TW202317775A - Method for differentiating fibroadenoma from phyllodes tumor - Google Patents

Abstract

The present invention provides a method for detecting a gene that relates to fibroadenoma or phyllodes tumor in a sample, said method comprising a step for detecting (1) at least one selected from the group consisting of PIK3CA and ERBB2, or (2) at least one selected from the group consisting of PTEN, NF1, CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU, in a sample derived from a subject.

Description

How to differentiate between fibroadenomas and phyllodes tumors

The present invention broadly relates to methods for identifying fibroadenomas and phyllodes tumors.

Fibroadenoma (Fa) is a benign epithelial mesenchymal tumor of the breast with a high frequency. It is divided into intracanalicular type, pericanalicular type, organoid type, and breast cancer. Type 4 of mastopathic type. Fibroadenoma is a typical benign tumor that occurs frequently in people in their early 20s. It sometimes grows in size and becomes a candidate for resection due to cosmetic concerns, but it is basically a tumor that does not require treatment.

Among them, mammary gland type is a subtype that is easily misdiagnosed as breast cancer in needle biopsy, but the WHO (World Health Organization, World Health Organization) breast tumor classification does not specify the subtype corresponding to mammary gland fibroadenoma. , considered to be a subtype unique to Japan.

One example of a disease similar to mammary fibroadenoma is Phyllodes Tumor (PT). Pathologically, phyllodes tumors are classified into benign, borderline malignant, and malignant. Malignant phyllodes tumors are one of the most difficult-to-treat tumors and show the same development pattern as cancer, such as leading to distant metastasis.

Thus, the biological characteristics of fibroadenomas and phyllodes tumors are significantly different, but pathological identification may be difficult. Prior technical literature non-patent literature

Non-patent document 1: Nagazawa Hui et al., Somatic variation analysis of phyllodes tumors and fibroadenoma using next-generation sequencing, Journal of Santa Maria Medical University, Vol. 42, pp. 129–140, 2014

[Problem to be solved by the invention]

In view of the above situation, the purpose of the present invention is to provide a method for identifying fibroadenomas and phyllodes tumors. [Technical means to solve problems]

To differentiate fibroadenomas from phyllodes tumors, by using somatic variant analysis using next-generation sequencing, we identified missense variants in LPP, GATA3, and AMER1 that are unique to phyllodes tumors and that are unique to fibroadenomas. There are missense mutations in PAX5, but it has been reported that there are no mutations in genes such as PIK3CA or p53, which are highly mutated in cancer (Non-patent Document 1).

The present inventors conducted a new cancer gene panel examination to study whether genes related to cancer manifestations are involved in the proliferation of fibroadenoma and phyllodes tumors. As a result, they found that new mutations in PIK3CA etc. can be used to identify fibroadenoma and phyllodes tumors. The present invention was completed by identifying important genetic mutations in tumor-like tumors.

That is, this case includes the following inventions. [1] A method for detecting genes related to fibroadenoma or phyllodes tumor in a sample, which includes the following steps: In the sample derived from the subject, detect 1) at least one or more selected from the group consisting of PIK3CA and ERBB2, or 2) selected from the group consisting of PTEN, NF1, CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1 At least one of the group consisting of , NF2, TGFBR2 and SUFU. [2] A method for identifying fibroadenoma and phyllodes tumor in samples from subjects suspected of suffering from fibroadenoma or phyllodes tumor, which includes the following steps: 1) When any one or more of the following mutations are detected, the sample is determined to be derived from fibroadenoma: PIK3CA has a deletion of four amino acids (VGNR) from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histidine; and/or 2) When any one or more of the following mutations are detected, the sample is determined to be derived from phyllodes tumor: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. [3] The method described in [2], wherein the sample is derived from breast tissue. [4] A method for detecting the presence or risk of fibroadenoma in a sample derived from a subject, which includes the following steps: When any one or more of the following mutations are detected in the sample, it is determined that the sample contains fibroadenoma or is at risk: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine. [5] A method for detecting the presence or risk of phyllodes in a sample derived from a subject, which includes the following steps: When any one or more of the following mutations are detected in the sample, it is determined that the sample contains phyllodes or is at risk: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. [6] A primer set, which includes: 1 or a plurality of primers used to specifically amplify a region containing any one or more of the following mutations: The gene encoding the mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108 of PIK3CA, A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced by histidine; and One or more primers used to specifically amplify a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN is mutated into a stop codon. Frame shift mutation in isoleucine at position 679 of NF1, Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. [7] A probe set comprising: 1 or a plurality of probes that specifically bind to a region containing any one or more of the following variations: The gene encoding the mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108 of PIK3CA, A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced by histidine; and One or more probes that specifically bind to a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN is mutated into a stop codon. Frame shift mutation in isoleucine at position 679 of NF1, Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. [8] A kit for distinguishing fibroadenomas and phyllodes tumors, which includes a primer set as described in [6] and a probe set as described in [7]. [9] A diagnostic marker for fibroadenoma, which includes an amino acid sequence or a base sequence encoding the region containing any one or more of the following mutations: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine. [10] A diagnostic marker for fibroadenoma, which includes an amino acid sequence or a base sequence encoding the region containing any one or more of the following mutations: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; A frame-shift variant in isoleucine at position 679 of NF1; and Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. [Effects of the invention]

According to the present invention, fibroadenomas and phyllodes tumors, which were previously difficult to differentiate pathologically, can be distinguished through specific gene mutations. In addition, the information on gene mutation can also be used as auxiliary information to differentiate between fibroadenoma and phyllodes tumor.

Hereinafter, embodiments of the present invention (hereinafter referred to as "this embodiment") will be described. However, the scope of the present invention is not limited to the following embodiments.

(Detection method) In a first form, a method for detecting genes related to fibroadenomas or phyllodes tumors in a sample is provided, and includes the following steps: in a sample derived from a subject, detecting 1) a gene selected from PIK3CA and ERBB2 At least one or more of the group consisting of, or 2) at least one or more of the group consisting of PTEN, NF1, CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

As used in this specification, "gene" includes DNA (Deoxyribonucleic Acid, deoxyribonucleic acid) encoding a specific protein, or its amino acid sequence including a leader sequence or a secretory sequence, such as its proprotein, preproprotein, etc. regions, non-coding DNA regions containing 5' non-coding sequences or 3' non-coding sequences before and after these coding regions, or a part thereof. That is, genes are not limited to exons, but sometimes also refer to introns. In addition, genes are not limited to those specifically mentioned, and transcription products (such as mRNA, tRNA, rRNA, antisense RNA (ribonucleic acid, ribonucleic acid), etc.) are also included in the scope of genes. The DNA can be cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) in the blood.

The subjects can be humans or other non-human mammals, especially non-human primates. The sample may be from a healthy subject, a subject suspected of suffering from fibroadenoma or phyllodes tumor, for example, a subject diagnosed as suffering from fibroadenoma or phyllodes tumor or diagnosed as having the possibility of suffering from fibroadenoma or phyllodes tumor. Any one of the samples. There is no intention to limit, but examples of such samples include tissue samples such as biopsy samples, blood circulating cells, serum, plasma, blood, feces, urine, sputum, cerebrospinal fluid, pleural hydrops, and nipple aspiration. fluid, lymph fluid, cell culture fluid, and other tissues and fluids collected from patients. The sample is preferably derived from breast tissue. Samples can be collected through cell diagnosis (needle aspiration cell diagnosis) or tissue diagnosis, such as needle biopsy, Mammotome biopsy, VACORA biopsy, etc.

PIK3CA (PI3K catalytic subunit p110α) is equivalent to the catalytic subunit p110-alpha of phosphoinositide 3-kinase (PI3K), an important regulator of signal transmission pathways. PIK3CA has the amino acid sequence shown in SEQ ID NO: 1. The base sequence encoding the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.

ERBB2 (epidermal growth factor receptor 2, epithelial growth factor receptor 2) is a cell membrane receptor type tyrosine kinase and has the amino acid sequence shown in SEQ ID NO: 5. The base sequence encoding the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 6.

The PTEN (phosphatase and tensin homolog Deleted from Chromosome 10, phosphatase and tensin homolog deleted from Chromosome 10) gene is one of the cancer suppressor genes, and its expression product is known to cause phosphoinositol 3, which is an inositol phospholipid. Phosphate (PtdIns(3,4,5)P3) is dephosphorylated.

PtdIns(3,4,5)P3 is synthesized in cells by PI3 kinase (PI3K) and contributes to the expression of various biological activities by causing the activation of protein kinase B (PKB)/Akt. PTEN has weak phosphatase activity on proteins and is responsible for the dephosphorylation reaction of active inositol phospholipid PtdIns(3,4,5)P3, converting it into PtdIns(4,5)P2. By inhibiting PTEN, PtdIns(3,4,5)P3 is accumulated in cells and transmits signals related to carcinogenesis. In fact, in cancer cells, mutations and other abnormalities have been found in the PTEN gene.

PTEN has the amino acid sequence shown in SEQ ID NO: 9. The base sequence encoding the amino acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 10.

The NF1 (neurofibromatosis type 1) gene encodes a protein called neurofibromin that inhibitoryly controls the RAS/MAPK pathway and is associated with the pathogenesis of neurofibromatosis type 1. NF1 has the amino acid sequence shown in SEQ ID NO: 13, and the gene encoding NF1 has the base sequence shown in SEQ ID NO: 14.

Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a cancer suppressor protein encoded by the CDKN2A (p16) gene. It is known that p16 plays an important role in the regulation of the cell cycle, and mutations in p16 increase the risk of various cancers, especially melanoma.

CDKN2A has the amino acid sequence shown in SEQ ID NO: 15. The base sequence encoding the amino acid sequence of SEQ ID NO: 15 is shown in SEQ ID NO: 16.

As a cyclin-dependent inhibitor similar to CDKN2A, there is cyclin-dependent kinase inhibitor 2B (cyclin-dependent kinase inhibitor 2A: CDKN2B). CDKN2B is also known as MTS-2 or p15 ^INK4b . The region of the CDKN2B gene that is frequently mutated or deleted in various tumors is adjacent to the tumor suppressor gene CDKN2A. This gene forms a complex with CDK4 or CDK6 and blocks the activation of CDK kinase caused by cyclin D. Therefore, the encoded protein functions as a cell proliferation control factor that inhibits the progression of the G1 phase of the cell cycle.

The expression of CDKN2B gene is significantly induced by TGFβ, which suggests that this gene plays a role in inhibiting TGFβ-induced proliferation. Two splice variants encoding another protein have been reported in the CDKN2B gene.

CDKN2B has the amino acid sequence shown in SEQ ID NO: 17. The base sequence encoding the amino acid sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 18. When the CDKN2B gene or the protein encoded by it is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor.

In the case of PTEN gene deletion, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor.

The ATM gene encodes a 370 kDa giant protein with a serine/threonine kinase domain at the C terminus. It has been known so far that it is activated by double-strand breaks in DNA that occur due to radiation, chemicals, or normal intracellular metabolism, and damages DNA by phosphorylating intracellular matrices such as p53, Chk2, and BRCA1. It is one of the cancer suppressor genes that plays a central role in arresting the cell cycle or inducing apoptosis.

ATM has the amino acid sequence shown in SEQ ID NO: 19. The base sequence encoding the amino acid sequence of SEQ ID NO: 19 is shown in SEQ ID NO: 20.

TP53 is one of the cancer suppressor genes and has the function of controlling DNA repair or inhibiting cell proliferation cycles such as cell proliferation cessation and apoptosis in every cell. It is believed that if the function achieved by this gene is incomplete, cell apoptosis will occur when cells become cancerous. The protein product of TP53 is p53.

p53 has the amino acid sequence shown in SEQ ID NO: 21. The base sequence of TP53 encoding the amino acid sequence of SEQ ID NO: 21 is shown in SEQ ID NO: 22.

In the case of deletion of the NF1 gene, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor.

As a gene related to the onset of neurofibromatosis type II, there is the NF2 gene encoding Merlin, a protein that forms the cytoskeleton. Merlin is produced by the nervous system, especially the nerve sheath cells that surround and insulate nerves. Merlin helps regulate some of the major signaling pathways important in controlling cell shape, cell growth and how cells adhere to each other (cell adhesion), and functions as a tumor suppressor, preventing cells from rapidly proliferating or dividing.

Merlin has the amino acid sequence shown in SEQ ID NO: 23. The base sequence of NF2 encoding the amino acid sequence of SEQ ID NO: 23 is shown in SEQ ID NO: 24.

TGFβ2 (Transforming growth factor beta-2) is a cytokine belonging to the TGFβ family. It is transmitted through type I and type II receptors (TGFBR1 and TGFBR2) as transmembrane receptors and SMAD proteins as downstream effectors. Signals control cell proliferation, differentiation, adhesion, movement, etc. TGFBR2 has a protein kinase domain that combines with other receptor proteins to form a heterodimer complex. This complex phosphorylates the protein, which enters the nucleus and regulates the transcription of a subset of genes involved in cell proliferation. Mutations in the TGFBR2 gene can lead to Marfan syndrome, Leuditz aortic aneurysm syndrome, hereditary hemorrhagic vasodilation syndrome, or various tumors.

TGFBR2 has the amino acid sequence shown in SEQ ID NO: 25. The base sequence encoding the amino acid sequence of SEQ ID NO: 25 is shown in SEQ ID NO: 26.

One of the hedgehog signaling pathway genes, SUFU (suppressor of fused), is a component of the sonic hedgehog (SHH)/transmembrane protein receptor (PTCH) signaling pathway. Therefore, mutations in SUFU are detrimental to normal development and can lead to cancer diathesis syndromes (eg, holoprosencephaly HPE3, basal cell nevus cancer syndrome BCNS, and Greig extracranial polydactyly syndrome GCPS). SUFU is also known to play an important role in suppressing tumors.

SUFU has the amino acid sequence shown in SEQ ID NO: 27. The base sequence encoding the amino acid sequence of SEQ ID NO: 27 is shown in SEQ ID NO: 28.

Genetic testing can be performed by methods known to those skilled in the art. For example, next-generation sequencing can be used to directly confirm the presence of genes in the sample. Gene-specific forward primers and reverse primers can also be prepared and gene amplification methods such as PCR (polymerase chain reaction) can be used. The presence or absence of the gene is confirmed by sequencing the amplified product or analyzing the restriction enzyme fragment pattern. These technologies can be used individually or in combination.

In the samples in which the above genes are detected, genes known to be related to fibroadenoma or phyllodes tumor can be further detected. In addition, samples that detect genes related to fibroadenoma or phyllodes tumor can be used to detect fibroadenoma or phyllodes tumor in the sample, diagnose fibroadenoma or phyllodes tumor in the subject, etc. For such purposes, samples detecting the above-mentioned genes can be further analyzed.

(Identification method) In a second aspect, a method for identifying fibroadenoma and phyllodes tumor in a sample from a subject suspected of suffering from fibroadenoma or phyllodes tumor is provided. Fibroadenomas are roughly divided into four types: intraductal type, peritubular type, organoid type, and mastomatous type. The fibroadenoma to be identified is preferably the mastomatous type. Phyllodes tumors are pathologically classified into benign, borderline, and malignant.

The subject is defined as above, preferably a subject who is suspected of suffering from fibroadenoma or phyllodes tumor, such as a person who is diagnosed with fibroadenoma or phyllodes tumor, or who is diagnosed with the possibility of suffering from fibroadenoma or phyllodes tumor. Subjects with phyllodes tumor. The sample is defined as above.

The identification method may include the following steps: detecting one or more proteins selected from the group consisting of PIK3CA, ERBB2, CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU, or In the case of specific mutations in genes encoding these, it is determined that the sample originates from fibroadenoma or phyllodes tumor.

As an example of this step, the following steps can be cited: 1) When any one or more of the following mutations are detected, the sample is determined to be derived from fibroadenoma: PIK3CA has a deletion of four amino acids (VGNR) from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histidine; and/or 2) When any one or more of the following mutations are detected, it is determined that the sample originates from phyllodes tumor, especially malignant phyllodes tumor: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

In the sample derived from the subject, four amino acids (VGNR) from valine at position 105 to arginine at position 108 of the amino acid sequence encoding PIK3CA (sequence number 1) were deleted. In this case, it can be determined that the sample is derived from fibroadenoma, that is, the subject may have fibroadenoma. The mutated amino acid sequence and the base sequence encoding it are shown in SEQ ID NO: 3 and 4 respectively.

In the sample derived from the subject, the aspartic acid at position 1144 of the amino acid sequence encoding ERBB2 (SEQ ID NO: 5) is replaced by histidine, or the base sequence (SEQ ID NO: 6) When guanine at position 3430 is replaced by cytosine, it can be determined that the sample is derived from fibroadenoma, that is, the subject may have fibroadenoma. The mutated amino acid sequence and the base sequence encoding it are shown in SEQ ID NO: 7 and 8 respectively.

In a sample derived from a subject, the cytosine constituting the codon for glutamine at position 110 of the base sequence encoding PTEN (Sequence No. 10) is replaced with thymine, such as in CAA When it is replaced with a stop codon, such as TAA, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor. The amino acid sequence when CAA is mutated into TAA and the base sequence encoding it are shown in SEQ ID NO: 11 and 12, respectively. When the codon encoding glutamine is CAG, the mutated stop codon is TAG.

In the sample derived from the subject, the base (ATC) encoding isoleucine at position 679 of the amino acid sequence encoding NF1 (Sequence No. 13) is deleted or inserted into other bases, resulting in When there is a frame shift variation in the reading frame of amino acids after 1680, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor.

When the CDKN2A gene (SEQ ID NO: 16) or the protein encoded by it (SEQ ID NO: 15) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the CDKN2B gene (SEQ ID NO: 18) or the protein encoded by it (SEQ ID NO: 17) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the ATM gene (SEQ ID NO: 20) or the protein encoded by it (SEQ ID NO: 19) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When TP53 (SEQ ID NO: 22) or the protein p53 (SEQ ID NO: 21) encoded by it is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the NF1 gene (sequence number 15) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the NF2 gene (SEQ ID NO: 24) or the protein Merlin (SEQ ID NO: 23) encoded by it is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the TGFBR2 gene (SEQ ID NO: 26) or the protein encoded by it (SEQ ID NO: 25) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

When the SUFU gene (SEQ ID NO: 28) or the protein encoded by it (SEQ ID NO: 27) is deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may suffer from phyllodes tumor.

In the sample derived from the subject, at least one or more, preferably three or more genes selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1NF2, TGFBR2 and SUFU , preferably when all genes are deleted, it can be determined that the sample is derived from phyllodes tumor, that is, the subject may have phyllodes tumor.

Variation detection can be performed by methods known to those skilled in the art. For example, next-generation sequencing can be used to directly confirm the mutation, or mutation-specific forward primers and reverse primers can be prepared, and gene amplification methods such as PCR can be used to determine the sequence of the amplified product or determine the restriction enzyme fragment pattern. Analysis to detect variations. These technologies can be used individually or in combination.

From the viewpoint of improving the identification accuracy, the identification method may be combined with a known method for diagnosing fibroadenoma or phyllodes tumor.

The identification method is envisioned to be used in analytical tests such as a companion test, which studies one genetic mutation in one test, or a gene panel test, which comprehensively studies many genetic mutations at once. Therefore, the identification method may include steps required for such examinations, or treatment steps for applying therapeutic drugs determined based on the examination results to the subject. For example, for patients diagnosed with malignant phyllodes tumor, known treatment methods can be used. Known treatment methods are not particularly limited, and examples include, in addition to surgical resection, administration of therapeutic drugs (doxorubicin, ifosfamide), and the like. Regarding the administration method of the therapeutic drug, its usage is not limited, and known methods can be used.

(Methods to detect the presence or risk of fibroadenoma) In a third aspect, a method is provided for detecting the presence or risk of fibroadenoma in a sample derived from a subject.

When any one or more of the following mutations are detected in a sample derived from a subject, it can be determined that the sample contains fibroadenoma, especially mastomatous fibroadenoma, or is at risk: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine.

From the perspective of improving the accuracy of the determination step, other mutations specific to fibroadenoma can also be detected simultaneously with the detection of the above-mentioned mutations. In addition to the detection steps, steps required to detect the presence or risk of fibroadenoma may be included.

(Methods to detect the presence or risk of phyllodes) In a fourth aspect, a method is provided for detecting the presence or risk of phyllodes in a sample derived from a subject.

When any one or more of the following mutations are detected in a sample derived from a subject, it can be determined that the sample contains phyllodes tumors, especially malignant phyllodes tumors, or is at risk of them: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

From the viewpoint of improving the accuracy of the determination step, other mutations specific to phyllodes tumor can also be detected simultaneously with the above-mentioned mutation detection. In addition to the detection steps, steps required to detect the presence or risk of phyllodes may be included.

(introduction group) In the fifth form, an introduction set that can be used by the above methods is provided.

In one implementation, the primer set may include: One or more primers used to specifically amplify a region containing any one or more of the following variations: The gene encoding PIK3CA mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108; A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced with histidine.

In one implementation, the primer set may include: One or more primers used to specifically amplify a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

(probe set) In a sixth aspect, a probe that can be used by the above method or the like is provided.

In one embodiment, the probe set may include one or more probes that specifically bind to a region containing any one or more of the following variations: The gene encoding PIK3CA mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108; A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced with histidine.

In one embodiment, the probe set may include one or more probes that specifically bind to a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

(Diagnostic markers of fibroadenoma) In the seventh form, a diagnostic marker of fibroadenoma is provided.

Diagnostic markers for fibroadenoma, especially mammary fibroadenoma, detect the following mutations: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine.

Detection of diagnostic markers can be performed in any of DNA, cDNA, RNA, mRNA, DNA analogs, RNA analogs, amino acids, and proteins that reflect corresponding mutations. For example, the diagnostic marker may include an amino acid sequence or a base sequence encoding the region containing any one or more of the above variations.

(Diagnostic markers of phyllodes tumor) In the eighth pattern, a diagnostic marker of phyllodes is provided.

Diagnostic markers for phyllodes tumors, especially malignant phyllodes tumors, detect the following mutations: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.

Detection of diagnostic markers can be performed in any of DNA, cDNA, RNA, mRNA, DNA analogs, RNA analogs, amino acids, and proteins that reflect corresponding mutations. For example, the diagnostic marker may include an amino acid sequence or a base sequence encoding the region containing any one or more of the above variations. In the case of a deletion, the presence or absence of the deletion can be confirmed by confirming the base sequence of the region containing the binding of coding genes or non-coding genes before and after the deleted gene or the amino acid sequence encoded by it.

Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these. Example

Using the QIAamp DNA FFPE Tissue Kit (Qiagen), 3 cases of tumor tissue were pathologically diagnosed as fibroadenomas (one of which was breast type) and 3 cases of tumor tissue were diagnosed as phyllodes tumors (one was benign and one was DNA was extracted from formalin-fixed paraffin-embedded (FFPE) thin sections.

Regarding the extracted DNA, KAPA HyperPlus Liabrary Preparation Kit was used to fragment the DNA, perform end repair and dA addition to the fragmented DNA, and then add index sequences and sequences that can be used for base sequence analysis using Illumina's sequencer. the adapter sequence. After the DNA with the adapter sequence is generated by the magnetic bead method, 7 to 9 cycles of PCR amplification are performed.

The DNA appended with the amplified adapter sequence was purified using SPRIselect in such a way that 150 to 400 bp of DNA became the main component. The above DNA was hybridized with a biotinylated RNA bait hybridizing to the coding regions of 435 cancer-related genes included in the CANCERPLEX panel, and then purified using streptavidin-magnetic beads, using 12 cycles of PCR amplification was performed to create a gene library for NGS sequence analysis.

Regarding the gene bank DNA, NextSeq500 (Illumina) was used for sequence analysis, and the bioinformatics pipeline of KEW Inc. was used for analysis to detect genetic variations in each DNA. The mutation detection results of each specimen are shown in Table 1 and Table 2. [Table 1] fibroadenoma phyllodes L024 L027 L028 (mastopathy type) L025 (benign) L023 (marginal) MED12 - p.G44S - p.G44D p.G44D TERT - - - cC-124T cC-124T PIK3CA - - p.V105_R108del - - ERBB2 - - p.D1144H - - [Table 2] phyllodes L027 (malignant) PTEN p.Q110X NF1 p.I679fs CDKN2A Missing CDKN2B Missing ATM Missing TP53 Missing NF1 Missing NF2 Missing TGFBR2 Missing SUFU Missing

TW202317775A_111131381_SEQL.xml

Claims (10)

A method for detecting genes related to fibroadenoma or phyllodes tumor in a sample, which includes the following steps: In the sample derived from the subject, detect 1) at least one or more selected from the group consisting of PIK3CA and ERBB2, or 2) selected from the group consisting of PTEN, NF1, CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1 At least one of the group consisting of , NF2, TGFBR2 and SUFU. A method for identifying fibroadenoma and phyllodes tumor in samples from subjects suspected of suffering from fibroadenoma or phyllodes tumor, which includes the following steps: 1) When any one or more of the following mutations are detected, the sample is determined to be derived from fibroadenoma: PIK3CA has a deletion of four amino acids (VGNR) from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histidine; and/or 2) When any one or more of the following mutations are detected, the sample is determined to be derived from phyllodes tumor: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. The method of claim 2, wherein the sample is derived from breast tissue. A method for detecting the presence or risk of fibroadenoma in a sample derived from a subject, which includes the following steps: When any one or more of the following mutations are detected in the sample, it is determined that the sample contains fibroadenoma or is at risk: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine. A method for detecting the presence or risk of phyllodes in a sample derived from a subject, which includes the following steps: When any one or more of the following mutations are detected in the sample, it is determined that the sample contains phyllodes or is at risk: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; Frame shift mutation in isoleucine at position 679 of NF1; Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. A primer set, which includes: 1 or a plurality of primers used to specifically amplify a region containing any one or more of the following mutations: The gene encoding the mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108 of PIK3CA, A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced by histidine; and One or more primers used to specifically amplify a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN is mutated into a stop codon. Frame shift mutation in isoleucine at position 679 of NF1, Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. A probe set comprising: 1 or a plurality of probes that specifically bind to a region containing any one or more of the following variations: The gene encoding the mutant PIK3CA with a deletion of four amino acids from valine at position 105 to arginine at position 108 of PIK3CA, A gene encoding a mutant ERBB2 in which aspartic acid at position 1144 of ERBB2 is replaced by histidine; and One or more probes that specifically bind to a region containing a gene encoding any one or more of the following variants: The codon encoding glutamine at position 110 of PTEN is mutated into a stop codon. Frame shift mutation in isoleucine at position 679 of NF1, Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU. A kit for identifying fibroadenomas and phyllodes tumors, which includes the primer set of claim 6 and the probe set of claim 7. A diagnostic marker for fibroadenoma, which includes an amino acid sequence or a base sequence encoding the region containing any one or more of the following mutations: PIK3CA has a deletion of four amino acids from valine at position 105 to arginine at position 108; Aspartic acid at position 1144 of ERBB2 is replaced with histamine. A diagnostic marker for fibroadenoma, which includes an amino acid sequence or a base sequence encoding the region containing any one or more of the following mutations: The codon encoding glutamine at position 110 of PTEN was mutated into a stop codon; A frame-shift variant in isoleucine at position 679 of NF1; and Gene deletion selected from the group consisting of CDKN2A, CDKN2B, PTEN, ATM, TP53, NF1, NF2, TGFBR2 and SUFU.