WO2020013355A1 - Composition for predicting prognosis of malignant phyllodes tumor and kit comprising same - Google Patents

Composition for predicting prognosis of malignant phyllodes tumor and kit comprising same Download PDF

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WO2020013355A1
WO2020013355A1 PCT/KR2018/007798 KR2018007798W WO2020013355A1 WO 2020013355 A1 WO2020013355 A1 WO 2020013355A1 KR 2018007798 W KR2018007798 W KR 2018007798W WO 2020013355 A1 WO2020013355 A1 WO 2020013355A1
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malignant
prognosis
subtype
composition
gene
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French (fr)
Korean (ko)
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문형곤
윤지희
김종일
노동영
유태경
이은신
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서울대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a composition for predicting malignant frond prognosis and a kit comprising the same.
  • the present invention relates to a composition and a kit including the same for predicting the prognosis of malignant frondoma by measuring expression levels of specific marker genes.
  • breast cancers in female breasts start in cells that fill the ducts that serve to carry milk from milk producing glands or lobules. Such breast cancer is also called vascular cancer. Other breast cancers start in cells that fill the lobules and are called lobules.
  • a minority of breast cancers include stroma, which is the connective tissue, lymph vessels and blood vessels surrounding fat, ducts and lobules, and begins in other tissues, including the breast.
  • stroma is the connective tissue, lymph vessels and blood vessels surrounding fat, ducts and lobules, and begins in other tissues, including the breast.
  • Almost all breast cancers are cancers that start in epithelial cells that fill the tubes or lobules. Such cancers are known as ductal carcinoma and lobular carcinoma, respectively.
  • Breast cancer can also begin with sarcomas that form in connective tissue of the breast, including muscle, fat or blood vessels.
  • Fibroepithelial tumors of the breast are neoplasms, including fibroadenomas and foliomas, which have bilateral features that proliferate both epithelial and stromal cells of the mammary gland.
  • Malignant phyllodes tumor (MPT) accounts for 1% of all tumors in the breast.Front filaments are benign, malignant or borderline depending on the extent of mitosis and proliferation of the substrate. Tumors can be divided into 70-80% of benign tumors, about one third of 20-25% of malignant tumors and borderline tumors are rare. Fronds are rare cancers, but as many as 67% of patients with malignant fronds have local recurrence and 21% have distant metastasis, leading to death.
  • Korean Patent Registration KR 10-1058230 B1 discloses a marker composition for diagnosing metastatic breast cancer, which is a stage IV cancer using thioredoxin 1 as an active ingredient. Biomarkers for predicting prognosis are unknown.
  • biomarkers that can predict the prognosis of patients with malignant foliar tumors can be widely applied in related fields.
  • an aspect of the present invention is to provide a composition for predicting the prognosis of malignant frondoma that can predict the prognosis of malignant fronoma.
  • Another aspect of the present invention is to provide a kit for predicting malignant frond prognosis that can predict the prognosis of a patient with fronoma.
  • Another aspect of the invention relates to a method of providing information necessary for predicting the prognosis of a patient with frond.
  • composition for predicting malignant foliar prognosis comprising a gene of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as a biomarker.
  • kits for predicting malignant stromal prognosis comprising the composition.
  • methods are provided to provide the information necessary to predict the prognosis of malignant fronds.
  • an effective targeted treatment strategy for malignant stromal tumors with aggressive clinical features and no effective treatment has been proposed by classifying malignant stromal into two subtypes based on genomic expression patterns of malignant stromal tumors. Can be established.
  • FIG. 1 shows genetic modifications in malignant frondoma of nine samples.
  • 1A shows the rate of malignant phyllodes tumors measured in number of somatic mutations per Mb of covered target sequence in malignant phyllodes tumors (MPTs) compared to the Mann Whitney test.
  • FIG. 1B shows the somatic mutation characteristics of MPT indicated by pie plots indicating the effect of each mutation feature identified using RPT 'deconstrctSigs'
  • FIG. 1C compares APOBEC related mutation features between MPT and invasive catheter cancer. (Mann Whitney test)
  • Figure 1D shows the somatic mutation and copy number changes of cancer-related genes in MPT, DNAH11 gene was included because it is known that the mutation is repeated in fibroepithelial tumors. *: p ⁇ 0.05, ***: p ⁇ 0.001.
  • FIG. 2A is a heat map of sample-to-sample distances showing unique gene expression profiles of malignant lobe tumors (MPT) compared to typical breast and invasive catheter carcinoma tissues (yellow: common breast cancer, red: MPT, green: invasive catheter) heat map), which was derived from gene read counts normalized using the Deseq2 R package.
  • 2B shows a significantly unregulated pathway in MPT compared to normal breast tissue (top) and invasive catheter carcinoma tissue (bottom).
  • FIG. 3 shows the molecular subtypes of malignant fronds.
  • FIG. 3A shows the unsupervised clustering results of nine malignant lobe species (MPT) using genome-wide transcript data
  • FIG. 3B shows epithelial subtypes (left) and fibrous subtypes (right).
  • 3C and 3D show the expression levels of various collagen, claudin and CDH1 depending on the molecular subtype (Mann Whitney test)
  • FIG. 3E is related to specific pathways.
  • FIG. 3F shows unsupervised clustering of additional FFPE MPT samples using 21 gene markers
  • FIG. 3G shows mitosis index according to subtypes. The Chi-square test is shown
  • FIG. 3H shows the Kaplan-Meier survival curve (log rank test) showing the survival rate without recurrence of both types. . *: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.001.
  • FIG. 4 shows the PI3K / mTOR and PDGFRB targeting approaches in a patient-derived frond model.
  • 4A shows histological characteristics of primary tumor (2014-0916) and patient-derived xenograft tumor (MX-99)
  • FIG. 4B is a correlation plot showing mRNA expression levels of primary and xenograft tumors (two samples). All are FPKM ⁇ 1, log2 (FPKM))
  • FIG. 4C shows the distribution of somatic mutations in PDGFRB and PIK3R1
  • FIG. 4D shows PDGFRB mRNA expression levels in common breast tissue, invasive breast cancer tissue, and MPT, FIG.
  • FIG. 4F shows tumor growth in MX-99 xenograft model treated by vehicle, imetinib and adjusted p value derived from multiple t-test (FIG. 4F)
  • FIG. 4F shows vehicle, imetinib and PKI- Western blot analysis of downstream signal transduction pathway molecules in xenograft tumors when treated with 587 is shown.
  • FIG. 4G shows the quantitative analysis of Western blot results (Mann Whitney test). *: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.001.
  • the biomarker for predicting the prognosis of malignant frond tumors is identified by identifying the genomic and molecular biological characteristics of malignant frond tumor patients tissue through whole exome sequencing and transcriptome sequencing. Furthermore, a technique for classifying subtypes of fronds showing characteristic clinical features using such biomarkers using genome expression patterns is provided.
  • composition for predicting malignant foliar prognosis of the present invention comprises the genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as biomarkers.
  • the characteristics of genes having the nucleotide sequences of SEQ ID NOs: 1 to 12 are summarized as follows.
  • the gene name refers to the HUGO Gene Nomenclature Committee, the position refers to the Refseq, and SEQ ID NO. Indicates the NCBI Reference Sequence.
  • the numbers in parentheses below correspond to the sequence numbers of the present invention.
  • the genes correspond to the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 12, respectively.
  • the nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 8 and 10 to 12 is an mRNA sequence excluding the intron portion of the gene, and among them, shows only a coding sequence translated into a protein will be.
  • the CDH1 and ERBB3 genes of SEQ ID NO: 1 have a transcript variant that can be a variety of protein isoforms, and thus, the coding sequence of the entire gene sequence, not the mRNA sequence. Appeared.
  • prognosis refers to the progression and cure of malignant fronds, such as recurrence, metastatic spread, and the likelihood of malignant frond-mediated death or progression, including drug resistance.
  • prognosis refers to the possibility of bovine recurrence after the treatment of malignant frondoma, and preferably to predict whether to relapse within two years after surgery or chemotherapy of malignant frond.
  • the term "good prognosis” refers to the possibility that the disease of a patient with malignant foliar disease will be cured, and "unfavorable prognosis” refers to the relapse or recurrence, metastasis or death of the diseased malignant frond. It means that there is a possibility. Patients with malignant frondoma classified as having good results have no or decreasing disease in malignant fronds. Conversely, patients with malignant stromal tumors with poor outcomes lead to disease regeneration, tumor recurrence, metastasis or death. "Good prognosis” means that a patient with malignant foliar tumor may be left without disease for at least 2 years, more specifically at least 5 years. In another aspect of the present invention, "unfavorable prognosis” means that a patient with malignant stromal tumor may experience disease regeneration, tumor recurrence, metastasis, or death within less than 5 years, more specifically less than 2 years.
  • the term "prediction" means that the patient responds favorably or unfavorably to a therapy, such as chemotherapy or radiation therapy, such that the patient is removed by treatment, e.g., surgical treatment of a particular therapeutic agent, and / or primary tumor, And / or survival and / or likelihood after treatment with chemotherapy for a certain period of time without recurrence of cancer.
  • the predictive method of the present invention can be used clinically by selecting and applying the most appropriate treatment regimen for any particular malignant stromal patient.
  • Prediction methods of the present invention determine whether a patient responds favorably to a treatment such as a prescribed treatment regimen, including, for example, administration of a predetermined treatment or combination, surgical intervention, chemotherapy, or the like, or after a treatment regimen. Long term survival or systemic or local recurrence is predictable. It can also be planned to minimize unnecessary adjuvant chemotherapy or to use adjuvant chemotherapy for patients who are expected to have systemic or local recurrence.
  • prognostic composition for prognosis is to distinguish between patients with good prognosis and patients with poor prognosis after treatment for malignant edema, and predict the possibility of recurrence, CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, Genes of PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 are included as markers for prognostic prediction.
  • prognostic marker means prognosis including predicting recurrence after the treatment of malignant edema.
  • RNA expression level measurement is a process of confirming the presence and expression of RNA of a marker gene in a biological sample in order to predict the prognosis of malignant frondoma, and can be known by measuring the amount of RNA.
  • RT-PCR competitive RT-PCR
  • RNase protection assay RPA
  • northern blotting noden
  • FPKM fragments per kilobase of exon per million
  • composition for predicting malignant foliar prognosis of the present invention may further include an agent for measuring the expression level of RNA of the gene or a protein encoded by these genes, the agent for measuring the expression level of the RNA is It may include an antisense oligonucleotide, primer pair or probe that specifically binds to the gene, the agent for measuring the expression level of the protein may be an antibody specific for the protein encoded from the gene.
  • the agent for measuring the RNA level of the prognostic marker gene of malignant foliar tumor according to the present invention is, for example, an antisense oligonucleotide, a primer pair or a probe, and specifically targets specific regions of these genes based on the nucleotide sequence of the marker gene.
  • Primers or probes for amplification can be designed. Since the nucleotide sequences of the prognostic marker genes of malignant foliar tumors according to the present invention are known in the art, such as those registered in the GenBank, those skilled in the art will specifically amplify specific regions of these genes based on the nucleotide sequences. Primers or probes can be designed.
  • antisense refers to a backbone between nucleotide sequences and subunits in which antisense oligomers can hybridize with target sequences in RNA by Watson-Crick base pairing to form heterodimers with mRNA typically within the target sequence.
  • oligomer having The oligomer may have precise sequence complementarity or similar complementarity to the target sequence.
  • primer refers to template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperatures. It refers to a single stranded oligonucleotide that can act as a starting point. Appropriate length of the primer may vary depending on the purpose of use, but is typically 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.
  • the possibility of recurrence and the two-year survival prognosis can be predicted through the amplification of the PCR product. have.
  • probe refers to a nucleic acid fragment such as RNA or DNA corresponding to short bases of several hundred bases and hundreds of bases capable of specific binding with RNA.
  • Probes can be constructed in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like.
  • the prognosis including the recurrence of malignant frond tumor can be predicted through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
  • Antisense oligonucleotides, primers or probes according to the present invention may be chemically synthesized using methods well known in the art, including phosphoramidite solid support methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methylphosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
  • the term "measurement of expression level of protein” refers to a process of confirming the presence and expression level of a protein encoded from a marker gene in a biological sample in order to prognostically predict the possibility of recurrence of malignant stromal tumor.
  • the amount of the protein is determined using an antibody that binds specifically.
  • Electrophoresis immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.
  • an antibody is a term known in the art and means a specific protein molecule directed to an antigenic site.
  • an antibody refers to an antibody that specifically binds to a protein encoded from a marker gene of the present invention, which antibody is cloned into an expression vector according to a conventional method by the marker gene. After obtaining the protein to be encoded, it can be prepared by conventional methods from the obtained protein. It also includes peptide fragments that can be made from such proteins, and peptide fragments of the present invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids.
  • the antibody of the present invention is not particularly limited in form, and a part thereof is included in the antibody of the present invention as long as it is a polyclonal antibody, a monoclonal antibody or an antigen-binding agent, and all immunoglobulin antibodies are included.
  • polyclonal antibodies can be produced by methods well known in the art for injecting protein antigens encoded from the prognostic predictive marker genes of malignant frondoma described above into animals and collecting blood from the animals to obtain serum comprising the antibody.
  • Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.
  • Monoclonal antibodies are well known in the art by the hybridoma method (Kohler and Milstein, European Jounral of Immunology, 6: 511-519, 1976), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222 (58): 1-597, 1991).
  • Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
  • Antibodies of the present invention also include recombinant antibodies such as humanized antibodies.
  • Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule means a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 , Fv, and the like.
  • the present inventors have identified the genome and molecular biological characteristics of the tissues of patients with malignant fronds through whole exome sequencing and transcriptome sequencing, and showed subtypes of fronds with characteristic clinical features. (subtype) can be classified using genome expression patterns.
  • the inventors have divided the malignant fronds into two subtypes by using gene expression data characteristics of malignant fronds with unsupervised hierarchical clustering.
  • composition is divided into fibrous subtype first subtype when there are 6 or more genes of FPKM 1 or less in the gene, and malignant leaflets by dividing into epithelial subtype 2 subtype when less than 6 genes. It may be for predicting the prognosis of the species.
  • the first subtype is protein digestion and absorption, axon guidance, ECM-receptor interaction, focal adhesion, and stromal-related collagen such as various collagens. It was characterized by abnormal expression of stroma-related genes (FIGS. 3B and 3C), and the second subtype was cell metabolism, cell adhesion molecules, tight junctions, and multiple immunity. It was confirmed that the expression of genes involved in related processes is high, and the expression of genes reflecting the characteristics of epithelial cells such as CDH1, CLDN3, CLDN4, CLDN7, OCLN is significantly high (FIG. 3D).
  • malignant fronoma can be classified into two subtypes, so that the first subtype is 'Fibrous' subtype and the second subtype is 'Epithelial' (epithelial). Subtype). This classification may itself reflect the bilateral biological properties of fibroepithelial tumors.
  • ECM-receptor interaction pathways KEGG ID: hsa04512
  • cell adhesion molecular pathways KEGG ID: hsa045114
  • epithelial mesenchymal transition EMT
  • the two malignant frond subtypes classified as above showed distinct differences between the two subtypes in various clinical and pathologic characteristics.
  • the first fibrous subtype has a significantly higher mitotic index of known major prognostic factors than the second epithelial subtype, and the fibrous subtype when the patient's recurrence is tracked. Significantly more relapses occurred at. That is, the first subtype according to the present invention represents an "unfavorable prognosis", and the second subtype represents a "good prognosis”.
  • malignant filamentous tumors can be divided into two subtypes with distinct differences in clinical characteristics and therapeutic results, rather than a single disease, as is known in the past.
  • the prognosis can be predicted according to the subtype.
  • composition for predicting malignant frond prognosis of the present invention may be, for example, to predict the possibility of recurrence of malignant frondoma after two years.
  • kits for predicting malignant stromal prognosis comprising the composition for predicting malignant stromal prognosis.
  • the kit of the present invention detects markers by detecting RNA expression levels or protein expression levels of marker genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2 to detect markers. Prognosis can be predicted, including the possibility of relapse.
  • the marker detection kit of the present invention primers, probes, or the marker gene as mentioned in the composition for predicting the prognosis of malignant foliar prognosis in order to measure the expression level of the marker gene for predicting the prognosis of recurrence of malignant foliar tumor.
  • primers, probes, or the marker gene as mentioned in the composition for predicting the prognosis of malignant foliar prognosis in order to measure the expression level of the marker gene for predicting the prognosis of recurrence of malignant foliar tumor.
  • antibodies that selectively recognize proteins encoded therefrom one or more other component compositions, solutions or devices suitable for analysis may be included.
  • the kit for measuring the RNA expression level of the marker genes in the present invention may be a kit containing the essential elements required to perform RT-PCR.
  • the RT-PCR kit includes test tubes or other appropriate containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water and the like.
  • the kit of the present invention may be in the form of a microarray for prognostic predicting the possibility of recurrence of malignant frondoma comprising the marker genes according to the present invention.
  • the microarray may comprise a DNA or RNA polynucleotide probe.
  • the microarray may include a conventional microarray configuration except that the microarray includes a probe specific for the nucleotide sequence of the prognostic marker gene of the malignant frond tumor according to the present invention.
  • the microarray of the present invention can provide useful information for predicting the prognosis of recurrence of malignant frondoma by detecting the expression of the prognostic marker gene of malignant frondoma according to the present invention.
  • DNA microarrays include, but are not limited to, the method according to the present invention by a method using a micropipetting or pin type spotter using a piezoelectric method.
  • Probes for marker genes can be immobilized on a substrate.
  • the substrate of the microarray of the present invention is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but not limited thereto. It doesn't happen.
  • the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
  • hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art.
  • the detection involves labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, such as a substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating a signal from the labeling substance.
  • the hybridization result can be detected by detecting.
  • the kit for measuring the expression level of the protein encoded from the marker genes in the present invention is a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or fluorescent substance, chromogenic substrate, etc. for immunological detection of the antibody It may include.
  • a substrate a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, etc. may be used, and a peroxidase (peroxidase) may be used. ), Alkaline phosphatase and the like can be used.
  • ABTS 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)
  • OPD o-phenyl
  • Rendiamine TMB (tetramethyl benzidine) and the like can be used.
  • the present invention provides a method for providing information necessary for predicting the prognosis of a malignant frondoma, and the method for providing information necessary for predicting the prognosis of a malignant frondoma is provided by a sample of an isolated individual. Measuring the RNA expression levels of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2 from the FPKM values; And 2) classifying a fibrous subtype first subtype when there are 6 or more genes having a FPKM value of 1 or less, and dividing an epithelial subtype second subtype by less than 6 genes. will be.
  • the method for providing the information necessary for predicting the prognosis of the malignant frond tumor further comprises the step of determining a sample of an individual classified into the fibrous subtype first subtype as having a high probability of recurrence of the malignant frond tumor. can do.
  • the first fibrous subtype has a significantly higher mitotic index of known major prognostic factors than the second epithelial subtype, and the fibrous subtype when the patient's recurrence is tracked. Recurrences occur significantly in.
  • the present inventors performed whole-length exome sequencing using fresh frozen tumor tissues excised from nine malignant frond tumor patients secured in a biological sample repository. Compared with genotyping data generated in 22 common breast cancer tissues derived from mammary cells, malignant filamentous tissues showed significantly less genotypic mutations (0.56 / Mb for MPT and 2.42 / Mb for invasive ductal carcinoma, FIG. 1A). alc 1B). Alexandrov et al (Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al.
  • FIG. 1D The frequencies of genome structural and genetic somatic variations observed in the nine cases of malignant foliar tissues are summarized in FIG. 1D.
  • Somatic changes and various genome structural changes of the PIK3CA, PIK3R1, PDGFRA, PDGFRB, PTEN, and TP53 genes were also observed.
  • transcriptome sequencing was performed on 64 breast cancer tissues and 59 normal breast tissues obtained from 64 general breast cancer patients. Compared with the normal breast cancer tissues, it was confirmed that malignant fronds show a unique gene expression pattern (FIG. 2A). Genes involved in ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling were observed to have very frequent expression changes in malignant fronds (Fig. 2B). Cytoscape software was observed. The analysis of the networks used also showed that ECM-receptor interactions, PI3K-Akt signaling pathways, protein degradation and absorption, and pathways in cancer network abnormalities were significantly common.
  • the inventors were able to distinguish between two types of subtypes by surprisingly classifying them using unexpressed hierarchical clustering techniques using gene expression data characteristics for malignant frond tissues (FIG. 3A).
  • the first subtype is protein digestion and absorption, axon guidance, ECM-receptor interaction, focal adhesion, and stromal-related (eg, various collagen). It was characterized by abnormal expression of stroma-related genes (FIGS. 3B and 3C), and the second subtype was cell metabolism, cell adhesion molecules, tight junctions, and many immune related It was confirmed that the expression of genes involved in the process was high, and the expression of genes reflecting the characteristics of epithelial cells such as CDH1, CLDN3, CLDN4, CLDN7, OCLN was significantly high (FIG. 3D).
  • malignant fronoma can be classified into two subtypes, so that the first subtype is 'Fibrous' subtype and the second subtype is 'Epithelial' (epithelial). Subtype). This classification may itself reflect the bilateral biological properties of fibroepithelial tumors.
  • the present inventors further found that the ECM-receptor interaction pathway (KEGG ID: hsa04512), the cell adhesion molecule pathway (KEGG ID: hsa04514), and the EMT () may affect the biological properties of fibroepithelial tumors.
  • the overall expression pattern of genes belonging to the pathway of epithelial mesenchymal transition) was analyzed and the results showed that the same subtypes could be classified even in unsupervised clustering using the entire gene group (FIG. 3E).
  • the present inventors further performed transcriptome sequencing using paraffin embedded tissue of 28 cases of malignant fronoma.
  • Subtype classification of malignant mesenchymal patients was performed using gene expression signature consisting of dog genes (FIG. 3F).
  • the two malignant frond subtypes classified based on gene expression patterns showed distinct differences between the two subtypes in various clinical and pathologic characteristics.
  • the first fibrous subtype has a significantly higher mitotic index of known major prognostic factors compared to the second epithelial subtype, and the fibrous subtype is associated with patient recurrence. Significant relapses occurred in subtypes.
  • the present inventors derived the fact that malignant filamentous tumors can be divided into two subtypes with distinct differences in clinical characteristics and therapeutic results, as opposed to a single disease.
  • Table 1 shows the results of confirming the FPKM values of the respective genes in the fresh frozen tumor tissues resected from nine malignant frond tumor patients secured in the biological sample storage as described in 1.
  • Tables 2 and 2-1 below show the results of confirming the FPKM values of the respective genes in the paraffin-embedded tissues of the 28 cases of malignant fronoma.
  • the classification of these subtypes is FPKM 1 of the genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2, as can be seen in Tables 1, 2 and 2-1.
  • Tables 1, 2 and 2-1 When the following genes were 6 or more, it was confirmed that they could be classified into fibrous subtype first subtypes and less than 6 genes could be classified into epithelial subtype second subtypes.
  • a patient-derived xenograft model could be established by transplanting tissue from one patient (2014-0916) out of nine malignant mesenchymal tissues used in this study into NSG mice.
  • PIK3R1 is a regulatory subunit that regulates the function of PIK3CA and is known to play a role in inhibiting tumorigenesis in epithelial cells. Somatic variation of this gene is associated with activation of PI3K / Akt signaling in tumor tissues. have.
  • PDGFRB N666K somatic variation induces tumorigenesis in cells, and previous studies have shown that the somatic mutation maintains responsiveness to imatinib mesylate.
  • PDGFRB was also confirmed in the gene expression data, it was observed that the expression is relatively high in malignant mesenchymal tumors, especially in 2014-0916 tumors observed the highest expression phenomenon (Fig. 4D).

Abstract

The present invention relates to a composition for predicting the prognosis of a malignant phyllodes tumor and a kit comprising same. More specifically, provided in the present invention are: a composition for predicting the prognosis of a malignant phyllodes tumor, containing the genes CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as biomarkers; a kit for predicting the prognosis of a malignant phyllodes tumor, comprising the composition; and a method for providing information required for the prognosis prediction, of a malignant phyllodes tumor, using the composition.

Description

악성 엽상종 예후 예측용 조성물 및 이를 포함하는 키트Composition for predicting malignant stromal prognosis and kit comprising same
본 발명은 악성 엽상종 예후 예측용 조성물 및 이를 포함하는 키트에 관한 것으로 특이적인 마커 유전자의 발현 수준을 측정함으로써 악성 엽상종의 예후를 예측할 수 있도록 하는 조성물 및 이를 포함하는 키트에 관한 것이다.The present invention relates to a composition for predicting malignant frond prognosis and a kit comprising the same. The present invention relates to a composition and a kit including the same for predicting the prognosis of malignant frondoma by measuring expression levels of specific marker genes.
여성 유방의 대부분의 암은 우유를 생산하는 샘 또는 소엽으로부터 우유를 운반하는 역할을 하는 관(duct)을 가득 채우는 세포에서 시작된다. 그러한 유방암은 관암으로도 불리운다. 다른 유방암은 소엽을 가득 채우는 세포에서 시작되고 소엽암이라고 불리운다. 유방암의 소수는 지방, 관 및 소엽을 둘러싸는 연결 조직, 림프 혈관 및 혈관인 스트로마를 포함하고 유방을 포함하는 다른 조직에서 시작된다. 거의 모든 유방암은 관 또는 소엽을 가득 채우는 상피세포에서 시작되는 암이다. 그러한 암은 각각 관 암종 및 소엽 암종로 알려져 있다. 유방암은 또한 근육, 지방 또는 혈관을 포함하는 유방의 연결 조직에서 형성되는 육종으로 시작할 수 있다.Most cancers in female breasts start in cells that fill the ducts that serve to carry milk from milk producing glands or lobules. Such breast cancer is also called vascular cancer. Other breast cancers start in cells that fill the lobules and are called lobules. A minority of breast cancers include stroma, which is the connective tissue, lymph vessels and blood vessels surrounding fat, ducts and lobules, and begins in other tissues, including the breast. Almost all breast cancers are cancers that start in epithelial cells that fill the tubes or lobules. Such cancers are known as ductal carcinoma and lobular carcinoma, respectively. Breast cancer can also begin with sarcomas that form in connective tissue of the breast, including muscle, fat or blood vessels.
유방의 섬유상피종양은 섬유선종과 엽상종을 포함하는 신생물로 엽상종은 유선 조직의 상피세포와 기질세포가 모두 증식하는 양면적인 특성을 가지고 있다. 엽상종(Malignant phyllodes tumor, MPT)은 유방에 생기는 모든 종양의 1%를 차지하고 있으며, 나아가 엽상종은 조직 검사 결과 유사분열(mitosis)의 정도와 기질의 증식 정도에 따라 양성종양, 악성 또는 경계성 종양으로 나뉠 수 있으며 70-80% 정도가 양성종양, 약 1/3인 20~25% 정도가 악성종양으로 보고되고 경계성 종양은 드문 것으로 알려져 있다. 엽상종은 드문 암이지만 많게는 67%에 달하는 악성 엽상종 환자에서 국소 재발이 발생하고 21%에서는 원격전이가 발생하여 사망에 이르게 된다.Fibroepithelial tumors of the breast are neoplasms, including fibroadenomas and foliomas, which have bilateral features that proliferate both epithelial and stromal cells of the mammary gland. Malignant phyllodes tumor (MPT) accounts for 1% of all tumors in the breast.Front filaments are benign, malignant or borderline depending on the extent of mitosis and proliferation of the substrate. Tumors can be divided into 70-80% of benign tumors, about one third of 20-25% of malignant tumors and borderline tumors are rare. Fronds are rare cancers, but as many as 67% of patients with malignant fronds have local recurrence and 21% have distant metastasis, leading to death.
현재 받아들여지는 악성 엽상종의 표준 치료는 충분한 절제연을 확보하면서 외과적으로 절제하는 것이다. 수술 이후 추가적으로 방사선 치료를 시행하는 것은 생존율 향상이 입증되지 않았으며, 추가적인 항암치료나 표적치료제의 효과는 임상적으로 확인되지 않았다. 이와 같이 악성 엽상종은 그 빈도가 매우 드물고 생물학적으로도 연구가 충분하지 않아 높은 재발율에도 불구하고 효과적인 치료 전략이 수립되어 있지 않다. 최근 보고되기 시작한 엽상종의 유전체 연구를 통해 비로소 엽상종이라는 질환의 유전체적 특성에 대해 이해가 시작되었으며, 반복적인 크로모좀의 변화, 유전자 발현의 특성, 체성 변이의 패턴 등이 알려지고 있다. Currently accepted standard treatment for malignant fronds is surgical resection with sufficient margins. Additional postoperative radiotherapy has not been proven to improve survival, and the efficacy of additional chemotherapy or targeted therapies has not been clinically confirmed. Thus, malignant fronds are very rare, and biologically insufficient studies have not established effective treatment strategies despite high recurrence rates. A recent study on the genome of fronds has recently begun to understand the genotypic characteristics of a disease called fronds, and it is known that repeated changes in chromosomes, gene expressions, and patterns of somatic variation are known.
그러나 많은 경우 이러한 연구들이 양성 엽상종을 포함한 전체 엽상종에 대해서 이루어졌고 실제 연구에 포함된 환자들의 다수는 양성 및 경계성 엽상종인 경우들이 많다. 일반적인 유방암에 대한 유방암 진단용 마커로 한국 등록특허 KR 10-1058230 B1는 티오레독신 1의 핵산을 유효성분으로 하는 Ⅳ기 암인 전이성 유방암 진단용 마커 조성물을 개시하고 있으나, 엽상종, 특히 악성 엽상종에 있어서 예후를 예측하기 위한 바이오 마커는 알져지지 않았다. In many cases, however, these studies have been carried out on whole foliomas, including benign fronds, and many of the patients included in the actual studies are benign and borderline fronds. As a marker for diagnosing breast cancer for general breast cancer, Korean Patent Registration KR 10-1058230 B1 discloses a marker composition for diagnosing metastatic breast cancer, which is a stage IV cancer using thioredoxin 1 as an active ingredient. Biomarkers for predicting prognosis are unknown.
따라서, 악성 엽상종 환자의 예후를 예측할 수 있는 바이오 마커가 제시되는 경우 관련 분야에서 널리 적용될 수 있을 것으로 기대된다. Therefore, it is expected that biomarkers that can predict the prognosis of patients with malignant foliar tumors can be widely applied in related fields.
이에 본 발명의 한 측면은 악성 엽상종 환자의 예후를 예측할 수 있는 악성 엽상종 예후 예측용 조성물을 제공하는 것이다. Accordingly, an aspect of the present invention is to provide a composition for predicting the prognosis of malignant frondoma that can predict the prognosis of malignant fronoma.
본 발명의 다른 측면은 엽상종 환자의 예후를 예측할 수 있는 악성 엽상종 예후 예측용 키트를 제공하는 것이다. Another aspect of the present invention is to provide a kit for predicting malignant frond prognosis that can predict the prognosis of a patient with fronoma.
본 발명의 또 다른 측면은 엽상종 환자의 예후를 예측하는데 필요한 정보를 제공하는 방법에 관한 것이다. Another aspect of the invention relates to a method of providing information necessary for predicting the prognosis of a patient with frond.
본 발명의 일 견지에 의하면 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 유전자를 바이오 마커로 포함하는, 악성 엽상종 예후 예측용 조성물이 제공된다. According to one aspect of the present invention, there is provided a composition for predicting malignant foliar prognosis comprising a gene of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as a biomarker.
본 발명의 다른 견지에 의하면, 상기 조성물을 포함하는, 악성 엽상종 예후 예측용 키트가 제공된다. According to another aspect of the present invention, there is provided a kit for predicting malignant stromal prognosis comprising the composition.
본 발명의 다른 견지에 의하면, 분리된 개체의 시료로부터 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 RNA 발현수준을 FPKM 값으로 측정하는 단계; 및 2) 상기 유전자 중 FPKM 값이 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분하고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분하는 단계를 포함하는, 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법이 제공된다. According to another aspect of the invention, measuring the RNA expression level of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 from the sample of the isolated individual by FPKM value; And 2) classifying a fibrous subtype first subtype when there are 6 or more genes having a FPKM value of 1 or less, and dividing an epithelial subtype second subtype by less than 6 genes. In addition, methods are provided to provide the information necessary to predict the prognosis of malignant fronds.
본 발명에 의하면, 악성 엽상종의 유전체적 발현 양상에 기초하여 악성 엽상종을 두 가지 아형으로 분류함으로써, 공격적인 임상 양상을 보이며 종래에 효과적인 치료법이 제시되지 못한 악성 엽상종 종양에 대한 효과적인 표적치료 전략을 수립할 수 있다.According to the present invention, an effective targeted treatment strategy for malignant stromal tumors with aggressive clinical features and no effective treatment has been proposed by classifying malignant stromal into two subtypes based on genomic expression patterns of malignant stromal tumors. Can be established.
도 1은 9개 샘플의 악성 엽상종에서의 유전적 변형을 나타낸 것이다. 도 1A는 침윤성 도관 암(Mann Whitney test)과 비교하여 악성 엽상종(malignant phyllodes tumor, MPT)에서, 커버된 표적 서열의 Mb 당 체세포 돌연변이의 수로 측정되는 체세포 돌연변이의 발생률(malignant phyllodes tumor)을 나타내며, 도 1B는 RPT 'deconstrctSigs'를 사용하여 식별된 각 돌연변이 특징의 영향을 표시하는 파이 플롯으로 표시된 MPT의 체세포 돌연변이 특징을 나타내고, 도 1C는 MPT와 침윤성 도관암 사이의 APOBEC 관련 돌연변이 특징을 비교한 것이며(Mann Whitney test), 도 1D는 MPT에서 암 관련 유전자의 체세포 돌연변이와 카피 수 변화를 나타낸 것으로, DNAH11 유전자는 섬유 상피 종양에서 반복적으로 돌연변이가 일어나는 것으로 알려져 있으므로 포함되었다. * : p <0.05, *** : p <0.001.1 shows genetic modifications in malignant frondoma of nine samples. 1A shows the rate of malignant phyllodes tumors measured in number of somatic mutations per Mb of covered target sequence in malignant phyllodes tumors (MPTs) compared to the Mann Whitney test. FIG. 1B shows the somatic mutation characteristics of MPT indicated by pie plots indicating the effect of each mutation feature identified using RPT 'deconstrctSigs', and FIG. 1C compares APOBEC related mutation features between MPT and invasive catheter cancer. (Mann Whitney test), Figure 1D shows the somatic mutation and copy number changes of cancer-related genes in MPT, DNAH11 gene was included because it is known that the mutation is repeated in fibroepithelial tumors. *: p <0.05, ***: p <0.001.
도 2는 악성 엽상종의 유전자 발현 특성을 나타낸 것이다. 도 2A는 일반적인 유방 및 침윤성 도관암종 조직(황색: 일반적인 유방암, 적색: MPT, 녹색: 침윤성 도관암종)과 비교했을 때 악성 엽상종(MPT)의 독특한 유전자 발현 프로파일을 보여주는 표본간 거리의 히트 맵(heatmap)을 나타낸 것이며, 이 히트 맵은 Deseq2 R 패키지를 사용하여 정규화된 유전자 리드 카운트(read counts)로부터 도출되었다. 도 2B는 일반적인 유방 조직(상부) 및 침윤성 도관암종 조직(하부)과 비교하여 MPT에서 유의하게 조절되지 않는 경로를 나타내는 것이다.Figure 2 shows the gene expression characteristics of malignant frond. FIG. 2A is a heat map of sample-to-sample distances showing unique gene expression profiles of malignant lobe tumors (MPT) compared to typical breast and invasive catheter carcinoma tissues (yellow: common breast cancer, red: MPT, green: invasive catheter) heat map), which was derived from gene read counts normalized using the Deseq2 R package. 2B shows a significantly unregulated pathway in MPT compared to normal breast tissue (top) and invasive catheter carcinoma tissue (bottom).
도 3은 악성 엽상종의 분자 아형을 나타낸 것이다. 도 3A는 게놈-와이드 전사체 데이터를 이용한 9개의 악성 엽상종(MPT)의 감독되지 않는 클러스터링(Unsupervised clustering) 결과를 나타낸 것이며, 도 3B는 상피성 아형(좌) 및 섬유성 아형(우)에서의 상향조절된 유전자를 이용하는 주요 경로를 나타내는 것이고, 도 3C 및 3D는 분자 서브타입에 따른 다양한 콜라겐, 클라우딘(claudin) 및 CDH1의 발현 수준을 나타내며(Mann Whitney test), 도 3E는 특정 경로에 관련된 유전자를 기반으로 아형의 클러스터링을 보여주는 히트 맵이고, 도 3F는 21 개의 유전자 표지를 사용하여 추가 FFPE MPT 시료를 감독되지 않는 클러스터링(unsupervised clustering)을 나타낸 것이며, 도 3G는 아형에 따른 유사 분열 지수의 비교(Chi-square test)를 나타낸 것이고, 도 3H는 양 유형의 재발없는 생존율을 보여주는 Kaplan-Meier survival curve(log rank test)를 나타낸 것이다. * : p <0.05, ** : p <0.01, *** : p <0.001.3 shows the molecular subtypes of malignant fronds. FIG. 3A shows the unsupervised clustering results of nine malignant lobe species (MPT) using genome-wide transcript data, and FIG. 3B shows epithelial subtypes (left) and fibrous subtypes (right). 3C and 3D show the expression levels of various collagen, claudin and CDH1 depending on the molecular subtype (Mann Whitney test), and FIG. 3E is related to specific pathways. Heat map showing clustering of subtypes based on genes, FIG. 3F shows unsupervised clustering of additional FFPE MPT samples using 21 gene markers, and FIG. 3G shows mitosis index according to subtypes. The Chi-square test is shown, and FIG. 3H shows the Kaplan-Meier survival curve (log rank test) showing the survival rate without recurrence of both types. . *: p <0.05, **: p <0.01, ***: p <0.001.
도 4는 환자-유래 엽상종 모델에서 PI3K/mTOR 및 PDGFRB 타겟팅 접근을 나타낸 것이다. 도 4A는 원발 종양(2014-0916) 및 환자 유래 이종 이식 종양(MX-99)의 조직 학적 특징을 나타낸 것이며, 도 4B는 원발 종양 및 이종 이식 종양의 mRNA 발현 수준을 나타내는 상관관계 도표(두 샘플 모두 FPKM ≥1, log2 (FPKM))이고, 도 4C는 PDGFRB 및 PIK3R1에서 체세포 돌연변이의 분포를 나타낸 것이며, 도 4D는 일반적인 유방 조직, 침윤성 유방암 조직 및 MPT에서의 PDGFRB mRNA 발현 수준을 나타낸 것이며, 도 1E는 비히클, 이메티닙 및 PKI-587(adjusted p value derived from multiple t-test)에 의해 처리된 MX-99 이종 이식 모델의 종양 성장을 나타낸 것이고, 도 4F는 비히클, 이메티닙 및 PKI-587로 치료했을 때 이종 이식 종양에서 하류 신호 전달 경로 분자의 웨스턴 블럿 분석 결과를 나타낸 것이며, 도 4G는 웨스턴 블럿 결과의 정량 분석(Mann Whitney test)을 나타낸 것이다. *: p <0.05, **: p <0.01, *** : p <0.001.4 shows the PI3K / mTOR and PDGFRB targeting approaches in a patient-derived frond model. 4A shows histological characteristics of primary tumor (2014-0916) and patient-derived xenograft tumor (MX-99), and FIG. 4B is a correlation plot showing mRNA expression levels of primary and xenograft tumors (two samples). All are FPKM ≧ 1, log2 (FPKM)), and FIG. 4C shows the distribution of somatic mutations in PDGFRB and PIK3R1, FIG. 4D shows PDGFRB mRNA expression levels in common breast tissue, invasive breast cancer tissue, and MPT, FIG. 1E shows tumor growth in MX-99 xenograft model treated by vehicle, imetinib and adjusted p value derived from multiple t-test (FIG. 4F), FIG. 4F shows vehicle, imetinib and PKI- Western blot analysis of downstream signal transduction pathway molecules in xenograft tumors when treated with 587 is shown. FIG. 4G shows the quantitative analysis of Western blot results (Mann Whitney test). *: p <0.05, **: p <0.01, ***: p <0.001.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명에 의하면, 전장 엑솜 시퀀싱(whole exome sequencing)과 전사체 시퀀싱(transcriptome sequencing)을 통해서 악성 엽상종 환자 조직의 유전체적 및 분자생물학적 특성을 파악하여 악성 엽상종 예후 예측용 바이오 마커를 확인하고, 나아가 이러한 바이오 마커를 이용하여 특징적인 임상 양상을 보이는 엽상종의 아형(subtype)을 유전체 발현 패턴을 이용해서 분류하는 기술이 제공된다. According to the present invention, the biomarker for predicting the prognosis of malignant frond tumors is identified by identifying the genomic and molecular biological characteristics of malignant frond tumor patients tissue through whole exome sequencing and transcriptome sequencing. Furthermore, a technique for classifying subtypes of fronds showing characteristic clinical features using such biomarkers using genome expression patterns is provided.
보다 상세하게, 본 발명의 악성 엽상종 예후 예측용 조성물은 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 유전자를 바이오 마커로 포함하는 것이다. In more detail, the composition for predicting malignant foliar prognosis of the present invention comprises the genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as biomarkers.
본 발명에 따른 악성 엽상종의 예후 예측을 위한 마커 유전자로서 서열번호 1 내지 12의 염기서열을 갖는 유전자들의 특징을 하기와 같이 정리하였다. 이때 유전자명은 HUGO Gene Nomenclature Committee를 참고하였고, 위치는 Refseq을 참고하였으며, SEQ ID NO.는 NCBI Reference Sequence를 나타내는 것이다. 하기 괄호 안의 번호는 본 발명의 서열번호에 상응하는 것이다.As a marker gene for predicting the prognosis of malignant frondoma according to the present invention, the characteristics of genes having the nucleotide sequences of SEQ ID NOs: 1 to 12 are summarized as follows. The gene name refers to the HUGO Gene Nomenclature Committee, the position refers to the Refseq, and SEQ ID NO. Indicates the NCBI Reference Sequence. The numbers in parentheses below correspond to the sequence numbers of the present invention.
(1) CDH1  (1) CDH1
유전자명: cadherin 1Gene name: cadherin 1
위치: chr16:68771193-68869445Location: chr16: 68771193-68869445
Log2FC: 8.50Log2FC: 8.50
Adjusted P.vaule: 3.3E-48Adjusted P.vaule: 3.3E-48
NCBI SEQ ID NO,: NG_008021.1NCBI SEQ ID NO ,: NG_008021.1
(2) SOX10(2) SOX10
유전자명: SRY-box 10Gene name: SRY-box 10
위치: chr22:38368319-38380539Location: chr22: 38368319-38380539
Log2FC: 7.58Log2FC: 7.58
Adjusted P.vaule: 3.8E-31Adjusted P.vaule: 3.8E-31
NCBI SEQ ID NO,: NM_006941.3NCBI SEQ ID NO ,: NM_006941.3
(3) TACSTD2(3) TACSTD2
유전자명: tumor-associated calcium signal transducer 2Gene name: tumor-associated calcium signal transducer 2
위치: chr1:59041095-59043166Location: chr1: 59041095-59043166
Log2FC: 7.52Log2FC: 7.52
Adjusted P.vaule: 1.6E-99Adjusted P.vaule: 1.6E-99
NCBI SEQ ID NO,: NM_002353.2NCBI SEQ ID NO ,: NM_002353.2
(4) KRT5(4) KRT5
유전자명: keratin 5Gene name: keratin 5
위치: chr12:52908359-52914243Location: chr12: 52908359-52914243
Log2FC: 7.51Log2FC: 7.51
Adjusted P.vaule: 3.2E-39Adjusted P.vaule: 3.2E-39
NCBI SEQ ID NO,: NM_000424.3NCBI SEQ ID NO ,: NM_000424.3
(5)BBOX1(5) BBOX1
유전자명: gamma-butyrobetaine hydroxylase 1Gene name: gamma-butyrobetaine hydroxylase 1
위치: chr11:27062509-27149354Location: chr11: 27062509-27149354
Log2FC: 7.47Log2FC: 7.47
Adjusted P.vaule: 3.0E-19Adjusted P.vaule: 3.0E-19
NCBI SEQ ID NO,: NM_003986.2NCBI SEQ ID NO ,: NM_003986.2
(6)COL17A1(6) COL17A1
유전자명: collagen type XVII alpha 1 chainGene name: collagen type XVII alpha 1 chain
위치: chr10:105791046-105845638Location: chr10: 105791046-105845638
Log2FC: 7.41Log2FC: 7.41
Adjusted P.vaule: 3.2E-109Adjusted P.vaule: 3.2E-109
NCBI SEQ ID NO,: NM_000494.3NCBI SEQ ID NO ,: NM_000494.3
(7)PRSS8(7) PRSS8
유전자명: serine protease 8Gene name: serine protease 8
위치: chr16:31142754-31147083Location: chr16: 31142754-31147083
Log2FC: 7.38Log2FC: 7.38
Adjusted P.vaule: 1.9E-27Adjusted P.vaule: 1.9E-27
NCBI SEQ ID NO,: NM_002773.4NCBI SEQ ID NO ,: NM_002773.4
(8) PRR15L(8) PRR15L
유전자명: proline rich 15 likeGene name: proline rich 15 like
위치: chr17:46029334-46035243Location: chr17: 46029334-46035243
Log2FC: 7.34Log2FC: 7.34
Adjusted P.vaule: 7.3E-33Adjusted P.vaule: 7.3E-33
NCBI SEQ ID NO,: NM_024320.3NCBI SEQ ID NO ,: NM_024320.3
(9)ERBB3(9) ERBB3
유전자명: erb-b2 receptor tyrosine kinase 3Gene name: erb-b2 receptor tyrosine kinase 3
위치: chr12:56473809-56497291Location: chr12: 56473809-56497291
Log2FC: 6.59Log2FC: 6.59
Adjusted P.vaule: 1.5E-36Adjusted P.vaule: 1.5E-36
NCBI SEQ ID NO,: NG_011529.1NCBI SEQ ID NO ,: NG_011529.1
(10)KRT14(10) KRT14
유전자명: keratin 14Gene name: keratin 14
위치: chr17:39738531-39743147Location: chr17: 39738531-39743147
Log2FC: 6.51Log2FC: 6.51
Adjusted P.vaule: 6.2E-15Adjusted P.vaule: 6.2E-15
NCBI SEQ ID NO,: NM_000526.4NCBI SEQ ID NO ,: NM_000526.4
(11) RAB25 (11) RAB25
유전자명: RAB25, member RAS oncogene familyGene name: RAB25, member RAS oncogene family
위치: chr1:156030940-156040305Location: chr1: 156030940-156040305
Log2FC: 6.39Log2FC: 6.39
Adjusted P.vaule: 8.2E-38Adjusted P.vaule: 8.2E-38
NCBI SEQ ID NO,: NM_020387.3NCBI SEQ ID NO ,: NM_020387.3
(12) AGR2(12) AGR2
유전자명: anterior gradient 2, protein disulphide isomerase family memberGene name: anterior gradient 2, protein disulphide isomerase family member
위치: chr7:16832264-16844738Location: chr7: 16832264-16844738
Log2FC: 6.32Log2FC: 6.32
Adjusted P.vaule: 1.1E-16Adjusted P.vaule: 1.1E-16
NCBI SEQ ID NO,: NM_006408.3NCBI SEQ ID NO ,: NM_006408.3
상기 유전자는 각각 서열번호 1 내지 서열번호 12의 염기서열에 상응하는 것이다.The genes correspond to the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 12, respectively.
이때, 상기 서열번호 2 내지 서열번호 8 및 10 내지 12의 염기서열은 해당 유전자 중 인트론(intron) 부분이 제외된 mRNA 서열이며, 그 중에서도 단백질로 번역(translation)되는 코딩 서열(coding sequence)만을 나타낸 것이다. 다만, 서열번호 1의 CDH1 및 열번호 9의 ERBB3 유전자는 여러 단백질 아이소폼(isoform)이 될 수 있는 전사 변종(transcript variant)을　가지고 있어,　mRNA 서열이 아닌 전체 유전자 서열 중 코딩 서열(coding sequence)을　나타냈다.In this case, the nucleotide sequence of SEQ ID NO: 2 to SEQ ID NO: 8 and 10 to 12 is an mRNA sequence excluding the intron portion of the gene, and among them, shows only a coding sequence translated into a protein will be. However, the CDH1 and ERBB3 genes of SEQ ID NO: 1 have a transcript variant that can be a variety of protein isoforms, and thus, the coding sequence of the entire gene sequence, not the mRNA sequence. Appeared.
본 발명에서 용어 "예후"는 악성 엽상종의, 예를 들어 재발, 전이성 확산, 및 약물 내성을 비롯한 악성 엽상종-기인성 사망 또는 진행의 가능성 등의 병의 경과 및 완치 여부를 의미한다. 본 발명의 목적상 예후는 악성 엽상종 치료 후 소 재발 가능성을 의미하며, 바람직하게는 악성 엽상종의 수술 또는 항암 화학요법을 시술 받은 후 2년 이내에 재발할지의 여부를 예측하는 것을 의미한다. As used herein, the term “prognosis” refers to the progression and cure of malignant fronds, such as recurrence, metastatic spread, and the likelihood of malignant frond-mediated death or progression, including drug resistance. For the purposes of the present invention, prognosis refers to the possibility of bovine recurrence after the treatment of malignant frondoma, and preferably to predict whether to relapse within two years after surgery or chemotherapy of malignant frond.
본 발명에서 용어 "양호한 예후"는 악성 엽상종 환자의 질병이 완치될 가능성을 의미하고, "양호하지 않은 예후"는 투병 중인 악성 엽상종의 재생(relapse) 또는 재발(recurrence), 전이 또는 사망할 가능성이 있음을 의미한다. 양호한 결과를 갖는 것으로 분류된 악성 엽상종 환자는 투병 중인 악성 엽상종이 없거나 감소되고 있는 상태이다. 이와는 반대로, 양호하지 않은 결과의 악성 엽상종 환자는 질병의 재생, 종양 재발, 전이 또는 사망에 이른다. "양호한 예후"는 악성 엽상종 환자가 투병 중인 악성 엽상종이 적어도 2년, 보다 구체적으로는 적어도 5년 이상 동안 없는 상태로 있을 수 있음을 의미한다. 본 발명의 다른 측면에서, "양호하지 않은 예후"는 악성 엽상종 환자가 5년 미만, 보다 구체적으로는 2년 미만 이내에 질병 재생, 종양 재발, 전이 또는 사망을 경험할 수 있음을 의미한다. As used herein, the term "good prognosis" refers to the possibility that the disease of a patient with malignant foliar disease will be cured, and "unfavorable prognosis" refers to the relapse or recurrence, metastasis or death of the diseased malignant frond. It means that there is a possibility. Patients with malignant frondoma classified as having good results have no or decreasing disease in malignant fronds. Conversely, patients with malignant stromal tumors with poor outcomes lead to disease regeneration, tumor recurrence, metastasis or death. "Good prognosis" means that a patient with malignant foliar tumor may be left without disease for at least 2 years, more specifically at least 5 years. In another aspect of the present invention, "unfavorable prognosis" means that a patient with malignant stromal tumor may experience disease regeneration, tumor recurrence, metastasis, or death within less than 5 years, more specifically less than 2 years.
본 발명에서 용어, "예측"이란 환자가 화학요법 또는 방사선 치료 등의 치료법에 대해 선호적으로 또는 비선호적으로 반응하여 환자가 치료, 예를 들어 특정 치료제, 및/또는 원발성 종양의 수술로 제거, 및/또는 암의 재발 없이 특정 시기 동안 화학요법으로 치료된 후의 생존 여부 및/또는 가능성과 관련된다. 본 발명의 예측방법은 임의의 특정 악성 엽상종 환자에 대한 가장 적절한 치료방식을 선택하여 적용함으로써 임상적으로 사용될 수 있다. 본 발명의 예측방법은 환자가, 예를 들어 소정의 치료제 또는 조합물, 외과적 개입, 화학요법 등의 투여를 비롯한 소정의 치료 처방과 같은 치료법에 선호적으로 반응하는지를 확인하거나, 치료 처방 후 환자의 장기 생존 또는 전신 또는 국소 재발이 가능한지를 예측할 수 있다. 또한 이를 통하여 불필요한 보조 항암요법을 최소화하거나 전신 또는 국소 재발이 예측되는 환자에게는 더욱 효과적인 보조 항암요법을 사용할 수 있도록 계획할 수 있다.As used herein, the term "prediction" means that the patient responds favorably or unfavorably to a therapy, such as chemotherapy or radiation therapy, such that the patient is removed by treatment, e.g., surgical treatment of a particular therapeutic agent, and / or primary tumor, And / or survival and / or likelihood after treatment with chemotherapy for a certain period of time without recurrence of cancer. The predictive method of the present invention can be used clinically by selecting and applying the most appropriate treatment regimen for any particular malignant stromal patient. Prediction methods of the present invention determine whether a patient responds favorably to a treatment such as a prescribed treatment regimen, including, for example, administration of a predetermined treatment or combination, surgical intervention, chemotherapy, or the like, or after a treatment regimen. Long term survival or systemic or local recurrence is predictable. It can also be planned to minimize unnecessary adjuvant chemotherapy or to use adjuvant chemotherapy for patients who are expected to have systemic or local recurrence.
본 발명에서 용어 "예후 예측용 조성물"은 악성 엽상종에 대한 치료 후 예후가 양호한 환자와 양호하지 않은 환자를 구별하여 재발 가능성을 예측할 수 있는 것으로, CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 유전자를 예후 예측용 마커로서 포함한다.In the present invention, the term "prognostic composition for prognosis" is to distinguish between patients with good prognosis and patients with poor prognosis after treatment for malignant edema, and predict the possibility of recurrence, CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, Genes of PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 are included as markers for prognostic prediction.
본 발명에서 용어 "예후 예측용 마커", "예후 예측을 위한 마커" 또는 "예후 예측 마커(prognosis marker)"란 악성 엽상종 치료 후 재발 여부를 비롯한 예후를 예측할 수 있는 것을 의미한다. 본 발명의 목적상 악성 엽상종 진단 후 수술 또는 화학요법을 시술을 받고 2년 이내에 전신 또는 국소 재발 여부를 결정하여 예후가 양호한 환자와 양호하지 않은 환자를 구별할 수 있는 마커를 의미한다.In the present invention, the term "prognostic marker", "prognostic marker" or "prognosis marker" means prognosis including predicting recurrence after the treatment of malignant edema. For the purposes of the present invention, a marker capable of distinguishing between a patient with a good prognosis and a patient with a poor prognosis by determining whether systemic or local recurrence is performed within 2 years after undergoing surgery or chemotherapy after diagnosis of malignant frondoma.
본 발명에서 악성 엽상종의 예후 예측 마커로 선택된 상기 유전자들과 악성 엽상종 또는 이의 재발과의 관련성 및 악성 엽상종에 있어서 상기 유전자들의 구체적인 기능에 대해서는 현재까지 보고된 바 없다.In the present invention, the association between the genes selected as prognostic markers of malignant fronds and malignant fronds or their recurrences and the specific function of the genes in malignant fronds have not been reported to date.
본 발명에서 용어 "유전자 또는 전사체(transcript)"는 혼용하여 사용될 수 있다.In the present invention, the term "gene or transcript" may be used interchangeably.
본 발명에서 용어 "RNA 발현수준 측정"은 악성 엽상종의 예후를 예측하기 위하여 생물학적 시료에서 마커 유전자의 RNA 존재 여부와 발현 정도를 확인하는 과정으로, RNA의 양을 측정함으로써 알 수 있다. 이를 위한 분석방법으로는 RT-PCR, 경쟁적 RT-PCR(competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), DNA 마이크로어레이 칩, FPKM(Fragments Per Kilobase of exon per Million) 등이 있으나, 이들로 한정되는 것은 아니다. In the present invention, the term "RNA expression level measurement" is a process of confirming the presence and expression of RNA of a marker gene in a biological sample in order to predict the prognosis of malignant frondoma, and can be known by measuring the amount of RNA. For this, RT-PCR, competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), northern blotting (northern) blotting), DNA microarray chips, and fragments per kilobase of exon per million (FPKM), but are not limited thereto.
본 발명의 악성 엽상종 예후 예측용 조성물은 상기 유전자의 RNA 또는 이들의 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 추가로 포함할 수 있으며, 상기 RNA의 발현수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 포함하는 것일 수 있고, 상기 단백질의 발현수준을 측정하는 제제는 상기 유전자로부터 코딩되는 단백질에 특이적인 항체일 수 있다. The composition for predicting malignant foliar prognosis of the present invention may further include an agent for measuring the expression level of RNA of the gene or a protein encoded by these genes, the agent for measuring the expression level of the RNA is It may include an antisense oligonucleotide, primer pair or probe that specifically binds to the gene, the agent for measuring the expression level of the protein may be an antibody specific for the protein encoded from the gene.
본 발명에 따른 악성 엽상종의 예후 마커 유전자의 RNA 수준을 측정하는 제제는 예를 들어 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이며, 상기 마커 유전자의 염기서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 프라이머 또는 프로브를 고안할 수 있다. 본 발명에 따른 악성 엽상종의 예후 마커 유전자의 염기서열은 유전자뱅크(GenBank)에 등록되는 등 당해 분야에 공지된 상태이므로, 당업자는 상기 염기서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭할 수 있는 프라이머 또는 프로브를 디자인할 수 있다.The agent for measuring the RNA level of the prognostic marker gene of malignant foliar tumor according to the present invention is, for example, an antisense oligonucleotide, a primer pair or a probe, and specifically targets specific regions of these genes based on the nucleotide sequence of the marker gene. Primers or probes for amplification can be designed. Since the nucleotide sequences of the prognostic marker genes of malignant foliar tumors according to the present invention are known in the art, such as those registered in the GenBank, those skilled in the art will specifically amplify specific regions of these genes based on the nucleotide sequences. Primers or probes can be designed.
본 발명에서 용어, "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어 표적 서열 내에서 전형적으로 mRNA와 헤테로이중체를 형성할 수 있는 뉴클레오티드 염기서열 및 서브유닛간 백본을 갖는 올리고머를 지칭한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 유사 상보성을 가질 수 있다. 이 안티센스 올리고머는 mRNA의 번역을 차단 또는 저해하고 mRNA의 스플라이스 변이체를 생산하는 mRNA의 프로세싱 과정을 변화시킬 수 있다. As used herein, the term “antisense” refers to a backbone between nucleotide sequences and subunits in which antisense oligomers can hybridize with target sequences in RNA by Watson-Crick base pairing to form heterodimers with mRNA typically within the target sequence. Refers to an oligomer having The oligomer may have precise sequence complementarity or similar complementarity to the target sequence. These antisense oligomers can alter or process the processing of mRNA, which blocks or inhibits translation of mRNA and produces splice variants of the mRNA.
본 발명에서 용어, "프라이머"는 적절한 완충액 중의 적절한 조건(예를 들면, 4개의 다른 뉴클레오시드 트리포스페이트 및 DNA, RNA 폴리머라제 또는 역전사 효소와 같은 중합제) 및 적절한 온도 하에서 주형-지시 DNA 합성의 시작점으로서 작용할 수 있는 단일 가닥 올리고뉴클레오티드를 말한다. 상기 프라이머의 적절한 길이는 사용 목적에 따라 달라질 수 있으나, 통상 15 내지 30개 뉴클레오티드이다. 짧은 프라이머 분자는 일반적으로 주형과 안정한 혼성체를 형성하기 위해서 더 낮은 온도를 필요로 한다. 프라이머 서열은 주형과 완전하게 상보적일 필요는 없으나, 주형과 혼성화할 정도로 충분히 상보적이어야 한다. 본 발명에서는 본 발명에 따른 악성 엽상종의 예후 마커 유전자에 대한 정방향 및 역방향 프라이머를 사용하여 PCR 증폭을 수행한 후 PCR 생성물의 증폭 여부를 통해 악성 엽상종의 재발 가능성 및 2년 생존 예후를 예측할 수 있다.As used herein, the term “primer” refers to template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperatures. It refers to a single stranded oligonucleotide that can act as a starting point. Appropriate length of the primer may vary depending on the purpose of use, but is typically 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. In the present invention, after performing PCR amplification using the forward and reverse primers for the prognostic marker genes of malignant frondoma according to the present invention, the possibility of recurrence and the two-year survival prognosis can be predicted through the amplification of the PCR product. have.
본 발명에서 용어, "프로브"는 RNA와 특이적 결합을 이룰 수 있는 짧게는 수개 염기 내지 길게는 수백 개 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미한다. 프로브는 올리고 뉴클레오티드 프로브, 단일 가닥 DNA 프로브, 이중 가닥 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 본 발명에 따른 마커 유전자에 대해 상보적인 프로브를 이용하여 혼성화를 실시한 후, 혼성화 여부를 통해 악성 엽상종의 재발 여부를 비롯한 예후를 예측할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당해 분야에 공지된 것을 기초로 변형할 수 있다.As used herein, the term “probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to short bases of several hundred bases and hundreds of bases capable of specific binding with RNA. Probes can be constructed in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, after hybridization is performed using a probe complementary to the marker gene according to the present invention, the prognosis including the recurrence of malignant frond tumor can be predicted through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
본 발명에 따른 안티센스 올리고뉴클레오티드, 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법을 비롯한 당해 분야에 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡핑, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Antisense oligonucleotides, primers or probes according to the present invention may be chemically synthesized using methods well known in the art, including phosphoramidite solid support methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methylphosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
한편, 본 발명에서 용어, "단백질의 발현수준 측정"은 악성 엽상종 재발 가능성을 예후 예측하기 위하여 생물학적 시료에서 마커 유전자로부터 코딩된 단백질의 존재 여부와 발현수준을 확인하는 과정으로, 상기 단백질에 대하여 특이적으로 결합하는 항체를 이용해 단백질의 양을 확인한다. 이를 위한 분석방법으로는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(radioimmunoassay), 방사면역확산법(radioimmunodiffusion), 오우크레로니(Ouchterlony) 면역확산법, 로케트(Rocket) 면역전기영동, 면역조직화학염색, 면역침전분석(immunoprecipitation assay), 보체고정분석(complete fixation assay), FACS, 단백질 칩(protein chip) 등이 있으나, 이들로 한정되는 것은 아니다.Meanwhile, in the present invention, the term "measurement of expression level of protein" refers to a process of confirming the presence and expression level of a protein encoded from a marker gene in a biological sample in order to prognostically predict the possibility of recurrence of malignant stromal tumor. The amount of the protein is determined using an antibody that binds specifically. Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunity Electrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.
본 발명에서 용어, "항체"는 당해 분야에 공지된 용어로서, 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커 유전자로부터 코딩된 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝 하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻은 후, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 펩티드 단편도 포함되며, 본 발명의 펩티드 단편으로는, 최소한 7개 아미노산, 바람직하게는 9개 아미노산, 더욱 바람직하게는 12개 이상의 아미노산을 포함한다. 본 발명의 항체는 그 형태가 특별히 제한되지 않으며, 다중클론 항체, 단일클론 항체 또는 항원 결합성을 갖는 것이라면 그것의 일부도 본 발명의 항체에 포함되고, 모든 면역글로불린 항체가 포함된다. As used herein, the term "antibody" is a term known in the art and means a specific protein molecule directed to an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein encoded from a marker gene of the present invention, which antibody is cloned into an expression vector according to a conventional method by the marker gene. After obtaining the protein to be encoded, it can be prepared by conventional methods from the obtained protein. It also includes peptide fragments that can be made from such proteins, and peptide fragments of the present invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The antibody of the present invention is not particularly limited in form, and a part thereof is included in the antibody of the present invention as long as it is a polyclonal antibody, a monoclonal antibody or an antigen-binding agent, and all immunoglobulin antibodies are included.
상기한 바와 같이 본 발명에 의하면 악성 엽상종의 예후 예측 마커 유전자가 규명되었으므로, 이를 이용하여 항체를 생성하는 것은 당업계에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있다. 다중클론 항체는 상기한 악성 엽상종의 예후 예측 마커 유전자로부터 코딩되는 단백질 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 당해 분야에 널리 공지된 방법에 의해 생산할 수 있다. 이러한 다중클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소 개 등의 임의의 동물 종 숙주로부터 제조 가능하다. 단클론 항체는 당해분야에 널리 공지된 하이브리도마 방법(hybridoma method)(Kohler 및 Milstein, European Jounral of Immunology, 6: 511-519, 1976), 또는 파지 항체 라이브러리(Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222(58): 1-597, 1991) 기술을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제할 수 있다.As described above, according to the present invention, since the prognostic predictive marker gene of malignant frond tumor has been identified, the production of antibodies using the same can be easily prepared using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for injecting protein antigens encoded from the prognostic predictive marker genes of malignant frondoma described above into animals and collecting blood from the animals to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog. Monoclonal antibodies are well known in the art by the hybridoma method (Kohler and Milstein, European Jounral of Immunology, 6: 511-519, 1976), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222 (58): 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
본 발명의 항체에는 인간화 항체 등의 재조합 항체도 포함된다. 본 발명에 사용되는 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2, Fv 등이 있다.Antibodies of the present invention also include recombinant antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule means a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 , Fv, and the like.
나아가, 본 발명자들은 악성 엽상종 환자의 조직을 전장 엑솜 시퀀싱(whole exome sequencing)과 전사체 시퀀싱(transcriptome sequencing)을 통해서 그 유전체적 및 분자생물학적 특성을 파악하고 특징적인 임상 양상을 보이는 엽상종의 아형(subtype)을 유전체 발현 패턴을 이용해서 분류할 수 있도록 하였다. Furthermore, the present inventors have identified the genome and molecular biological characteristics of the tissues of patients with malignant fronds through whole exome sequencing and transcriptome sequencing, and showed subtypes of fronds with characteristic clinical features. (subtype) can be classified using genome expression patterns.
본 발명자들은 감독되지 않는 계층적 클러스터링(Unsupervised hierarchical clustering) 기법으로 악성 엽상종 조직에 대한 유전자 발현 데이터 특성을 이용하여 악성 엽상종을 두 종류의 아형으로 구분하였다.The inventors have divided the malignant fronds into two subtypes by using gene expression data characteristics of malignant fronds with unsupervised hierarchical clustering.
보다 상세하게, 상기 조성물은 상기 유전자 중 FPKM 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분하고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분하여 악성 엽상종의 예후를 예측하기 위한 것일 수 있다. More specifically, the composition is divided into fibrous subtype first subtype when there are 6 or more genes of FPKM 1 or less in the gene, and malignant leaflets by dividing into epithelial subtype 2 subtype when less than 6 genes. It may be for predicting the prognosis of the species.
상기 제1 아형은 단백질 분해와 흡수(protein digestion and absorption), 엑손 가이던스(axon guidance), ECM-수용체 상호작용(ECM-receptor interaction), 국소 접착(focal adhesion), 그리고 다양한 콜라겐과 같은 스트로마-관련(stroma-related) 유전자의 발현 이상의 특징을 나타내었고(도 3B 및 3C), 제2 아형은 세포 대사(cell metabolism), 세포 부착 분자(cell adhesion molecules), 치밀이음(tight junction) 및 다수의 면역 관련 프로세스에 관여하는 유전자의 발현이 높고, CDH1, CLDN3, CLDN4, CLDN7, OCLN 과 같은 상피세포의 특성을 반영하는 유전자의 발현이 현저히 높음을 확인하였다(도 3D). The first subtype is protein digestion and absorption, axon guidance, ECM-receptor interaction, focal adhesion, and stromal-related collagen such as various collagens. It was characterized by abnormal expression of stroma-related genes (FIGS. 3B and 3C), and the second subtype was cell metabolism, cell adhesion molecules, tight junctions, and multiple immunity. It was confirmed that the expression of genes involved in related processes is high, and the expression of genes reflecting the characteristics of epithelial cells such as CDH1, CLDN3, CLDN4, CLDN7, OCLN is significantly high (FIG. 3D).
이와 같은 유전자 발현의 특성을 반영하여 본 발명자들은 악성 엽상종을 두 가지 아형으로 분류할 수 있음을 확인하여, 제1 아형을 'Fibrous(섬유성)' 아형 그리고 제2 아형을 'Epithelial(상피성)' 아형으로 명명하였다. 이러한 분류는 그 자체로 섬유상피 종양의 양면적인 생물학적 특성을 반영할 수 있다. Reflecting the characteristics of this gene expression, the present inventors confirmed that malignant fronoma can be classified into two subtypes, so that the first subtype is 'Fibrous' subtype and the second subtype is 'Epithelial' (epithelial). Subtype). This classification may itself reflect the bilateral biological properties of fibroepithelial tumors.
이와 같은 개별 유전자 발현의 특성 외에도 섬유상피종양의 생물학적 특성에 영향을 미칠 수 있는 ECM-수용체 상호작용 경로(KEGG ID: hsa04512), 세포 부착 분자 경로(KEGG ID: hsa04514), EMT(epithelial mesenchymal transition)의 경로에 속하는 유전자의 총체적 발현 패턴을 분석한 결과, 유전자 집단 전체를 이용한 감독되지 않는 클러스터링(Unsupervised clustering)에서도 동일한 아형의 분류가 가능한 것을 확인하였다 (도 3E). In addition to these individual gene expression characteristics, ECM-receptor interaction pathways (KEGG ID: hsa04512), cell adhesion molecular pathways (KEGG ID: hsa04514), and epithelial mesenchymal transition (EMT) may affect the biological properties of fibroepithelial tumors. As a result of analyzing the overall expression pattern of genes belonging to the path of, it was confirmed that the same subtype can be classified even in unsupervised clustering using the entire gene population (FIG. 3E).
이와 같이 분류된 두 가지 악성 엽상종 아형은 다양한 임상병리적 특성에서도 두 아형간 뚜렷한 차이를 보였다. The two malignant frond subtypes classified as above showed distinct differences between the two subtypes in various clinical and pathologic characteristics.
제1 섬유성(Fibrous) 아형은 제2 상피성(Epithelial) 아형에 비해 기존에 알려진 주요 예후 인자의 유사분열 인덱스(mitotic index)가 현저히 높으며, 환자의 재발을 추적하였을 때 섬유성(Fibrous) 아형에서 유의하게 재발이 많이 발생함을 확인할 수 있었다. 즉, 본 발명에 따른 상기 제1 아형은 "양호하지 않은 예후"를 나타내고, 상기 제2 아형은 "양호한 예후"를 나타낸다.The first fibrous subtype has a significantly higher mitotic index of known major prognostic factors than the second epithelial subtype, and the fibrous subtype when the patient's recurrence is tracked. Significantly more relapses occurred at. That is, the first subtype according to the present invention represents an "unfavorable prognosis", and the second subtype represents a "good prognosis".
이러한 결과를 토대로 본 발명자들은 악성 엽상종은 과거에 알려진 것처럼 하나의 질환이 아니라, 임상적 특성 및 치료성적 측면에서 뚜렷한 차이를 보이는 두 가지 아형으로 나뉘어질 수 있음을 확인하고, 이를 구분하고, 구분된 아형에 따라 예후를 예측할 수 있다는 사실을 확인하였다.Based on these results, the present inventors have identified that malignant filamentous tumors can be divided into two subtypes with distinct differences in clinical characteristics and therapeutic results, rather than a single disease, as is known in the past. The prognosis can be predicted according to the subtype.
상기 본 발명의 악성 엽상종 예후 예측용 조성물은 예를 들어 2년 후 악성 엽상종이 재발할 가능성을 예측하기 위한 것일 수 있다.The composition for predicting malignant frond prognosis of the present invention may be, for example, to predict the possibility of recurrence of malignant frondoma after two years.
본 발명의 다른 견지에 의하면 상기 악성 엽상종 예후 예측용 조성물을 포함하는, 악성 엽상종 예후 예측용 키트가 제공된다. According to another aspect of the present invention, there is provided a kit for predicting malignant stromal prognosis, comprising the composition for predicting malignant stromal prognosis.
본 발명의 키트는 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 마커 유전자의 RNA 발현수준 또는 단백질의 발현수준을 확인하여 마커를 검출함으로써 악성 엽상종 재발 가능성 등 예후를 예측할 수 있다. The kit of the present invention detects markers by detecting RNA expression levels or protein expression levels of marker genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2 to detect markers. Prognosis can be predicted, including the possibility of relapse.
한편, 본 발명의 마커 검출용 키트에는 악성 엽상종 재발 여부를 예후 예측할 수 있는 마커 유전자의 발현 수준을 측정하기 위하여 상기 악성 엽상종 예후 예측용 조성물에서 언급한 바와 같이 프라이머, 프로브, 또는 상기 마커 유전자로부터 코딩된 단백질을 선택적으로 인지하는 항체뿐만 아니라 분석에 적합한 1종 이상의 다른 구성 성분 조성물, 용액 또는 장치가 포함될 수 있다.On the other hand, the marker detection kit of the present invention, primers, probes, or the marker gene as mentioned in the composition for predicting the prognosis of malignant foliar prognosis in order to measure the expression level of the marker gene for predicting the prognosis of recurrence of malignant foliar tumor. As well as antibodies that selectively recognize proteins encoded therefrom, one or more other component compositions, solutions or devices suitable for analysis may be included.
구체적인 일례로서, 본 발명에서 상기 마커 유전자들의 RNA 발현수준을 측정하기 위한 키트는 RT-PCR을 수행하는데 요구되는 필수요소를 포함하는 키트일 수 있다. RT-PCR 키트는 마커 유전자에 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소 및 역전사효소와 같은 효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다. As a specific example, the kit for measuring the RNA expression level of the marker genes in the present invention may be a kit containing the essential elements required to perform RT-PCR. In addition to each primer pair specific to the marker gene, the RT-PCR kit includes test tubes or other appropriate containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water and the like.
또한, 본 발명의 키트는 본 발명에 따른 마커 유전자들을 포함하는 악성 엽상종의 재발 가능성을 예후 예측하기 위한 마이크로어레이 형태일 수 있다. 상기 마이크로어레이는 DNA 또는 RNA 폴리뉴클레오티드 프로브를 포함하는 것일 수 있다. 상기 마이크로어레이는 본 발명에 따른 악성 엽상종의 예후 마커 유전자의 염기서열에 특이적인 프로브를 포함하는 것을 제외하고는 통상적인 마이크로어레이의 구성을 포함할 수 있다. 본 발명의 마이크로어레이는 본 발명에 따른 악성 엽상종의 예후 마커 유전자의 발현 경향을 검출하여 악성 엽상종의 재발 여부를 예후 예측하는데 유용한 정보를 제공할 수 있다. In addition, the kit of the present invention may be in the form of a microarray for prognostic predicting the possibility of recurrence of malignant frondoma comprising the marker genes according to the present invention. The microarray may comprise a DNA or RNA polynucleotide probe. The microarray may include a conventional microarray configuration except that the microarray includes a probe specific for the nucleotide sequence of the prognostic marker gene of the malignant frond tumor according to the present invention. The microarray of the present invention can provide useful information for predicting the prognosis of recurrence of malignant frondoma by detecting the expression of the prognostic marker gene of malignant frondoma according to the present invention.
본 발명에 따른 악성 엽상종의 예후 마커 유전자에 대한 프로브를 기판 상에 고정화하여 마이크로어레이를 제조하는 방법은 당해 분야에 잘 알려져 있다. 예컨대, DNA 마이크로어레이는, 이들로 한정되는 것은 아니지만, 파이조일렉트릭(piezoelectric) 방식을 이용한 마이크로피펫팅(micropipetting) 또는 핀(pin) 형태의 스폿터(spotter)를 이용한 방법에 의해 본 발명에 따른 마커 유전자에 대한 프로브를 기판 상에 고정화시킬 수 있다. 본 발명의 마이크로어레이의 기판은 아미노-실란(amino-silane), 폴리-L-라이신(poly-L-lysine) 및 알데히드(aldehyde)로 이루어진 군에서 선택되는 활성기가 코팅된 것이 바람직하나, 이에 한정되는 것은 아니다. 또한 상기 기판은 슬라이드 글래스, 플라스틱, 금속, 실리콘, 나일론 막 및 니트로셀룰로스 막(nitrocellulose membrane)으로 이루어진 군에서 선택되는 것이 바람직하나, 이들로 한정되는 것은 아니다.Methods of preparing microarrays by immobilizing probes on prognostic marker genes of malignant frondoma according to the present invention on a substrate are well known in the art. For example, DNA microarrays include, but are not limited to, the method according to the present invention by a method using a micropipetting or pin type spotter using a piezoelectric method. Probes for marker genes can be immobilized on a substrate. The substrate of the microarray of the present invention is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but not limited thereto. It doesn't happen. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
또한, 마이크로어레이 상에서의 핵산의 혼성화 및 혼성화 결과의 검출은 당해분야에 잘 알려져 있다. 상기 검출은 핵산 시료를 형광물질, 예를 들면, Cy3 및 Cy5와 같은 물질을 포함하는 검출가능한 신호를 발생시킬 수 있는 표지물질로 표지한 다음, 마이크로어레이 상에 혼성화하고 상기 표지물질로부터 발생하는 신호를 검출함으로써 혼성화 결과를 검출할 수 있다.In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection involves labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, such as a substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating a signal from the labeling substance. The hybridization result can be detected by detecting.
또한, 본 발명에서 상기 마커 유전자들로부터 코딩된 단백질의 발현수준을 측정하기 위한 키트는 항체의 면역학적 검출을 위하여 기재, 적당한 완충용액, 발색효소 또는 형광 물질로 표지된 2차 항체, 발색기질 등을 포함할 수 있다. 상기에서 기재로는 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96-웰 플레이트, 폴리스틸렌 수지로 합성된 96-웰 플레이트, 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소로는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있다. 또한, 형광물질로는 FITC, RITC 등이 사용될 수 있고, 발색기질로는 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)), OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘) 등이 사용될 수 있다.In addition, the kit for measuring the expression level of the protein encoded from the marker genes in the present invention is a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or fluorescent substance, chromogenic substrate, etc. for immunological detection of the antibody It may include. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, etc. may be used, and a peroxidase (peroxidase) may be used. ), Alkaline phosphatase and the like can be used. In addition, FITC, RITC, etc. may be used as the fluorescent substance, and ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid)) and OPD (o-phenyl) may be used as a chromogenic substrate. Rendiamine), TMB (tetramethyl benzidine) and the like can be used.
본 발명의 다른 견지로, 본 발명에 의하면 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법이 제공되며, 상기 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법은 분리된 개체의 시료로부터 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 RNA 발현수준을 FPKM 값으로 측정하는 단계; 및 2) 상기 유전자 중 FPKM 값이 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분하고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분하는 단계를 포함하는 것이다. In another aspect of the present invention, the present invention provides a method for providing information necessary for predicting the prognosis of a malignant frondoma, and the method for providing information necessary for predicting the prognosis of a malignant frondoma is provided by a sample of an isolated individual. Measuring the RNA expression levels of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2 from the FPKM values; And 2) classifying a fibrous subtype first subtype when there are 6 or more genes having a FPKM value of 1 or less, and dividing an epithelial subtype second subtype by less than 6 genes. will be.
나아가, 상기 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법은 섬유성(Fibrous subtype) 제1 아형으로 구분된 개체의 시료를 악성 엽상종의 재발 가능성이 높은 것으로 판정하는 단계를 추가로 포함할 수 있다. Furthermore, the method for providing the information necessary for predicting the prognosis of the malignant frond tumor further comprises the step of determining a sample of an individual classified into the fibrous subtype first subtype as having a high probability of recurrence of the malignant frond tumor. can do.
제1 섬유성(Fibrous) 아형은 제2 상피성(Epithelial) 아형에 비해 기존에 알려진 주요 예후 인자의 유사분열 인덱스(mitotic index)가 현저히 높으며, 환자의 재발을 추적하였을 때 섬유성(Fibrous) 아형에서 유의하게 재발이 많이 발생한다. The first fibrous subtype has a significantly higher mitotic index of known major prognostic factors than the second epithelial subtype, and the fibrous subtype when the patient's recurrence is tracked. Recurrences occur significantly in.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples are merely examples to help understanding of the present invention, but the scope of the present invention is not limited thereto.
실시예Example
1. 악성 1. malicious 엽상종의Frond 유전체적Genomic 특성 characteristic
본 발명자들은 생체시료저장소에 확보된 아홉 명의 악성 엽상종 환자에서 절제한 신선동결 종양조직을 이용하여 전장 엑솜 분석(whole exome sequencing)을 시행하였다. 유선세포에서 유래한 일반적인 유방암 조직 22개에서 생성한 유전체변이 데이터와 비교해 보았을 때 악성 엽상종 조직은 현저히 적은 유전체 체성변이 빈도를 보였다(0.56/Mb for MPT and 2.42/Mb for invasive ductal carcinoma, 도 1A alc 1B). Alexandrov et al(Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer. Nature 2013;500(7463):415-21 doi 10.1038/nature12477.)가 제시한 유전체변이 분류 중 악성 엽상종에서 가장 흔히 관찰되는 유전체 변이는 내재적인 DNA 손상과정에 의해 유발되는 C·G→ T·A 치환이었으며 일반적인 유방암에서 흔히 관찰되는 APOBEC 에디팅 관련 특징(editing-related signature)은 매우 낮은 빈도로 발견되는 점을 감안하면 악성 엽상종은 일반적인 유방암과는 전혀 다른 발병기전을 가지는 것을 유추할 수 있다(도 1C). The present inventors performed whole-length exome sequencing using fresh frozen tumor tissues excised from nine malignant frond tumor patients secured in a biological sample repository. Compared with genotyping data generated in 22 common breast cancer tissues derived from mammary cells, malignant filamentous tissues showed significantly less genotypic mutations (0.56 / Mb for MPT and 2.42 / Mb for invasive ductal carcinoma, FIG. 1A). alc 1B). Alexandrov et al (Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer.Nature 2013; 500 (7463): 415-21 doi 10.1038 / nature12477 Among the genotypes presented by.), The most common genotypes found in malignant fronds were C · G → T · A substitutions caused by intrinsic DNA damage processes, and APOBEC editing-related features commonly found in common breast cancers. Considering the fact that -related signatures are found at a very low frequency, it can be inferred that malignant filamentous tumors have a completely different pathogenesis than common breast cancer (FIG.
상기 아홉 케이스의 악성 엽상종 조직에서 관찰되는 유전체 구조변이와 유전자 체성변이의 빈도는 도 1D에 정리하였다. 아홉 케이스 중 네 케이스(44.4%)에서는 1q 개인(gain)을 관찰할 수 있었고, 여섯 케이스(66.7%)에서는 EGFR 증폭 또는 돌연변이(amplifications or mutations)를 관찰할 수 있었다. 또한 PIK3CA, PIK3R1, PDGFRA, PDGFRB, PTEN, 및 TP53 유전자의 체성변이 및 다양한 유전체 구조변화 역시 관찰할 수 있었다.The frequencies of genome structural and genetic somatic variations observed in the nine cases of malignant foliar tissues are summarized in FIG. 1D. Four of the nine cases (44.4%) were able to observe 1q gain, and six cases (66.7%) were able to observe EGFR amplifications or mutations. Somatic changes and various genome structural changes of the PIK3CA, PIK3R1, PDGFRA, PDGFRB, PTEN, and TP53 genes were also observed.
2. 악성 2. Malicious 엽상종의Frond 유전자 발현 특성 Gene expression characteristics
악성 엽상종의 유전자 발현 특성을 파악하기 위해 64명의 일반적 유방암 환자에서 확보한 64개의 유방암 조직 및 59개의 정상유방조직에 대해 전사체 시퀀싱(transcriptome sequencing)을 시행하였다. 일반적인 유방암 조직과 비교하였을 때 악성 엽상종은 독특한 유전자 발현 패턴을 보이는 것을 확인할 수 있었다(도 2A). ECM-수용체 상호작용, 국소 접착(focal adhesion) 및 PI3K-Akt 시그널링에 관여하는 유전자들이 악성 엽상종에서는 매우 빈번하게 발현 변화가 생기는 현상을 관찰하였으며(도 2B), 사이토스케이프 소프트에어(Cytoscape software)를 이용한 네트워크 분석에서도 ECM- 수용체 상호작용, PI3K-Akt 시그널링 경로, 단백질 분해와 흡수(digestion and absorption) 및 종양에서의 경로들(pathways in cancer) 네트워크의 이상이 유의하게 흔히 발생함을 확인할 수 있었다. To characterize the gene expression of malignant fronds, transcriptome sequencing was performed on 64 breast cancer tissues and 59 normal breast tissues obtained from 64 general breast cancer patients. Compared with the normal breast cancer tissues, it was confirmed that malignant fronds show a unique gene expression pattern (FIG. 2A). Genes involved in ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling were observed to have very frequent expression changes in malignant fronds (Fig. 2B). Cytoscape software was observed. The analysis of the networks used also showed that ECM-receptor interactions, PI3K-Akt signaling pathways, protein degradation and absorption, and pathways in cancer network abnormalities were significantly common.
3. 악성 3. Malicious 엽상종의Frond 분자생물학적 아형 분류 Molecular Biological Subtype Classification
본 발명자들은 감독되지 않는 계층적 클러스터링(Unsupervised hierarchical clustering) 기법으로 악성 엽상종 조직에 대한 유전자 발현 데이터 특성을 이용하여 분류하였을 때 놀랍게도 악성 엽상종을 두 종류의 아형으로 구분할 수 있었다 (도 3A).The inventors were able to distinguish between two types of subtypes by surprisingly classifying them using unexpressed hierarchical clustering techniques using gene expression data characteristics for malignant frond tissues (FIG. 3A).
제1 아형은 단백질 분해와 흡수(protein digestion and absorption), 엑손 가이던스(axon guidance), ECM-수용체 상호작용(ECM-receptor interaction), 국소 접착(focal adhesion), 그리고 다양한 콜라겐과 같은 스트로마-관련(stroma-related) 유전자의 발현 이상의 특징을 나타내었고(도 3B 및 3C), 제2 아형은 세포 대사(cell metabolism), 세포 부착 분자(cell adhesion molecules), 치밀이음(tight junction) 및 다수의 면역 관련 프로세스에 관여하는 유전자의 발현이 높고, CDH1, CLDN3, CLDN4, CLDN7, OCLN 과 같은 상피세포의 특성을 반영하는 유전자의 발현이 현저히 높음을 확인하였다(도 3D). The first subtype is protein digestion and absorption, axon guidance, ECM-receptor interaction, focal adhesion, and stromal-related (eg, various collagen). It was characterized by abnormal expression of stroma-related genes (FIGS. 3B and 3C), and the second subtype was cell metabolism, cell adhesion molecules, tight junctions, and many immune related It was confirmed that the expression of genes involved in the process was high, and the expression of genes reflecting the characteristics of epithelial cells such as CDH1, CLDN3, CLDN4, CLDN7, OCLN was significantly high (FIG. 3D).
이와 같은 유전자 발현의 특성을 반영하여 본 발명자들은 악성 엽상종을 두 가지 아형으로 분류할 수 있음을 확인하여, 제1 아형을 'Fibrous(섬유성)' 아형 그리고 제2 아형을 'Epithelial(상피성)' 아형으로 명명하였다. 이러한 분류는 그 자체로 섬유상피 종양의 양면적인 생물학적 특성을 반영할 수 있다.Reflecting the characteristics of this gene expression, the present inventors confirmed that malignant fronoma can be classified into two subtypes, so that the first subtype is 'Fibrous' subtype and the second subtype is 'Epithelial' (epithelial). Subtype). This classification may itself reflect the bilateral biological properties of fibroepithelial tumors.
이와 같은 개별 유전자 발현의 특성 외에도 본 발명자들은 추가적으로 섬유상피종양의 생물학적 특성에 영향을 미칠 수 있는 ECM-수용체 상호작용 경로(KEGG ID: hsa04512), 세포 부착 분자 경로(KEGG ID: hsa04514), EMT(epithelial mesenchymal transition)의 경로에 속하는 유전자의 총체적 발현 패턴을 분석하였으며 그 결과 위의 유전자 집단 전체를 이용한 감독되지 않는 클러스터링(Unsupervised clustering)에서도 동일한 아형의 분류가 가능한 것을 확인하였다 (도 3E). In addition to the characteristics of such individual gene expression, the present inventors further found that the ECM-receptor interaction pathway (KEGG ID: hsa04512), the cell adhesion molecule pathway (KEGG ID: hsa04514), and the EMT () may affect the biological properties of fibroepithelial tumors. The overall expression pattern of genes belonging to the pathway of epithelial mesenchymal transition) was analyzed and the results showed that the same subtypes could be classified even in unsupervised clustering using the entire gene group (FIG. 3E).
4. 본 발명에 따른 악성 4. Malicious according to the present invention 엽상종의Frond 아형 분류 Subtype classification
이처럼 유전체 발현 양상으로 악성 엽상종이 두 가지 아형으로 분류될 수 있음을 확인한 후 본 발명자들은 추가로 28 케이스의 악성 엽상종 환자의 파라핀 포매조직을 이용하여 전사체 시퀀싱(transcriptome sequencing)을 시행하였고 특히 21개의 유전자로 이루어진 유전자 발현 특성(signature)을 이용하여 악성 엽상종 환자의 아형 분류를 진행하였다(도 3F). After confirming that malignant fronoma can be classified into two subtypes by genome expression, the present inventors further performed transcriptome sequencing using paraffin embedded tissue of 28 cases of malignant fronoma. Subtype classification of malignant mesenchymal patients was performed using gene expression signature consisting of dog genes (FIG. 3F).
이처럼 유전자 발현 패턴을 기반으로 분류된 두 가지 악성 엽상종 아형은 다양한 임상병리적 특성에서도 두 아형간 뚜렷한 차이를 보였다. The two malignant frond subtypes classified based on gene expression patterns showed distinct differences between the two subtypes in various clinical and pathologic characteristics.
제1 섬유성(Fibrous) 아형은 제2 상피성(Epithelial) 아형에 비해 기존에 알려진 주요 예후 인자의 유사분열 인덱스(mitotic index)가 현저히 높았으며, 환자의 재발을 추적하였을 때 섬유성(Fibrous) 아형에서 유의하게 재발이 많이 발생함을 확인할 수 있었다. The first fibrous subtype has a significantly higher mitotic index of known major prognostic factors compared to the second epithelial subtype, and the fibrous subtype is associated with patient recurrence. Significant relapses occurred in subtypes.
이러한 결과를 토대로 본 발명자들은 악성 엽상종은 과거에 알려진 것처럼 하나의 질환이 아니라, 임상적 특성 및 치료성적 측면에서 뚜렷한 차이를 보이는 두 가지 아형으로 나뉘어질 수 있다는 사실을 도출하였다.Based on these results, the present inventors derived the fact that malignant filamentous tumors can be divided into two subtypes with distinct differences in clinical characteristics and therapeutic results, as opposed to a single disease.
보다 상세하게 하기 표 1은 1.에서 언급한 바와 같은 생체시료저장소에 확보된 아홉 명의 악성 엽상종 환자에서 절제한 신선동결 종양조직을 대상으로 하여 상기 각 유전자의 FPKM 값을 확인한 결과를 나타낸 것이다. In more detail, Table 1 shows the results of confirming the FPKM values of the respective genes in the fresh frozen tumor tissues resected from nine malignant frond tumor patients secured in the biological sample storage as described in 1.
[표 1]TABLE 1
Figure PCTKR2018007798-appb-I000001
Figure PCTKR2018007798-appb-I000001
하기 표 2 및 2-1은 상기 28 케이스의 악성 엽상종 환자의 파라핀 포매조직을 대상으로 하여 상기 각 유전자의 FPKM 값을 확인한 결과를 나타낸 것이다.Tables 2 and 2-1 below show the results of confirming the FPKM values of the respective genes in the paraffin-embedded tissues of the 28 cases of malignant fronoma.
[표 2] TABLE 2
Figure PCTKR2018007798-appb-I000002
Figure PCTKR2018007798-appb-I000002
[표 2-1] TABLE 2-1
Figure PCTKR2018007798-appb-I000003
Figure PCTKR2018007798-appb-I000003
한편, 이러한 아형의 분류는 상기 표 1, 2 및 2-1에서 확인할 수 있는 바와 같이 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 유전자 중 FPKM 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분되고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분될 수 있음을 확인하였다. On the other hand, the classification of these subtypes is FPKM 1 of the genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2, as can be seen in Tables 1, 2 and 2-1. When the following genes were 6 or more, it was confirmed that they could be classified into fibrous subtype first subtypes and less than 6 genes could be classified into epithelial subtype second subtypes.
5. 악성 5. Malicious 엽상종의Frond 환자 유래 이종이식 모델을 이용한 표적 치료 가능성의 확인 Confirmation of Targeted Treatment Potential Using Patient-derived Xenograft Model
본 연구에 이용된 9개의 악성엽상종 조직 중 한 환자의 조직(2014-0916)을 NSG 마우스에 이식하여 환자 유래 이종이식 모델을 수립할 수 있었다. A patient-derived xenograft model could be established by transplanting tissue from one patient (2014-0916) out of nine malignant mesenchymal tissues used in this study into NSG mice.
상기 종양은 이식 후 16주가 지나자 임상적으로 촉진가능한 종양을 형성하였으며 이 PDX 모델(MX-99)은 원발암의 조직학적 특성, 유전체 변이 종류, 그리고 유전자 발현 패턴을 잘 유지하였고(도 4A 및 4B), 특히 원발암과 PDX 종양 모두에서 PIK3R1 (M1fs) 및 PDGFRB (N666K)의 유전자변이를 동반하고 있는 것으로 나타났다(도 4C). PIK3R1은 PIK3CA의 기능을 조절하는 조절 서브유닛(regulatory subunit)이며 상피 세포에서 종양발생을 억제시키는 역할을 하는 것으로 알려져 있고, 이 유전자의 체성변이는 종양조직에서의 PI3K/Akt 시그널링의 활성화와 연관되어 있다. 악성 엽상종의 유전자발현 자료에서 PI3K/Akt/mTOR 시그널링의 활성도가 정상유방조직에 비해 현저히 변화가 생기는 것을 관찰할 수 있었다(도 2B). PDGFRB N666K 체성변이는 세포에서 종양발생을 유도하고 그 체성변이가 있더라도 이메티닙 메실레이트(imatinib mesylate)에 대한 반응성은 유지됨을 기존 연구에서 확인할 수 있었다. PDGFRB 역시 유전자발현 데이터에서 확인한 결과 악성 엽상종에서 그 발현이 상대적으로 높다는 것을 관찰하였고 특히 2014-0916 종양에서 가장 발현이 높은 현상을 관찰하였다 (도 4D). The tumors formed clinically palpable tumors 16 weeks after transplantation, and this PDX model (MX-99) maintained well the histological characteristics, genome variant types, and gene expression patterns of primary cancers (FIGS. 4A and 4B). ), Especially in both primary and PDX tumors, with mutations in PIK3R1 (M1fs) and PDGFRB (N666K) (FIG. 4C). PIK3R1 is a regulatory subunit that regulates the function of PIK3CA and is known to play a role in inhibiting tumorigenesis in epithelial cells. Somatic variation of this gene is associated with activation of PI3K / Akt signaling in tumor tissues. have. In gene expression data of malignant mesenchymal species, it was observed that the activity of PI3K / Akt / mTOR signaling was significantly changed compared to normal breast tissue (FIG. 2B). PDGFRB N666K somatic variation induces tumorigenesis in cells, and previous studies have shown that the somatic mutation maintains responsiveness to imatinib mesylate. PDGFRB was also confirmed in the gene expression data, it was observed that the expression is relatively high in malignant mesenchymal tumors, especially in 2014-0916 tumors observed the highest expression phenomenon (Fig. 4D).
이러한 결과를 토대로 본 발명자들은 듀얼 PI3K-mTOR 인히비터(PKI-587)와 PDGFRB 인히비터(이메티닙 메실레이트)의 종양억제 효과를 악성 엽상종 PDX 모델에서 확인해보기로 하였다. 이메티닙 메실레이트와 PKI-587 모두 악성 엽상종 PDX 모델에서 종양의 성장을 효과적으로 억제할 수 있었으며 특히 PKI-587이 더 높은 종양억제효과를 보였다(도 4E). 상기 약제들로 치료받은 종양을 절제하여 PDGFRB 및 PIK3R1의 공통된 다운스트림 분자(downstream molecule)인 Akt 및 mTOR의 활성도를 측정하였을 때 두 약제 모두 그 활성도를 억제함을 관찰할 수 있었고 PDGFR의 인산화 역시 이메티닙에 의해 효과적으로 억제되었다(도 4F 및 4H). 따라서 악성 엽상종의 유전체적 특성에 입각한 표적치료제의 효과를 확인할 수 있었다.Based on these results, we determined the tumor suppression effects of dual PI3K-mTOR inhibitors (PKI-587) and PDGFRB inhibitors (imetinib mesylate) in the malignant foliar PDX model. Imetinib mesylate and PKI-587 were both able to effectively inhibit tumor growth in malignant foliar PDX model, especially PKI-587 showed higher tumor suppression effect (FIG. 4E). When the tumors treated with the drugs were resected and the activities of Akt and mTOR, which are common downstream molecules of PDGFRB and PIK3R1, were measured, it was observed that both drugs inhibited the activity. It was effectively inhibited by methinib (FIGS. 4F and 4H). Therefore, the effects of targeted therapies based on the genomic characteristics of malignant fronds were confirmed.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.
(1) CDH1 (1) CDH1
유전자명: cadherin 1Gene name: cadherin 1
위치: chr16:68771193-68869445Location: chr16: 68771193-68869445
Log2FC: 8.50Log2FC: 8.50
Adjusted P.vaule: 3.3E-48Adjusted P.vaule: 3.3E-48
NCBI SEQ ID NO,: NG_008021.1NCBI SEQ ID NO ,: NG_008021.1
(2) SOX10(2) SOX10
유전자명: SRY-box 10Gene name: SRY-box 10
위치: chr22:38368319-38380539Location: chr22: 38368319-38380539
Log2FC: 7.58Log2FC: 7.58
Adjusted P.vaule: 3.8E-31Adjusted P.vaule: 3.8E-31
NCBI SEQ ID NO,: NM_006941.3NCBI SEQ ID NO ,: NM_006941.3
(3) TACSTD2(3) TACSTD2
유전자명: tumor-associated calcium signal transducer 2Gene name: tumor-associated calcium signal transducer 2
위치: chr1:59041095-59043166Location: chr1: 59041095-59043166
Log2FC: 7.52Log2FC: 7.52
Adjusted P.vaule: 1.6E-99Adjusted P.vaule: 1.6E-99
NCBI SEQ ID NO,: NM_002353.2NCBI SEQ ID NO ,: NM_002353.2
(4) KRT5(4) KRT5
유전자명: keratin 5Gene name: keratin 5
위치: chr12:52908359-52914243Location: chr12: 52908359-52914243
Log2FC: 7.51Log2FC: 7.51
Adjusted P.vaule: 3.2E-39Adjusted P.vaule: 3.2E-39
NCBI SEQ ID NO,: NM_000424.3NCBI SEQ ID NO ,: NM_000424.3
(5) BBOX1(5) BBOX1
유전자명: gamma-butyrobetaine hydroxylase 1Gene name: gamma-butyrobetaine hydroxylase 1
위치: chr11:27062509-27149354Location: chr11: 27062509-27149354
Log2FC: 7.47Log2FC: 7.47
Adjusted P.vaule: 3.0E-19Adjusted P.vaule: 3.0E-19
NCBI SEQ ID NO,: NM_003986.2NCBI SEQ ID NO ,: NM_003986.2
(6) COL17A1(6) COL17A1
유전자명: collagen type XVII alpha 1 chainGene name: collagen type XVII alpha 1 chain
위치: chr10:105791046-105845638Location: chr10: 105791046-105845638
Log2FC: 7.41Log2FC: 7.41
Adjusted P.vaule: 3.2E-109Adjusted P.vaule: 3.2E-109
NCBI SEQ ID NO,: NM_000494.3NCBI SEQ ID NO ,: NM_000494.3
(7) PRSS8(7) PRSS8
유전자명: serine protease 8Gene name: serine protease 8
위치: chr16:31142754-31147083Location: chr16: 31142754-31147083
Log2FC: 7.38Log2FC: 7.38
Adjusted P.vaule: 1.9E-27Adjusted P.vaule: 1.9E-27
NCBI SEQ ID NO,: NM_002773.4NCBI SEQ ID NO ,: NM_002773.4
(8) PRR15L(8) PRR15L
유전자명: proline rich 15 likeGene name: proline rich 15 like
위치: chr17:46029334-46035243Location: chr17: 46029334-46035243
Log2FC: 7.34Log2FC: 7.34
Adjusted P.vaule: 7.3E-33Adjusted P.vaule: 7.3E-33
NCBI SEQ ID NO,: NM_024320.3NCBI SEQ ID NO ,: NM_024320.3
(9) ERBB3(9) ERBB3
유전자명: erb-b2 receptor tyrosine kinase 3Gene name: erb-b2 receptor tyrosine kinase 3
위치: chr12:56473809-56497291Location: chr12: 56473809-56497291
Log2FC: 6.59Log2FC: 6.59
Adjusted P.vaule: 1.5E-36Adjusted P.vaule: 1.5E-36
NCBI SEQ ID NO,: NG_011529.1NCBI SEQ ID NO ,: NG_011529.1
(10) KRT14(10) KRT14
유전자명: keratin 14Gene name: keratin 14
위치: chr17:39738531-39743147Location: chr17: 39738531-39743147
Log2FC: 6.51Log2FC: 6.51
Adjusted P.vaule: 6.2E-15Adjusted P.vaule: 6.2E-15
NCBI SEQ ID NO,: NM_000526.4NCBI SEQ ID NO ,: NM_000526.4
(11) RAB25 (11) RAB25
유전자명: RAB25, member RAS oncogene familyGene name: RAB25, member RAS oncogene family
위치: chr1:156030940-156040305Location: chr1: 156030940-156040305
Log2FC: 6.39Log2FC: 6.39
Adjusted P.vaule: 8.2E-38Adjusted P.vaule: 8.2E-38
NCBI SEQ ID NO,: NM_020387.3NCBI SEQ ID NO ,: NM_020387.3
(12) AGR2(12) AGR2
유전자명: anterior gradient 2, protein disulphide isomerase family memberGene name: anterior gradient 2, protein disulphide isomerase family member
위치: chr7:16832264-16844738Location: chr7: 16832264-16844738
Log2FC: 6.32Log2FC: 6.32
Adjusted P.vaule: 1.1E-16Adjusted P.vaule: 1.1E-16
NCBI SEQ ID NO,: NM_006408.3NCBI SEQ ID NO ,: NM_006408.3

Claims (14)

  1. CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 유전자를 바이오 마커로 포함하는, 악성 엽상종 예후 예측용 조성물.A composition for predicting malignant foliar prognosis comprising a gene of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2 as a biomarker.
  2. 제1항에 있어서, 상기 유전자는 서열번호 1 내지 서열번호 12의 염기서열로 이루어진, 악성 엽상종 예후 예측용 조성물.According to claim 1, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 12, composition for predicting the prognosis of malignant frond species.
  3. 제1항에 있어서, 상기 유전자의 RNA 또는 이들의 유전자에 의해 코딩되는 단백질의 발현수준을 측정하는 제제를 추가로 포함하는, 악성 엽상종 예후 예측용 조성물.The composition of claim 1, further comprising an agent for measuring the expression level of RNA of the gene or a protein encoded by the gene thereof.
  4. 제3항에 있어서, 상기 RNA의 발현수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 포함하는, 악성 엽상종 예후 예측용 조성물.The composition of claim 3, wherein the agent for measuring the expression level of RNA comprises an antisense oligonucleotide, a primer pair, or a probe specifically binding to the gene.
  5. 제3항에 있어서, 상기 단백질의 발현수준을 측정하는 제제는 상기 유전자로부터 코딩되는 단백질에 특이적인 항체인, 악성 엽상종 예후 예측용 조성물.According to claim 3, wherein the agent for measuring the expression level of the protein is a composition specific for predicting malignant frond prognosis, the antibody specific for the protein encoded from the gene.
  6. 제1항에 있어서, 상기 조성물은 상기 유전자 중 FPKM 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분하고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분하여 악성 엽상종의 예후를 예측하기 위한 것인, 악성 엽상종 예후 예측용 조성물.The method of claim 1, wherein the composition is classified into a fibrous subtype first subtype when there are six or more genes having FPKM 1 or less in the gene, and is classified into an epithelial subtype second subtype when less than six. It is for predicting the prognosis of malignant frondoma, The composition for predicting malignant frond prognosis.
  7. 제1항에 있어서, 상기 조성물은 2년 후 악성 엽상종이 재발할 가능성을 예측하기 위한 것인, 악성 엽상종 예후 예측용 조성물.According to claim 1, wherein the composition is for predicting the possibility of recurrence of malignant frondoma after 2 years, malignant frond tumor prognostic composition.
  8. 제1항 내지 제7항 중 어느 한 항의 조성물을 포함하는, 악성 엽상종 예후 예측용 키트.A kit for predicting malignant frond tumor prognosis comprising the composition of any one of claims 1 to 7.
  9. 제8항에 있어서, 상기 키트는 RT-PCR 키트, 마이크로어레이 칩 키트 또는 단백질 칩 키트인, 악성 엽상종 예후 예측용 키트.The kit of claim 8, wherein the kit is an RT-PCR kit, a microarray chip kit, or a protein chip kit.
  10. 제8항에 있어서, 상기 RT-PCR 키트가 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 12개 유전자에 특이적인 프라이머 쌍을 포함하는, 악성 엽상종 예후 예측용 키트.The malignant foliar of claim 8, wherein the RT-PCR kit comprises primer pairs specific for 12 genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2. Species prediction kit.
  11. 제8항에 있어서, 상기 마이크로어레이 칩 키트가 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 12개 유전자에 특이적인 프로브를 포함하는, 악성 엽상종 예후 예측용 키트.The malignant frondoma according to claim 8, wherein the microarray chip kit comprises probes specific for 12 genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 and AGR2. Prognostic Kit.
  12. 제8항에 있어서, 상기 단백질 칩 키트가 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 12개 유전자로부터 코딩되는 단백질에 특이적인 항체를 포함하는, 악성 엽상종 예후 예측용 키트.The protein chip kit of claim 8, wherein the protein chip kit comprises an antibody specific for a protein encoded from 12 genes of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2. Kit for predicting malignant frond prognosis.
  13. 분리된 개체의 시료로부터 CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 및 AGR2의 RNA 발현수준을 FPKM 값으로 측정하는 단계; 및Measuring RNA expression levels of CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25, and AGR2 from the samples of the isolated individuals by FPKM values; And
    2) 상기 유전자 중 FPKM 값이 1 이하인 유전자가 6개 이상일 때 섬유성(Fibrous subtype) 제1 아형으로 구분하고, 6개 미만일 때 상피성(Epithelial subtype) 제2 아형으로 구분하는 단계2) dividing the gene into a fibrous subtype first subtype when there are 6 or more genes having a FPKM value of 1 or less, and dividing it into an epithelial subtype second subtype when less than 6 genes
    를 포함하는, 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법.A method for providing information necessary for predicting the prognosis of malignant frondoma, comprising.
  14. 제13항에 있어서, 섬유성(Fibrous subtype) 제1 아형으로 구분된 개체의 시료를 악성 엽상종의 재발 가능성이 높은 것으로 판정하는 단계를 추가로 포함하는, 악성 엽상종의 예후를 예측하는데 필요한 정보를 제공하는 방법.The method of claim 13, further comprising determining a sample of an individual classified into a fibrous subtype first subtype as having a high likelihood of recurrence of malignant frond tumor. How to give it.
PCT/KR2018/007798 2018-07-10 2018-07-10 Composition for predicting prognosis of malignant phyllodes tumor and kit comprising same WO2020013355A1 (en)

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