KR102448586B1 - Biomarker for predicting the response of a patient with pancreatic cancer to anti-cancer drugs - Google Patents

Abstract

The present invention relates to a biomarker for predicting anticancer drug reactivity, and more specifically, a marker composition for predicting anticancer drug reactivity comprising a specific gene or a protein encoding the same in a pancreatic cancer patient, a composition for predicting anticancer drug treatment reactivity, predicting anticancer drug reactivity It relates to a kit for use, and a method for providing information for predicting anticancer drug treatment responsiveness. As a result of analyzing the protein in exosomes isolated from the plasma of pancreatic cancer patients, the present inventors found that the sensitivity to the therapeutic effect of an anticancer agent, specifically, a combination of gemcitabine and nappaclitaxel, was significantly high depending on the expression level of a specific gene. It is expected that it will be usefully used for predicting the effectiveness of the therapeutic effect of anticancer drugs in pancreatic cancer patients.

Description

Marker for predicting chemotherapy reactivity of pancreatic cancer patients {Biomarker for predicting the response of a patient with pancreatic cancer to anti-cancer drugs}

The present invention relates to a biomarker for predicting anticancer drug reactivity, and more specifically, a marker composition for predicting anticancer drug reactivity comprising a specific gene or a protein encoding the same in a pancreatic cancer patient, a composition for predicting anticancer drug treatment reactivity, predicting anticancer drug reactivity It relates to a kit for use, and a method for providing information for predicting anticancer drug treatment responsiveness.

Pancreatic cancer is a malignant tumor originating from the pancreas, and it is known that the 5-year survival rate is less than 10% because most patients are found to have advanced cancer. Although the incidence of pancreatic cancer in Korea ranks eighth, cancer-related deaths account for just after lung cancer, liver cancer, stomach cancer, and colorectal cancer. Symptoms of pancreatic cancer include symptoms found in various pancreatic diseases, and abdominal pain, loss of appetite, weight loss, and jaundice are the most common symptoms. Jaundice occurs in most cases.

Currently, well-known anticancer agents for the treatment of pancreatic cancer are known as gemcitabine, 5-fluorouracil (5-FU), and the like. Treatment with these anticancer drugs is observed by observing the therapeutic response, and if the drug reactivity is good, it is continued, and if there is no drug reactivity, the anticancer drug is changed. In addition, the response to chemotherapy varies among patients with similar clinical characteristics, and even in patients with the same stage, there is a significant difference in patient survival. Moreover, the therapeutic response to anticancer drugs varies from individual to individual, so the concept of personalized treatment in which an anticancer drug is selected and used according to a patient's genetic status is emerging.

Korean Patent No. 2100557 discloses a method for predicting the therapeutic reactivity of oxaliplatin by detecting the expression level of SLC22A18 in colorectal cancer patients, and Korean Patent No. 1601940 discloses KDM1B (Lysine-Specific Demethylase 1B) Disclosed is a composition for predicting the responsiveness of an ovarian cancer patient to an anticancer drug treatment by detecting the expression level of .

However, the development of markers capable of predicting the reactivity of anticancer treatment in pancreatic cancer patients is still insufficient.

In order to solve the above problems, the present inventors collected plasma from normal persons and pancreatic cancer patients in order to discover gene markers that can predict the chemotherapy reactivity in pancreatic cancer patients, and separated exosomes from them to prepare exosome proteins. As a result of the analysis, it was confirmed that there is a correlation between the expression level of a specific gene and the anticancer drug reactivity, and based on this, the present invention was completed.

Accordingly, the present invention provides a gene comprising KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2) It provides a marker composition for predicting the anticancer drug response of a pancreatic cancer patient, comprising a protein encoded by the mRNA or the gene.

In addition, the present invention includes KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2). It provides a composition for predicting an anticancer drug response of a pancreatic cancer patient, and a kit for predicting an anticancer drug response of a pancreatic cancer patient comprising the composition, comprising an agent for measuring the level of mRNA or protein encoded by the gene.

In addition, in the present invention, KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277) in specimen-derived biological samples. 2) It provides an information providing method for predicting the anticancer drug response of pancreatic cancer, comprising the step of measuring the level of the mRNA of the gene or the protein encoded from the gene.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2) provides a marker composition for predicting an anticancer drug response of a pancreatic cancer patient, comprising the mRNA of the gene or the protein encoded by the gene.

In one embodiment of the present invention, the marker composition is MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2, NM_001354643. 1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated) membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit) mRNA of one or more genes selected from the group consisting of beta type-2; NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) may include more.

In another embodiment of the present invention, the anticancer agent may be gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel).

In addition, the present invention includes KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2). It provides a composition for predicting an anticancer drug response of a pancreatic cancer patient, and a kit for predicting an anticancer drug response of a pancreatic cancer patient comprising the same, comprising an agent for measuring the mRNA level of a gene or a protein level encoded by the gene.

In one embodiment of the present invention, the composition for predicting the anticancer drug response of the pancreatic cancer patient is MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2, NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP2) -2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3) ), PSMB2 (Proteasome subunit beta type-2; NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) mRNA of one or more genes selected from the group consisting of It may further comprise an agent for measuring the level of the protein encoded by the gene.

In another embodiment of the present invention, the anticancer agent may be gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel).

In addition, the present invention provides KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277) in a subject-derived biological sample. .2) provides an information providing method for predicting the anticancer drug response of pancreatic cancer, comprising the step of measuring the level of the mRNA of the gene or the protein encoded from the gene.

In one embodiment of the present invention, the method comprises MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2, NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome) -associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 ( mRNA of one or more genes selected from the group consisting of proteasome subunit beta type-2; NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) It may further comprise the step of measuring the protein level.

In another embodiment of the present invention, the anticancer agent may be gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel).

In another embodiment of the present invention, the biological sample may be exosomes derived from blood or plasma.

In another embodiment of the present invention, the mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction ( Real-time PCR), RNase protection assay (RPA), microarray, and northern blotting (northern blotting) can be measured through one or more methods selected from the group consisting of.

In another embodiment of the present invention, the protein level is determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. , flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and at least one method selected from the group consisting of a protein chip can be measured through

As a result of analyzing the protein in exosomes isolated from the plasma of pancreatic cancer patients, the present inventors found that the sensitivity to the therapeutic effect of an anticancer agent, specifically, a combination of gemcitabine and nappaclitaxel, was significantly high depending on the expression level of a specific gene. It is expected that it will be usefully used for predicting the effectiveness of the therapeutic effect of anticancer drugs in pancreatic cancer patients.

1 to 13 are analyzes of proteins in exosomes isolated from the plasma of pancreatic cancer patients administered with anticancer drugs, the bars shown in the figure represent the quantitative protein amount of each sample, among which the blue bar represents the average value of each group sample and standard deviation is shown.
1 is an analysis of the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, and the expression level of KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3) was confirmed according to drug reactivity. the results are shown.
Figure 2 shows the result of confirming the expression level of GSDMA (Gasdermin-A; NCBI accession number: NM_178171.5) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent. will be.
3 shows the results of confirming the expression level of LACRT (Extracellular glycoprotein lacritin; NCBI accession number: NM_033277.2) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent. will be.
4 shows the results of confirming the expression level of MMP9 (Matrix metalloproteinase-9; NCBI accession number: NM_004994.3) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent. it has been shown
5 is an analysis of the proteins in the exosomes isolated from the plasma of pancreatic cancer patients administered with anticancer drugs, according to drug reactivity, MME (Neprilysin; NCBI accession number: NM_000902.5, NM_007288.3, NM_007289.4, NM_007287 .4, NM_001354642.2, NM_001354643.1) shows the results of checking the expression level.
Figure 6 shows the results of confirming the expression level of LYZ (Lysozyme C; NCBI accession number: NM_000239.3) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of pancreatic cancer patients administered with the anticancer agent. .
7 is an analysis of the protein in the exosomes isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, according to drug reactivity, GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NCBI accession) number: NM_001007240.3, NM_001502.4 , NM_001007241.3, NM_001007242.3) shows the results of checking the expression level.
8 is an analysis of the protein in the exosomes isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, according to drug reactivity, LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NCBI accession number: NM_001122606.1 ) shows the results of checking the expression level.
9 shows the results of confirming the expression level of CTSD (Cathepsin D; NCBI accession number: NM_001909.5) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent. .
10 is an analysis of the protein in exosomes isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, and the expression level of CD81 (CD81 antigen; NCBI accession number: NM_004356.4, NM_001297649.2) according to drug reactivity was confirmed. the results are shown.
11 is a result of confirming the expression level of LAMB1 (Laminin subunit beta-1; NCBI accession number: NM_002291.3) according to drug reactivity by analyzing the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent. is shown.
12 is an analysis of the proteins in the exosomes isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, and PSMB2 (Proteasome subunit beta type-2; NCBI accession number: NM_002794.5, NM_001199779.2, NM_001199780.2) shows the result of checking the expression level.
13 is an analysis of the protein in the exosome isolated from the plasma of a pancreatic cancer patient administered with an anticancer agent, and the expression level of AZGP1 (Zinc-alpha-2-glycoprotein; NCBI accession number: NM_001185.4) according to drug reactivity. The confirmed results are shown.

The present inventors collected plasma from normal persons and pancreatic cancer patients to discover gene markers that can predict chemotherapy reactivity in pancreatic cancer patients, and separated exosomes from them to analyze exosome proteins. As a result, expression of specific genes It was confirmed that there is a correlation between the level and the anticancer drug reactivity, and based on this, the present invention was completed.

Accordingly, the present invention includes KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2). It provides a marker composition for predicting the anticancer drug response of a pancreatic cancer patient, comprising the mRNA of the gene or the protein encoded by the gene.

In the present invention, the marker composition is MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2, NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2 ; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit beta type- 2; mRNA of one or more genes selected from the group consisting of NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) or a protein encoded by the gene. can

As used herein, the term “pancreatic cancer” refers to an aggressive characteristic in which pancreatic cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and a metastatic (metastatic) characteristic that spreads to other parts of the body Metastatic) refers to diseases caused by cells with characteristics.

In the present invention, the term “marker for predicting anticancer drug response” is a substance used to predict whether anticancer drug administration can be useful for the treatment of cancer before administration, and is used to predict reactivity to anticancer drug by measuring its expression level do. Such markers may include organic biomolecules such as nucleic acids, polypeptides, proteins, lipids or sugars, and the like. For the purpose of the present invention, the marker for predicting anticancer drug treatment reactivity is a nucleic acid or polypeptide marker that can predict anticancer drug treatment reactivity in pancreatic cancer patients.

In the present invention, the anticancer agent may be gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel), but is not limited thereto.

The present inventors have identified the use of a specific gene according to the present invention as a novel marker for predicting the prognosis of anticancer drug responsiveness in pancreatic cancer through specific examples.

In an embodiment of the present invention, specific genes (KRT73, GSDMA, LACRT, MMP9, MME, LYZ, GP2, LAMP2, CTSD, CD81, LAMB1, It was confirmed that the expression of PSMB2 and AZGP1) was significantly increased (see Example 2).

Through the above results, specific genes (KRT73, GSDMA, LACRT, MMP9, MME, LYZ, GP2, LAMP2, CTSD, CD81, LAMB1, PSMB2, and AZGP1) or proteins encoded by the genes are used for predicting anticancer drug reactivity in pancreatic cancer patients. It can be inferred that it can be used as a marker.

As another aspect of the present invention, the present invention is KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277. 2) It provides a kit for predicting the anticancer drug response of a pancreatic cancer patient, including a composition for predicting an anticancer drug response of a pancreatic cancer patient, and a composition for predicting an anticancer drug response in a pancreatic cancer patient, including an agent for measuring the mRNA or protein level of the gene comprising the gene .

In the present invention, the composition for predicting the anticancer drug response of the pancreatic cancer patient is MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2) , NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 mRNA of one or more genes selected from the group consisting of (Proteasome subunit beta type-2; NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) or encoded from the gene It may further include an agent for measuring the protein level.

The kit for prediction of the present invention consists of one or more other component compositions, solutions or devices suitable for the analysis method.

For example, the kit of the present invention contains genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of a DNA polymerase, a dNTP mixture, a PCR buffer, and water to perform PCR. It may be a kit comprising The PCR buffer may contain KCl, Tris-HCl and MgCl ₂ . In addition, components necessary for performing electrophoresis that can confirm whether the PCR product is amplified may be additionally included in the kit of the present invention.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included. In addition, a primer pair specific for a gene used as a quantitative control may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA corresponding to a gene or fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative structural gene or fragment thereof. In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

In addition, the kit of the present invention may be a kit characterized in that it includes essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for a marker protein, and an agent for measuring the protein level. The ELISA kit may include a reagent capable of detecting an antibody that has formed an "antigen-antibody complex", for example, a labeled secondary antibody, chromopores, an enzyme, and a substrate thereof. In addition, an antibody specific for the quantitative control protein may be included.

As another aspect of the present invention, the present invention provides KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT in a subject-derived biological sample. (Extracellular glycoprotein lacritin; NM_033277.2) It provides an information providing method for predicting an anticancer drug response in pancreatic cancer, including measuring the level of mRNA of a gene or a protein encoded from the gene.

In the present invention, the method is MMP9 (Matrix metalloproteinase-9; NM_004994.3), MME (Neprilysin; NM_000902.5, NM_007288.3, NM_007289.4, NM_007287.4, NM_001354642.2, NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3, NM_001502.4, NM_001007241.3, NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4, NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit beta type-2) Measuring the mRNA level of one or more genes selected from the group consisting of NM_002794.5, NM_001199779.2, NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) or a protein level encoded by the gene may further include.

The term "information providing method for predicting anticancer drug response of pancreatic cancer" used in the present invention is a preliminary step for predicting whether anticancer drug administration can be useful for the treatment of cancer. of clinical judgment or findings are excluded.

The subject-derived biological sample may include tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, preferably blood or plasma, and more preferably plasma-derived exosome. However, the present invention is not limited thereto.

The mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray (microarray), and may be measured through one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.

The protein level was determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, and immunohistochemistry. It may be measured through one or more methods selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip, but is limited thereto it's not going to be

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Specific protein analysis of exosomes in plasma

1-1. Exosome sample preparation

Blood from normal and pancreatic cancer patients is collected in BD Vacutainer Plastic K2EDTA Tube (BD, Cat.no 367525), and plasma is separated by centrifugation within 2 hours at 2,500 rpm and 4°C for 20 minutes. The supernatant plasma was aliquoted into Protein LoBind Tubes (Eppendorf, Cat.no 0030108116) by 500 μl and stored at -80°C.

Plasma samples were centrifuged at 3,000 g and 4 °C for 15 minutes to remove cells and cell debris in the plasma. Then, 250 μl of Exo2D ^TM for Protein assay (EXOSOMEplus) was added to 500 μl of plasma sample at a ratio of 2:1 to isolate exosomes based on the Aqueous Two-Phase System, Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-Free ( The exosome samples were prepared by resuspending the exosomes in 200 μl of PBS with the addition of Thermo scientific). Samples were stored at -80 °C until analysis.

1-2. Exosome protein quantitative analysis

30 μl of RIPA buffer containing protease and phosphatase (1X) was added to 30 μl of the plasma exosome sample prepared in Example 1-1, mixed sufficiently, and then centrifuged. The extracted exosome protein was subjected to concentration measurement and quantitative analysis using Bovine Serum Albumin (BSA) Standard Set (BIO-RAD) and Pierce ^TM BCA Protein Assay (Thermo scientific).

Then, 20 μg of exosomal protein was mixed with SDS loading buffer and electrophoresed using 10% Sodium Dodecyl Sulfate Polyacrylamide Gel Eletrophoresis (SDS-PAGE) technique to separate proteins according to molecular weight size. After immersing the electrophoresed gel in InstantBlue™ (Coomassie blue stanning) reagent and staining at room temperature for 15 minutes, protein detection and distribution were first checked.

1-3. Protein digestion using S-Trap™ Mini Spin Column (ProtiFi)

(1) 5% SDS, 50 mM TAEB (pH7.55), and 1x PPIC were added to the sample and dissolved. The pH value was adjusted using phosphoric acid.

(2) 300 μg of a protein sample quantified using BCA assay was taken. DTT was added to the protein to be digested to a final concentration of 20 mM, followed by alkylation at 95 °C for 10 minutes.

(3) After cooling at room temperature, IAA was added to a final concentration of 40 mM and reduced for 30 minutes in the dark. Then, 12% phosphoric acid was mixed to make 1.2% phosphoric acid.

(4) 350 μl of S-Trap binding buffer (90% methanol, 100 mM TEAB (pH 7.1)) was added, and all were transferred to an S-Trap spin column. Then, centrifugation was performed at 4,000 g for 30 seconds using a centrifuge.

(5) 400 μl of S-Trap buffer was added to the S-Trap spin column and centrifuged at 4,000 g for 30 seconds using a centrifuge. This was repeated 3 times.

(6) Transfer the S-Trap spin column to a new 2 ml sample tube, dissolve the protein and trypsin in a 1:25 ratio in 125 μl of 50 mM TEAB buffer, add it to the S-Trap spin column, and incubate at 37°C for 16 hours. did.

(7) 80 μl of 50 mM TEAB buffer was added and centrifuged at 1,000 g for 60 seconds. Then, 80 μl of 0.2% FA was added and centrifuged at 1,000 g for 60 seconds. Finally, 80 μl of elution buffer (50% ACN, 0.2% FA) was added, centrifuged at 4,000 g for 60 seconds, and the eluted sample was freeze-dried.

1-4. Proteomics analysis

Protein peptide samples prepared through the digestion process were analyzed for proteomic bodies using Q Exactive Plus LC-MS/MS System mass spectrometer (Thermo Scientific). Each sample was measured in duplicate, and the data generated by the instrument was analyzed using Proteome Discoverer ^TM 2.2 (Thermo Scientific) software for database search, comprehensive protein and peptide identification, characterization and quantitative analysis and statistical analysis. proceeded.

Example 2. Confirmation of specific gene expression according to anticancer drug reactivity in pancreatic cancer patients

Plasma was obtained from 4 patients with pancreatic cancer (2 patients with good reactivity and 2 patients with poor reactivity) administered in combination with gemcitabine and lead paclitaxel, and plasma exosomal protein was extracted through the method of Example 1, followed by anticancer drug Genes showing differences in expression were analyzed according to their reactivity.

As a result, as shown in FIGS. 1 to 13, the expression of KRT73, GSDMA, LACRT, MMP9, MME, LYZ, GP2, LAMP2, CTSD, CD81, LAMB1, PSMB2 and AZGP1 genes was significant only in the sample of the patient group showing good anticancer drug reactivity. It was confirmed that there was a positive increase.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (13)

mRNA of a gene comprising KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2) or the above As a marker composition for predicting anticancer drug response of a patient with degenerative pancreatic cancer, comprising a protein encoded by the gene,
The anticancer agent is gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel), characterized in that the marker composition. According to claim 1,
The marker composition is MMP9 (Matrix metalloproteinase-9; NCBI accession number: NM_004994.3), MME (Neprilysin; NM_000902.5), MME (NM_007288.3), MME (NM_007289.4), MME (NM_007287). 4), MME (NM_001354642.2), MME (NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3), GP2 (NM_001502.4), GP2 (NM_001007241.3), GP2 (NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4) ), CD81 (NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit beta type-2; NM_002794.5), PSMB2 (NM_001199779.2), PSMB2 (NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) of one or more genes selected from the group consisting of mRNA or a protein encoded by the gene, characterized in that it further comprises a marker composition. delete mRNA of a gene comprising KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2) or the above As a composition for predicting the anticancer drug response of a patient with degenerative pancreatic cancer, comprising an agent for measuring the protein level encoded by the gene,
The anticancer agent is gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel), characterized in that the composition. 5. The method of claim 4,
The composition is MMP9 (Matrix metalloproteinase-9; NCBI accession number: NM_004994.3), MME (Neprilysin; NM_000902.5), MME (NM_007288.3), MME (NM_007289.4), MME (NM_007287.4) ), MME (NM_001354642.2), MME (NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3), GP2 (NM_001502.4), GP2 ( NM_001007241.3), GP2 (NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4) , CD81 (NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit beta type-2; NM_002794.5), PSMB2 (NM_001199779.2), PSMB2 (NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) Anticancer drug response of a patient with degenerative pancreatic cancer, characterized in that it further comprises an agent for measuring the mRNA of one or more genes selected from the group consisting of or a protein level encoded from the gene Predictive composition. delete A kit for predicting the anticancer drug response of a patient with degenerative pancreatic cancer, comprising the composition of claim 4 . KRT73 (Keratin, type II cytoskeletal 73; NCBI accession number: NM_175068.3), GSDMA (Gasdermin-A; NM_178171.5) and LACRT (Extracellular glycoprotein lacritin; NM_033277.2) genes in a biological sample derived from a subject As an information providing method for predicting anticancer drug response of degenerative pancreatic cancer, comprising measuring the level of mRNA or protein encoded from the gene,
The anticancer agent is gemcitabine (Gemcitabine) and lead paclitaxel (Nab-paclitaxel), characterized in that the information providing method. 9. The method of claim 8,
The method is MMP9 (Matrix metalloproteinase-9; NCBI accession number: NM_004994.3), MME (Neprilysin; NM_000902.5), MME (NM_007288.3), MME (NM_007289.4), MME (NM_007287.4) ), MME (NM_001354642.2), MME (NM_001354643.1), LYZ (Lysozyme C; NM_000239.3), GP2 (Pancreatic secretory granule membrane major glycoprotein GP2; NM_001007240.3), GP2 (NM_001502.4), GP2 ( NM_001007241.3), GP2 (NM_001007242.3), LAMP2 (Isoform LAMP-2C of Lysosome-associated membrane glycoprotein 2; NM_001122606.1), CTSD (Cathepsin D; NM_001909.5), CD81 (CD81 antigen; NM_004356.4) , CD81 (NM_001297649.2), LAMB1 (Laminin subunit beta-1; NM_002291.3), PSMB2 (Proteasome subunit beta type-2; NM_002794.5), PSMB2 (NM_001199779.2), PSMB2 (NM_001199780.2) and AZGP1 (Zinc-alpha-2-glycoprotein; NM_001185.4) characterized in that it further comprises the step of measuring the mRNA level of one or more genes selected from the group consisting of or a protein encoded from the gene, the information providing method. delete 9. The method of claim 8,
The biological sample is characterized in that the exosomes derived from blood or plasma, information providing method. 9. The method of claim 8,
The mRNA level is determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase protection assay; RPA), microarray (microarray), characterized in that measured through one or more methods selected from the group consisting of northern blotting (northern blotting), information providing method. 9. The method of claim 8,
The protein level was determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, and immunohistochemistry. Information, characterized in that it is measured by at least one method selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip. How to provide.

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