WO2020091367A1

WO2020091367A1 - Biomarker for breast cancer diagnosis and use thereof

Info

Publication number: WO2020091367A1
Application number: PCT/KR2019/014342
Authority: WO
Inventors: 조제열; 이강훈
Original assignee: 서울대학교산학협력단
Priority date: 2018-10-30
Filing date: 2019-10-29
Publication date: 2020-05-07
Also published as: KR102138249B1; KR20200048788A

Abstract

According to a composition and a kit for breast cancer diagnosis, and a method for breast cancer diagnosis or a method for providing information for breast cancer diagnosis using same according to one aspect, breast cancer can be diagnosed at an early stage. In addition, mammary gland carcinoma can be diagnosed in a companion animal, and the likelihood of a human guardian of the companion animal developing breast cancer can be predicted thereby.

Description

Biomarker for breast cancer diagnosis and use thereof

The present invention relates to a composition, kit for diagnosing breast cancer or mammary cancer, and a method for predicting the likelihood of diagnosis or development of breast cancer using the same.

Human breast or mammary cancer is a malignant tumor that ranks second in the incidence of female cancer. There are many causes of breast cancer, but the known cause is only about 20%. About 80% of the remaining cause is unknown, but the opinion is that it is most likely due to the external environment. Diseases caused by the external environment are so diverse that it is difficult to explain them as one cause. Therefore, a complex external environment is considered to be the main cause of breast cancer. Diseases caused by external environmental causes cannot be predicted and prevented in advance, and since the treatment process is performed only after the diagnosis of the disease, there is a problem that it takes a considerable amount of time, cost, and recovery.

Companion animals, for example, companion dogs, are exposed to the same space and environment as humans, similarly receiving the external environment they receive. Dogs have a faster metabolism than humans and have higher susceptibility to the external environment, so they can first express certain diseases that can occur in humans (eg, guardians of dogs) to the outside. For example, the occurrence of mammary cancer in a dog can mean the possibility of breast cancer in humans.

Therefore, it is necessary to develop a method for predicting the likelihood of human breast cancer caused by the environment by first diagnosing mammary cancer of a companion dog living in the same environment as a person and applying it to a person.

Provide a composition for diagnosing breast cancer,

A kit for diagnosing breast cancer is provided.

It provides a method of diagnosing breast cancer or providing information to diagnose breast cancer.

One aspect is dematin (DMTN), myo-inositol oxygenase (MIOX), alpha-hemoglobin-stabilizing protein (AHSP), hemoglobin subunit mu (HBM), 5-aminolevulinate synthase (ALAS2), ENSCAFG00000032136, SNX5 (Sorting) nexin-5), ENSCAFG00000037058, ENSCAFG00000024346, MATR3 (Matrin-3), IL12B (Interleukin-12 subunit beta), CD36 (cluster of differentiation 36), EMD (Emerin), NECTIN2 (nectin cell adhesion molecule 2), GP9 (Platelet glycoprotein IX), ITGB1 (Integrin beta-1), ITGA7 (Integrin alpha-7), PVR (Poliovirus receptor (NK cell)), NRG1 (Neuregulin 1), MARCKSL1 (MARCKS-related protein), SPP1 (secreted phosphoprotein 1) , SGCD (Delta-sarcoglycan), CAPN2 (Calpain-2 catalytic subunit), MMP14 (Matrix metalloproteinase-14), MMP19 (Matrix metalloproteinase-19), SH3PXD2B (SH3 and PX domain-containing protein 2B), TIMP1 (Metalloproteinase inhibitor 1 ), And an agent for measuring the expression level of a gene selected from the group consisting of ENG (Endoglin) Provide water.

The blood may be isolated from individuals suspected of breast cancer.

The blood-derived sample may be peripheral blood mononuclear cells (PBMC) isolated from blood. The PBMC may include T-cells, B-cells, natural killer (NK) cells, monocytes, macrophages, dendritic cells, or combinations thereof. The sample may include a transcript or protein.

The DMTN (dematin) may also be called DMT, EPB49, or dematin actin binding protein. The DMTN is Uniprot No. Amino acid sequence represented by Q08495 may be included. The DMTN is a Uniprot No. Amino acid sequence represented by J9P207 or F1PDQ2.

The myo-inositol oxygenase (MIOX) may also be referred to as inositol oxygenase, ALDRL6, KSP32, or RSOR. The MIOX may function as an enzyme that oxidizes myo-inositol to glucuronic acid. The MIOX is Uniprot No. Amino acid sequence represented by Q9UGB7 may be included. The MIOX is a Uniprot No. And an amino acid sequence represented by E2QTD8 or J9P8P0.

The alpha-hemoglobin-stabilizing protein (AHSP) may also be referred to as an Erythroid differentiation-related factor (EDRF) or an Erythroid-associated factor (ERAF). AHSP can function as a chaperone that prevents abnormal aggregation of alpha-hemoglobin during normal red blood cell development. The AHSP is Uniprot No. Amino acid sequence represented by Q9NZD4 may be included. The AHSP is Uniprot No. It may include an amino acid sequence represented by E2RSC2.

The HBM (Hemoglobin Subunit Mu) may also be called a hemoglobin mu chain or a mu-globin. The HBM is a Uniprot No. Amino acid sequence represented by Q6B0K9 may be included. The HBM is a Uniprot No. Amino acid sequence represented by J9NVU7 may be included.

The 5-aminolevulinate synthase (ALAS2) may also be referred to as ALAS-E, ALASE, ANH1, ASB, XLDPP, XLEPP, XLSA, or SIDBA1. ALAS2 can function as a protein that catalyzes the first step in the heme biosynthetic pathway. The ALAS2 is a Uniprot No. Amino acid sequence represented by P22557 may be included. The ALAS2 is a Uniprot No. Amino acid sequence represented by F1P796 may be included.

The ENSCAFG00000032136 is a protein whose function has not been confirmed. The ENSCAFG00000032136 is an Ensembl access number. The ENSCAFG00000032136 is Uniprot No. Amino acid sequence represented by J9NYD3 may be included.

The sorting nexin-5 (SNX5) is one of the sorting nexin families, and may contain a phox (PX) domain, which is a phosphoinositide binding domain. The SNX5 may be involved in intracellular mass transport. The SNX5 is Uniprot No. Amino acid sequence represented by Q9Y5X3 or Q5QPE4. The SNX5 is a Uniprot No. Amino acid sequence represented by E2RAE6 may be included.

The ENSCAFG00000037058 is a gene whose function has not been confirmed. The ENSCAFG00000037058 may include a long intergenic non-coding RNA (LincRNA) transcript represented by ENSCAFT00000051478.1 in dog cases.

The ENSCAFG00000024346 is a gene whose function has not been confirmed. The ENSCAFG00000024346 is Uniprot No. Amino acid sequence represented by F1P6J5 may be included.

The MATR3 (Matrin-3) may also be called ALS21, MPD2, or VCPDM. MATR3 can function as a protein located in the nuclear matrix. The MATR3 is Uniprot No. Amino acid sequence represented by P43243 may be included. The MATR3 is Uniprot No. Amino acid sequence represented by Q8K310 may be included.

The IL12B (Interleukin-12 subunit beta) may also be called CLMF, CLMF2, IL-12B, IMD28, NKSF, NKSF2, IMD29, or interleukin 12B. IL12B can function as a subunit of interleukin 23. The IL12B is Uniprot No. Amino acid sequence represented by P29460 may be included. The IL12B is Uniprot No. Amino acid sequence represented by F1PK70 may be included.

The CD36 (cluster of differentiation 36) may also be called BDPLT10, CHDS7, FAT, GP3B, GP4, GPIV, PASIV, or SCARB3. The CD36 may function to deliver fatty acids into cells. The CD36 is Uniprot No. Amino acid sequence represented by P16671 may be included. The CD36 is Uniprot No. Amino acid sequence represented by D5IGC7 may be included.

The EMD (Emerin) may also be referred to as EDMD, LEMD5, or STA. EMD is a serine-rich nuclear membrane protein and is a member of the nuclear lamina-associated protein family. The EMD is Uniprot No. Amino acid sequence represented by P50402 may be included. In the case of the dog, the EMD is Uniprot No. Amino acid sequence represented by J9P3Z7 may be included.

The Nectin2 (nectin cell adhesion molecule 2) may also be referred to as CD112, HVEB, PRR2, PVRR2, or PVRL2 (Poliovirus receptor-related 2). NECTIN2 is one of the components of the plasma membrane of the adherens junction. The NECTIN2 is Uniprot No. Amino acid sequence represented by Q92692 may be included. The NECTIN2 is Uniprot No. Amino acid sequence represented by F1PWL4 may be included.

The platelet glycoprotein IX (GP9) may also be called CD42a, GPIX, glycoprotein IX, or glycoprotein IX platelets. GP9 may be a small membrane glycoprotein present on the platelet surface. The GP9 is a Uniprot No. Amino acid sequence represented by P14770 may be included. The GP9 is a Uniprot No. Amino acid sequence represented by F1PV29 may be included.

The ITGB1 (Integrin beta-1) may also be called CD29, FNRB, GPIIA, MDF2, MSK12, VLA-BETA, VLAB, or integrin subunit beta 1. ITGB1 can bind integrin alpha 1 and 2 to form integrin complexes that function as collagen receptors. The ITGB1 is Uniprot No. Amino acid sequence represented by P05556. The ITGB1 is Uniprot No. Amino acid sequence represented by E2RT60 may be included.

The ITGA7 (Integrin alpha-7) may play an important role in regulating cell-matrix interaction. The ITGA7 is Uniprot No. Amino acid sequence represented by Q13683 may be included. The ITGA7 is Uniprot No. E2R064 or may include an amino acid sequence represented by F1PIU6.

The PVR (Poliovirus receptor (NK cell)) may also be called CD155, HVED, NECL5, Necl-5, PVS, or TAGE4. PVR is a type I transmembrane glycoprotein of the immunoglobulin superfamily. The PVR is a Uniprot No. It may include an amino acid sequence represented by P15151. The PVR is a Uniprot No. Amino acid sequence represented by F1PVS7 may be included.

The NRG1 (Neuregulin 1) may also be called ARIA, GGF, GGF2, HGL, HRG, HRG1, HRGA, MST131, MSTP131, NDF, NRG1-IT2, or SMDF. NRG1 can function in synaptic plasticity. The NRG1 is Uniprot No. Amino acid sequence represented by Q02297 or Q7RTW3. The NRG1 is Uniprot No. Amino acid sequence represented by F1Q0Y7 may be included.

The MARCKSL1 (MARCKS-related protein) may also be referred to as F52, MACMARCKS, MLP, MLP1, MRP, MARCKS-like 1, or MARCKS-like 1. MARCKSL1 can function in cytoskeletal regulation, protein kinase C signaling and calmodulin signaling. The MARCKSL1 is Uniprot No. It may include an amino acid sequence represented by P49006. The MARCKSL1 is Uniprot No. Amino acid sequence represented by F6Y4A3 may be included.

The secreted phosphoprotein 1 (SPP1) may also be called BNSP, BSPI, ETA-1, OPN, or Osteopontin. The SPP1 is Uniprot No. It may include an amino acid sequence represented by P10451. The SPP1 is Uniprot No. It may include the amino acid sequence represented by E2R161.

The Delta-sarcoglycan (SGCD) may also be called 35DAG, CMD1L, DAGD, SG-delta, SGCDP, SGD, sarcoglycan delta, or LGMDR6. SGCD is one of the components of the sarcoglycan complex. The SGCD is Uniprot No. Amino acid sequence represented by Q92629 may be included. The SGCD is Uniprot No. And an amino acid sequence represented by E2RJC4.

The CAPN2 (Calpain-2 catalytic subunit) can also be called CANP2, CANPL2, CANPml, mCANP, or calpain 2. CAPN2 is a non-lysosomal intracellular cysteine protease. The CAPN2 is Uniprot No. Amino acid sequence represented by P17655 may be included. The CAPN2 is Uniprot No. Amino acid sequence represented by F1P975 may be included.

The matrix metalloproteinase-14 (MMP14) may also be called MMP-14, MMP-X1, MT-MMP, MT-MMP 1, MT1-MMP, MT1MMP, MTMMP1, WNCHRS, or matrix metallopeptidase 14 Can be. MMP14 is one of the matrix metalloproteinases (MMPs) involved in the breakdown of extracellular matrix in diseases such as arthritis or cancer metastasis, as well as normal physiological processes such as embryonic development, reproduction, and tissue remodeling. The MMP14 is Uniprot No. Amino acid sequence represented by P50281 may be included. The MMP14 is Uniprot No. Amino acid sequence represented by F1PXM2 may be included.

The matrix metalloproteinase-19 (MMP19) may also be referred to as MMP18, RASI-1, CODA, or substrate metallopeptidase 19. MMP19 is one of the MMPs. The MMP19 is Uniprot No. Amino acid sequence represented by Q99542 may be included. The MMP19 is Uniprot No. It may include an amino acid sequence represented by E2QW82.

The SH3PXD2B (SH3 and PX domain-containing protein 2B) may also be referred to as FAD49, KIAA1295, or TKS4. SH3PXD2B is a Uniprot No. for humans. Amino acid sequence represented by A1X283. The SH3PXD2B is a Uniprot No. It may include an amino acid sequence represented by F1PDY3.

The TIMP1 (Metalloproteinase inhibitor 1) may also be called CLGI, EPA, EPO, HCI, TIMP, TIMP-1, or TIMP metallopeptidase inhibitor 1. The TIMP1 is Uniprot No. It may include an amino acid sequence represented by P01033 or Q6FGX5. The TIMP1 is Uniprot No. Amino acid sequence represented by P81546 may be included.

The ENG (Endoglin) may be also called Eng, AI528660, AI662476, CD105, Endo, S-endoglin, END, HHT1, ORW1, or endoglin. ENG is a type I membrane glycoprotein located on the cell surface and is a component of the TGF beta receptor complex. ENG can function in tumor proliferation, survival, and metastasis. The above ENG is Uniprot No. Amino acid sequence represented by P17813 or Q96CG0. The ENG is a Uniprot No. Amino acid sequence represented by F1P847 may be included.

The term “gene” is a unit of genetic information and refers to a nucleic acid sequence that encodes a substance having a function. The gene may include an open reading frame (ORF). The gene may include an ORF as well as a regulatory sequence comprising a promoter.

The expression level of the gene may be an expression level of a transcript transcribed or translated from the gene and a protein or fragment thereof translated therefrom. The transcript may include mRNA, non-coding RNA, or complementary DNA (cDNA) thereof. The fragment may be an immunogenic polypeptide as part of the protein.

The term "expression level" means the amount of protein or the amount of transcript. The expression level may be a relative proportion of protein or transcript. For example, an increase in the expression level may be an increase in the amount of protein or transcript compared to the negative control.

The expression level of the gene selected from the group consisting of IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and ENG is breast-specific. May have an immune response.

The agent may be an antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof expressed by the gene, or an aptamer. The antibody can be a polyclonal antibody or a monoclonal antibody. The term "antibody" can be used interchangeably with the term "immunoglobulin". The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a surface of a metal chip, plate, or well. The aptamer may be a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) or peptide that specifically binds to a protein or fragment thereof expressed by the gene.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to the nucleic acid sequence of the gene. The nucleic acid can be a primer, probe, or antisense oligonucleotide. The primer, probe, or antisense oligonucleotide may be labeled with a fluorescent substance, chemiluminescent or radioactive isotope on its terminal or inside.

The term "breast cancer" refers to cancer that occurs in the breast, and can also be interchangeably referred to as "mammary tumor." For example, it can be called breast cancer in humans and mammary cancer in dogs and cats. The breast cancer may include mammary gland breast cancer, lobule breast cancer, or a combination thereof. The breast cancer includes cancer that occurs in the epithelium of the ducts and leaves and cancer that occurs in the matrix.

The term "diagnosis" refers to determining the disease name and may include the name of the breast cancer, the condition of the disease, the stage, the etiology, the presence or absence of complications, the prognosis, and recurrence.

The composition may be for simultaneously or sequentially diagnosing a dog's mammary tumor and a human's breast cancer. For example, the composition can be used to diagnose canine cancer in dogs and then to diagnose breast cancer in humans.

Other aspects include DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSLG, SP1 , CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and provides a kit for diagnosing breast cancer, comprising an agent that measures the expression level of a gene selected from the group consisting of ENG.

The blood-derived samples, DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SPP1, , MMP14, MMP19, SH3PXD2B, TIMP1, ENG, gene, expression level, agent, breast cancer, and diagnosis are as described above.

The kit may further include a sample necessary for diagnosing breast cancer. The kit may include a solid support, an antibody, an antigen-binding fragment, or a substrate for immunological detection of an aptamer, a suitable buffer solution, a chromogenic enzyme, a secondary antibody labeled with a fluorescent material, or a chromogenic substrate. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Other aspects include DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR in blood-derived samples isolated from suspected breast cancer , NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, measuring the expression level of the gene selected from the group consisting of SH3PXD2B, TIMP1, and ENG; And

It provides a method for diagnosing breast cancer, comprising comparing the measured expression level with the expression level of a gene in a normal control.

The method is DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR , NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and measuring the expression level of the gene selected from the group consisting of ENG.

The individual can be a mammal, for example, a human, dog, cat, mouse, rabbit, horse, sheep, hamster, hedgehog, ferret, or guinea pig. The individual may be an individual suffering from or suspected of having breast cancer.

The blood-derived sample may be peripheral blood mononuclear cells (PBMC).

The measuring step may include incubating the blood-derived sample with a nucleic acid containing a polynucleotide identical to or complementary to the nucleic acid sequence of the gene.

The measuring may include incubating the biological sample with an antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof encoded from the gene, or an aptamer.

The measuring steps include electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, microarray, northern blotting, polymerase amplification reaction ( polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCT) polymerase amplification reaction, real-time PCR, or a combination thereof.

The method includes comparing the measured expression level with the expression level of a gene in a normal control.

The term "normal control" can be used interchangeably with the term "negative control". The normal control group may be an individual who has never had breast cancer or a healthy individual.

In the above method, when the measured expression level of a gene selected from the group consisting of the DMTN, MIOX, AHSP, HBM, and ALAS2 is decreased compared to the expression level measured in a normal control group, the subject has a probability of developing breast cancer or breast cancer. And determining to be high.

In the above method, when the measured expression level of a gene selected from the group consisting of the ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, and MATR3 is increased compared to the expression level measured in the normal control group, the subject is likely to develop breast cancer or breast cancer. And determining to be high.

The method has the expression level of a gene selected from the group consisting of IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and ENG. When increased compared to the expression level measured in a normal control, the subject may further comprise determining to exhibit a breast cancer-specific immune response.

It provides a method for providing information for diagnosing breast cancer, comprising comparing the measured expression level with the expression level of a gene in a normal control.

The method may further include predicting the likelihood of developing the breast cancer of the second individual through the diagnosis of the breast cancer of the individual. The individual (ie, the first individual) may be a companion animal and the second individual may be a human. The companion animal may refer to an animal living in the same living environment with a person. The companion animal may be a dog, cat, mouse, rabbit, hamster, hedgehog, ferret, or guinea pig. Because one of the major causes of breast cancer is environmental factors, it is possible to diagnose breast cancer in a companion animal living in the same environment as a person and to provide early diagnosis of breast cancer in a person who is a guardian of the companion animal or provide information necessary for breast cancer diagnosis. have. For example, a diagnosis of mammary cancer in a dog can be diagnosed, and through this, a person who is a guardian of the dog can predict the possibility of developing breast cancer.

According to a composition, kit for diagnosing breast cancer, and a method for diagnosing breast cancer using the same, or a method for providing information for diagnosing breast cancer, breast cancer may be diagnosed early. In addition, it is possible to diagnose mammary cancer in a companion animal and predict the likelihood of breast cancer in a person who is a guardian of the companion animal.

Figure 1a is a graph analyzing the transcripts of the dog's peripheral blood monocytes by volcano plot (x-axis: log value of fold change (FC), y-axis: -log value of p-value), Figure 1b is a diagram analyzed by a Venn diagram by dividing the gene increased or decreased in cancer significantly more than 4 times into mammary cancer, benign tumor, and other cancers.

2A and 2B are graphs showing the expression patterns of genes significantly increased and decreased in the mammary cancer group (B: benign tumor, C: mammary cancer, N: normal, O: other cancer, y axis: expression).

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

실시예 1. 유선암 특이적 바이오마커의 확인Example 1. Identification of mammary cancer specific biomarkers

1. 반려견 유선암 혈액 시료에서 유선암 특이적으로 차등발현된 바이오마커의 선별1. Screening of biomarkers specifically differentiated from mammary cancer from blood samples of dog mammary cancer

Dogs with mammary cancer (n = 40), dogs with benign tumor adenoma (n = 25), and other cancers without mammary cancer (including tongue, bladder, liver, fat, spleen, or lung mass) About 3 ml to about 4 ml of blood was obtained from a total of 100 dogs of the dog (n = 14) and the normal dog (n = 21) (provided by Seoul National University Animal Hospital and Haemaru Animal Hospital). The obtained blood was added to a tube containing EDTA.

Subsequently, Ficoll centrifugation was performed to obtain peripheral blood mononuclear cells (PBMC) from the blood. RNA was obtained from PBMC and transcripts were analyzed by next-generation sequencing.

For transcript analysis, a volcano plot was performed, and the results are shown in FIG. 1A. Genes increased or decreased in cancer significantly more than 4 times were selected through volcano plotting.

The selected genes were divided into mammary cancer, benign tumor, and other cancers, and analyzed by a ben diagram, and the results are shown in FIG. 1B. As shown in FIG. 1B, 84 genes specifically increasing and 59 genes decreasing were selected.

When heatmap clustering was performed on selected genes and all samples tested, it was possible to distinguish cancer from normal samples. However, as a result of heat map clustering, it was confirmed that some normal samples were included in the cancer sample group, and further analysis was performed.

2. 유전자 온톨로지 분석 및 경로 분석2. Gene ontology analysis and pathway analysis

Cancer specific immune cell responses were analyzed through gene ontology analysis and cell pathway analysis.

Functional analysis was performed using the Cyego program provided by cytoscape ver.3.6.1 for gene groups specifically increased in the blood of benign tumors, other cancers, or mammary cancers, respectively.

As a result of the functional analysis, the highest number of gene groups specifically increased for benign tumors was obtained with 328, but the functional networks shown in GO / path analysis were few and short. Particularly, a large group of genes involved in mast cell activation were gathered. On the other hand, the mammary cancer-specifically increased gene group showed that 84 genes were obtained, but formed a very clear network in GO / path analysis. It was confirmed that most of the selected genes are involved in the regulation of immune cells acting on cancer. In addition, it has been confirmed that cancer-specific immune cell transcripts are involved in interleukin-4 (IL) -4 and IL-13 signaling, ECM-receptor interaction, and cell junction organization. However, functional specificity was not confirmed in other cancer-specific gene groups.

The gene group specific for mammary cancer was extracted to select the mammary cancer-specific immune response gene as follows.

1) IL12B (Interleukin-12 subunit beta)

2) CD36 (cluster of differentiation 36)

3) EMD (Emerin)

4) NECTIN2 (Upon binding to CD226, stimulates T-cell proliferation and cytokine production)

5) GP9 (Platelet glycoprotein IX)

6) ITGB1 (Integrin beta-1)

7) ITGA7 (Integrin alpha-7)

8) PVR (Poliovirus receptor (NK cell))

9) NRG1 (Pro-neuregulin-1, membrane-bound isoform)

10) MARCKSL1 (MARCKS-related protein)

11) SPP1 (Osteopontin)

12) SGCD (Delta-sarcoglycan)

13) CAPN2 (Calpain-2 catalytic subunit)

14) MMP14 (Matrix metalloproteinase-14)

15) MMP19 (Matrix metalloproteinase-19)

16) SH3PXD2B (SH3 and PX domain-containing protein 2B)

17) TIMP1 (Metalloproteinase inhibitor 1)

18) ENG (Endoglin)

These 18 gene groups were selected as biomarkers for finding cancer-specific immune action in blood cells.

3. 유선암 특이적 바이오마커의 선별3. Selection of specific biomarkers for mammary cancer

When comparing normal and mammary cancer, genes with high expression in normal but low expression in mammary cancer were selected as follows.

DMTN, MIOX, AHSP, HBM, and ALAS2

Conversely, genes that had low expression in normal but high expression in mammary cancer were selected as follows.

ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, and MATR3

Expression patterns were confirmed in the 100 mammary cancer, benign tumor, and other cancer samples described in Example 1.1, and the results are shown in FIGS. 2A and 2B (B: benign tumor, C: mammary cancer, N: normal, O: other cancer) , y-axis: amount of expression (expressed by the voom function of log2 for the number of RNA fragments identified in one million reads).

These were selected as biomarkers that can distinguish mammary cancer from normal using blood. In particular, MATR3 showed a significant difference from benign tumors, and thus was confirmed to be a biomarker for selecting normal, mammary cancer, and benign tumors.

Claims

In blood-derived samples, DMTN (dematin), MIOX (myo-inositol oxygenase), AHSP (Alpha-hemoglobin-stabilizing protein), HBM (Hemoglobin Subunit Mu), ALAS2 (5-aminolevulinate synthase), ENSCAFG00000032136, SNX5 (Sorting nexin-5) ), ENSCAFG00000037058, ENSCAFG00000024346, MATR3 (Matrin-3), IL12B (Interleukin-12 subunit beta), CD36 (cluster of differentiation 36), EMD (Emerin), NECTIN2 (nectin cell adhesion molecule 2), GP9 (Platelet glycoprotein IX) , ITGB1 (Integrin beta-1), ITGA7 (Integrin alpha-7), PVR (Poliovirus receptor (NK cell)), NRG1 (Neuregulin 1), MARCKSL1 (MARCKS-related protein), SPP1 (secreted phosphoprotein 1), SGCD ( Delta-sarcoglycan (CAPN2), Calpain-2 catalytic subunit (CAPN2), Matrix metalloproteinase-14 (MMP14), Matrix metalloproteinase-19 (MMP19), SH3 and PX domain-containing protein 2B (SH3PXD2B), Metalloproteinase inhibitor 1 (TIMP1), and A composition for diagnosing breast cancer, comprising an agent that measures the expression level of a gene selected from the group consisting of ENG (Endoglin).
The composition according to claim 1, wherein the blood-derived sample is peripheral blood mononuclear cell (PBMC).
The method according to claim 1, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and the expression of a gene selected from the group consisting of ENG The level is a composition that exhibits a breast cancer-specific immune response.
The method according to claim 1, wherein the formulation

Antibodies or antigen-binding fragments thereof, or aptamers that specifically bind to the protein expressed by the gene or fragments thereof; or

A composition comprising a polynucleotide identical to or complementary to the nucleic acid sequence of the gene.
The composition according to claim 4, wherein the antibody is a polyclonal antibody or a monoclonal antibody.
The composition of claim 4, wherein the nucleic acid is a primer, probe, or antisense oligonucleotide.
The composition according to claim 1, wherein the composition is for simultaneously or sequentially diagnosing dog mammary tumor and human breast cancer.
In blood-derived samples, DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SPP1, SPG1, A kit for diagnosing breast cancer, comprising an agent that measures the expression level of a gene selected from the group consisting of MMP14, MMP19, SH3PXD2B, TIMP1, and ENG.
DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, from blood-derived samples isolated from suspected breast cancer Measuring the expression level of a gene selected from the group consisting of MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and ENG; And

Comprising the step of comparing the measured expression level with the expression level of the gene of the normal control, diagnostic method of breast cancer.
The method of claim 9, wherein the individual is a human, dog, cat, mouse, rabbit, horse, sheep, hamster, hedgehog, ferret, or guinea pig.
The method according to claim 9, wherein the blood-derived sample is peripheral blood mononuclear cells (PBMC).
The method of claim 9, wherein the measuring step comprises incubating the blood-derived sample with a nucleic acid comprising a polynucleotide identical to or complementary to the nucleic acid sequence of the gene.
The method according to claim 9, wherein the measuring step is electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay: ELISA), immunohistochemical staining, protein chip, immunoprecipitation, microarray, Northern blotting, A method that is performed by polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCT) polymerase amplification reaction, real-time PCR, or a combination thereof.
The method according to claim 9, the measured expression level of the gene selected from the group consisting of the DMTN, MIOX, AHSP, HBM, and ALAS2 is reduced compared to the expression level measured in the normal control group; or

When the measured expression level of the gene selected from the group consisting of the ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, and MATR3 increased compared to the expression level measured in the normal control group,

The method further comprises determining that the individual has breast cancer or has a high probability of having breast cancer.
The method according to claim 9, The IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and the expression of a gene selected from the group consisting of ENG If the level is increased compared to the expression level measured in the normal control group,

The method further comprising determining that the subject has a breast cancer-specific immune response.
DMTN, MIOX, AHSP, HBM, ALAS2, ENSCAFG00000032136, SNX5, ENSCAFG00000037058, ENSCAFG00000024346, MATR3, IL12B, CD36, EMD, NECTIN2, GP9, ITGB1, ITGA7, PVR, NRG1, from blood-derived samples isolated from suspected breast cancer Measuring the expression level of a gene selected from the group consisting of MARCKSL1, SPP1, SGCD, CAPN2, MMP14, MMP19, SH3PXD2B, TIMP1, and ENG; And

A method of providing information for diagnosing breast cancer, the method comprising comparing the measured expression level with the expression level of a gene in a normal control.
The method of claim 16, further comprising predicting the likelihood of developing a breast cancer in a second subject through diagnosis of the breast cancer in the subject.
The method of claim 16, wherein the subject is a companion animal and the second subject is a human.