, and b) Protein levels in the test sample are PIGR, VIME, CTNB1, CAYP 1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, MX1 If it is higher than the baseline control level and lower than the CAPG baseline control level, then EEC and Steps to establish the prognosis of EC by distinguishing NEEC, Includes, i) The subject has endometrial cancer or is suspected of having endometrial cancer. If the patient is diagnosed with EEC and differential diagnosis is made, lymph node dissection may be performed. The EEC medical regimen, which is complemented by a total hysterectomy and bilateral adnexectomy Starting is recommended, ii) If the subject has endometrial cancer or is suspected of having endometrial cancer If diagnosed and differential diagnosis indicates that the patient has NEEC, a total hysterectomy and bilateral hysterectomy will be performed. Adnexectomy, pelvic and para-aortic lymph node dissection, omentectomy, and peritoneal dissection. Initiation of a NEEC medical regimen, consisting of oneal biopsy, is recommended.

[0232] Should a medical regimen be initiated for patients with endometrial cancer in a state of established prognosis? In another detailed embodiment of the method for determining or recommending whether or not the subject is affected by the EEC If diagnosed, adjuvant therapy selected from radiation therapy, chemotherapy, and combinations thereof is recommended. Recommended. In yet another detailed embodiment, the subject is diagnosed with NEEC. In such cases, chemotherapy is recommended as adjunctive therapy.

[0233] Another aspect of the present invention is a diagnostic and / or prognostic method defined in the above aspects and embodiments. The objective is to provide an algorithm for carrying out one of the following:

[0234] In a detailed embodiment, the algorithm is for the diagnosis and / or prognosis of EC. In particular, for determining the EC subtype, especially EEC or NEEC, in order to assess the prognosis of the disease. This is a computer implementation method. This algorithm allows samples to be infected with EC. Whether the sample originates from an infected subject or an uninfected subject, or whether the sample is infected with EC If the subject is affected, it is necessary to determine whether they have EEC or NEEC. This is possible. In a more detailed embodiment, the algorithm provides a recommended treatment. Furthermore, a computer implementation method for carrying out the method defined above is also provided, and this method Then, after determining the expression level of one or more proteins for the diagnosis and / or prognosis of EC A value and / or score are assigned to the level, and a mathematical formula is arbitrarily used to obtain the calculated value. In the functions of level, score and / or calculated value, if you are suffering from or have suffered from EC The decision is made between the options that are not affected, and / or between the options that are affected, across different EC subtypes. A decision is made.

[0235] In other words, in another detailed embodiment of the algorithm of the present invention, the diagnosis of EC and / or, after determining the expression level of one or more proteins for prognosis, the level is given a value and / or The score is given and is arbitrarily calculated using a formula to obtain the calculated value, and the level, score and / Or, in the function of calculating values, between the options of being infected with EC or not being infected, A decision is made between the options for which the affected EC subtype is affected and / or between different EC subtypes.

[0236] This invention distinguishes endometrioid endometriotic carcinoma (EEC) from non-endometrioid endometriotic carcinoma (NEEC). A method for determining the prognosis of endometrial cancer by using a sample isolated from the female reproductive tract. This method includes determining the expression level of one or more of the following PIGR and VIME proteins. This includes laws.

[0237] The inventors discovered that these two proteins are released from a sample of the female reproductive tract. We were the first to discover that it is useful for distinguishing EC subtypes. The samples were particularly useful for the uterine tract. This method can be selected from tissue biopsy of the organs and fluid from the reproductive tract (i.e., uterine aspirate). , CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3 , LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD 6, IF2B3, PLD3, MX1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS The expression level of one or more proteins selected from the group consisting of NGAL and LDHA Further detection is also included. The expression level is particularly important for antibodies or anti-proteins that specifically bind to proteins. This is determined by body fragments, particularly at the protein level. The kit of parts and means used is also part of the present invention.

[0238] The assay conducted by the present inventors identified proteins that provide important information about EC. This is observed to be differentially expressed in EEC samples and NEEC(SEC) samples. This is the case with VIME. Therefore, this protein is used for the classification of EC subtypes. Furthermore, it also becomes possible to confirm the diagnosis. The invention was a method for diagnosing and / or prognosing EC. In particular, determining the expression level of VIME in female isolated samples from the female reproductive tract. Includes methods.

[0239] Furthermore, the assayed samples reveal the difference between EC and endometrial hyperplasia in the female reproductive tract. This allows for the determination of differential expression of several proteins in liquid samples. Endometrium Hyperplasia is a thickening of the endometrium caused by excessive strogen stimulation. It is a benign condition. However, it should be considered a precursor lesion to EC and should be clearly diagnosed. Includes EC samples (any subtype, EEC and NEEC) and hyperplasia cases (inc.) In an assay using non-EC samples (ludig), 16 proteins were differentially identified. It has been found to be abundant in hyperplasia and other non-EC controls (normal endometrium or fibroids) The adjusted p-value was <0.05 among women with benign conditions other than hyperplasia such as polyps. The magnification change exceeded 2x. Of these proteins, four were NAMPT, ENOA, and CATD. , and GSTP1 showed an AUC higher than 0.85. After proper validation, these were found to be EC This leads to the diagnosis of hyperplasia. Therefore, the present invention relates to the diagnosis of endometrial hyperplasia in the subject. Methods for diagnosis and / or prognosis, particularly in the following cases in isolated female samples from the female reproductive tract: Proteins: Expression levels of NAMPT, ENOA, CATD, and GSTP1 of 1 or higher This also relates to methods, including the determination process. It involves the early detection of hyperplasia, a precursor lesion of EC. This will increase patient follow-on to detect malignant tumors as early as possible. It is possible. In relation to this aspect, the present invention relates to NAMPT, ENOA, CAT Monitoring medical regimens for hyperplasia using one or more D and GSTP1 markers We also provide a method to bring the marker to a normal level (i.e., to the level of a control without hyperplasia). A decrease or return to indicates that the patient responded favorably to the medical regimen, and therefore, the regimen It can be shown that the men are active. The marker level does not change significantly, or rises. If this occurs, it means the regimen is ineffective and / or the hyperplasia is progressing to EC. This indicates a high level of suction. Surgical treatment is necessary in these cases.

[0240] The in vitro method of the present invention provides diagnostic and / or prognostic information. In one embodiment, The method of the present invention comprises (i) the step of collecting diagnostic and / or prognostic information, and (ii) the step of The process further includes the step of storing information on a data carrier.

[0241] In the sense of the present invention, "data carrier" refers to paper or other materials used for the diagnosis and / or prognosis of endometrial cancer. It should be understood as any means containing meaningful information data for the purpose of carrying out the carrier. This could be any element or device capable of accommodating prognostic data. For example, the carrier could be: ROM, such as CD-ROM or semiconductor ROM, or magnetic recording media, such as floppy disks. - May include a storage medium such as a disk or hard disk. Furthermore, the carrier may be electrically or Electric or optical signals may be transmitted via optical cables, or wirelessly or by other means. It can be a carrier capable of transmitting signals, etc. Diagnostic / prognostic data can be transmitted via cable or other devices. If the signal is represented by a chair or means that can be directly transported, the carrier is such It may consist of a cable or other device or means. Other carriers are USB devices. It relates to chairs and computer archives. Examples of suitable data carriers include paper, CDs, This can be done via USB, a PC computer archive, or voice registration using the same information.

[0242] Throughout the specification and claims, the term "comprise" and the following The variation of the term is intended to exclude other technical features, additives, ingredients, or steps. No. Furthermore, the term "comprise" means "consist of." This includes the case of "ting of". Additional objects, advantages and features of the present invention are discussed in the specification. This will become apparent to those skilled in the art, or it can be learned through the implementation of the present invention. The examples are provided for illustrative purposes only and are not intended to limit the present invention. No. Furthermore, the present invention does not apply to all possible combinations of the specific preferred embodiments described herein. It covers all combinations. [Examples]

[0243] Example 1: Biomedical analysis of EC in exosomes derived from uterine fluid and uterine fluid -ka

[0244] 1. Samples, reagents, and methods 1.1 Description of patients and samples The patient was treated at three different institutions: HUVH (Hospital University V all d'Hebron, Barcelona, ​​Spain), HUAV (Hospital U Niversitari Arnau de Vilanova, Lleida, Spain) and UMCF (University Medical Center of Freib Applications were accepted from Freiburg, Germany. Each participating institution obtained approval through an ethical review process. Samples were collected after participants signed informed consent forms.

[0245] Uterine aspirate (UA) is a sample from Cornier Pipelle (Gynetics Med). The sample was obtained by aspiration using ical products. The sample was placed in a 1.5 mL tube. Then, phosphate-buffered saline (PBS) was added in a 1:1 ratio (v / v), and the sample was gently prepared. Smooth pipetting, F45-30-11 rotor (Eppendorf Microc Centrifuge (Entrifuge 5417R) at 2500 g (4°C) for 20 minutes. The entire process, including separation, was kept on ice to remove the cell fraction. Next, the remaining supernatant (S The N) fraction was dispensed and frozen at -80°C until needed.

[0246] In the isolation of exosomes (abbreviated as ELV), ELVs were previously described by Thery et al. ELV isolation protocol http: / / www.ncbi.nlm.nih.gov Following the modified method in / pubmed / 18228490, fractional centrifugation was performed from the SN of UA. Obtained. Briefly, SN was thawed and diluted with PBS to a final volume of 25 mL. 10,0 The centrifugal separation step was performed at 00 × g (4°C) for 30 minutes using Thermo Scientific. Heraeus MultifugeX3R Centrifugal Separator (FiberLite Rotor F The procedure was performed using 15-8x-50c) and cell fragments, macroparticles, and apoptotic bodies were removed. The resulting pellet, in which V has been concentrated, is resuspended in 50 μL of PBS and frozen at -80°C. The condensation was then performed. Next, the supernatant was transferred to an ultracentrifuge tube (Beckman Coulter) and PBS was used. The Thermo Scientific Sorva, equipped with an AH-629 rotor, meets the requirements. 2 hours of centrifugation at 100,000 × g (4°C) using a WX UltraSeries centrifuge. During this time, the first ultracentrifugation step was performed. The supernatant from this second centrifugation is the soluble fraction. It was frozen at -80°C. This first pellet was resuspended in PBS, and 100,000 The pellet was centrifuged again at ×g (4℃) for 1 hour. The final pellet with concentrated ELV was (miniature (Abstract cells (MVs) and some remaining apoptotic bodies) were resuspended in 50 μL of PBS. 5 μL of MV and ELV pellets were taken for quantification by particle size distribution and NTA at -8 The samples were stored at 0°C, and the remaining samples were frozen at -80°C for protein extraction.

[0247] 1.2 Analysis of Tan by Parallel Reaction Monitoring (LC-PRM) of Liquid Chromatography Protein extraction and identification At the time of discovery, the solution used was 40 mM Tris pH 8, 300 mM NaCl, and 10 mM ED. TA, 2% Triton X-100 and 1:100 protease inhibitor (Sigma- Resuspend ELV in a 1:1 ratio (v / v) in a lysis buffer composed of Aldrich (Aldrich). Next, the sample was frozen at -20°C for at least 8 hours, then thawed on ice, and the amplitude was measured. 5 seconds of ultrasonic treatment at 100% (Labsonic M, Sartorius Sted The membrane was disrupted by cycling through the im Biotech (im Biotech) process five times. The extracted protein... It was stored at -20°C until needed.

[0248] The verification phase was performed on the ELV and uterine aspirate (UA) without the need to isolate the ELV. During the verification phase, in order to extract the protein from ELV, the wash contained in the lysis buffer was used. The agent was changed to NP-40 at a concentration of less than 1%, and protein extraction was performed directly by in-solution digestion and LC-MS / The MS was already prepared to be suitable. The remaining procedure was kept the same. The liquid fraction of UA To extract proteins, microvesicles, protein aggregates and / or other components present in the sample are needed. To break down the mucus, each UA is subjected to ultrasonic treatment (Labson) at 100% amplitude for 5 seconds. We performed five cycles (ic M, Sartorius Stedim Biotech). Next, Albumin & IgG depletion spin trap kit (GE Healthcare) Use according to the manufacturer's instructions to extract albumin from samples. Min and immunoglobulin G were removed. All extracted proteins were kept at -2 until needed. Stored at 0°C. Uterine fluid sample without removal of albumin and immunoglobulin G. Diagnostic and prognostic data were obtained even in cases of ELV (endoscopic lung vein) or UA (urinary anesthetic) without ELV isolation.

[0249] At the discovery stage, the peptide dissolved in 0.1% formic acid was first placed in an in-house packed column with an inner diameter of 1 50μm ID×20cm nano-LC(Jupiter C18,3μm, 300Å (Added to Phenomenex, Torrance, California) EASY na Separation was performed using the noLC system (Thermo Scientific). Peptides were separated. For elution, use a constant flow rate of 600 nL / min with 5-40% ACN (0.2% FA) over a 1-hour period. A gradient was used. The LC system was QExactive plus Orbitrap Connected to a Thermo Fisher Scientific mass spectrometer, R AW files can be converted to XCalibur software (Thermo Fisher Sci. The tandem mass spectrum was captured by (ntific). The tandem mass spectrum is a precursor of m / z 2.0. Using a precursor isolation window The Top-12 method was used. The resolution for the search scan was 70,000 at m / z 400. AGC 1e 6 (Maximum injection time 200 ms, scan range m / z 300-2000), In the MS / MS spectrum, it was 17.500 (5e 5 AGC in this context, maximum injection time 50ms The scan range was m / z 200-2000). Normalized collision energy (NCE) The value was set to 25, and the exclusion time was set to 10 seconds.

[0250] For protein identification, MaxQuant (Varginine) is used for SILAC quantification. The RAW files were analyzed using version 1.3.0.3. The default parameters used were... The meter has a tolerance of 20 ppm for MS / MS fragments and carbamide methylation (Car bamidomethylatin) (C) fixation, oxidation (M) and variable modification It was cetyl (protein N-terminus). Severe labeling included arginine (Arg10) and lysine. Select (Lys8) and apply the "match between run" option. The RAW files were searched in the HUMAN SwissProt database. In essence / absence analysis, PEAKS7.0 (http: / / www.b Label-free analysis was performed using ioinfor.com / . Database search was performed. The Human Uniprot database, which contains 37,254 entries, contains RAW files. I searched for the virus. For the search parameters, I set the error tolerance for the precursor to 10 ppm. For MS2 fragments, the acceptable value was 0.01 Da. Maximum 2 of trypsin One miscut was acceptable: carbamide methylation (C), deamidation (NQ), and oxidation (M). The ) was a variable modification. Only peptides with a false discovery rate (FDR) of less than 1% were examined. To validate the biomarkers, PEAKS results were processed using Scaffold proteome software. ProteomeSoftware (http: / / www.proteomesoftware.com / ) I uploaded it.

[0251] A statistical analysis of the discovery stage yielded two different results: (1) different subgroups of patients Qualitative data consisting of proteins that are present or absent and (2) each protein and internal Quantitative data was obtained consisting of expression measurements derived from the SILAC ratio, which represents the standard relative abundance. .

[0252] For qualitative data, Fisher's exact test was applied to each protein, and the glue The presence / absence of the substance was compared between the two (p-value < 0.001). For quantitative data, differential analysis was performed. To select the protein expressed, Student's t-test was performed between each pair in the group. The test was performed on proteins present in more than four individuals within each group. Only this was done. The problem of multiple comparisons arising from performing many tests (one per protein) was addressed. To address this, the Benjamini-Hockberg method is used to adjust the p-value and F The dynamic range (DR) was strongly controlled (adjusted p-value < 0.25 and FC > 1.3).

[0253] All analyses were performed using the statistical program "R" (R version 3.2, (C)2015 The R Foundation for Statistical Computing) This was done using [the specified method].

[0254] Along with five candidates selected from the inventors' group's previous results, the results of the discovery stage (4 Based on 9), a total of 54 tanks were selected for the target experiment using Selective Response Monitoring (SRM). Proteins were selected. Two unique peptides were selected for each protein and then subjected to previous mass spectrometry analysis. Based on the detectability in the experiment, monitoring was performed by SRM. For each selected peptide... Regarding the isotope-labeled version ( 15 N2, 13 C6-lysine, 15 N4, 13 C6-A We purchased ruginine and spiked each sample to use it as an internal standard. The internal standard was: Based on the experimental dilution curve, the concentration within the linear response range established for each individual peptide was spalled. I came (data hidden). In parallel, I mixed in isotope-labeled peptides and performed an MS2 spectrum analysis. It was used to generate a knowledge database of libraries and retention times.

[0255] A total of 85 endogenous peptides and their corresponding internal standards were identified in independent cohorts of 107 patients. SRM was measured in Lys-C and trypsin-digested samples from the phlebotomy tube. The five strongest transitions per petitone have an inner diameter of 75 μM and a 1.9 μM C ​​content. A 25cm reverse-phase chromatography column packed with 18 particles (Nikkyo Technos Co., Ltd.) (This is the main column) and a 2cm pre-column (Acclaim PepMap 100, C18, 15μM) Equipped with a triple quadrupole mass spectrometer (5500Q-Trap, AB Sci) and 100A, Monitoring was conducted at ex Instruments (Foster, California, USA). Loading buffer: H2O containing 0.1% formic acid; Eluent buffer: ACN, 0.1% formic acid The flow rate used was 250 nL / min, and the elution buffer amounted to 5-40% in 40 minutes. A chromatography gradient was used to avoid sample carryover. A blank test was performed between SRM measurements of the samples. Measurements were performed with a 300-second window and 1 The process was performed in scheduled SRM mode, using a target cycle time of 0.5 seconds.

[0256] Transition groups corresponding to target peptides for data analysis in the validation phase. Transition traces associated with the target peptide, in both light and heavy forms. Co-elution of -; based on the correlation between light SRM relative intensity and heavy SRM relative intensity, Sky Evaluation was performed using line v2.5. All peptide peaks were visually inspected. MSstat The linear mixed-effects model implemented in the Skyline plug-in (v3.3) As input, use the entire transition area between different sample groups. The change in protein magnification and the adjusted p-value were calculated.

[0257] In developing predictor variables, MSstats was used to obtain log2-transformed transitions. The amount of protein present in all samples is estimated based on area, and the defined glue The classification ability of each protein was used as an input variable for a logistic regression model between proteins. The performance metric used is the Area Under the Curve (AUC), and evaluation is performed using a method that covers no more than two-thirds of the dataset. The most distinctive high-value protein was selected as the initial classification metric. The AUC value was increased. While maintaining a constant temperature (ΔAUC > 0.02), most characteristic proteins were repeatedly added. Classification evaluation The evaluation procedure was repeated 500 times, using a different subset of patients in each iteration. The sample subset was created by randomly selecting patients from the original cohort without substitution. Therefore, it was generated and balanced for each subgroup of samples. Final consensus mode The sequence is composed of combinations of proteins, with the most frequently selected from 500 repeats. We fitted Dell to the entire dataset and used the area under the ROC curve, sensitivity, specificity, and precision. Prediction accuracy was quantified. The pROC package of the statistical program "R" was used to create ROCs. Used in the calculation of AUC and other performance values ​​(i.e., sensitivity, specificity, and accuracy) The "optimal cutoff" is considered to be the point at which the sum of sensitivity and specificity reaches its maximum value, and these are obtained. Ta.

[0258] 2.Results 2.1 Discovery Stage In the discovery phase of this project, 10 EEC (also abbreviated as EC1) patients were identified as having NEE Uterine aspiration was performed on 10 C (also abbreviated as EC2, especially SEC) patients and 10 control patients. ELV was isolated using a sample size (N=30).

[0259] After confirming the purity of the isolated vesicles, immediately before separation by 1D-SDS-PAGE, 30 Each of the following samples (EEC n=10, NEEC n=10, and CTRL n=10) With a 2:1 heavy / light ratio of equal amounts of Super-SILAC mix (internal standard), spa I came. Each gel lane was sliced ​​into 10 bands, and each band was digested with trypsin. The proteins were extracted and 300 individual LC-MS / MS analyses were performed. Only those peptides were identified by at least one unique peptide, and these peptides were present in less than 1% of cases. Assuming it has a false discovery rate (FDR), the protein database of MS / MS data The search results identified a total of 2138 proteins. MS / MS spectra were analyzed using software. Further processing with the software MaxQuant allows for the quantification, i.e., identification, of 920 proteins. This led to a total quantification rate of 43% of the proteome.

[0260] A total of 152 proteins showed adjusted p-values ​​<0.25 and multipliers less than -1.3 or greater than 1.3. The changes resulted in differential expression. From these, 147 proteins were identified as potential diagnostic biomergers. It is a difference from the comparison of CTRL and EC, and 28 proteins are potential prognostic bioma -Ka (difference from a comparison between EEC and NEEC). In parallel with quantitative analysis, it was not possible to quantify... The presence / absence test was performed to consider the protein in question because that protein has There are no heavy counterparts, but they exist in certain groups. This is because it is still relevant for the purpose of... The proteins were re-analyzed using a sieve and Fisher's test was performed to identify proteins that were significantly present within the group. Selected (p-value < 0.001). Following this analysis, 29 potential diagnostic biomarkers and 9 This included a total of 30 candidates that corresponded to potential prognostic biomarkers.

[0261] Always respecting the above statistical limitations, (i) relative quantitative analysis, (ii) presence / absence test and (iii) Proteins or cancers in which the family is over-presented in the candidate list By combining biological criteria such as the elimination of saturated proteins, a total of 54 candidate finalists were identified. A list was generated. Of these 54 candidate biomarkers, 50 correspond to diagnostic biomarkers. Of these, 15 correspond to prognostic biomarkers, of which 37 only have diagnostic potential, and 2 are prognostic. While 13 possessed only retrospective potential, 13 possessed both diagnostic and prognostic potential.

[0262] 2.2 Validation of protein biomarkers in UA ELV Having obtained the list of 54 candidates described in the previous section, we conducted LC- in a new, larger patient cohort. The aim was to validate the potential of the candidate using SRM. This involved discovery-stage testing and Similarly, the ELV fraction of UA was the selected sample for analysis. EC was diagnosed and histological sub In addition to evaluating the individual potential of each marker that distinguishes the types, here we have different candidates We attempted to generate diagnostic and prognostic models by combining supplementary data. Furthermore, we considered U in an independent cohort. The classification accuracy of each candidate in the overall liquid fraction of A was evaluated.

[0263] In the validation phase, a total of 107 patients were recruited (Cohort B), and EEC or EC1 (n=4 5) The three groups are NEEC or EC2 (n=21), and CTRL (n=41). They were separated. In particular, NEEC corresponded to patients diagnosed with serous endometrial cancer (SEC). Therefore, as seen in the previous section, the ELV from the UA is isolated and characterized. (Data hidden). List of 54 protein candidate biomarkers generated at the discovery stage. This was verified by LC-SRM. To verify, two unique triglycerides were used for each protein. We select the psin peptide and then schedule a total of 85 endogenous peptides. Monitoring was performed by RM. However, 85 peptides corresponding to 51 proteins were used. Of these, only 69 were detected. Three candidates (TNR6, CLH1, and PSB) 3) was not detected in any of the samples.

[0264] As a result of this SRM experiment, 43 out of 48 potential diagnostic biomarkers (89.6%) %) (i.e., a significant difference in abundance was observed between CTRL and EC), even at this verification stage. The results were found to be statistically significant (adjusted p-value < 0.01), therefore, individual diagnostic biomarkers Its potential as such was confirmed. Of the 45 quantified proteins, a total of 29 were found to be EC It demonstrated high accuracy in individually distinguishing between cases and CTRL cases (highlighted in bold in Table 6). (AUC value higher than 0.75). The five most important individual biomarkers are AGRIN(A UC=0.90, CI95:0.85-0.96), TACD2(AUC=0.87, C I95:0.81-0.94), SORT(AUC=0.86, CI95:0.79-0 .93), MVP (AUC=0.86, CI95:0.78-0.93) and FAS (A The results were UC=0.85 and CI95:0.78-0.92. Table 2.1 below shows the ELV. This shows a list of biomarkers selected for internal EC diagnostics.

[0265] Potential prognostic biomarkers (i.e., a comparison between EC1 (EEC) and EC2 (NEEC)) Therefore, only 5 of the 15 candidate proteins are differentially expressed during the validation stage. This was found (adjusted p-value < 0.01). The biomarkers examined were CLD6 and IF2B3. These were BCAM, PLD3, and MX1. Of the 45 quantified proteins, a total of 4 were E It demonstrated high accuracy in individually distinguishing between C1 and EC2 cases, with an AUC value of 0.75:CLD6 (AUC=0.88, CI95:0.76-1.00), BCAM(AUC=0.87, CI95:0.76-0.97), IF2B3(AUC=0.80, IC95:0.68 -0.93) and PLD3 (AUC=0.79, IC95:0.66-0.93) The following Table 2.2 shows a list of biomarkers selected for the EC prognosis of ELV. vinegar.

[0266] 2.3 Validation of candidate UA in all liquids using targeted proteomics The use of these biomarkers is more readily available and does not require the isolation of extracellular vesicles. To understand whether or not it can be transferred to the sample, it is identified in the exosomes of the entire liquid fraction of UA. The objective was to conduct trials of the identified biomarkers. To that end, a total of 67 patients (co Select Hort C), EEC (n=22), NEEC (n=20), CTRL (n=25 They were divided into three groups.

[0267] In this part of the study, a total of 51 proteins were analyzed. Each protein had two unique properties. We selected trypsin peptides and from these, we ultimately scheduled a total of 75 endogenous peptides. Monitoring was performed using SRM. Only 37 proteins in total were detected, followed by AU We quantified C to estimate it.

[0268] The results obtained from this verification test indicate that ELV-based biomarkers can be transferred to the entire liquid of UA. To understand the possibilities, we evaluated them by comparing them with the results of previous studies. First, in the entire liquid of UA The detectability of the ELV biomarker is non-detectable compared to the detectability when analyzing ELV. It has always been noted that the number of biomarkers detected in ELV is limited, and of the 51 biomarkers detected in ELV, UA Only 34 (66.7%) were detected.

[0269] Tables 1.1 and 1.2 below show the EC diagnostics for the entire liquid fraction of UA, and UA Important biomarkers for EC prognosis in the total liquid fraction (comparison of EEC and NEEC) A list.

[0270] [Table 1]

[0271] [Table 2]

[0272] Example 2: EEC (EC1) cases in a cohort of 116 patients using uterine aspirate. Prognosis of EC by differentiating it from NEEC (EC2) cases.

[0273] 1. Samples, reagents, and methods 1.1 Description of patients and samples From 2012 to 2015, Vall Hebron University Hospital (Barcelona, ​​Spain), Hospital University ari Arnau de Vilanova (Lieda, Spain) and Univers At ity Medical Center Freiburg (Freiburg, Germany) A total of 116 women were recruited. Informed consent was approved by the ethics committee of each hospital. All patients signed the consent form. All women were suspected of having an EC, i.e., transvaginal ultrasound. Based on the test results, the appearance and / or closure of abnormal uterine bleeding (AUB). Endometrial thickness of 4 mm or more in postmenopausal women and 8 mm or more in premenopausal women is determined by EC The diagnostic process began. Of the 116 women, 69 were diagnosed with endometrioid EC (EEC). This included 49 patients with ) and 20 patients with non-endomedical serous EC (SEC or NEEC). 47 women were diagnosed with either a normal endometrium or a benign disorder (including endometrial hyperplasia). She was a non-EC female. The patient's clinicopathological characteristics are described in the following table.

[0274] Table 3 Clinical characteristics of women enrolled in the study *One case with an undetermined grade. [Table 3]

[0275] Uterine aspirated material Cornier Pipelle (Eurogine Ref. 0304 The sample was collected by aspirating with 0200 and transferred to a 1.5 ml microcentrifuge tube. Saltwater is added in a 1:1 (v / v) ratio, and in order to separate the liquid fraction from the cell fraction, 2, The sample was centrifuged at 500xg for 20 minutes. The uterine aspirate fluid, ranging from 100 μl to 1 ml, was then extracted. It was kept at -80°C until use.

[0276] 1.2 Liquid chromatography parallel reaction monitoring (LC-PRM) analysis Simple preparation Sample preparation for LC-PRM analysis is described by Martinez-Garcia E et al. “De velopment of a sequential workflow based on LC-PRM for the verification of endom etrial cancer protein biomarkers in utero ine aspirate samples”,Oncotarget-2016,vo This was performed as previously described in l.no. 7(33), pp.: 53102-53114 (cited above) . Briefly, the liquid fraction from uterine aspirates was sonicated and processed with an Albumi n&IgG depletion spin trap kit (GE Healthca re) to remove albumin and immunoglobulin G proteins from 50 μl of each sample . Total protein concentration was estimated by Bradford assay. Next, all 116 samples were divided into two equal aliquots of 12.5 μg, and technical replicates were used for subsequent steps and analysis. After denaturing, reducing and alkylating the samples , two-step sequential proteolysis was performed, first with Lys-C endoproteinase MS grade (Thermo Scienti fic) at a protease / total protein mass ratio of 1 / 150 (w / w) for 4 hours at 37°C , followed by trypsin (Promega) at a protease / total protein mass ratio of 1 / 50 (w / w) overnight at 37°C. A mixture of stable isotope-labeled synthetic peptides (Thermo Fisher, crude quality) was spiked into each sample (C-terminal arginine 13 C6, 15 N4, Δm=10Da, C-terminal lysine 13 C6, 15 N2, Δm=8Da, or heavy leucine 13 C6, 15 N1, Δm=7Da, or if this is not available for phenylal anine 13 C9, 15 N1, Δm=10Da). Finally, the sam ples were purified by solid-phase extraction (Sep Pak tC18, 50 mg, Waters), vacu um-dried, and resuspended in 0.1% formic acid before LC-PRM analysis. As in Example 1, Even in uterine fluid samples (UA) where albumin and immunoglobulin G were not removed, the prognostic values ​​were not good. I obtained the data.

[0277] 1.3 Preparation of LC-PRM Peptide separation was performed using Dionex Ultimate 3000RSLC chromatography. The Fee system was operated in column switching mode. 0.05% triphosphate in water A mobile phase of fluoroacetic acid and 1% acetonitrile was used at a flow rate of 5 μl / min in the digestion sump. A 250ng equivalent is placed in a trap column (75μm x 2cm, C18pepmap100) It was injected and added to a 3 μm (3 μm) solution. Starting with 2% solvent A, a linear gradient was applied to 35% solvent B. The peptide was then analyzed at a rate of 300 nl / min over 48 minutes using an analytical column (75 μm × 15 cm, C). Further elution was performed in 18pepmap100 (2μm). Solvent A is 0.1% formic acid in water. Solvent B was 0.1% formic acid in acetonitrile. Q Exactive plus PRM analysis was performed using a mass spectrometer (Thermo Scientific). The process involves a full MS1 scan performed at a resolution of 70,000 (at 200 m / z), and (2 (At 00 m / z) The normalized collision energy obtained with a resolution of 35,000 is 20, time setting It consisted of a targeted MS2 scan. The quadrupole isolation window of the precursor ion was defined in MS2. The event is set to 1 m / z units, and the time settings for each pair of endogenous and isotope-labeled peptides are set accordingly. The window duration was set to 2 minutes.

[0278] 1.4. PRM Data Processing The PRM data was processed as described by Martinez et al. (see above). It contains the five most potent fragment ions (i.e., PRM transitions) of each precursor. The area of ​​the extracted ion chromatogram (XIC) is calculated using the Skyline program (v3.1). Extracted using (McCoss Lab, University of Washington, USA). Peptide identity The peak area of ​​the five transitions of the reference (synthesis without biological matrix) PRM acquisition of peptide mixes and corresponding endogenous and heavy peptides in clinical samples The cosθ of the spectral contrast angle calculated between the transition area This was confirmed using a spectral matching method based on the y-based approach. The endogenous nature of the peptide and the same If both cosθ values ​​of the topologically labeled version exceed 0.98, the peptide can be detected and the same The test was passed. A score of less than 0.98 was generated due to MS measurements falling below the limits of quantification. In such cases, the area values ​​were replaced with background estimates.

[0279] 1.5 Statistical analysis The light / heavy area ratio of each peptide was extracted from the Skyline, and the average across replications was calculated. Statistical analysis was performed using SPSS (v20.0) (IBM, Armonk, New York, USA) and Graph Pad Prism(v.6.0)(GraphPad Software The study was conducted in La Jolla, California, USA. The data did not follow a normal distribution. Therefore, using the non-parametric Mann-Whitney U test, the patient's group We compared the levels of peptides monitored between the two groups. Benjamini-Hoc The hberg FDR method was used to adjust p-values ​​for multiple comparisons. <0.05 Adjusted p-values ​​were considered statistically significant. Receiver Operational Characteristics (ROC) analysis was used. The specificity and sensitivity of biomarkers were evaluated, and the area under the ROC curve (A) was calculated for each individual protein. UC was estimated.

[0280] 1.6 Development of classification indicators The capabilities of various protein combinations are evaluated, and samples are classified into two clinical categories. Therefore, we adjusted the logistic regression model to fit the data. ROC curves were generated, along with AUC and the "optimal" cutoff for distinguishing between groups. Sensitivity and specificity were obtained at the point. The optimal cutoff is the identity line (diagonal). This corresponded to the threshold that maximized the distance at [location]. The optimality criterion was: max(sensitivity + specificity). The AUC 95% confidence interval (CI) was calculated using the Delong method. Sensitivity and characteristics The 95% confidence interval for the opposite sex is determined by bootstrap resampling and Fawcett's method. The calculations were performed using the averaging method. All ROC analyses were performed using the R package "pROC". (Robin et al., “pROC: and open-source e package for R and S+to analyze and com pare ROC curves”,BMC Bioinformatics-2011 (vol.no.12:77). To evaluate the robustness of each protein panel, data Each sample in the set was adjusted for the remaining samples in the dataset using logistics. By applying a regression model, we can use the "leave-one-out" method. )" Cross-validation procedure is performed to obtain a new ROC curve, and then the usual ROC analysis is performed went.

[0281] 2.Results EEC is the most common histological type in EC, and compared to non-endometrioid EC cases (NEEC) In this case, the prognosis is good. NEEC accounts for approximately 20% of all EC cases, but EC recurrence and It accounts for over 50% of deaths. Among NEECs, serous EC (SEC) is the most common. It is a subtype. In a cohort of 49 EEC cases and 20 SEC cases, 51 tans After investigating the amount of protein present, uterine aspirate samples from EEC patients were collected as shown in Table 4. In this study, the levels of 11 proteins were significantly elevated (adjusted p-value < 0.05). Among them, Six proteins exhibited a magnification change of more than 2 and showed the highest individual AUC values:PI GR 0.85(95%CI, 0.734-0.958), CAYP1 0.83(95 %CI, 0.725-0.942), CTNB1 0.78(95%CI, 0.670- 0.895), SG2A1 0.77(95%CI, 0.661-0.880), VIM E 0.76 (95% CI, 0.645-0.881) and WFDC2 0.74 (95% CI) (%CI, 0.624-0.855).

[0282] Table 4 Proteins used to differentiate between EEC and SEC (NEEC) [Table 4]

[0283] In addition to determining the expression levels of one or more of the proteins in Table 4, the inventors also considered other factors. We found that analyzing the protein levels improved the robustness of the classification. In particular, X PO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP1, GT R1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA (one or more of these) Bell.

[0284] Following the same procedure as above, 2 to 12 proteins between diagnostic and predictive biomarkers, We evaluated all possible combinations of 2 and 3 proteins to determine the individual biomarkers. A panel of proteins that improve results was identified from CTNB1, XPO2, and CAPG. The combination of these three proteins has an AUC of 0.99 (95% CI, 0.90-1). The panel has the best performance for distinguishing between EEC and SEC in the liquid of uterine aspirate samples. This panel had a sensitivity of 95% (95% CI, 85%-100%) and 95%. A specificity of 9% (95% CI, 89.8%-100%) was achieved (Figure 1). After the "leave-one-out" cross-validation is complete, the values ​​are 95% sensitivity and 89%. The specificity was 8%.

[0285] As can be inferred from Figure 1, both markers CTNB1 and CAPG are from SEC. This provided interesting sensitivity and specificity values ​​for the differential diagnosis of EEC, but surprisingly, The combination of CTNB1, CAPG, and XPO2 (panel) is a model of high sensitivity and specificity. A different diagnostic value was given. Therefore, the panel is used not only as a diagnostic panel, but also as an EC subtype It should also be understood as a reliable prognostic panel that enables classification. The sooner the classification is done, the better; basically, the correct medical register should be applied as soon as possible. For men, this leads to an increase in survival rate.

[0286] In this study, we will use a fluid biopsy (i.e., uterine aspirate) obtained from the female reproductive tract. This discloses a highly reliable method for distinguishing between EEC and SEC organizations. True improvement is predicated on the current diagnostic procedure having limited cell content in this sample. Based on histological examination of the quantity, significant shortcomings: an average of 22% of patients remain undiagnosed, and up to 50% of E-clinical cases are diagnosed. This is because it can lead to incorrect histological type and / or grade assignment in C cases.

[0287] The clinical strength of this study is that the women enrolled in this trial are women participating in the EC diagnostic process. It included a wide range of diversity. Regarding EC patients, there are two most common tissue structures. The study included EEC and SEC cases. Non-EC patients were mainly AUB or intrauterine. This study included all women suspected of having EC due to membrane thickening. This included benign conditions (primarily This includes women with polyps, fibroids, and endometrial hyperplasia, as well as women with a normal endometrium. Furthermore, this study included premenopausal women with functional endometrium and primarily atrophic endometrium. The study included both postmenopausal women and women exhibiting endometrial characteristics.

[0288] The role of proteins tested to improve the assignment of risk groups for EC patients Regarding this matter, the European Society for Medical Oncology (EUR) Current major risk stratification systems such as the ESMO classification are based on tumor The focus is on the histological structure, grade, myometrial invasion, and lymphatic invasion of the tumor. Since the information is not available preoperatively, the histological subtype and grade are allocated to the risk group. This is an important factor in determining the scope of the procedure for diagnosis and surgical staging. The combination of three proteins (CTNB1, XPO2, and CPG) results in 95.0% With sensitivity and specificity of 95.9%, it correctly distinguishes between EEC and NEEC(SEC) tissue EC types. I was able to distinguish them clearly.

[0289] These three proteins were previously associated with EC. Beta-catenin (CTNB) 1) is an essential gene responsible for cell proliferation and differentiation in the Wnt signaling pathway, as well as for intercellular adhesion of epithelial cells. It plays an important role in gene transcription. According to observations in current assays, CTNB1 It is described in EEC tissue specimens, but not in NEEC tumors. Macrophages Actin capping protein (CAPG) plays a role in reconstructing actin filaments. Therefore, it regulates cell motility. It is involved in the cell migration and invasiveness of several types of cancer. Unlike CTNB1, compared to EEC cases at the tissue level, SEC is more invasive. Tumors have been reported to have higher levels of CAPG, which is the result of the uterine aspiration reported here. This is consistent with the last known cell apoptosis-sensitive protein, E. Sportin-2 (XPO2) plays a role in the mitotic spindle checkpoint. Removal of XPO2 leads to cell cycle arrest, which is consequently associated with tumor growth. However, it is also associated with tumor invasion and metastasis. XPO2 is more prevalent in many cancer types, including EC. This is observed at a high level and is clearly associated with higher cancer grades and poorer outcomes in patients. In this study, no significant difference in XPO2 levels was observed between EEC and SEC cases. Although it wasn't present, when included in a panel formed by CTNB1 and CAPG, its performance was... It has improved in terms of intention.

[0290] Notably, this study addresses a critical clinical need of the EC as described herein. The proteomics approach based on uterine aspirates described here is more clinically relevant to EC tumors. The same proteomics signature classifies patients into risk groups and helps predict surgical treatment. This is to pave the way to determination. Therefore, to accurately classify the most common organizations, The first step was achieved using Necha. Overall, a uterine aspiration-based biomass The development of the Kasigna is expected to improve the management of EC patients and save significant healthcare costs. It is being done.

[0291] As explained, the specific protein sets in Tables C and D are listed below.

[0292]

number

[0293]

number

[0294] The invention is also related to the following sections.

[0295] Item 1: A method for determining the prognosis of endometrial cancer, wherein the following are found in a uterine fluid sample from the female reproductive tract: Proteins PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, C ADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL , expression levels of one or more of MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 A method that includes determining the bell. Item 2: The uterine fluid sample is a uterine aspirate sample from the female reproductive tract, as described in Item 1. method. Item 3: The following proteins: PIGR, VIME, CTNB1, CAYP1, SG2A1 , and determining the expression level of WFDC2 and one or more, in any of items 1 to 2. Method of description. Item 4 The following proteins: XPO2, PRDX1, CLIC1, PDIA1, KPYM , ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, TPIS, NGA The following items further include determining the expression levels of one or more proteins of L and LDHA. The method described in any of the three points. Section 5 XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GST P1, GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA Item 1 further includes determining the expression level of one protein selected from the group. The method described in any of the following four ways. Section 6 CLD6, BCAM, in exosome-containing fractions isolated from uterine aspirates Expression of one or more proteins selected from the group consisting of IF2B3, PLD3, and MX1. The method described in any of items 1 to 5, further comprising determining the level. Item 7 LAMP, MMP9, PIGR;AGRIN, MMP9, PIGR;AGR2, PIGR, PLD3; AGR2, BCAM, PODXL; BCAM, PODXL; PIG R, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6, PODXL; B CAM, RL29; BCAM, PODXL; CLD6, PPIA, AGRIN, BCAM ;ANXA, BCAM;BCAM, RAB8A;BCAM, SYIC;CLD6, IFB 3; and the sets listed in Table C The expression levels of at least one set of proteins selected from the group consisting of the following will be determined. The method described in any of items 1 to 6, including the above. Section 8 The prognosis of endometrial cancer distinguishes endometrioid endometrial cancer from non-endometrioid endometrial cancer. The method described in any of items 1 to 7, which is determined accordingly. Item 9: The method described in any of items 1-8, in which the expression level is determined at the protein level. . Item 10 Protein levels are measured in immunoassays, bioluminescence assays, fluorescence assays, and chemical Selected from the group consisting of bioluminescence assays, electrochemical assays, mass spectrometry, and combinations thereof. The method described in any of items 1 to 9, as determined by assay or technique. Item 11 The expression level of the protein is determined by the antibody or other substance that can bind to the protein. The method described in any of items 9-10, determined using the fragment. Item 12 The method according to item 11, wherein an antibody or a fragment thereof forms part of the kit. Item 13 In vitro for the prognosis of endometrial cancer in uterine fluid samples from the female reproductive tract PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODX From the group consisting of L, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 Use of one or more selected proteins. Section 14 The method described in any one of Sections 1 to 13 for the prognosis of endometrial cancer , PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1 , CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP Selected from the group consisting of 9, CD59, CLD6, IF2B3, PLD3, and MX1. Use of 1 or more proteins. Item 15 Use of a kit for the prognosis of endometrial cancer, comprising a solid carrier and the following tan Protein PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CAD H1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, M Expression levels of MP9, CD59, CLD6, IF2B3, PLD3, and MX1 of 1 or higher The means for detection, and optionally the following proteins: XPO2, PRDX1, CLIC1, PD IA1, KPYM, ENOA, GSTP1, GTR1, CH10, MIF, PEBP1, A kit comprising means for detecting one or more expression levels of TPIS, NGAL, and LDHA. Use. Item 16 Solid carriers and LAMP, MMP9, PIGR; AGRIN, MMP9, PI GR;AGR2, PIGR, PLD3;AGR2, BCAM, PODXL;BCAM, P ODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6 , PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AG RIN, BCAM;ANXA, BCAM;BCAM, RAB8A;BCAM, SYIC; CLD6, IFB3; and at least one set selected from the group consisting of the sets listed in Table C. Use of the kit described in item 15, which includes means for detecting the expression level of the protein in the kit. Item 17 Means for detecting protein expression levels include immunoassays, bioluminescence assays, etc. (i) Fluorescence assay, chemiluminescence assay, electrochemical assay, mass spectrometry, and combinations thereof. A means of carrying out an assay or technique selected from the group, any of items 15-16. Use the kit described in the instructions. Item 18 The means for detecting the expression level of a protein is an antibody or a fragment thereof. Use as described in any of items 15-17. Item 19 Solid carriers and PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCA M, PODXL, MMP9, CD59, CLD6, BCAM, IF2B3, PLD3, M X1, XPO2, PRDX1, CLIC1, PDIA1, KPYM, ENOA, GSTP 1. From GTR1, CH10, MIF, PEBP1, TPIS, NGAL, and LDHA A kit comprising means for detecting the expression level of one or more proteins selected from a group. Item 20 Solid carriers and LAMP, MMP9, PIGR; AGRIN, MMP9, PI GR;AGR2, PIGR, PLD3;AGR2, BCAM, PODXL;BCAM, P ODXL; PIGR, PLD3; BCAM, PIGR; CLD6, RAB8A; CLD6 , PODXL; BCAM, RL29; BCAM, PODXL; CLD6, PPIA, AG RIN, BCAM;ANXA, BCAM;BCAM, RAB8A;BCAM, SYIC; CLD6, IFB3; and at least one set selected from the group consisting of the sets listed in Table C. A kit including means for detecting the expression level of a protein. Section 20 MMP9, PODXL, RAB8A; MMP9, PODXL, RSSA; AG RIN, MMP9, PODXL; MMP9, PODXL, VAMP8; MMP9, MX1 ;MMP9, RSSA;MMP9, MVP;MMP9, RAB8A;MMP9, VAMP 8;BCAM, MMP9;MMP9, AGRIN;AGRIN, CD81, TERA;A GRIN, CD59, MVP; AGR2, AGRIN, CD81; AGRIN, CD16 6, MVP; AGRIN, CD81; AGRIN, CD166; AGRIN, CD59; AGRIN, MMP9; and the set of proteins listed in Table D A method for detecting the expression levels of at least one set of proteins selected from the group consisting of the following: The kit described in item 19, further including the steps. Item 21 The means for detecting the expression level of the protein is an immunoassay, bioluminescence assay, etc. Fluorescence assays, chemiluminescence assays, electrochemical assays, mass spectrometry, and combinations thereof A means of carrying out an assay or technique selected from the group consisting of the following, items 19-20 The kit is described in either of the following. Item 22 The means for detecting the expression level of a protein is an antibody or a fragment thereof. A kit as described in any of items 19-21. Item 23 is a kit for performing enzyme-linked immunosorbent assays, as described in items 19-22. The kit is described in either of the following. Item 24 further includes panel diagrams for classifying individual samples, as per item 19. Kits listed in any of the ~23 items. Item 25: A computer implementation method for carrying out any of the methods described in items 1 to 12. In such cases, the expression level of one or more proteins was determined for the diagnosis and / or prognosis of EC. Then, a value and / or score is assigned to the level, and a formula is arbitrarily used to calculate the calculated value. In the functions of level, score and / or calculated value, if you are suffering from or have suffered from EC Among the options that are not affected, and / or among the options that are affected, across different EC subtypes. The method by which a decision is made.

[0296] References -DeSouza LV,et al,“Endometrial cancer b iomarker discovery and verification usin g differentially tagged clinical samples with multidimensional liquid chromatogr aphy and tandem mass spectrometry”,Mol C ell Proteomics MCP-2007,vol.no.6,pp.:117 0-8. -Kemik P,et al.“Diagnostic and prognost ic values of preoperative serum levels o f YKL-40,HE-4and DKK-3in endometrial can cer”,Gynecol Oncol-2016;vol.no.140,pp.:6 4-9. -Martinez-Garcia E,et al.“Development o f a sequential workflow based on LC-PRM for the verification of endometrial canc er protein biomarkers in uterine aspirat e samples”,Oncotarget-2016,vol.no.7(33), pp.:53102-53114 -Robin et al.,“pROC:and open-source pac kage for R and S+to analyze and compare ROC curves”,BMC Bioinformatics-2011,vol. no.12:77

Claims

1. A method for diagnosing and / or prognosing endometrial cancer (EC), comprising determining the expression levels of PIGR and one or more of the following proteins: VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 in a uterine fluid sample from the female reproductive tract.

2. A method for determining or recommending whether or not to initiate a medical regimen for a patient with endometrial cancer (EC) in terms of prognosis, wherein the method is: a) A step of in vitro determining the expression level of PIGR and one or more proteins selected from the group consisting of VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 in a uterine fluid sample from the female reproductive tract of the subject, and b) In order to establish the prognosis of endometrial cancer (EC) by distinguishing between endometrioid endometrial cancer (EEC) and non-endometrioid endometrial cancer (NEEC), the protein expression levels of the proteins in the test sample are compared to the reference control levels based on whether the protein levels are higher than the reference control levels for PIGR, VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1, and lower than the reference control level for CAPG. Methods that include...

3. The method according to claim 1 or 2, wherein the uterine fluid sample is a uterine aspirate sample from the female reproductive tract.

4. The method according to any one of claims 1 to 3, comprising determining the expression levels of at least one set of proteins selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; PIGR, PLD3; BCAM, PIGR.

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

6. The method according to claim 5, wherein the protein level is determined by an assay or technique selected from the group consisting of immunoassays, bioluminescence assays, fluorescence assays, chemiluminescence assays, electrochemical assays, mass spectrometry, and combinations thereof.

7. The method according to any one of claims 5 to 6, wherein the expression level of the protein is determined using an antibody or a fragment thereof that can bind to the protein.

8. The method according to claim 7, wherein the antibody or fragment thereof forms part of the kit.

9. The method according to claim 1, for the diagnosis and / or prognosis of endometrial cancer (EC), using PIGR and one or more proteins selected from the group consisting of VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3 and MX1.

10. The method according to claim 1 or 2, using means for detecting the expression level of PIGR and one or more proteins of VIME, CTNB1, CAYP1, SG2A1, WFDC2, CADH1, CD44, LEG3, LEG1, CAPG, AGR2, BCAM, PODXL, MMP9, CD59, CLD6, IF2B3, PLD3, and MX1 for the diagnosis and / or prognosis of endometrial cancer (EC).

11. A kit for the diagnosis and / or prognosis of endometrial cancer (EC), comprising a solid carrier and at least one set of protein expression level detection means selected from the group consisting of LAMP, MMP9, PIGR; AGRIN, MMP9, PIGR; AGR2, PIGR, PLD3; PIGR, PLD3; BCAM, PIGR; and the set listed in Table C. Table 1

12. Use of a kit for the diagnosis and / or prognosis of endometrial cancer (EC), wherein the kit comprises a solid carrier 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.

13. The kit according to claim 11, wherein the means for detecting the expression level of the protein is an antibody or a fragment thereof.

14. Use of the kit according to claim 12, wherein the means for detecting the expression level of the protein is an antibody or a fragment thereof.

15. The method according to any one of claims 1 to 8, wherein after determining the expression level of one or more of the proteins, a value and / or score is assigned to the expression level.

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