Method for inferring clinical outcome of endometrial cancer patient, inferring device, inferring program, test reagent and test kit

Abstract

To provide a new POLE-mut based method for inferring the clinical outcome of an endometrial cancer patient.SOLUTION: A method for inferring a clinical outcome of a patient having endometrial cancer, comprises: obtaining information associated with DNA polymerase ε exonuclease domain mutation (POLE-EDM) from an endometrial cancer sample collected from the patient; determining that the clinical outcome of the patient is a good prognosis if POLE-EDM is present; and / or determining that the clinical outcome of the patient is not a good prognosis if POLE-EDM is not present. The problem is solved by the method.SELECTED DRAWING: None

Description

[Technical field]

[0001] Disclosed herein are a method, a prediction device, a prediction program, a test reagent, and a test kit for predicting the clinical outcome of an endometrial cancer patient. [Background technology]

[0002] The molecular classification of endometrial cancer includes four subtypes: (i) DNA polymerase epsilon mutation (POLE-mut), (ii) mismatch repair protein deficiency (MMR-D), (iii) abnormal expression of protein 53 (p53abn), and (iv) no specific molecular profile (NSMP) (Non-Patent Document 1). Several mutations have been reported as POLE-mut in Non-Patent Documents 2 to 11. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Leon-Castillo A, et al., J Pathol. 2020;250:312-22. [Non-Patent Document 2] Shuangni Yu, et al., J Gynecol Oncol. 2022 May;33(3):e38. doi: 10.3802 / jgo.2022.33.e38. [Non-Patent Document 3] https: / / cancer.sanger.ac.uk / cosmic / mutation / overview?id=107938599 [Non-Patent Document 4] https: / / www.mycancergenome.org / content / alteration / pole-s297f / [Non-Patent Document 5] https: / / cancer.sanger.ac.uk / cosmic / mutation / overview?id=107933199 [Non-Patent Document 6] Marina Stasenko, et al., Gynecol Oncol. 2020; 156(1): 194-202. [Non-Patent Document 7] Leon-Castillo, et al., J Pathol. 2020;250:323-33 [Non-Patent Document 8] Eduard Dorca, et al., Virchows Archiv, 2023; 483: 787-794 [Non-Patent Document 9] https: / / www.mycancergenome.org / content / alteration / pole-s459f / [Non-Patent Document 10] https: / / acsjournals.onlinelibrary.wiley.com / doi / 10.1002 / cncr.29046 [Non-Patent Document 11] Caroline C. Billingsley., Cancer, 2015: 121(3): 386-394 Summary of the Invention [Problem to be solved by the invention]

[0004] An objective of the present invention is to provide a novel POLE-mut-based method for predicting the clinical outcome of endometrial cancer patients. [Means for solving the problem]

[0005] The following embodiments are disclosed in this specification. Section 1. 1. A method for predicting a clinical outcome in a patient having endometrial cancer, the method comprising: Obtaining information related to DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) from an endometrial cancer sample collected from the patient; determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is non-favorable; The method comprising: The method according to the present invention does not include the following A) or B): A) NCBI Reference Sequence: In the amino acid sequence of POLE represented by NP_006222.2, a single mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination. Section 2. The POLE-EDM is At least one selected from the group consisting of M at the 299th position, N at the 363rd position, F at the 367th position, W at the 369th position, S at the 459th position, and A at the 463rd position from the N-terminus in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2; or a mutation corresponding to said mutation in the amino acid sequence of a POLE isoform; The method according to item 1. Section 3. The POLE-EDM is At least one selected from M299L, N363D, F367C, W369* (* represents a stop codon), S459del, and A463V in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2, or a mutation corresponding to said mutation in the amino acid sequence of a POLE isoform; The method according to item 2. Section 4. 1. A predictive device for predicting a clinical outcome in a patient having endometrial cancer, comprising: The prediction device includes a control unit, The control unit acquires information regarding DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) detected in an endometrial cancer sample collected from the patient, determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is unfavorable; The POLE-EDM is a prediction device that does not include the following A) or B): A) NCBI Reference Sequence: In the amino acid sequence of POLE represented by NP_006222.2, a single mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination. Section 5. When the program is executed by a computer, The control unit acquires information regarding DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) detected in an endometrial cancer sample collected from a patient having endometrial cancer, determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is unfavorable; A predictive program for predicting a clinical outcome of the patient, comprising: The POLE-EDM does not include A) or B). A) NCBI Reference Sequence: In the amino acid sequence of POLE represented by NP_006222.2, a single mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination. Section 6. A reagent for carrying out the method according to item 1, comprising a primer, a primer set, and / or a probe for detecting DNA polymerase epsilon exonuclease domain mutation (POLE-EDM) in an endometrial cancer sample collected from a patient having endometrial cancer. Section 7. A test kit for carrying out the method according to claim 1, comprising the reagent according to claim 6. Section 8. Item 2. A method for treating a patient with endometrial cancer, comprising the steps of: not administering chemotherapy, molecular targeted therapy, and radiation therapy to the patient in addition to surgical treatment if POLE-EDM is present in the method described in item 1; or administering chemotherapy, molecular targeted therapy, and / or radiation therapy to the patient in addition to surgical treatment if POLE-EDM is present. Effect of the Invention

[0006] It is possible to predict the clinical outcome of patients with endometrial cancer. [Brief description of the drawings]

[0007] [Figure 1] 1 shows a hardware configuration of a prediction device 10. [Diagram 2] 13 shows the flow of processing by a prediction program 104b. [Diagram 3]The relapse-free survival and overall survival of NCCH patients with POLE-EDM (POLE-EDM) and patients without POLE-EDM (Non-POLE-EDM) are shown. [Figure 4] The figures show relapse-free survival in (i) the POLE-EDM-positive group, (ii) the mismatch repair deficient (MMR-D)-positive group, (iii) the p53abn-positive group, and (iv) the no specific molecular profile (NSMP) group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] 1. How to predict clinical outcomes in patients with endometrial cancer In one embodiment, a method for predicting clinical outcome of a patient having endometrial cancer (hereinafter also referred to simply as "prediction method") includes obtaining information related to DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) from an endometrial cancer sample taken from the patient.

[0009] The method further includes determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is non-favorable.

[0010] POLE (human DNA polymerase ε) is a protein having an amino acid sequence represented by, for example, NCBI Reference Sequence: NP_006222.2 (SEQ ID NO: 1). POLE includes a DNA polymerase domain and an exonuclease domain. The exonuclease domain of POLE is represented by amino acid sequences from 268 to 471 in the amino acid sequence represented by NP_006222.2 (SEQ ID NO: 1). DNA polymerase ε exonuclease domain mutation (POLE-EDM) refers to a mutation in the exonuclease domain of POLE, and the exonuclease domain of POLE may include one or more mutations. The mRNA sequence encoding the amino acid sequence represented by SEQ ID NO: 1 is registered in NCBI as, for example, Reference Sequence: NM_006231.4 (SEQ ID NO: 2).

[0011] As used herein, amino acids may be represented by one-letter abbreviations as follows: alanine: A, arginine: R, asparagine: N, aspartic acid: D, cysteine: C, glutamine: Q, glutamic acid: E, glycine: G, histidine: H, isoleucine: I, leucine: L, lysine: K, methionine: M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

[0012] POLE-EDM does not include a single mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V in the amino acid sequence of POLE represented by NP_006222.2, or a single mutation corresponding to the above mutation in the amino acid sequence of a POLE isoform, or a single mutation corresponding to the above mutation in the amino acid sequence of a POLE isoform, or a combination of the above mutations. POLE isoforms include, but are not limited to, XP_054228285.1 or XP_011533097.1. In this specification, for example, P286C is a mutation in which the 286th P from the N-terminus is replaced with C.

[0013] Preferably, in the present specification, POLE-EDM is at least one selected from the group consisting of M at the 299th position, N at the 363rd position, F at the 367th position, W at the 369th position, S at the 459th position, and A at the 463rd position from the N-terminus in the amino acid sequence of POLE represented by NP_006222.2, or a mutation corresponding to the above mutations in the amino acid sequence of a POLE isoform.

[0014] More preferably, POLE-EDM as used herein is at least one selected from M299L, N363D, F367C, W369* (* represents a stop codon), S459del (del represents a deletion), and A463V in the amino acid sequence of POLE represented by NP_006222.2, or a mutation corresponding to said mutation in the amino acid sequence of a POLE isoform.

[0015] Endometrial cancer is also called uterine cancer. As used herein, the sample is not limited as long as it is collected from a patient. The sample may include endometrial cancer tissue or cells derived from endometrial cancer tissue.

[0016] The endometrial cancer tissue may be, for example, a surgically excised material, a biopsy material, etc. The endometrial cancer tissue may be a live tissue, or may be frozen, fixed, embedded in paraffin, or the like.

[0017] Cells derived from endometrial cancer tissues can be collected, for example, by smearing the uterine epithelium, preferably the epithelium of the uterine body, with a collection tool (e.g., cotton swab, slide glass, etc.), or by smearing a surgically excised specimen or a biopsy specimen of endometrial cancer tissue with a collection tool (e.g., cotton swab, slide glass, etc.), or by imprinting the collection tool. Cells derived from endometrial cancer tissues may be live cells, or may be frozen, fixed, etc.

[0018] Information related to the POLE-EDM may include POLE amino acid sequence information and / or POLE nucleotide sequence information.

[0019] POLE-EDM is preferably detected by extracting DNA from a sample and analyzing the nucleotide sequence of the exonuclease domain of POLE. DNA extraction from a sample can be carried out by known methods or using a commercially available kit.

[0020] The nucleotide sequence of the exonuclease domain can be determined according to known methods. For example, the nucleotide sequence can be determined by sequencing, PCR, microarray, etc.

[0021] Sequencing may be next-generation sequencing or Sanger sequencing. In the case of next-generation sequencing, mutations can be detected by mapping the nucleotide sequence obtained by sequencing to a reference sequence. Sanger sequencing can be determined by sequence alignment of the nucleotide sequence obtained by sequencing with the sequence of the exonuclease domain derived from normal endometrial tissue. Sanger sequencing may be performed on the amplification product of the exonuclease domain using DNA extracted from the sample as a template. Alternatively, DNA extracted from the sample may be made into a library, and Sanger sequencing may be performed on the library.

[0022] By sequencing, if the nucleotide sequence of the exonuclease domain of the DNA extracted from the sample is different from the reference sequence or the normal sequence, it can be determined that there is a mutation, and if the nucleotide sequence of the exonuclease domain of the DNA extracted from the sample is not different from the reference sequence or the normal sequence, it can be determined that there is no mutation.

[0023] Mutation detection by PCR can be performed by performing an amplification reaction using DNA extracted from a sample as a template with a primer that binds to the mutant sequence, and detecting the amplified product. If an amplified product is obtained with the primer that binds to the mutant sequence, it can be determined that there is a mutation at the site where the primer anneals. If no amplified product is obtained with the primer that binds to the mutant sequence, it can be determined that there is no mutation at the site where the primer anneals.

[0024] Detection of mutations using microarrays can be performed by detecting hybridization of DNA extracted from a sample with a probe that binds to the mutant sequence and that has been previously bound to a microarray substrate. At this time, the DNA extracted from the sample is fragmented and labeled with a fluorescent dye. These methods are publicly known. If a fluorescent signal of DNA extracted from the sample is observed on the probe that binds to the mutant sequence, it can be determined that there is a mutation at the site where the probe that binds to the mutant sequence hybridizes. If a fluorescent signal of DNA extracted from the sample is not observed on the probe that binds to the mutant sequence, it can be determined that there is no mutation at the site where the probe that binds to the mutant sequence hybridizes.

[0025] The mutation sequence detected as a nucleotide sequence can be converted into a mutation sequence in an amino acid sequence according to a general human codon table. The mutation sequence can be converted into an amino acid sequence by a known method.

[0026] The sequence obtained by sequencing may be directly linked to a label indicating the amino acid mutation corresponding to the mutant nucleotide sequence registered in advance (e.g., a text label such as "M299L"). Since primers and probes can be associated with amino acid sequences in advance for PCR and microarray, the PCR amplification results and the detection results of fluorescent signals by microarray may be directly linked to amino acid mutations.

[0027] In this specification, the patient with endometrial cancer is not limited as long as it is a patient with endometrial cancer.In addition, the patient may be a patient who has not been diagnosed with endometrial cancer when the sample is collected, and is diagnosed with endometrial cancer after the sample is collected.Endometrial cancer may be primary or recurrent.

[0028] Clinical outcome refers to the prognosis of a patient. Prognosis can be evaluated by survival time or recurrence-free survival time. If survival time or recurrence-free survival time is 3 years or more, preferably 5 years or more, the prognosis can be determined to be good.

[0029] When POLE-EDM is detected by the above method, it is suggested that the clinical outcome of the patient is prognostic. Alternatively, when POLE-EDM is detected, it can be determined that the clinical outcome of the patient is prognostic. Alternatively, when POLE-EDM is detected, it can be determined that chemotherapy, molecular targeted therapy, and radiation therapy are not applied to the patient in addition to surgical treatment. When POLE-EDM is not detected, it can be determined that the clinical outcome of the patient is not prognostic. Alternatively, when POLE-EDM is not detected, it can be determined that chemotherapy, molecular targeted therapy, and / or radiation therapy are applied to the patient in addition to surgical treatment.

[0030] 2. Testing reagents and kits One embodiment relates to a test reagent comprising a primer, a primer set, and / or a probe for detecting DNA polymerase epsilon exonuclease domain mutation (POLE-EDM) in an endometrial cancer sample obtained from a patient having endometrial cancer. The primers and primer sets are used in the above-mentioned PCR, and the probes are used as capture probes in the above-mentioned microarray.

[0031] The sequence of the primer that anneals to the POLE is, for example, 50 mer or less, preferably 30 mer or less, and more preferably about 15 to 25 mer. The site of the primer that is complementary to the mutant nucleotide is preferably the 3' end of the primer. The primer may contain a sequence that does not anneal to the POLE. The primer may be labeled with a fluorescent dye or the like. The test reagent may be in a dry state, or may be a solution in which the primer or primer set is dissolved in a buffer or the like, or a frozen product thereof.

[0032] The probe has a sequence that hybridizes to the POLE of, for example, 50 mer or less, preferably 30 mer or less, more preferably about 15 to 25 mer. The probe may contain a sequence that does not hybridize to the POLE. The probe is preferably immobilized on an array substrate.

[0033] The kit includes the test reagent. The kit may include the test reagent and a package insert or the like that includes a URL of a web page that describes how to use the test reagent. When the test reagent is a primer or a primer set, the kit may include a heat-resistant DNA polymerase, a buffer, dNTPs, and the like used in PCR. When the test reagent is a probe, the kit may include a hybridization buffer, a washing buffer, and the like.

[0034] 3. Predictive devices for predicting clinical outcomes in patients with endometrial cancer (1) Hardware configuration One embodiment relates to a prediction device 10 for predicting clinical outcomes of endometrial cancer patients (hereinafter, simply referred to as "prediction device 10"). The prediction device 10 may be communicatively connected to a sequencer, a PCR device, or a microarray reader via wire or wirelessly.

[0035] FIG. 1 shows the hardware configuration of a prediction device 10. The prediction device 10 may be connected to an input unit 111, an output unit 112, and a storage medium 113.

[0036] In the prediction device 10, a control unit 101, a main memory unit 102, a ROM (read only memory) 103, an auxiliary memory unit 104, and an interface (I / F) 105 are connected to each other via a bus 109 so as to be able to perform data communication with each other. The main memory unit 102 and the auxiliary memory unit 104 may be collectively referred to simply as a memory unit. The memory unit volatilely or non-volatilely stores an operating system (OS) 104a, a prediction program 104b for predicting clinical outcomes of patients with endometrial cancer (described below) (hereinafter also simply referred to as "prediction program 104b"), labels indicating amino acid mutations, and the like.

[0037] The control unit 101 is a CPU of the prediction device 10. The control unit 101 may cooperate with a GPU. The control unit 101 cooperates with an operating system (OS) 104a stored in the auxiliary storage unit 104 or the ROM 103 to execute a prediction program 104b and process acquired data, whereby the computer functions as the prediction device 10. The control unit 101 may cooperate with an MPU 101.

[0038] The ROM 103 is composed of a mask ROM, PROM, EPROM, EEPROM, etc., and stores the prediction program 104b executed by the control unit 101 and data used therein. The ROM 103 stores a boot program executed by the control unit 101 when the prediction device 10 is started up, and programs and settings related to the operation of the hardware of the prediction device 10.

[0039] The main memory unit 102 is configured by a RAM (Random access memory) such as an SRAM or a DRAM. The main memory unit 102 is used to read out the prediction programs 104b recorded in the ROM 103 and the auxiliary memory unit 104. The main memory unit 102 is also used as a working area when the control unit 101 executes these prediction programs 104b.

[0040] The auxiliary storage unit 104 is composed of a hard disk, a semiconductor memory element such as a flash memory, an optical disk, etc. The auxiliary storage unit 104 stores various prediction programs 104b to be executed by the control unit 101, such as an operating system and application programs, and various setting data used for executing the prediction programs 104b. Specifically, for example, the auxiliary storage unit 104 stores labels indicating amino acid mutations corresponding to mutations in a nucleotide sequence.

[0041] The I / F 105 is composed of serial interfaces such as USB, IEEE1394, RS-232C, parallel interfaces such as SCSI, IDE, IEEE1284, analog interfaces including a D / A converter and an A / D converter, a network interface controller (NIC), etc. The I / F 105 inputs and outputs data, commands, etc. from a sequencer, a PCR device, a microarray reader, a network, an input unit 111, an output unit 112, and a storage medium 113. The input unit 111 is composed of a touch panel, a keyboard, a mouse, a pen tablet, a microphone, etc., and is used to input characters or voice to the prediction device 10.

[0042] The output unit 112 is composed of, for example, a display, a printer, and the like.

[0043] The storage medium 113 is configured by a removable memory such as a USB memory or an external hard disk, a removable hard disk, a CD-ROM, a DVD-ROM, or the like.

[0044] The control unit 101 may obtain application software and various settings necessary for controlling the prediction device 10 via a network instead of reading them from the ROM 103 or the auxiliary storage unit 104. The application program may be stored in an auxiliary storage unit of a server computer on the network, and the prediction device 10 may access this server computer to download the prediction program 104b and store it in the ROM 103 or the auxiliary storage unit 104.

[0045] In addition, an operating system that provides a graphical user interface environment, such as Windows (registered trademark) manufactured and sold by Microsoft Corporation, is installed in the ROM 103 or the auxiliary storage unit 104. A prediction program for uterine glandular tumor lesions, which will be described later, is assumed to operate on the operating system. In other words, the prediction device 10 may be a personal computer or the like.

[0046] (2) Processing flow of prediction program 104b 2 shows a flow of processing by the prediction program 104b. When an operator inputs a command to start processing from the input unit 111, the control unit 101 of the prediction device 10 starts processing for predicting the clinical outcome of a patient.

[0047] In step S11, the control unit 101 acquires information about POLE-EDM (hereinafter, also simply referred to as "information") by receiving it from a sequencer, a PCR device, a microarray reader, a network, or a storage medium 113. Alternatively, the control unit 101 may acquire information by accepting information input by an operator from the input unit 111.

[0048] In step S12, the control unit 101 compares the reference sequence of POLE, or the normal nucleotide sequence information; or the normal amino acid sequence information of POLE with the information acquired in step S11. The reference sequence of POLE, or the normal nucleotide sequence information; or the normal amino acid sequence information of POLE may be stored in the auxiliary storage unit 104, or may be stored in an external sequence database other than the prediction device 10.

[0049] In step S13, the control unit 101 determines whether or not a POLE-EDM exists.

[0050] If control unit 101 determines in step S13 that a POLE-EDM is present, the process proceeds to step S14 (YES), where it outputs a label indicating the amino acid mutation to output unit 112. If control unit 101 determines in step S13 that a POLE-EDM is not present, the process proceeds to step S15 (NO), where it outputs a label indicating that a POLE-EDM is not present to output unit 112 (for example, a symbol label such as "-" or a text label such as "normal").

[0051] 4. Storage medium for storing the prediction program An embodiment of this embodiment relates to a program product, such as a storage medium, that stores the prediction program 104b. That is, the prediction program 104b can be stored in a storage medium, such as a hard disk, a semiconductor memory element such as a flash memory, or an optical disk. The storage medium may also be a computer, such as a server device. The format of recording the program in the storage medium is not limited as long as the prediction device 10 can read the program. It is preferable that the recording in the storage medium is non-volatile. EXAMPLES

[0052] The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0053] 1. Study design This study consisted of two independent retrospective cohorts of 1,029 Japanese patients with primary endometrial cancer. The first cohort was the National Cancer Center Hospital (NCCH) cohort, which included 265 patients, all of whom underwent primary surgery, 99 (37.4%) received adjuvant therapy, and 73 (27.6%) experienced recurrence or progression after primary surgery. The second cohort was the Center for Cancer Genomics and Advanced Therapeutic (C-CAT) cohort, which included 764 patients, all of whom had recurrence or progression. 553 patients (72.4%) underwent primary surgery, but data on the primary treatment regimen used was unavailable in 211 patients. Among the two cohorts, the median number of chemotherapy regimens for the 705 patients with available treatment records was 2 regimens (range: 1-9 regimens). One patient who overlapped with both cohorts was excluded. Patient characteristics for each cohort are summarized in Table 1.

[0054] [Table 1]

[0055] 2. Characteristics of the National Cancer Center Hospital (NCCH) cohort Of all patients who underwent primary surgery at NCCH between 1997 and 2023 and had a pathological diagnosis of endometrial cancer, 515 were included in the study. Patients who received neoadjuvant chemotherapy were excluded (Figure 1[1]). All cases were reviewed by at least two gynecologic pathologists, and pathological diagnoses were confirmed according to the 2020 World Health Organization Classification of Tumors. Clinicopathological data, including age and disease stage (as defined by the International Federation of Gynecology and Obstetrics [FIGO] in 2008), were retrospectively obtained for each patient.

[0056] 3. Characteristics of the C-CAT cohort In June 2019, insurance coverage for comprehensive genomic profiling (CGP) testing was introduced in Japan for solid tumor patients for whom standard treatment is not possible or expected to be completed. C-CAT was established under the National Health Insurance System to collect, store, and utilize mutation data and medical information of patients who underwent CGP testing such as FoundationOne® CDx and NCC Oncopanel (https: / / for-patients.c-cat.ncc.go.jp / ). By the end of April 2022, data on more than 32,000 patients with advanced cancer had been collected.

[0057] In this study, genomic profiles of 922 advanced endometrial cancer cases were obtained by controlled access to the C-CAT database for this study. The profiles were obtained with two comprehensive genomic profiling tests approved by the Pharmaceuticals and Medical Devices Agency: FoundationOne® CDx, which can detect somatic mutations in 324 genes, and NCC Oncopanel, which can detect somatic and germline mutations in 114 genes (Figure 1[2]). The protocol of this study was approved by the Institutional Review Board of the National Cancer Center Research Institute (approval number 2020-067) and then by the C-CAT Data Usage Review Board (approval number CDU2021-001N), and the study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki.

[0058] 4. DNA Preparation and Next Generation Sequencing Genomic DNA was extracted from 515 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues using the QIAamp® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA (50 ng) from the tumor tissues was analyzed using Ion AmpliSeq. TM Cancer Hotspot Panel v2 and Ion AmpliSeq TMLibraries were constructed using a Custom Panel (Thermo Fisher Scientific, Waltham, MA, USA). TM Ion AmpliSeq Designer (https: / / www.ampliseq.com) can detect POLE (coverage rate: 100%). TM A Custom Panel was created (Solution ID: IAD191594_167). For sample quality control, samples with an average read depth coverage of more than 1000, a base quality score of 20 (i.e., 1% or less probability of being inaccurate), and accounting for 90% (Cancer Hotspot Panel) / 75% (Custom Panel) of all reads were selected.

[0059] The COSMIC (version 87) and Clinver (20190114) databases were used to annotate each gene abnormality. Somatic mutations were counted if they were defined as "pathogenic / likely pathogenic" or "oncogenic / likely oncogenic."

[0060] 5. Classification of molecular subtypes using immunohistochemistry Using molecular classification, the patients were classified into four groups: (i) POLE-EDM, (ii) mismatch repair deficient (MMR-D), (iii) p53abn, and (iv) patients with the no specific molecular profile (NSMP). The immunohistochemistry (IHC) methods for detecting MMR-D and p53abn are described below. In this study, we used Ion AmpliSeq TMWe sequenced all exons of POLE using a custom panel, and oncogenic / pathogenic, nonsense, and in-frame deletion variants detected within the exonuclease domain of POLE, which were reported as somatic hotspots, were defined as POLE-EDM. Oncogenic / pathogenic, nonsense, and in-frame deletion variants not involving the exonuclease domain were defined as non-POLE-EDM. Molecular classification was performed as follows: First, cancer tissues were evaluated for POLE-EDM. Second, IHC was used to evaluate the presence / absence of MMR proteins. Finally, tumors were evaluated for p53 abnormalities using IHC, resulting in four subgroups: POLE-EDM, MMR-D, p53abn, and NSMP. In this study, double / dual-classified endometrial cancers were classified into upstream clades as previously reported. Due to poor DNA quality of FFPE samples, frozen tumor tissue specimens separate from tumor sections were analyzed for MMR-D and p53 IHC in 18 of 265 cases.

[0061] 6. Evaluation using IHC for p53 and MMR All surgically resected specimens were fixed in 10% neutral buffered formalin for 24–72 h and embedded in paraffin. Representative sections of all 4-μm-thick sections were analyzed by IHC using the following antibodies: anti-p53 (DO7, undiluted; Dako, Glostrup, Denmark), anti-hMLH1 (ES05, ​​1:200 dilution; Dako), anti-hMSH2 and anti-hMSH6 (SP93, 1:200 dilution; Spring Bioscience, CA, USA), and anti-hPMS2 (A16-4, 1:200 dilution; Biocare Medical, CA, USA). All IHC assays were performed using a Dako autostainer (Dako) according to the manufacturer's instructions. After deparaffinization, tissue sections were stained with the above antibodies and counterstained with hematoxylin. Abnormal p53 staining patterns were defined as a strong and diffuse nuclear staining pattern (>80% of cancer cells) or a completely negative ("null pattern") staining of cancer cells, with appropriate staining of surrounding nontumor cells as internal positive controls. Weak and heterogeneous staining patterns of tumor cells were classified as wild type. As IHC of PMS2 and MSH6 alone has been reported to be able to replace a four-antibody panel (consisting of MLH1, MSH2, MSH6, and PMS2) for MMR deficiency screening, MMR deficiency status was defined as a complete loss of nuclear staining of PMS2 and / or MSH6 proteins. Adjacent normal mucosa, stromal cells, and inflammatory cells with intact nuclear staining served as internal positive controls.

[0062] 7. Classification of molecular subtypes using next-generation sequencing On April 24, 2022, the C-CAT database (ver.20220406) was accessed to obtain clinical and genomic data for 764 well-defined endometrial cancer patients, excluding sarcoma patients. C-CAT cases of endometrial cancer were divided into four distinct molecular subtypes based on genomic abnormalities: (i) POLE-EDM, (ii) microsatellite instability (MSI) high (MSI-H), (iii) TP53 oncogene mutation (TP53mut), and (iv) NSMP. In endometrial cancer (corpus cancer), evaluation of p53 using IHC is an excellent surrogate marker for TP53 mutation status determined by sequencing, with comparable performance and good reproducibility between pathologists. Double / double-classified endometrial cancers were classified into the upstream branch group, similar to the NCCH cohort. The molecular classification methods by cohort are summarized below.

[0063] 8. Molecular taxonomy in each cohort Molecular classification was determined based on IHC in the NCCH cohort and sequencing data provided by the Foundation (FoundationOne® CDx or NCC Oncopanel) in the C-CAT cohort. The molecular classification for each cohort is summarized in Table 2.

[0064] [Table 2]

[0065] 9. Clinical outcomes In the NCCH cohort, the primary endpoint was recurrence-free survival (RFS), measured from the date of randomization to the date of proven recurrence or death, whatever the cause. Patients who were alive and recurrence-free at last follow-up were censored. The secondary endpoint was overall survival (OS), measured from the date of randomization to the date of death, regardless of cause. Six patients whose tumors were not completely resected at initial surgery were excluded from the survival analysis. Patients who were alive at the time of analysis were censored at the date of last follow-up. In the C-CAT cohort, the primary endpoint was overall survival (OS). Forty-six patients whose survival was unknown because the date of diagnosis or last follow-up was unknown were excluded from the survival analysis.

[0066] 10.Statistical analysis Statistical analysis was performed using R software version 4.1.2 (R Foundation, Vienna, Austria) and JMP version 16.0.0 (SAS Institute, New York, USA). Variables that showed statistically significant differences in univariate analysis were subsequently included in multivariate analysis. The statistical significance level was set at P < 0.05. Cumulative survival was estimated using the Kaplan-Meier method, and the difference in survival rates between the two groups was analyzed using the log-rank test. The effect of variables on overall survival (OS) or RFS was determined by univariate and multivariate analysis using the Cox proportional hazards model with JMP software.

[0067] 11. Detected POLE-EDM The POLE-EDMs detected in this study are shown in Table 3.

[0068] [Table 3]

[0069] 12.Comparison of survival time We compared relapse-free survival and overall survival between patients with POLE-EDM (POLE-EDM) and patients without POLE-EDM (Non-POLE-EDM) in NCCH.

[0070] The results are shown in Figure 3. Figure 3 shows the results for 265 patients out of all patients who underwent initial surgery at NCCH between 1997 and 2019 and received a pathological diagnosis of endometrial cancer (corpus cancer).

[0071] Patients with POLE-EDM had significantly better relapse-free survival and overall survival than patients without POLE-EDM, and there were no deaths during the study period.

[0072] The 259 patients in the NCCH cohort were classified into four groups: (i) POLE-EDM positive group, (ii) mismatch repair deficient (MMR-D) positive group, (iii) p53abn positive group, and (iv) no specific molecular profile (NSMP) group, and relapse-free survival was compared. The results are shown in Figure 4. The POLE-EDM positive group had a significantly longer relapse-free survival than the other groups. In addition, even patients who were both POLE-EDM and p53abn positive had a good relapse-free survival.

[0073] These results indicate that all POLE-EDMs detected in this study have a good prognosis. [Explanation of symbols]

[0074] 10 Prediction Device 101 Control section

Claims

1. 1. A method for predicting a clinical outcome in a patient having endometrial cancer, the method comprising: Obtaining information related to DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) from an endometrial cancer sample collected from the patient; determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is non-favorable; The method comprising: The POLE-EDM method does not include the following A) or B): A) A mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof, in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination.

2. The POLE-EDM is At least one selected from the group consisting of M at the 299th position, N at the 363rd position, F at the 367th position, W at the 369th position, S at the 459th position, and A at the 463rd position from the N-terminus in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2; or a mutation corresponding to said mutation in the amino acid sequence of a POLE isoform; The method of claim 1.

3. The POLE-EDM is At least one selected from M299L, N363D, F367C, W369* (* represents a stop codon), S459del, and A463V in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2, or a mutation corresponding to said mutation in the amino acid sequence of a POLE isoform; The method of claim 2.

4. 1. A predictive device for predicting a clinical outcome in a patient having endometrial cancer, comprising: The prediction device includes a control unit, The control unit obtains information regarding DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) detected in an endometrial cancer sample collected from the patient, determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is unfavorable; The POLE-EDM is a prediction device that does not include A) or B) below: A) A mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof, in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination.

5. When the program is executed by a computer, The control unit acquires information regarding DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) detected in an endometrial cancer sample collected from a patient having endometrial cancer, determining that if POLE-EDM is present, the patient's clinical outcome is favorable, and / or determining that if POLE-EDM is absent, the patient's clinical outcome is unfavorable; A predictive program for predicting a clinical outcome of the patient, comprising: The POLE-EDM does not include A) or B), the prediction program: A) A mutation selected from P286C, P286R, S297Y, S297F, V411L, A426V, P436R, M444K, A456P, S459F, and A465V, or a combination thereof, in the amino acid sequence of POLE represented by NCBI Reference Sequence: NP_006222.2; B) Mutations corresponding to the above mutations in the amino acid sequence of a POLE isoform, either alone or in combination.

6. A reagent for carrying out the method of claim 1, comprising a primer, a primer set, and / or a probe for detecting DNA polymerase epsilon exonuclease domain mutations (POLE-EDM) in an endometrial cancer sample taken from a patient with endometrial cancer.

7. A test kit for carrying out the method of claim 1, comprising the reagent of claim 6.

Images