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WO2020013355A1 - Composition pour prédire le pronostic d'une tumeur phyllode maligne et kit la comprenant - Google Patents

Composition pour prédire le pronostic d'une tumeur phyllode maligne et kit la comprenant 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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Korean (ko)
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문형곤
윤지희
김종일
노동영
유태경
이은신
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SNU R&DB Foundation
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Seoul National University R&DB Foundation
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    • 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).

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  • Oncology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention porte sur une composition pour prédire le pronostic d'une tumeur phyllode maligne et un kit la comprenant. Plus spécifiquement, la présente invention concerne : une composition pour prédire le pronostic d'une tumeur phyllode maligne, contenant les gènes CDH1, SOX10, TACSTD2, KRT5, BBOX1, COL17A1, PRSS8, PRR15L, ERBB3, KRT14, RAB25 et AGR2 en tant que biomarqueurs ; un kit pour prédire le pronostic d'une tumeur phyllode maligne, comprenant la composition ; et un procédé, destiné à fournir des informations requises pour la prédiction de pronostic d'une tumeur phyllode maligne, utilisant la composition.
PCT/KR2018/007798 2018-07-10 2018-07-10 Composition pour prédire le pronostic d'une tumeur phyllode maligne et kit la comprenant Ceased WO2020013355A1 (fr)

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PCT/KR2018/007798 WO2020013355A1 (fr) 2018-07-10 2018-07-10 Composition pour prédire le pronostic d'une tumeur phyllode maligne et kit la comprenant

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PCT/KR2018/007798 WO2020013355A1 (fr) 2018-07-10 2018-07-10 Composition pour prédire le pronostic d'une tumeur phyllode maligne et kit la comprenant

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WO2020013355A1 true WO2020013355A1 (fr) 2020-01-16

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065583A2 (fr) * 2003-01-15 2004-08-05 Genomic Health, Inc. Marqueurs d'expression genique pour le pronostic du cancer du sein
WO2005123945A2 (fr) * 2004-06-21 2005-12-29 Epigenomics Ag Marqueurs epigenetiques destines au traitement du cancer du sein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065583A2 (fr) * 2003-01-15 2004-08-05 Genomic Health, Inc. Marqueurs d'expression genique pour le pronostic du cancer du sein
WO2005123945A2 (fr) * 2004-06-21 2005-12-29 Epigenomics Ag Marqueurs epigenetiques destines au traitement du cancer du sein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENG, J. M.: "Loss of RAB25 expression in breast cancer", INT. J. CANCER, 15 June 2006 (2006-06-15), pages 2957 - 2964, XP055675601 *
SALMANS M. L.: "The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker", BREAST CANCER RES, 24 April 2013 (2013-04-24), XP055675588 *
ZHANG, Q.: "Differentially expressed mitochondrial genes in breast cancer cells: Potential new targets for anti-cancer therapies", GENE, vol. 596, 6 October 2016 (2016-10-06), pages 42 - 52, XP029805506 *

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