Method for detecting genes associated with malignant transformation of endodermal cysts

Abstract

To provide a method for detecting a gene associated with malignant alteration of endodermal cysts.SOLUTION: The present invention provides a method for detecting a gene associated with malignant alteration of an endodermal cyst in a sample. The method includes a step of detecting, in a subject-derived sample, KRAS, and at least one selected from a group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention broadly relates to a method for detecting genes associated with the malignant transformation of endodermal cysts, and the like. [Background technology]

[0002] Neuroenteric cysts, also known as enteric cysts, endodermal cysts, enteric cysts, respiratory cysts, or bronchogenic cysts, present with benign histopathology consisting of well-differentiated simple columnar ciliated epithelium or mucus-producing epithelium. Neuroenteric cysts usually present with benign histopathological findings and rarely show malignant transformation. Secondary malignant transformation of endodermal cysts such as neuroenteric cysts is extremely rare, and the mechanism behind this is yet to be elucidated. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rim et al., "Mucinous adenocarcinoma arising from a residual supratentorial neurenteric cyst and expressing mutated KRAS: a case report", Human Pathology (2016) 58, 146-151 Summary of the Invention [Effects of the Invention]

[0004] According to the present invention, it is possible to identify malignant endodermal cysts, which have conventionally been difficult to pathologically distinguish from benign endodermal cysts. [Problem to be solved by the invention]

[0005] In view of the above circumstances, an object of the present invention is to provide a method for detecting genes associated with the malignant transformation of endodermal cysts, and the like. [Means for solving the problem]

[0006] One gene associated with the malignant transformation of endodermal cysts is the KRAS gene, a known oncogene (Rim et al., "Mucinous adenocarcinoma arising from a residual supratentorial neurenteric cyst expressing mutated KRAS: a case report", Human Pathology (2016) 58, 146-151).

[0007] It has been found that detection of KRAS and one or more specific proteins in samples from subjects is associated with malignant transformation of endodermal cysts.

[0008] That is, the present application includes the following inventions. [1] A method for detecting a gene associated with the malignant transformation of an endodermal cyst in a sample, comprising: A method comprising the step of detecting KRAS and at least one or more selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B in a sample from a subject. [2] The method according to [1], wherein the endodermal cyst is a neuroenteric cyst. [3] The method according to [1] or [2], wherein the sample is derived from a cyst. [4] 1. A method of testing a sample from a subject suspected of having an endodermal cyst, comprising: A method for detecting KRAS and at least one or more genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B in a sample from a subject, 1) The presence of a mutation that enhances the growth signaling activity of the KRAS protein and the presence of a mutation in at least one gene selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduces the function of each gene or the protein it encodes indicates the presence of a malignant endodermal cyst in the sample or the potential for the malignant transformation of an endodermal cyst; or 2) the presence of a mutation that enhances the growth signaling activity of the KRAS protein and the absence of mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduce the function of each gene or the protein it encodes indicates the presence of a benign endodermal cyst in the sample; method. [5] The method according to [4], further comprising a second detection step of detecting KRAS and at least one or more selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B in another sample from a subject who provided a sample in which a mutation that enhances the growth signaling activity of the KRAS protein was detected in the first detection step, but no mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduce the function of each gene or the protein encoded thereby were detected. [6] The method according to [5], further comprising a third or subsequent detection step. [7] the mutation in the KRAS protein is a substitution of glycine at position 12 or 13 with aspartic acid; Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include: ARID1A deletion; CDKN2A deletion; CDKN2B deletion; PTCH1 deletion; TSC1 deletion; PTEN deletion; TP53 deletion; FLCN deletion; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletion; SUFU deletion; and CDKN1B deletion, The method according to any one of [4] to [6], wherein [8] one or more primers for detecting mutations that enhance the growth signaling activity of KRAS protein; The following mutations: and one or more primers for detecting mutations in at least one gene selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B, which mutations reduce the function of each gene or the protein encoded by it. [9] one or more probes for detecting mutations that enhance the growth signaling activity of the KRAS protein; The following mutations: and one or more probes for detecting mutations in at least one gene selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B, which mutations reduce the function of each gene or the protein encoded by it.

[10] A kit comprising the primer set according to [8] and the probe set according to [9].

[11] The amino acid sequence of the region containing a mutation that enhances the proliferation signaling activity of the KRAS protein or the base sequence that it encodes, A diagnostic marker for endodermal cysts, which is a mutation in at least one gene selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B, and which consists of the amino acid sequence of a region containing a mutation that reduces the function of each gene or the protein it encodes, or the nucleotide sequence encoding said region. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows magnetic resonance images at the time of onset (A), first recurrence (B), and second recurrence (C, D): (A) T2-weighted image showing a cystic lesion compressing the medulla oblongata from the dorsal side (black arrow). (B) T2-weighted image showing cystic regrowth on the dorsal side of the medulla oblongata (white arrow). (C) T2-weighted image shows a de novo mass with a cystic component in the trigone of the left lateral ventricle (black arrow). (D) Post-contrast T1-weighted image (white arrow) shows gadolinium-enhanced signal intensity. [Figure 2] Figure 2 shows the histopathological features of specimens from the first (A, B) and third (CF) surgeries. (A) Low-power view showing a pseudopapillary pattern of single- or multilayered cuboidal epithelial cells. (B) High-power view of cells without malignant features. (C) Patternless proliferation of cells. (D) Areas with tightly arranged, hyperchromatic, atypical nuclei. (E) Cells labeled with epithelial cell membrane antigen. (F) Cells typically labeled with Ki-67. (A-D) Hematoxylin and eosin staining. (E, F) Immunological examination. Bars in the figure: 50 μm (A, C), 17 μm (B, D), 100 μm (E, F). [Figure 3] Figure 3 shows the results of sequencing the first sample. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0011] (Detection method) In a first aspect, a method for detecting genes associated with the malignant transformation of endodermal cysts in a sample is provided, the method comprising detecting KRAS and at least one or more genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B one or more times in a sample derived from a subject.

[0012] As used herein, the term "gene" includes a DNA region encoding a specific protein or its amino acid sequence, such as a proprotein or preproprotein, including a leader sequence or secretory sequence, as well as a non-coding DNA region including 5' or 3' non-coding sequences preceding or following the coding region, or a portion thereof. In other words, a gene is not limited to exons and may also refer to introns. Furthermore, a gene is not limited to these specifically mentioned exons; transcription products (e.g., mRNA, tRNA, rRNA, antisense RNA, etc.) are also included within the scope of a gene. DNA may be cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA).

[0013] The subject may be a human or other non-human mammal, particularly a non-human primate. The sample may be derived from either a healthy individual or a subject suspected of malignant transformation of an endodermal cyst, e.g., a biological sample exhibiting pathological findings characteristic of an endodermal cyst, e.g., a subject exhibiting a cystic mass. Such samples include, but are not limited to, tissue samples such as biopsies, circulating cells, serum, plasma, blood, feces, urine, sputum, cerebrospinal fluid, pleural fluid, lymphatic fluid, cell culture media, and other tissues and fluids obtained from patients. Preferably, the sample is derived from a cyst. The sample may be obtained by cytology (fine-needle aspiration) or histology, e.g., needle biopsy.

[0014] Preferably, the endodermal cyst is a neuroenteric cyst.

[0015] KRAS is a downstream factor of receptor tyrosine kinases such as EGFR, and mutations in this gene can often be a driver gene that contributes to carcinogenesis.

[0016] Gene detection can be performed by methods known to those skilled in the art. For example, the presence or absence of a gene in a sample can be directly confirmed using a next-generation sequencer or the like, or the presence or absence of a gene can be confirmed by preparing forward and reverse primers specific to the gene and using a gene amplification method such as PCR, and then determining the sequence of the amplified product or analyzing the restriction fragment pattern. These techniques can be used alone or in combination.

[0017] After detecting the genes, it may be possible to confirm whether or not each gene contains a mutation associated with the malignant transformation of endodermal cysts. Because the above genes may change over time, even if no mutation associated with the malignant transformation of endodermal cysts is found in a single detection, it is preferable to periodically detect the above genes or their mutations.

[0018] In a sample in which the above-mentioned genes have been detected, genes already known to be associated with the malignant transformation of endodermal cysts can be further detected. Furthermore, a sample in which genes associated with the malignant transformation of endodermal cysts have been detected can be used to detect malignant endodermal cysts in a sample, diagnose the malignant transformation of endodermal cysts in a subject, etc. For these uses, a sample in which the above-mentioned genes have been detected can be further analyzed.

[0019] (Testing method) In a second aspect, a method is provided for testing a sample from a subject suspected of having an endodermal cyst, such as a neurenteric cyst.

[0020] The subject is defined as above, but preferably the subject is a subject suspected of having an endodermal cyst, for example, a subject having a biological sample in which pathological findings specific to an endodermal cyst are observed, for example, a subject having a cystic mass. The sample is defined as above.

[0021] In the testing method, KRAS and at least one or more selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B are detected once or multiple times.

[0022] The presence or absence of mutations in these genes in a sample from a subject can determine the presence of malignant endodermal cysts, the possibility of endodermal cysts becoming malignant, and the presence of benign endodermal cysts. Even if no mutations associated with the malignant transformation of endodermal cysts are found in a single detection, it is preferable to periodically detect the above genes from the perspective of early detection of malignant endodermal cysts.

[0023] When KRAS protein undergoes mutation and constitutively activates downstream signaling pathways, it promotes cell proliferation, leading to the development of cancer cells, etc. Mutations that promote cell proliferation signaling activity are known, and examples include mutations of the 12th or 13th glycines from the N-terminus of KRAS protein, and mutations of the 34th, 35th, 37th, and 38th guanines in the gene encoding KRAS protein. More specific examples include G12D(g35a), G12S(g34a), G12C(g34t), G12R(g34c), G12V(g35t), G12A(g35c), G13D(g38a), G13S(g37a), G13C(g37t), G13R(g37c), G13V(g38t), G13A(g38c), etc. Glycine to aspartic acid substitutions, G12D or G13D, are preferred.

[0024] The presence of a mutation that enhances the growth signaling activity of the KRAS protein, and the presence of a mutation in at least one gene selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduces the function of each gene or the protein it encodes, is an indicator of the presence of a malignant endodermal cyst, the potential for an endodermal cyst to become malignant, or the presence of a benign endodermal cyst.

[0025] Examples of mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes that reduce the function of the genes or the proteins they encode include deletions of the bases shown below. ARID1A Chr 1 ln.26718016_28043054del CDKN2A Chr 9 ln.133756118_13500615del CDKN2B Chr 9 ln.133756118_13500615del PTCH1 Chr 9 ln.133756118_13500615del TSC1 Chr 9 ln.138129095_133759286del PTEN Chr 10 ln.57148002_135524247del TP53 Chr 17 ln.21437953_501del FLCN Chr 17 ln.21437953_501del PTPRD Chr 9 ln.13500114_438569del SUFU Chr 10 ln. 57148002_135524247del CDKN1B Chr 12 ln.12028193_12871912del

[0026] In a sample from a subject, if the KRAS gene or the protein encoded thereby has a mutation that enhances growth signaling activity, and the ARID1A gene or the protein encoded thereby has a mutation that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 26718016 to 28043054 on chromosome 1, the sample can be determined to contain a malignant endodermal cyst.

[0027] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the CDKN2B gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 133756118 to 13500615 on chromosome 9, the sample can be determined to contain a malignant endodermal cyst.

[0028] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the CDKN2B gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 133756118 to 13500615 on chromosome 9, the sample can be determined to contain a malignant endodermal cyst.

[0029] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the PTCH1 gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if bases 133756118 to 13500615 on chromosome 9 are deleted, the sample can be determined to contain a malignant endodermal cyst.

[0030] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the TSC1 gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 138129095 to 133759286 on chromosome 9, the sample can be determined to contain a malignant endodermal cyst.

[0031] In a sample from a subject, if the KRAS gene or the protein encoded thereby has a mutation that enhances growth signaling activity, and the PTEN gene or the protein encoded thereby has a mutation that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 133756118 to 13500615 on chromosome 10, the sample can be determined to contain a malignant endodermal cyst.

[0032] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the TP53 gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 57148002 to 135524247 on chromosome 17, the sample can be determined to contain a malignant endodermal cyst.

[0033] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the FLCN gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 21437953 to 501 on chromosome 17, the sample can be determined to contain a malignant endodermal cyst.

[0034] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity and a mutation in the BAP1 gene or the protein encoded thereby that reduces, preferably eliminates, their functions, the sample can be determined to contain a malignant endodermal cyst. Known examples of such mutations include the substitution of phenylalanine at position 170 of the BAP1 gene with another base, such as leucine (F170L).

[0035] In a sample from a subject, if the KRAS gene or the protein encoded thereby has a mutation that enhances growth signaling activity, and the PTPRD gene or the protein encoded thereby has a mutation that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 13500114 to 438569 on chromosome 9, the sample can be determined to contain a malignant endodermal cyst.

[0036] In a sample from a subject, if the KRAS gene or the protein encoded thereby has a mutation that enhances growth signaling activity, and the SUFU gene or the protein encoded thereby has a mutation that reduces, preferably eliminates, their function, for example, if there is a deletion of bases 57148002 to 135524247 on chromosome 10, the sample can be determined to contain a malignant endodermal cyst.

[0037] If a sample from a subject contains a mutation in the KRAS gene or the protein encoded thereby that enhances growth signaling activity, and a mutation in the CDKN1B gene or the protein encoded thereby that reduces, or preferably eliminates, their function, for example, if there is a deletion of bases 12028193 to 12871912 on chromosome 12, the sample can be determined to contain a malignant endodermal cyst.

[0038] In one embodiment, the method of testing a sample from a subject suspected of having an endodermal cyst includes detecting at least one gene selected from the group consisting of KRAS and ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B.

[0039] In the first detection step, 1) the presence of a mutation that enhances the growth signaling activity of the KRAS protein and a mutation in at least one or more genes, preferably two or more genes, more preferably all genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B, which mutation reduces, preferably eliminates, the function of each gene or the protein it encodes, may indicate the presence of a malignant endodermal cyst in the sample or the potential for malignant transformation of an endodermal cyst; or 2) The presence of mutations that enhance the growth signaling activity of the KRAS protein and the absence of mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B that reduce, preferably eliminate, the function of the respective gene or the protein it encodes may indicate the presence of a benign endodermal cyst in the sample.

[0040] To determine the presence of malignant endodermal cysts, the potential for endodermal cysts to become malignant, or the presence of benign endodermal cysts, it is preferable to detect the above-mentioned mutations in all of the genes ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B or the proteins they encode.

[0041] In a preferred embodiment, the mutation in the KRAS protein is a substitution of glycine to aspartic acid at position 12 or 13, Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include: ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; is.

[0042] If a mutation that enhances the growth signaling activity of the KRAS protein is detected in the first detection step, but no mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduce the function of each gene or the protein it encodes are detected, a second, third, or subsequent detection step may be performed after a certain period of time has passed.

[0043] In the second detection step, KRAS and at least one or more selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B are detected in another sample from the subject who provided the sample in the first detection step. Subsequent detection steps are also similarly performed.

[0044] In a preferred embodiment, the mutation in the KRAS protein is a substitution of glycine to aspartic acid at position 12 or 13, Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include: ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; is.

[0045] Mutations can be detected by methods known to those skilled in the art. For example, mutations can be directly confirmed using a next-generation sequencer or the like, or mutations can be detected by preparing forward and reverse primers specific to the mutation, using a gene amplification method such as PCR, and determining the sequence of the amplified product or analyzing the restriction fragment pattern. These techniques may be used alone or in combination.

[0046] From the viewpoint of enabling more accurate differentiation, other gene mutations already known for benign or malignant endodermal cysts can also be detected.

[0047] The differentiation method is expected to be used in profiling tests such as companion tests that examine one type of genetic mutation in a single test, or gene panel tests that comprehensively examine multiple genetic mutations at once. Therefore, the differentiation method may include the steps required for these tests and a treatment step for administering a therapeutic drug to the subject based on the test results. For example, known treatment methods can be used for patients determined to have a malignant endodermal cyst. Known treatment methods include, but are not limited to, excision of the affected area.

[0048] (Method for detecting the presence or risk of malignant endodermal cysts) In a third aspect, a method is provided for detecting the presence or risk of malignant transformation of an endodermal cyst, such as a neurenteric cyst, in a sample from a subject.

[0049] In a sample from a subject, the presence of a mutation that enhances the growth signaling activity of the KRAS protein and the presence of a mutation in at least one or more genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU and CDKN1B that reduces the function of each gene or the protein it encodes indicates the presence of a malignant endodermal cyst in the sample or the possibility that an endodermal cyst will become malignant.

[0050] In a preferred embodiment, the mutation in the KRAS protein is a substitution of glycine to aspartic acid at position 12 or 13, Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include: ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; is.

[0051] From the viewpoint of increasing the accuracy of the determination, other mutations specific to malignant endodermal cysts may be detected in addition to the detection of the above mutations. In addition to the detection step, steps necessary for detecting the presence of malignant endodermal cysts or the risk thereof may be included.

[0052] (primer set) In a fourth aspect, a primer set that can be used in the above method and the like is provided.

[0053] In one embodiment, the primer set comprises: one or more primers for detecting mutations that enhance the growth signaling activity of KRAS protein; The following mutations: and one or more primers for detecting mutations in at least one or more genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B, which mutations reduce the function of each gene or the protein encoded by it.

[0054] In a preferred embodiment, the primer set comprises: A substitution of glycine at position 12 or 13 of the KRAS protein with aspartic acid; ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; and one or more primers for detecting the nucleotide sequence of the target gene.

[0055] (probe set) In a sixth aspect, a probe usable in the above method is provided.

[0056] In one embodiment, the probe set comprises one or more probes for detecting mutations that enhance the growth signaling activity of the KRAS protein. The following mutations: The method may include one or more probes for detecting mutations in at least one or more genes selected from the group consisting of ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B, which mutations reduce the function of each gene or the protein encoded by it.

[0057] In a preferred embodiment, the probe set detects a glycine to aspartic acid substitution at position 12 or 13 of the KRAS protein, ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; and one or more probes for detecting the nucleotide sequence.

[0058] (Diagnostic marker for fibroadenoma) In a seventh aspect, there is provided a diagnostic marker for endodermal cysts, such as neuroenteric cysts.

[0059] Diagnostic markers for endodermal cysts include: A substitution of glycine at position 12 or 13 of the KRAS protein with aspartic acid; ARID1A deletions, e.g., deletion of bases 26718016 to 28043054 on chromosome 1; CDKN2A deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; CDKN2B deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; PTCH1 deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 9; TSC1 deletions, e.g., deletion of bases 138129095 to 133759286 on chromosome 9; PTEN deletions, e.g., deletion of bases 133756118 to 13500615 on chromosome 10; TP53 deletions, e.g., deletion of bases 57148002 to 135524247 on chromosome 17; FLCN deletions, e.g., deletion of bases 21,437,953 to 501 on chromosome 17; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletions, e.g., deletion of bases 13500114 to 438569 on chromosome 9; SUFU deletions, such as deletions of bases 57148002 to 135524247 on chromosome 10; and CDKN1B deletion, e.g., deletion of bases 12028193 to 12871912 on chromosome 12; This relates to detecting

[0060] Diagnostic markers can be detected using DNA, cDNA, RNA, mRNA, DNA analogs, RNA analogs, amino acids, or proteins that reflect the corresponding mutations. The DNA can be cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). The diagnostic marker can include the amino acid sequence of a region containing one or more of the above mutations or the nucleotide sequence encoding the amino acid sequence.

[0061] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples. [Example]

[0062] A 65-year-old female subject was diagnosed with a cystic mass on the dorsal surface of the medulla oblongata by magnetic resonance imaging (MRI) (Figure 1A). Three months later, the subject presented with gait disturbance due to the growth of the cystic mass. A partial resection and biopsy of the cyst was performed. The gait disturbance resolved after the initial surgery. However, 15 months after the initial surgery, the cystic mass regrew (Figure 1B).

[0063] Partial resection of the cyst was performed again, and contrast-enhanced MRI revealed a mass with cystic components in the left lateral ventricular trigone 2 years after the second surgery ( Fig. 1C, D ), but the cystic mass on the dorsal side of the medulla oblongata had not recurred.

[0064] The patient then underwent partial resection of the identified mass. For residual mass and histopathological malignancy, whole-brain irradiation (36 Gy / 18 Fr) followed by intensity-modulated radiotherapy (24 Gy / 12 Fr) was performed.

[0065] However, four months later, the mass recurred and performance status progressively worsened. Eleven months after the third surgery, nearly five years after the onset of the cystic mass, the subject died of his disease.

[0066] Surgical specimens obtained at each operation were fixed in 10% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-μm-thick sections stained with hematoxylin and eosin. Immunohistochemical staining was performed using monoclonal antibodies against epithelial membrane antigen (EMA) (clone E29; Dako; 1:100), cytokeratin (clone CAM5.2; Becton Dickinson), and Ki-67 (clone MIB-1; Dako; 1:100). Bound antibodies were visualized using the peroxidase and polymer-based Histofine Simple Stain MAX-PO Kit (Nichirei).

[0067] Histopathological analysis of the first and second surgical specimens revealed a wall composed of pseudostratified cuboidal epithelium without nuclear or cellular atypia, similar to gastrointestinal mucosa (Figure 2A, B), and the specimens were determined to have neurenteric cysts. The third surgical specimen showed clear malignant features of epithelial cells with elongated, hyperchromatic nuclei, a few mitotic figures, small necroses, and a patternless or sheet-like growth pattern (Figure 2C, D). This specimen was immunopositive for epithelial membrane antigen (EMA) (Figure 2E) and cytokeratin, with a Ki-67 labeling index of approximately 20% (Figure 2F). Therefore, the neurenteric cyst was determined to have undergone malignant transformation.

[0068] Three surgical specimens were analyzed by next-generation sequencing. Sanger sequencing of the KRAS gene (codons 12 and 13) was performed on all specimens. The primers used were as follows: 5'-TGT GTG ACA TGT TCT AAT ATA GTC ACA T-3' Forward: SEQ ID NO: 1) and 5'-GGT CCT GCA CCA GTA ATA TGC-3' (reverse: SEQ ID NO: 2) It was.

[0069] All specimens were found to have a KRAS p.G12D mutation (Figure 3). Furthermore, genetic profiles were analyzed using CANCERPLEX® (Kew Inc.), an NGS panel of 435 active cancer-related genes. The KRAS p.G12D mutation was detected in all specimens, similar to the results of Sanger sequencing. The third specimen harbored 12 de novo mutations: ARID1A deficiency, BAP1 p.F170L, CDKN1B deficiency, CDKN2A deficiency, CDKN2B deficiency, FLCN deficiency, PTCH1 deficiency, PTEN deficiency, PTPRD deficiency, SUFU deficiency, TP53 deficiency, and TSC1 deficiency. The results are shown in the table below.

[0070] [Table 1]

[0071] The above results suggested that KRAS mutations are associated with the development of neurilemma cysts and that additional genetic alterations contribute to malignant transformation.

Claims

1. 1. A method for detecting a mutation associated with malignant transformation of an endodermal cyst in a sample, comprising: The method comprises detecting, in a sample derived from a subject, a mutation that enhances the proliferation signal transduction activity of KRAS protein and a mutation in the genes ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B; the mutation that enhances the growth signaling activity of the KRAS protein is a substitution of glycine at position 12 or 13 with aspartic acid; Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include the following: ARID1A deletion; CDKN2A deletion; CDKN2B deletion; PTCH1 deletion; TSC1 deletion; PTEN deletion; TP53 deletion; FLCN deletion; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletion; SUFU deletion; and CDKN1B deletion, That's the method.

2. The method of claim 1, wherein the endodermal cyst is a neuroenteric cyst.

3. 3. The method of claim 1 or 2, wherein the sample is derived from a cyst.

4. 1. A method of testing a sample from a subject suspected of having an endodermal cyst, comprising: The method comprises the steps of: detecting a mutation that enhances the growth signal transduction activity of KRAS protein and a mutation in the genes ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B in a sample derived from a subject; 1) the presence of a mutation that enhances the growth signaling activity of the KRAS protein and the presence of a mutation in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes that reduces the function of each gene or the protein it encodes indicates the presence of a malignant endodermal cyst in the sample or the likelihood that an endodermal cyst will become malignant; or 2) the presence of a mutation that enhances the growth signaling activity of the KRAS protein and the absence of mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduce the function of each gene or the protein it encodes indicates the presence of a benign endodermal cyst in the sample; the mutation that enhances the growth signaling activity of the KRAS protein is a substitution of glycine at position 12 or 13 with aspartic acid; Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include the following: ARID1A deletion; CDKN2A deletion; CDKN2B deletion; PTCH1 deletion; TSC1 deletion; PTEN deletion; TP53 deletion; FLCN deletion; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletion; SUFU deletion; and CDKN1B deletion, That is, method.

5. The method of claim 4, further comprising a second detection step of detecting mutations that enhance the growth signaling activity of the KRAS protein and mutations in the genes ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B in another sample derived from a subject who provided a sample in which a mutation that enhances the growth signaling activity of the KRAS protein was detected in the first detection step, but mutations in ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B that reduce the function of each gene or the protein encoded thereby were not detected.

6. The method described in claim 5, further comprising repeating the first and second detection steps after a certain period of time has elapsed.

7. one or more primers for detecting mutations that enhance the growth signaling activity of the KRAS protein; The following mutations: one or more primers for detecting mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes, which mutations reduce the function of each gene or the protein encoded by it; the mutation that enhances the growth signaling activity of the KRAS protein is a substitution of glycine at position 12 or 13 with aspartic acid; Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include the following: ARID1A deletion; CDKN2A deletion; CDKN2B deletion; PTCH1 deletion; TSC1 deletion; PTEN deletion; TP53 deletion; FLCN deletion; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletion; SUFU deletion; and CDKN1B deletion, This is the primer set.

8. one or more probes for detecting mutations that enhance the growth signaling activity of the KRAS protein; The following mutations: one or more probes for detecting mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes, which mutations reduce the function of each gene or the protein encoded by it; the mutation that enhances the growth signaling activity of the KRAS protein is a substitution of glycine at position 12 or 13 with aspartic acid; Mutations in the ARID1A, CDKN2A, CDKN2B, PTCH1, TSC1, PTEN, TP53, FLCN, BAP1, PTPRD, SUFU, and CDKN1B genes include the following: ARID1A deletion; CDKN2A deletion; CDKN2B deletion; PTCH1 deletion; TSC1 deletion; PTEN deletion; TP53 deletion; FLCN deletion; a phenylalanine to leucine substitution at position 170 of the BAP1 protein; PTPRD deletion; SUFU deletion; and CDKN1B deletion, , the probe set.

9. A kit comprising the primer set according to claim 7 and the probe set according to claim 8.

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