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WO2016060382A1 - Composition pour le diagnostic de cancer du pancréas, et procédé pour le diagnostic de cancer du pancréas à l'aide de cette dernière - Google Patents

Composition pour le diagnostic de cancer du pancréas, et procédé pour le diagnostic de cancer du pancréas à l'aide de cette dernière Download PDF

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Publication number
WO2016060382A1
WO2016060382A1 PCT/KR2015/009827 KR2015009827W WO2016060382A1 WO 2016060382 A1 WO2016060382 A1 WO 2016060382A1 KR 2015009827 W KR2015009827 W KR 2015009827W WO 2016060382 A1 WO2016060382 A1 WO 2016060382A1
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Prior art keywords
pancreatic cancer
protein
lrg1
ttr
expression level
Prior art date
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PCT/KR2015/009827
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English (en)
Korean (ko)
Inventor
최용환
남궁정현
이성곤
한상조
장진영
박태성
김영수
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SK Telecom Co Ltd
SNU R&DB Foundation
Original Assignee
SK Telecom Co Ltd
Seoul National University R&DB Foundation
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Priority claimed from KR1020150092393A external-priority patent/KR20160045547A/ko
Priority to KR1020157036434A priority Critical patent/KR101858717B1/ko
Priority to CN201580002481.2A priority patent/CN106796239B/zh
Priority to EP15850998.4A priority patent/EP3208614B1/fr
Priority to CN201811471602.XA priority patent/CN109576368B/zh
Priority to JP2016518429A priority patent/JP6415547B2/ja
Application filed by SK Telecom Co Ltd, Seoul National University R&DB Foundation filed Critical SK Telecom Co Ltd
Priority to KR1020157029860A priority patent/KR101837672B1/ko
Priority to US14/915,658 priority patent/US9983208B2/en
Publication of WO2016060382A1 publication Critical patent/WO2016060382A1/fr
Anticipated expiration legal-status Critical
Priority to US15/988,027 priority patent/US10215756B2/en
Ceased legal-status Critical Current

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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
    • 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

  • pancreatic cancer diagnostic composition and pancreatic cancer diagnostic method using the same
  • the present invention is a pancreatic cancer diagnostic composition and kit comprising an agent for measuring the protein expression level or mRNA expression level of a specific marker gene, which can be used to determine the onset or the likelihood of pancreatic cancer, and pancreatic cancer using the same
  • pancreatic cancer does not feel much at an early stage, and after general metastasis has already occurred. Pain, weight loss, and symptoms usually appear, and the healing rate is lower, so regular diagnosis is very important. Most clinical symptoms are slow onset, fragile, and loss of appetite and weight loss are the most common symptoms. Pancreatic cancer is a fatal cancer with a 5-year survival rate of 1-4% and a median survival of 5 months. It has the poorest prognosis among human cancers. In addition, since 80% to 90% of patients are found in a state where curative resection is not possible to expect a cure, the prognosis is poor and treatment mainly depends on chemotherapy. have.
  • pancreatic cancer The effectiveness of some anticancer drugs, including gemcitabine and tarceva, is extremely low, and the response rate to chemotherapy is only around 15%, which is more effective to improve the prognosis of pancreatic cancer patients.
  • the development of early diagnostics and treatments is urgently needed. Proper diagnosis and treatment of pancreatic cancer in the pancreatic cancer, a stage prior to the development of fatal pancreatic cancer, It is very important to improve your grades.
  • Diagnosis of pancreatic or pancreatic progenitor lesions includes blood tests (CA 19-9), X-ray imaging of the stomach and duodenum, biliary tract imaging through the skin and liver, and retrograde endoscopy.
  • Biliary grafting is used. Although disease lesions have been discovered by these methods, ultrasonography and computed tomography are most commonly used in recent years. More accurate biopsies can be performed to obtain relatively accurate results. However, the diagnosis method is very inconvenient to perform the method, such as inaccuracy or pain to the patient, the subjects are reluctant to do this. Therefore, there has been a demand for the development of a test method for diagnosing pancreatic cancer or pancreatic cancer precursor lesions easily and quickly. Abdomen for recent medical examination or diagnosis of diseases other than pancreas
  • cystic lesions of the pancreas are malignant tumors, and if so, whether they are still in the precancerous stage or already have malignant lesions.
  • Korean Patent Publication No. 2009-0003308 discloses a method for diagnosing pancreatic cancer by detecting the expression level of REG4 protein in an individual's blood sample
  • Korean Patent Application Publication No. 2012-0009781 discloses information necessary for diagnosing pancreatic cancer in an individual.
  • Analytical method for measuring the expression level of XIST RNA in cancer tissue isolated from the individual to provide a Korean Patent Application Publication No. 2007-01 19250 is a normal human pancreatic tissue and In comparison, a novel gene LBFL313, which is expressed differently in human pancreatic cancer tissue, is disclosed, and US Patent Application Publication No.
  • 201 1/0294136 discloses a method for diagnosing pancreatic cancer using biomarkers such as keratin 8 protein.
  • biomarkers such as keratin 8 protein.
  • each marker shows a great difference in the diagnosis efficiency and accuracy, it is necessary to find a marker that is more effective and to develop a diagnostic method using the same.
  • An object of the present invention is to provide a pancreatic cancer diagnostic marker that can easily diagnose the onset, the likelihood or the risk of pancreatic cancer.
  • An object of the present invention is to provide a pancreatic cancer diagnostic composition that can easily diagnose the onset, the likelihood or the risk of pancreatic cancer.
  • Another object of the present invention to provide a pancreatic cancer diagnostic kit comprising the composition.
  • Still another object of the present invention is to provide a method for diagnosing pancreatic cancer using the diagnostic composition or kit, or providing a method for diagnosing pancreatic cancer.
  • An object of the present invention is to provide a pancreatic cancer diagnostic use of the pancreatic cancer diagnostic marker that can easily diagnose the onset, likelihood or risk of pancreatic cancer.
  • the present invention is a pancreatic cancer or a prognostic diagnostic composition for pancreatic cancer comprising a pancreatic cancer diagnostic marker protein expression level or a preparation for measuring the expression level of mRNA, kit for diagnosing pancreatic cancer comprising the diagnostic composition And a method for determining the presence or the malignancy of pancreatic cancer in a subject having or suspected of having pancreatic cancer.
  • the composition or kit for diagnosing pancreatic cancer is one or more additional ingredients, solutions or The apparatus may further include.
  • the pancreatic cancer diagnosis is distinguished from the normal group .
  • Pancreatic cancer may be selectively detected, pancreatic cancer may be selectively detected among various cancers, or pancreatic cancer having a level of CA19-9 of less than 37 U /.
  • pancreatic cancer diagnostic composition or kit not only detects or diagnoses a pancreatic cancer group by distinguishing between a normal group and a pancreatic cancer patient group, but also detects or diagnoses stage 1 and 2 pancreatic cancers (PDAC) that are relatively difficult to diagnose early.
  • Pancreatic cancer can be screened to distinguish from various pancreas-related diseases other than cancer, such as pancreatitis and cholecystitis, and to selectively pancreatic cancer to distinguish from other types of cancer, such as breast cancer, colon cancer and / or thyroid cancer.
  • pancreatic cancer markers such as pancreatic cancer with a CA19-9 of less than 37 U / ml
  • pancreatic related diseases such as normal, pancreatitis and cholecystitis with a CA19-9 of less than 37 U / ml. Can be detected distinctly.
  • the marker gene is a combination of at least three kinds or more marker genes, specifically
  • c at least one marker gene selected from the group consisting of TTR (Transthyretin, ATTR, Prealbumin, TBPA), Complement Clr subcomponent precursor (ClR), Cluster preproprotein (CLU) and Plasma Kallikrein protein (KLKB 1).
  • TTR Transthyretin, ATTR, Prealbumin, TBPA
  • ClR Complement Clr subcomponent precursor
  • CLU Cluster preproprotein
  • KLKB Plasma Kallikrein protein
  • the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition.
  • the present invention comprises the steps of obtaining a sample from a subject to diagnose whether the pancreatic cancer;
  • the present invention relates to a method for providing pancreatic cancer diagnosis or pancreatic cancer diagnosis, comprising determining a subject group having a high probability of pancreatic cancer or a disease.
  • the expression level of CA19-9 in the subject was higher than the expression level in the normal control
  • the expression level of LRG1 was higher than the expression level in the normal control
  • the expression level or the expression level of the mRNA of the gene encoding the TTR protein is lower than the expression level in the normal control
  • the expression level of the C1R protein or the expression level of the mRNA of the gene encoding the C1R protein is higher than the expression level in the normal control.
  • Elevated, or the expression level of the CLU protein or mRNA of the gene encoding the CLU protein is lower than the expression level in the normal control, or the expression level of the KLKB 1 protein or the mRNA of the gene encoding the KLKB 1 protein This is normal
  • It provides a method for diagnosing pancreatic cancer or providing information for diagnosing pancreatic cancer, the method comprising determining that the pancreatic cancer is more likely to develop when the expression level is lower than that in the control group.
  • An additional embodiment of the present invention is an intraductal papillary mucinous neoplasm (IPMN) marker protein including LRGl (Leucine-rich alpha-2-glycoprotein 1, LRG1) or an expression level of mRNA of a gene encoding the same. It relates to a composition for screening diagnosis of high-risk group IPMN, including an agent for measuring the amount. remind
  • the IPMN marker may further comprise one or more markers selected from the group consisting of CA19-9 (carbohydmte antigen 19-9), TTR, C1R, CLU and KLKB l.
  • the IPMN marker may be one marker selected from the group consisting of TTR, CIR, CLU, and KLKB1 and markers including LRG1, or TTR, C1R, CLU, and
  • It may be a combination marker comprising LRG1 and two markers selected from the group consisting of KLKB 1.
  • CLU Clusterin preproprotein
  • LRG1 Leucine-rich alpha-2-glycoprotein 1
  • CA 19-9 carbohydrate antigen 19-9
  • Transthyretin ATTR, Prealbumin, TBPA (TLR), Complement Clr subcomponent precursor (ClR) and KLKB l ( Plasma Kallikrein protein;) of the IPMN marker protein or a gene encoding the same comprising at least one marker selected from the group consisting of including the agent measuring the expression level of mRNA, it relates to a diagnostic composition i screening of high risk IPMN.
  • the IPMN marker is a combination marker including one type of marker selected from the group consisting of LRG1, CA19-9, TTR, C1R and KLKB1 and CLU, or LRG1, CA19-9, TTR, C1R and KLKB 1 It may be a combination marker comprising LRG1 and two markers selected from the group.
  • One embodiment according to the present invention for the sample of the subject, measuring the expression level of the IPMN marker proteins or mRNA expression level of the gene encoding the same, respectively,
  • the present invention can significantly predict or identify the incidence of pancreatic cancer, early diagnosis, and degree of disease, and can be used for tumorigenesis research of pancreatic cancer.
  • the diagnostic method of the present invention can non-invasively diagnose early pancreatic cancer simply from blood or the like.
  • 1 is a graph showing the expression level of CA19-9 protein in the control group and pancreatic cancer patient group by chemiluminescent enzyme immunoassay (CLEIA).
  • 3 is a control and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis
  • FIG. 6 is a graph showing the expression level of KLKB1 protein in the control group and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis.
  • FIG. 7 is a graph showing the expression level of LRG1 protein in the control group and pancreatic catheter adenocarcinoma patient group by ELISA quantitative analysis.
  • FIG. 8 is a graph showing the expression level of TTR protein of the control group and pancreatic catheter adenocarcinoma patient group by ELISA quantitative analysis.
  • FIG. 9 is a graph showing the expression level of CLU protein of the control group and pancreatic catheter adenocarcinoma patient group by ELISA quantitative analysis.
  • FIG. 10 is a graph showing the expression level of the TTR protein of the control group and pancreatic catheter adenocarcinoma patient group by immunoturbidimetric assay.
  • CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 13 shows diagnostic performance for the separation of pancreatic cancer and other cancers when CA19-9, LRG1 and TTR proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. And detection sensitivity).
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by MRM quantitative analysis.
  • FIG. 14 shows the experimental group having a CA19-9 value of 37 U / m £ or less, when CA19-9, LRG1 and TTR proteins were used simultaneously, and CA19-9 protein alone and CA19-9 and TTR. Comparison of pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using a combination of markers.
  • the CA19-9 protein is chemiluminescent enzyme immunity By assay (CLEIA), LRGl and TTR proteins were measured by MRM quantitative analysis. ⁇
  • CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by ELISA method.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 16 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer when CA19-9, LRG1 and TTR proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. It is a comparison.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by ELISA method.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. ⁇ shows diagnostic performance for the separation of pancreatic and other cancers when CA19-9, LRG1 and TTR proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. And detection sensitivity).
  • the CA19-9 protein was measured by chemical light photoenzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were determined by ELISA method.
  • FIG. 18 shows experimental groups with CA19-9 levels of 37 U / m £ or less, when CA19-9, LRG1 and TTR proteins were used simultaneously, and CA19-9 protein alone and CA19-9 and TTR. Comparison of pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using a combination of markers.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • LRG1 and TTR proteins were measured by ELISA method. 19 compares pancreatic cancer diagnostic performance (AUC and detection sensitivity) when CA19-9, LRG1 and TTR proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. will be.
  • the CA19-9 protein was measured by chemiluminescence enzyme immunoassay (CLEIA), the LRG1 protein was determined by ELISA, and the TTR protein was determined by immunobidification.
  • CA19-9 protein is By chemiluminescent enzyme immunoassay (CLEIA), LRG1 protein was measured by ELISA method, TTR protein was measured by immunobidification method.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 21 shows diagnostic performance for distinguishing pancreatic cancer from other cancers when CA19-9, LRG1 and TTR proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. And detection sensitivity).
  • the CA19-9 protein was measured by chemiluminescence enzyme immunoassay (CLEIA), the LRG1 protein was determined by ELISA, and the TTR protein was determined by immunobidification.
  • FIG. 22 shows two groups, CA19-9 and TTR, using CA19-9 protein alone and CA19-9, LRG1, and TTR protein simultaneously, with a CA19-9 level of 37 U / m or less.
  • Pancreatic cancer diagnostic performance (AUC and detection sensitivity) when used in combination.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • LRG1 protein was determined by ELISA method and TTR protein was determined by immunobidification method.
  • CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and C1R proteins were determined by MRM quantitative analysis. . .
  • Figure 24 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer when using CA19-9, LRG1 and C1R proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. It is a comparison.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and C1R proteins were determined by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 25 shows diagnostic performance for distinguishing pancreatic cancer from other cancers when using CA19-9, LRG1, and C1R proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. And detection sensitivity).
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and C1R proteins were determined by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 26 shows the use of CA19-9, LRG1, and C1R proteins simultaneously and CA19-9 protein alone in an experimental group with CA19-9 levels of 37 U / or less. Comparison of pancreatic cancer diagnostic performance (AUC and detection sensitivity) using a combination of two markers, CA19-9 and TTR.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • FIG. 27 compares pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using CA19-9, LRG1 and CLU proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 28 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer when using CA19-9, LRG1 and CLU proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. It is a comparison.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 30 illustrates an experimental group having a CA19-9 value of 37 U / or less.
  • pancreatic cancer diagnostic performance (AUC and detection sensitivity) when LRG1 and CLU proteins were used simultaneously and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • FIG. 31 compares pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using CA19-9, LRG1 and CLU proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by ELISA method.
  • FIG. 32 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer when CA19-9, LRG1 and CLU proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. It is a comparison.
  • CA19-9 protein ⁇ chemiluminescent enzyme immunoassay (CLEIA)
  • LRG1 and CLU proteins were measured by ELISA method.
  • CA19-9JLRG1 and CLU proteins show diagnostic performance (AUC and detection) for differentiating pancreatic cancer and other cancers when CA19-9JLRG1 and CLU proteins are used simultaneously and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. Sensitivity).
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by ELISA method.
  • FIG. 34 is a diagram showing the use of CA19-9, LRG1, and CLU proteins simultaneously and CA19-9 protein alone and two markers of CA19-9 and TTR. Comparison of pancreatic cancer diagnostic performance (AUC and detection sensitivity) when used in combination.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • LRG1 and CLU proteins were measured by ELISA method. 35 compares pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using CA19-9, LRG1 and KLKB1 proteins simultaneously and using CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. will be.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were measured by MRM quantitative analysis.
  • CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • FIG. 37 shows diagnostic performance for distinguishing pancreatic cancer from other cancers when CA19-9, LRG1 and KLKB1 proteins are used simultaneously, and CA19-9 protein alone and a combination of two markers, CA19-9 and TTR. And detection sensitivity).
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were measured by MRM quantitative analysis.
  • FIG. 38 shows the experimental group with CA19-9 levels of 37 U / in £ or less, when CA19-9, LRG1 and KLKB1 proteins were used simultaneously, and CA19-9 protein alone and CA19-9 and TTR. Comparison of pancreatic cancer diagnostic performance (AUC and detection sensitivity) when using a combination of markers.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • FIG. 39 compares the diagnostic performance (AUC and detection sensitivity) of high risk group IPMN with LRG1 protein and CA19-9 protein alone.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 protein was determined by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • the 40-44 show two combination proteins of (LRGl + CA19-9), (LRG1 + TTR), (LRG1 + CLU), (LRG1 + C1R), or (LRGl + KLKB1) simultaneously and simultaneously.
  • the diagnostic performance (AUC and detection sensitivity) of the high risk group IPMN when using CA19-9 protein alone is compared.
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • the diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs when three combinations of (LRG1 + C1R + KLKB 1) were used simultaneously and when CA19-9 protein was used alone was compared.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1, TTR, CLU, C 1 R and KLKBl proteins were measured by MRM quantitative analysis. 55 to 57 show that when two combination proteins of (CLU + CA19-9), (CLU + TTR) and (CLU + KLKB 1) are used simultaneously and CA19-9 protein alone.
  • the diagnostic performance (AUC and detection sensitivity) of the high-risk IPMNs in use is compared.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the TTR, CLU and KLKB1 proteins were measured by MRM quantitative analysis.
  • the diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs when three combinations of (CLU + C1R + KLKB1) were used simultaneously and when CA19-9 protein was used alone was compared.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the L G1, TTR, CLU, C1R and KLKBl proteins were measured by MRM quantitative analysis.
  • FIG. 64 compares the diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs distinguished from low-risk IPMNs using LRG1 protein and CA19-9 protein alone.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 protein was measured by MRM quantitative analysis.
  • Figures 65-69 show CA19- and two combination marker proteins using (LRG1 + CA19-9), (LRG1 + TTR), (LRG1 + CLU), (LRG1 + C1R), and (LRG1 + KLKB1).
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRGI TTR, CLU, C1R and KLKB1 proteins were measured by MRM quantitative analysis.
  • the diagnostic performance (AUC and detection sensitivity) of the high risk IPMN which is distinguished from the low risk IPMN when using the three combination marker proteins (LRG1 + C1R + KLKB1) and the CA19-9 protein alone, is compared.
  • the CA19-9 protein is chemiluminescent By enzymatic immunoassay (CLEIA), LRG1, TTR, CLU, C1R and KLKBl proteins
  • CA19-9 The CA19-9 protein is chemiluminescent enzyme immunity
  • the diagnostic performance (AUC and detection sensitivity) of the high risk IPMN which is distinguished from the low risk IPMN when using the three combination marker proteins (CLU + C1R + KLKB1) and the CA19-9 protein alone, is compared.
  • the CA19-9 protein was chemiluminescent enzyme immunoassay (CLEIA), LRG1, TTR, CLU, C1R and KLKB1 protein
  • FIG. 90 compares the diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs distinguished from low-risk IPMNs using LRG1 protein and CA19-9 protein alone.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 protein was determined by ELISA assay.
  • CLIA chemiluminescent enzyme immunoassay
  • the diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs which is distinct from low-risk IPMNs when using two combinatorial marker proteins and CA19-9 protein alone, is compared.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1, TTR, CLU, C1R and KLKBl proteins were measured by ELISA assay.
  • High risk group distinguished from low risk group IPMN when using combination marker protein of (LRG 1 + C A 19-9 + TTR + CLU) and (LRG1 + TTR + CLU) and CA19-9 protein alone
  • the diagnostic performance (AUC and detection sensitivity) of IPMN is compared.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1, TTR, CLU, C1R and KLKBl proteins were measured by ELISA analysis. 98-100 show (CLU + CA19-9), (CLU + TTR) and
  • the CA19-9 protein is chemiluminescent enzyme immunity
  • LRG1, TTR, CLU, C1R and KLKB1 proteins were measured by ELISA assay.
  • pancreatic cancer refers to a cancer (carcinoma) or a tumor originating from pancreatic cells.There are various types of pancreatic cancer, and the prognosis from benign tumors that can be treated by surgical resection is very poor.
  • pancreatic adenocarcinoma Pancreatic ductal adenocarcinoma
  • pancreatic cancer accounts for about 90% of pancreatic cancer, and pancreatic cancer is generally used in a narrow sense to mean pancreatic adenocarcinoma, but other types of pancreatic cancer such as neuroendocrine tumors and Including acinar cell tumors, as another example, a representative cystic tumor.
  • the pancreatic cancer may be pancreatic adenocarcinoma or IPMN
  • the pancreatic adenocarcinoma may be caused by various causes, for example, may be pancreatic adenocarcinoma derived from IPMN or may be independent of IPMN, or
  • IPMN-derived pancreatic adenocarcinoma can also be excluded.
  • IPMN as a tumor that occurs in the main pancreatic duct or its main branches, and the epithelium grows into the papillary into the lumen, secretes mucus to varying degrees, and expands the pancreatic duct to cystic features.
  • IPMN is characterized morphologically by diffuse dilatation of the main pancreatic duct, atypical shading defects caused by mucus or mass, cystic dilatation of the pancreatic duct, enlargement of the papillary opening, and finding that a large amount of mucus is discharged from the papillary opening.
  • IPMN can be divided into main duct type, branch duct type, and mixed type according to the location of tumor and the extent of lesion.
  • IPMN Factors that determine the prognosis of IPMN are invasiveness, lymph node metastasis, vascular invasion, histological findings and histological findings of resection margin. IPMN before invasive cancer is a curable disease, but IPMN after invasive cancer occurs in 50-90% of recurrent pancreas or extra-pancreatic tissue. Since IPMN can recur on the remaining pancreas after curative resection, long-term follow-up is necessary and appropriate measures are needed to improve survival.
  • the evaluation of malignancy of IPMN is very important in the prognostic observation and selection of treatment methods, and usually, the degree of malignancy of IPMN is classified by postoperative microscopic biopsy, and Low, Intermediate, High grade dysplasia and IPMN associated with an invasive It is divided into carcinoma and malignancy increases in the order listed.
  • Part of the invasive carcinoma with infiltration of the surrounding stroma is ductal adenocarcinomas, the most common form of carcinoma in pancreatic cancer. It may be present in the form or form of mucinous noncystic carcinoma (colloid carcinoma).
  • the high grade dysplasia and invasive type are defined as malignant subtype or high risk IPMN according to the malignancy and / or invasiveness of the IPMN, and low and intermediate grade dysplasia are defined as low risk IPMN.
  • the pancreatic cancer-related markers according to the present invention can select high risk group IPMN by distinguishing it from low risk group IPMN.
  • the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition.
  • the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
  • MRM multiple reaction monitoring
  • diagnosis refers to a particular disease or condition
  • Determining the susceptibility of a subject, wherein the subject has a specific disease or condition To determine whether they currently have, to determine the prognosis of a subject with a particular disease or disorder (e.g., to identify a pre-metastatic or metastatic cancer state, to determine the stage of the cancer or to determine the response of the cancer to treatment) Doing or
  • Therametrics eg, monitoring the condition of an object to provide information about treatment efficacy.
  • the diagnosis in the present specification is to determine whether pancreatic cancer develops or the possibility (danger).
  • marker biomarker As used herein, the terms “marker biomarker” or “diagnostic marker” can distinguish between normal or pathological conditions or predict treatment response.
  • the protein expression level or gene expression level is significantly increased or decreased in individuals with pancreatic cancer or at risk of developing pancreatic cancer compared to normal controls (individuals not pancreatic cancer).
  • an organic biomolecule such as a polypeptide or a nucleic acid (eg, m NA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, lygosaccharides, etc.).
  • the present invention provides an agent for measuring the expression level of (a) carbohydrate antigen 19-9 (CA19-9), (2) an agent for measuring the expression level of Leucine-rich alpha-2-glycoprotein 1 (LRGl), and (3 ) TTR (Transthyretin, ATTR, Prealbumin, TBPA); C 1 R (Complement Clr
  • pancreatic cancer diagnostic markers or a gene encoding the protein, comprising an agent measuring the expression level of any one or more markers selected from the group consisting of subcomponent precursors), CLU (Clusterin preproprotein) and KLKB 1 (Plasma Kallikrein protein) It provides a pancreatic cancer diagnostic composition comprising an agent for measuring the expression level of mRNA.
  • the present invention provides a combination of at least three or more types of markers, for example (a) CA19-9; (b) LRGl; And (c) TTR, C 1R, CLU, and KLKB1 in combination with any one of the marker genes selected from the group, it is possible to effectively diagnose the onset or possibility of the development of pancreatic cancer from the subject.
  • markers for example (a) CA19-9; (b) LRGl; And (c) TTR, C 1R, CLU, and KLKB1 in combination with any one of the marker genes selected from the group, it is possible to effectively diagnose the onset or possibility of the development of pancreatic cancer from the subject.
  • the CA19-9 protein used as a marker for diagnosing pancreatic cancer in the present invention may be used as a pancreatic cancer marker that is conventionally used for tumor marker examination for prognosis of cancer of the digestive system.
  • LRG1 protein It is involved in angiogenesis and is associated with endometrial cancer and lung cancer.
  • LRG1 may be NCBI Accession # NP—443204.1.
  • Markers for diagnosing pancreatic cancer that can be used in the present invention are TTR, C1R, CLU and
  • TTR protein is related to the subunit consisting of 127 amino acid residues, etc.
  • C1R protein is the first of its kind to be found in the complement system, which is associated with immune function in the body and with Alzheimer's and kidney cancer.
  • CLU proteins are known to play a role in the clotting of proteins in the blood, and are associated with Alzheimer's and lung cancer.
  • KLKB1 protein is involved in hematology and is associated with hypertension and lung cancer.
  • TTR may be NCBI Accession # NP # 000362 ⁇ l
  • ClR may be NCBI Accession # NP_001724.3
  • CLU may be NCBI Accession # NP_001822.3
  • KLKB1 may be NCBI Accession #NP — 000883.2.
  • PD AC pancreatic ductal adenocarcinoma
  • IPMN intraductal papillary mucinous neoplasm
  • KLKB1 KLKB1
  • the term "measurement of expression level of protein” refers to a process for confirming the presence and expression level of a pancreatic cancer diagnostic marker (protein) or a gene encoding the same in a biological sample to diagnose pancreatic cancer.
  • the expression level measurement or comparative analysis of the protein may include protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and SELDI-TOF (Sulface).
  • MALDI-TOF Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry
  • SELDI-TOF SELDI-TOF (Sulface).
  • Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase
  • LC-MS Liquid chromatography-mass spectrometry
  • LC-MS / MS liquid chromatography-mass spectrometry / mass spectrometry
  • ELISA enzyme—linked immunosorbentassay
  • CA19-9 protein In pancreatic cancer diagnostic composition according to the invention, CA19-9 protein, LRG1 protein,
  • Agents that measure the expression level of TTR protein, C1R protein, CLU protein or KLKB1 protein may be antibodies, oligopeptides, specific binding to CA19-9 protein, LRG1 protein, TTR protein, C1R protein, CLU protein or KLKB1 protein, respectively. It may include a ligand, PNA (peptide nucleic acid) or aptamer (aptamer).
  • an antibody refers to a substance that specifically binds to an antigen and results in an antigen-antibody reaction.
  • an antibody refers to an antibody that specifically binds to CA19-9 protein, LRG1 protein, TTR protein, C1R protein, CLU protein or KLKB1 protein, respectively.
  • Antibodies of the invention include all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Such antibodies can be readily prepared using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, including the steps of injecting the pancreatic cancer marker protein antigen into an animal and collecting blood from the animal to obtain serum comprising the antibody.
  • polyclonal antibodies can be prepared from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like.
  • monoclonal antibodies are widely used in the art Known hybridoma methods (see hybridoma method; Kohler and Milstein (1976) European Journal of Immunology 6:51 1-519), or phage antibody library techniques (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 invention include functional fragments of antibody molecules, as well as complete forms with 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 and Fv.
  • DNA has a phosphate-ribose sugar backbone
  • ⁇ has a repeating ⁇ - (2-aminoethyl) -glycine backbone linked by peptide bonds, which greatly increases binding and stability to DNA or RNA, leading to molecular biology. It is used in diagnostic analysis and antisense therapies.
  • ⁇ is described in Nielsen ⁇ , Egholm ⁇ , Berg RH, Buchardt 0 (December 1991).
  • “Aptamers” in the present invention are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med.
  • measurement of expression level of mRNA means measuring the amount of mRNA by confirming the presence and expression level of mRNA of genes encoding the pancreatic cancer diagnostic protein in a biological sample in order to diagnose pancreatic cancer. .
  • RT-PCR reverse transcriptase
  • RPA RNase protection assay
  • the agent for measuring the expression level of mRNA of a gene encoding a CA19-9, LRG1, TTR, C1R, CLU or KLKB1 protein may be CA19-9, LRG1, TTR, C 1R, CLU or Primers, probes, or antisenses, each specifically binding to mRNA of a gene encoding KLKB 1 protein
  • KL B 1 protein The information of KL B 1 protein is known from UniProt et al.
  • Primers, probes or antisense nucleotides that bind will be readily designed. .
  • primer refers to a fragment that recognizes a target gene sequence, which includes primer pairs in the forward and reverse directions, but is preferably a primer pair that provides an analysis result with specificity and sensitivity. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that only the target gene sequence containing the complementary primer binding site is amplified and does not cause nonspecific amplification. .
  • probe refers to a substance that can specifically bind to a target substance to be detected in a sample.
  • it means a substance capable of confirming the presence of a target substance in a sample.
  • the type of probe is a material commonly used in the art, but there is no limitation, but preferably, PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide,
  • the probe may be a polypeptide, protein, RNA or DNA, most preferably PNA. More specifically, the probe is a bio-material, including or derived from an organism, or produced in vitro, for example, enzymes, proteins, antibodies, microorganisms > flora and fauna cells and organs, neurons > 1 ⁇ , And RNA, wherein the DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like. .
  • antisense means that an antisense oligomer is localized with a target sequence in RNA by Watson-Crick base pairing, allowing formation of mRNA and RNA: oligomeric heterodimers typically within the target sequence.
  • Oligomer having a sequence of nucleotide bases and an intersubunit backbone.
  • the oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.
  • the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition.
  • the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
  • MRM multiple reaction monitoring
  • pancreatic cancer diagnostic composition or a diagnostic kit comprising the same according to the present invention by using pancreatic cancer markers including any one or more markers selected from the group consisting of CA19-9 and LRG1 and TTR, C 1R, CLU and KLKB 1,
  • the diagnostic performance is very good compared to a composition or kit using a single marker of CA19-9 or a composition or kit using two markers of CA19-9 and LRG1.
  • the pancreatic cancer diagnostic kit may further include one or more other component compositions, solutions or devices suitable for the analysis method.
  • the diagnostic kit may further include essential elements necessary for performing reverse transcription polymerase reaction.
  • the reverse transcription polymerase kit includes primer pairs specific for the gene encoding the marker protein.
  • the primer has a sequence specific to the nucleic acid sequence of the gene
  • nucleotide As a nucleotide, it may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include primers specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits may be used for the test lubrication or other suitable container, reaction complete fluid (pH and magnesium concentrations vary),
  • Enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases,
  • DNA needle kit includes a substrate to which a cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and a fluorescent label Reagents, preparations, enzymes, and the like, for making probes.
  • the substrate may also comprise cDNA or oligonucleotide corresponding to the control gene or fragment thereof.
  • the diagnostic kit of the present invention may include necessary elements necessary to perform an ELISA.
  • ELISA kits contain antibodies specific for the protein. Antibodies are antibodies that have high specificity and affinity for marker proteins and have little cross reaction to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies.
  • the ELISA kit can also include antibodies specific for the control protein.
  • Other ELISA kits are capable of binding reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.
  • composition for diagnosing pancreatic cancer according to the present invention or a diagnostic kit including the same may be used for distinguishing a patient from a pancreatic cancer early stage (stage 1 or 2) and a normal person, in addition to distinguishing a pancreatic cancer patient from a normal person.
  • the present invention comprises (a) an agent for measuring the expression level of CA19-9, (b) an agent for measuring the expression level of LRGl, and (C) TTR, C 1 R, CLU and KLKB 1
  • pancreatic cancer for diagnosing pancreatic cancer at an early stage of pancreatic cancer, such as stage 1 or stage 2, comprising an agent measuring the expression level of any one or more markers selected from the group
  • composition for diagnosing pancreatic cancer according to the present invention or a diagnostic kit including the same may be used for selectively detecting a pancreatic cancer patient from other cancer patients, in addition to distinguishing a pancreatic cancer patient from a normal person.
  • the present invention is directed to any one selected from the group consisting of (a) an agent measuring the expression level of CA19-9, (b) an agent measuring the expression level of LRGl, and (C) a TTR, C 1R, CLU and KLKB 1
  • compositions for distinguishing pancreatic cancer from other cancers including agents that measure the expression level of one or more markers.
  • the present invention provides a method for diagnosing pancreatic cancer of a subject or providing information for pancreatic cancer diagnosis using the pancreatic cancer diagnostic composition or the pancreatic cancer diagnostic kit.
  • the diagnostic method includes the following steps:
  • Measuring protein expression level or mRNA expression level of pancreatic cancer markers comprising any one or more markers selected from the group consisting of KLKB1; (A) CA19-9, (b) LRG1 and (c) TTR, C1R, CLU and
  • pancreatic cancer markers comprising any one or more markers selected from the group consisting of KLKB1 with the expression levels of the markers in the normal control sample, respectively; And determining whether the subject has pancreatic cancer using a result of comparing the expression levels of the markers.
  • the "subject" applied to a method for diagnosing pancreatic cancer or a method for providing information for diagnosing pancreatic cancer according to the present invention includes a patient, a patient suspect group, or a normal group as a subject to diagnose whether pancreatic cancer develops.
  • a healthy person who has not been diagnosed with cancer and who wants to diagnose PDAC early in the medical examination or includes a subject for regular examination for diagnosing allogeneic cancer recurrence in patients with pancreatic cancer who have been surgically removed.
  • the subject may mean a mammal, and in another embodiment, a human.
  • the expression level is higher than that in the normal control group, and the expression level of the LRG1 protein or the mNRA expression level of the gene encoding the LRG1 protein is normal.
  • the expression level of the TTR protein or the mRNA of the gene encoding the TTR protein is lower than the expression level in the normal control, the expression level of the C1R protein or the mRNA of the gene encoding the C1R protein
  • the expression level of is higher than the expression level in the normal control, or the expression level of the CLU protein or mRNA of the gene encoding the CLU protein is lower than the expression level in the normal control, the expression level of the KLKB1 protein or the KLKB1 protein. If the expression level of the mRNA of the gene coding for is lower than the expression level in the normal control it can be determined that the risk of pancreatic cancer is high.
  • a sample is a biological sample that is a tissue, cell, or tissue that has a difference in protein expression level or gene expression level due to pancreatic cancer. It means a sample such as blood, serum, plasma, saliva, cerebrospinal fluid or urine, and preferably means blood, serum or plasma.
  • MRNA of the CA19-9 and LRG1 protein or the gene encoding the protein is increased in the level of expression in pancreatic cancer patients compared to the expression level in the normal control, and the TTR, CLU and KLKB1 protein or the gene encoding the protein mRNA levels in pancreatic cancer patients decreased compared to expression levels in normal controls, while mRNA levels of C1R protein or genes encoding these proteins increased in pancreatic cancer patients compared to expression levels in normal controls. Therefore, the markers including any one selected from the group consisting of (a) CA19-9, (b) LRG1 and (c) TTR, C 1 R, CLU and KLKB1 in a subject to diagnose pancreatic cancer.
  • the likelihood of developing pancreatic cancer can be determined by measuring the mRNA expression level of the protein or gene encoding the protein.
  • the expression level in pancreatic cancer patients of CA19-9 and LRG1 proteins or mRNA of the gene encoding the protein increases compared to the expression level in the normal control, and the mRNA of the TTR protein or the protein encoding the protein
  • the subject may be determined to have a high probability of developing pancreatic cancer.
  • the expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the protein in a subject to be diagnosed with pancreatic cancer is higher than that of the normal control group when measured by various methods.
  • the expression level of CA19-9 protein or mRNA expression level of the gene encoding the protein in the subject to be diagnosed is more than 1.0 times, more than 1.5 times, and more than twice the expression level in the normal control group. By more than 3 times, by more than 5 times or by more than 10 times.
  • the expression applies equally when referring to the expression level of the LRG1 or C1R protein or the mRNA expression level of the gene encoding the protein.
  • the expression level of the TTR protein or the mRNA expression of the gene encoding the protein in a subject to diagnose whether the pancreatic cancer The level is lower than the expression level in the normal control group 'means that the expression level of the TTR protein or the mRNA expression level of the gene encoding the protein in the subject to be diagnosed with pancreatic cancer when measured by various methods in the normal control group. Less than 0.1 times, less than 0.2 times, less than 0.3 times, less than 0.5 times or less than 1 times the expression level of.
  • the expression applies equally when referring to the expression level of a CLU or KLKB 1 protein or the mRNA expression level of a gene encoding said protein.
  • the 'determining the high likelihood of developing pancreatic cancer' may be performed using a pancreatic cancer diagnostic function.
  • pancreatic cancer diagnostic function include the following formula (1):
  • X is a measure of expression level of pancreatic cancer diagnostic markers
  • ⁇ is the Lagrange multiplier in SVM
  • y is the separator of normal group / pancreatic cancer group
  • the function is derived from a support vector machine (SVM).
  • SVM is an algorithm that estimates a function that satisfies a given condition based on the Lagrangian optimization theory. Among them, the SVM supports the classification of the classification method using the maximum margin classifier. It is called (Support Vector Classification, SVC).
  • SVC Support Vector Classification
  • the method of the present invention assigns expression levels of any one of CA19-9 and LRG1, and TTR, C1R, CLU, and KLKB 1 protein or mRNA thereof into the pancreatic cancer diagnostic function, and as a result, the possibility of developing pancreatic cancer is immediately determined. Can be judged by There is no need for a doctor's clinical judgment.
  • the diagnostic function is constructed by SVM, but it can be implemented by various kinds of discriminant analysis methods, including various machine learning methods such as Neural Network and Random Forest, depending on the purpose.
  • X is a new measurement of the protein of any one of CA19-9 and LRG1, TTR, C1R, CLU and KLKB1, oti is the Lagrange multiplier in SVM, yi is
  • the delimiter of the normal / pancreatic cancer group, X i is the reference measurement, and b is the correction value.
  • the protein expression level can be measured and compared using an antibody that specifically binds to the protein of interest.
  • the antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the same is used.
  • antigen-antibody complex means a combination of a protein antigen and an antibody that recognizes it to identify the presence or absence of the gene of interest in a biological sample. Detection of such antigen-antibody complexes is known in the art. Detection can be made using methods as described, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.
  • Methods include protein chip analysis, immunoassays, ligand binding assays,
  • MALDI-TOF Matrix Assisted Laser Desorption / Ionization Time of Flight Mass
  • the LC-MRM method can be used to measure and compare the expression level of each of the CA19-9, LRG1, C1R, CLU and KLKB1 proteins themselves.
  • a solution of 5% acetonitrile, 0.1% formic acid and a solution of 5% distilled water, 95% acetonitrile and 1% formic acid was passed through the LC analytical column with a concentration gradient of 95: 5 to 15:85 for 50 minutes. Can be separated. Concentration gradients are carried out because the resolution for a specific substance may vary depending on the solution mixing ratio, and the above range is an optimal range for simultaneously separating various proteins.
  • Sol A 95% distilled water, 5% acetonitrile, 0.1% formic acid
  • Sol B 5% distilled water, 95% acetonitrile, 0.1% formic acid
  • MRM Multiple Reaction Monitoring
  • MS MS / MS mode
  • SIM Multiple Reaction Monitoring
  • MRM Multiple Reaction Monitoring
  • MRM selects one ion from one broken ion one more time to source another MS in series. It is a method of using ions obtained from these after passing through layers again.
  • SIM there is a problem that the selected quantitative ion may interfere with the quantification when the selected quantitative ion is an ion that is also detected in plasma.
  • MRM If ions are broken once more, the molecular structure is different and tends to be differentiated.
  • SIS stable isotope standard
  • the measurement or comparison of mRNA expression levels of the respective genes encoding CA19-9, LRG1, C1R, CLU and KLKB1 proteins may be reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcription. Enzyme polymerase reaction, RNase protection assay, Northern blotting or DNA chip, etc. may be used, but is not limited thereto.
  • the present invention to provide information for a method for diagnosing pancreatic cancer, the method comprising: (a) obtaining a sample from a subject to diagnose whether or not pancreatic cancer develops; (b) measuring the expression level of CA19-9 protein or mRNA expression level of gene encoding CA19-9 protein, and the expression level of LRG1 protein or mRNA expression level of gene encoding LRG1 protein, respectively, from the sample; (c) the above
  • pancreatic cancer markers the method comprising comparing with expression levels in a sample.
  • the pancreatic cancer may be IPMN, specifically, high risk group IPMN.
  • the composition may be selected from high-risk IPMN by distinguishing it from low-risk IPMN, or in patients with normal, IPMN and pancreatic adenocarcinoma except pancreatic adenocarcinoma, or low-risk.
  • High-risk IPMNs can be diagnosed selectively, distinguishing them from controls that are subjects with IPMN.
  • High risk group IPMN may include high grade dysplasia and invasive type IPMN, and the high risk IPMN may include IPMN-derived pancreatic adenocarcinoma.
  • the expression level of mRNA of the intraductal papillary mucinous neoplasm (IPMN) marker protein or gene encoding the same including LRGl (Leucine-rich alpha-2-glycoprotein 1, LRG1) It relates to a composition for screening diagnosis of high-risk group IPMN, including an agent for measuring the amount. remind
  • the IPMN marker may further comprise one or more markers selected from the group consisting of CA19-9 (carbohydmte antigen 19-9), TTR, C1R, CLU and KLKBl.
  • the IPMN marker is one marker selected from the group consisting of TTR, C1R, CLU and KLKB 1 and markers including LRG1, or two markers selected from the group consisting of TTR, C1R, CLU and KLKB 1 And LRG1.
  • CLU Clusterin preproprotein
  • LRG1 Leucine-rich alpha-2-glycoprotein 1, LRG1
  • CA 19-9 carbohydrate antigen 19-9
  • TTR Transthyretin, ATTR, Prealbumin, TBPA
  • ClR Complement Clr subcomponent precursor
  • KLKB l Plasma It relates to an IPMN marker protein comprising at least one marker selected from the group consisting of Kallikrein protein;), or an agent for measuring the expression level of mRNA of the gene encoding the same, a composition for screening diagnosis of high-risk group IPMN.
  • a method for diagnosing a high risk group IPMN comprising determining whether the subject has a high risk IPMN using the comparison result of the marker expression levels.
  • the method may further include determining whether the subject has IPMN.
  • the checking may be performed by imaging, histology or genetic markers. For example, abdominal ultrasonography, computed tomography (CT), endoscopic retrograde pancreatic duct angiography (endoscopic retrograde)
  • CT computed tomography
  • endoscopic retrograde pancreatic duct angiography endoscopic retrograde
  • cholangiopancreatography ERCP
  • magnetic resonance pancreatobiliary also not limited to one including angiography (magnetic resonance cholangiopancreatography, MRCP), the ultrasonic endoscope, test (Endoscopic ultrasonography, EUS) or the like.
  • diagnosis using a biomarker can be performed using a known pancreatic cancer diagnostic marker such as CA-19-9.
  • Biopsy methods include fine needle aspiration (FNA) biopsy.
  • FNA fine needle aspiration
  • the control group can diagnose high-risk IPMN in the normal group, patients with pancreatic disease suggesting IPMN and pancreatic adenocarcinoma, or patients with low-risk IPMN, using the IPMN marker to distinguish the normal and various pancreatic diseases.
  • the control group is a low-risk IPMN patient, the high-risk IPMN can be diagnosed selectively from the low-risk IPMN.
  • the method may further comprise performing a method of treatment, such as surgical resection, drug administration, etc., if determined as a high risk IPMN of the subject.
  • a method of treatment such as surgical resection, drug administration, etc.
  • the method may further comprise performing a treatment such as drug administration, prognosis monitoring, or the like, when determined as the subject's low risk IPMN.
  • a treatment such as drug administration, prognosis monitoring, or the like, when determined as the subject's low risk IPMN.
  • pancreatic cancer In order to identify protein combinations for the effective diagnosis of pancreatic cancer, pancreatic cancer, other cancers, pancreatitis and cholecystitis patients group (Table 2) and normal group (Table 3) with the consent of the patients of five hospitals as shown in Table 2 and Table 3 below It was configured.
  • Test 1 was a sample group for PDAC classification test, control group consisted of normal, pancreatitis and cholecystitis group, and experimental group consisted of pancreatic cancer (PDAC) group.
  • control group consisted of normal, pancreatitis and cholecystitis group
  • experimental group consisted of pancreatic cancer (PDAC) group.
  • Test 2 was a sample group for PDAC initial staging test, control group consisted of normal : pancreatitis and cholecystitis group, experimental group consisted of stage 1 and 2 of pancreatic cancer (PDAC).
  • control group consisted of normal : pancreatitis and cholecystitis group
  • experimental group consisted of stage 1 and 2 of pancreatic cancer (PDAC).
  • Test 3 was a sample group for cancer / pancreatic cancer selectivity test, control group consisted of other cancer groups, and experimental group consisted of pancreatic cancer groups. Other cancers include 52 breast cancers, 45 colon cancers and 52 thyroid cancers.
  • Test 4 is a sample group for PDAC discrimination without CA19-9 performing clinically.
  • the control group consisted of normal, pancreatitis and cholecystitis groups with CA19-9 levels below 37 U / m.
  • the experimental group had pancreatic cancer (PDAC) with levels lower than CA19-9 . It was composed of groups.
  • a peptide having a mass ratio (m / z) was selected (Q1), and a fragment ion having a characteristic m / z of the peptide was selected among the fragment ions generated when the peptide was fragmented by an electrical shock (Q3). ).
  • This combination of Q1 and Q3 is a combination of ions specific to a particular protein, termed a transition, and is a quantitative information on the signal obtained by sequentially passing ions through this characteristic Q1 and Q3 in a triple quadrupole mass spectrometer. Quantitative analysis was performed in terms of. The National Institute of Standards and NIST, using an open source software called SKYLine, developed by the MacCoss team at the University of Washington, USA
  • peptides with ms / ms data based on the peptide tandem mass spectra of the library up to 10 peptides per protein were selected in order of highest total intensity.
  • Peptides were selected from a minimum of 7 amino acids and a maximum of 24 amino acids.
  • ROS reactive oxygen species
  • @ Missed cleavage may occur if plin is present after R or K that can be cut by trypsin in the peptide.
  • Precursor charges select peptides with a +2 charge and ion
  • the charge used was +1 charge and the ion type used y-ion.
  • Unique transitions and peptides were selected using Protein Blast P and Skyline programs, and the final selected transitions were only those that were within the predicted retention time (RT) range.
  • RT retention time
  • 600 SIS peptide RM analyzes were performed, and a calibration curve based on the hydrophobicity scale and the RT chromatogram was calculated.
  • the LC used a 1260-capillary LC from Agilent and used a column of capillary RR 0.5 ⁇ 150 3.5 um for the separation of peptides.
  • the sample was injected with 5 ⁇ and the flow rate was set at 20 / min.
  • Peptides were added to a concentration gradient of 15:85 at 15:85 for 5 minutes.
  • Agilent's triple quadrupole 6490-QQQ instrument is used for MRM mode for transitions to selected proteins.
  • the spiked 5 fmol beta-galactosidase peptide (GDFQFNISR [C 13 N15], 547.3 / 646.4) was also monitored simultaneously.
  • the internal standard peptide beta-galactosidase peptide (GDFQFNISR [C13N15], 547.3 / 646.4 m / z) was 0.09, 0.27, 0.82, 2.5, 7.4, 22.2, 66.7 and 200 fm respectively.
  • 10 ug of plasma was added to the matrix in the same manner as the target peptide analysis conditions, and the analysis was performed. Also included in the analysis was the case where no internal standard peptide was added for the purpose of confirming the endogenous signal, and the standard curve was determined by quantitating MRM three times at all nine concentration points.
  • MRM results for each individual are correlated using Skyline (MacCoss Lab, verl .4.1). Extraction ion chromatography (XIC) of MRM transitions was generated and the peak areas of each transition were calculated and plotted again over time.
  • SIS Stable isotope standard peptides for each target peptide were synthesized and measured, and the ratio of the target peptide measurement was calculated to quantitatively analyze the amount of blood in each protein.
  • LRG1, TTR and CLU proteins were measured in the pancreatic cancer patients and normal groups by ELISA.
  • IBL's hLRGl ELISA kit was used
  • AssayPro's prialbumin ELISA kit was used
  • CLU R & D Systems' Human Clusterin Quantikine kit
  • the ELISA kit and the sample to be analyzed were left at room temperature prior to the experiment, and then the sample was diluted with a dedicated dilution (LRG1: 1,000 times; TTR: 80,000 times; CLU:
  • Each well was dispensed with 50 standard, control and sample samples, respectively. Each well was covered with a cover sealer and allowed to stand at room temperature for 2 hours. The solution in each well was discarded and washed four times with distilled water. Thereafter, 200 conjugates were dispensed into each well. Each well was covered with a fresh cover sealer, left at room temperature for 2 hours, and then washed four times with distilled water. Thereafter, the substrate solution of was dispensed into each well and then left at room temperature for 30 minutes. Then 50 stop solutions are added to each well After dispensing, absorbance was measured at 540 ⁇ or 570 ⁇ , and the results were summarized by calculating concentration values.
  • TTR protein The expression level of TTR protein in pancreatic cancer patients and normal patients
  • the device and the sample to be analyzed were placed at room temperature before the experiment, and then 50 samples were dispensed. Aliquoted samples were diluted 4-fold with dedicated dilutions. The diluted sample 200 was put into the apparatus, and the results were summarized by the concentration value output from the apparatus.
  • TTR protein was specifically reduced in the pancreatic cancer patient group compared to the normal group, which was MRM-MS and ELISA of Examples 2 and 3. Same as the result.
  • Example 5 Diagnostic Performance of CA19-9, LRG1 and TTR Combination Markers by MRM-MS Method
  • the ROC graph represents the relationship between sensitivity and specificity on a two-dimensional plane. The larger the area under the ROC graph ( ⁇ 1; 0 ⁇ ⁇ ; ⁇ ⁇ 1), the more accurate it is. can do.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.9312, and
  • 0 . 9 was 0.8250, indicating that pancreatic cancer was a very good diagnostic marker.
  • the combination of CA19-9, LRG1 and TTR according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using CA19-9, LRG1 and TTR alone or in combination of two.
  • Example 2 the diagnostic performance for the early stage division of pancreatic cancer of the combination marker of CA19-9, LRG1 and TTR was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and TTR proteins by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.9070, Sn
  • Sp 0 9 has been shown to be very good as a diagnostic marker for the early staging of pancreatic cancer.
  • the combination of CA19-9, LRG1, and TTR according to the present invention was confirmed that the early stage diagnosis of pancreatic cancer is superior to when using a commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • CA19-9 protein is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8994, Sn
  • Sp 0 9 was 0.8250, indicating a very good performance as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and TTR according to the present invention is a commercial pancreatic cancer diagnostic marker CA19-9 It was confirmed that the cancer / pancreatic cancer separation performance is better than when used alone.
  • the diagnostic performance for the pancreatic cancer classification of the combination marker of CA19-9, LRG1 and TTR was analyzed in the experimental group CA19-9 ⁇ 37 U / m £.
  • pancreatic cancer is determined when the CA19-9 axis value is over 37 U /. Therefore, in the experimental group CA19-9 ⁇ 37 U / ii, CA19-9 can not perform as a diagnostic marker for pancreatic cancer.
  • the CA19-9 protein is Roche
  • the graph is a representation of the relationship between sensitivity and specificity on a two-dimensional plane.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8295, Sn
  • Sp 9 is 0.5172 to diagnose pancreatic cancer
  • Example 6 Diagnosis of CA19-9, LRG1 and TTR Combination Markers by ELISA Method Combination of CA19-9, LRG1 and TTR using the MRM-MS method in Example 5
  • the diagnostic performance of the markers for pancreatic cancer classification was analyzed.
  • the ELISA method was used to determine whether the performance was reproducible.
  • the combination of CA19-9, LRG1, and TTR was confirmed to be excellent in pancreatic cancer diagnosis.
  • Sp 0 .
  • the measurement result of 9 is shown in Table 6 and FIG. 15.
  • the CA19-9 protein is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is ELISA
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.9144, Sn
  • Sp 0 9 was 0.7800, indicating a very good performance as a diagnostic marker for early staging of pancreatic cancer.
  • the combination of CA19-9, LRG1 and TTR according to the present invention can be confirmed that the early stage of pancreatic cancer diagnostic performance better than when using a single pancreatic cancer diagnostic marker CA19-9 alone
  • the CA19-9 protein is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is ELISA
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8981, Sn
  • Sp 0 . 9 is 0.8250, which is very good as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and TTR according to the present invention was confirmed that the cancer / pancreatic cancer discrimination performance is better than when using a commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • pancreatic cancer was determined when the CA19-9 level is 37 U /. Therefore, in the experimental group of # 19-9 ⁇ 371; / 111 £, CA19-9 could not exert the performance as a diagnostic marker for pancreatic cancer.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by ELISA method.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8287, Sn
  • the performance as a marker was found to be very good.
  • the combination of CA19-9, LRG1 and TTR according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 7 Diagnosis Performance of CA19-9, LRG1 and TTR Combination Markers by Immunodiluting Method Using the MRM-MS Method in Example 5, Diagnosis Performance of Pancreatic Cancer Classification of Combination Markers of CA19-9, LRG1 and TTR It was confirmed that the performance was reproduced by the immuno-binding method, the combination of CA19-9, LRG1 and TTR in the immuno-binding method was confirmed that the pancreatic cancer diagnostic performance is excellent.
  • CA19-9 0.5198 0.2414 As shown in Table 7 and FIG. 19, the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.9402, and Sn
  • Sp o. 9 , 8250, was shown to be very good as a diagnostic marker for pancreatic cancer. In particular, the combination of CA19-9, L G1 and TTR according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 4 the diagnostic performance for the early stage division of pancreatic cancer of the combination marker of CA19-9, LRG1 and TTR was analyzed.
  • the CA19-9 protein was measured by chemiluminescence enzyme immunoassay (CLEIA), the LRG1 protein was determined by ELISA, and the TTR protein was determined by immunobidification.
  • the measurement result of Sp 0.9 is shown in Table 7 and FIG. 20.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.9146 and Sn
  • Sp 0 9 was 0.7600, indicating a very good performance as a diagnostic marker for early staging of pancreatic cancer.
  • the combination of CA19-9, LRG1, and TTR according to the present invention was confirmed that the early stage diagnosis of pancreatic cancer is superior to when using a commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 4 the diagnostic performance for the cancer and pancreatic cancer classification of the combination markers of CA19-9, LRG1 and TTR was analyzed.
  • the CA19-9 protein was measured by chemiluminescence enzyme immunoassay (CLEIA), the LRG1 protein was determined by ELISA, and the TTR protein was determined by immunobidification.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8965, Sn
  • Sp 0 9 was 0.8250, which is very good as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and TTR according to the present invention was able to confirm that the cancer / pancreatic cancer discrimination performance is better than when using a commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • the diagnostic performance of the pancreatic cancer classification of the combination markers of CA19-9, LRG1 and TTR using the immunobinding method of Example 4 was performed in the experimental group having CA19-9 ⁇ 37 U / ⁇ .
  • pancreatic cancer is determined when the CA19-9 level is 37 U /. Therefore, in the experimental group CA19-9 ⁇ 37 U / CA19-9 can not perform as a diagnostic marker for pancreatic cancer.
  • the CA19-9 protein was measured by chemiluminescence enzyme immunoassay (CLEIA), the LRG1 protein by ELISA method, and the TTR protein by immunobinding method.
  • CLIA chemiluminescence enzyme immunoassay
  • Sp 0 .
  • the measurement result of 9 is shown in Table 7 and FIG. 22.
  • the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8439, Sn
  • Sp 0 9 is 0.5172 to diagnose pancreatic cancer
  • the combination of CA19-9, LRG1 and C1R according to the present invention has an AUC of 0.9296, and Sn
  • Sp 0 . 9 Pancreatic Cancer Diagnosis as ER8375
  • the performance as a marker was found to be very good.
  • the combination of CA19-9 : LRG1 and C1R according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 2 the diagnostic performance for the early stage pancreatic cancer classification of the combination markers of CA19-9, LRG1 and C1R was analyzed.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and C1R proteins by MM quantitative analysis.
  • the combination of CA19-9, LRG1 and C1R according to the present invention has an AUC of 0.9097, Sn
  • S p o. 9 is 0.7800, which is very good as a diagnostic marker for the early stage of pancreatic cancer.
  • the combination of CA19-9, LRG1 and C1R according to the present invention was confirmed to be superior to the early stage diagnosis of pancreatic cancer when using CA19-9, LRG1 and C1R alone or in combination of two.
  • Example 2 Diagnosis of CA19-9, LRG1 and C1R Combination Markers for Cancer / Pancreatic Cancer
  • the MRM-MS method of Example 2 was used to distinguish between cancer and pancreatic cancer of the combination markers of CA19-9, LRG1 and C1R.
  • the diagnostic performance was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA).
  • the LRG1 and C1R proteins were determined by MRM quantitative analysis.
  • Sp o.
  • the measurement result of 9 is shown in Table 10 and FIG. 25.
  • Sp 0 9 was 0.8375, which shows a very good performance as a diagnostic marker for cancer and pancreatic cancer.
  • the diagnostic performance for pancreatic cancer classification of the combination markers of CA19-9, LRG1 and C1R was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and C1R proteins were determined by MRM quantitative analysis.
  • the combination of CA19-9, LRG1 and C1R according to the present invention has an AUC of 0.8160, Sn
  • Sp 0 9 is 0.5517 to diagnose pancreatic cancer
  • the MRM-MS method of Example 2 the diagnostic performance for the early stage classification of pancreatic cancer of the combination marker of CA19-9, LRG1 and CLU was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and CLU proteins by ⁇ MRM quantitative analysis.
  • the measurement result of Sp 0.9 is shown in Table 12 and FIG.
  • CA19-9 0.5198 0.2414 As shown in Table 12 and Figure 28, the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9027, Sn
  • Sp 0 9 was 0.7800, indicating a very good performance as a diagnostic marker for early staging of pancreatic cancer.
  • the combination of the CA19-9, LRG1 and CLU according to the present invention was confirmed that the performance of diagnosing the early stage of pancreatic cancer is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • the MRM-MS method of Example 2 was analyzed to analyze the diagnostic performance of cancer and pancreatic cancer classification of the combination markers of CA19-9, LRG1 and CLU.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were determined by MRM quantitative analysis.
  • Sp 0 .
  • the measurement result of 9 is shown in Table 12 and FIG. 29.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9093, Sn
  • Sp o. 9 is 0.8500, which shows excellent performance as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and CLU according to the present invention was confirmed that the cancer / pancreatic cancer discrimination performance is better than when using the commercial pancreatic cancer marker CA19-9 alone.
  • Example 2 the diagnostic performance for pancreatic cancer classification of the combination markers of CA19-9, LRG1 and CLU was analyzed.
  • the CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.
  • the measurement result of Sp 0.9 is shown in Table 12 and FIG.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.8384, Sn
  • Sp 0 9 is 0.5862 to diagnose pancreatic cancer
  • the performance as a marker was found to be very good.
  • the combination of CA19-9, LRG1 and CLU according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 10 Diagnosis Performance of CA19-9, LRG1 and CLU Combination Markers by ELISA Method
  • Example 9 the diagnostic performance for the pancreatic cancer classification of the combination markers of CA19-9, LRG1 and CLU was examined using the MRM-MS method. It was confirmed whether the performance was reproduced by the ELISA method, the combination of CA19-9, LRG1 and CLU in the ELISA method was confirmed that the pancreatic cancer diagnostic performance is excellent.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9399, Sn
  • Sp 0.9 was 0.8000, indicating a very good performance as a diagnostic marker for pancreatic cancer.
  • CA19-9 according to the present invention Combination of LRG1 and CLU was confirmed that the pancreatic cancer diagnosis performance is better than when using CA19-9, LRG1 and CLU alone or in combination of two.
  • Example 3 the diagnostic performance of the early stage of pancreatic cancer classification of the combination marker of CA19-9, LRG1 and CLU was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were determined by ELISA quantitative analysis.
  • the measurement result of Sp 0.9 is shown in Table 14 and FIG.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9193, Sn
  • As Sp 0.9 was 0.6400, it was found to be very good as a diagnostic marker for early stage pancreatic cancer.
  • the combination of the CA19-9, LRG1 and CLU according to the present invention was confirmed that the performance of diagnosing the early stage of pancreatic cancer is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9079, Sn
  • Sp 0 9 was 0.8000, which shows a very good performance as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and CLU according to the present invention was confirmed that the cancer / pancreatic cancer discrimination performance is better than when using the commercial pancreatic cancer marker CA19-9 alone.
  • Example 3 the diagnostic performance for the pancreatic cancer classification of the combination marker of CA19-9, LRG1 and CLU was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA) and the LRG1 and CLU proteins were determined by ELISA quantitative analysis.
  • Sp o.
  • the measurement result of 9 is shown in Table 14 and FIG. 34.
  • the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.8435, Sn
  • CA19-9 was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis.
  • Sp 0 .
  • the measurement result of 9 is shown in Table 15 and FIG. 35.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention has an AUC of 0.9382, and Sn
  • Sp 09 was 0.8625, indicating a very good performance as a diagnostic marker for pancreatic cancer.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention was confirmed that the pancreatic cancer diagnostic performance is superior to when using CA19-9, LRG1 and KLKB1 alone or in combination of two.
  • the MRM-MS method of Example 2 the diagnostic performance of the early stages of pancreatic cancer classification of the combination markers of CA19-9, LRG1 and KLKB1 was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis.
  • CA19-9 0.5198 0.2414 As shown in Table 16 and FIG. 36 above, the combination of CA19-9, LRG1 and KLKB1 according to the present invention has an AUC of 0.9148 and Sn
  • Sp 0 9 was 0.8000, indicating a very good performance as a diagnostic marker for early staging of pancreatic cancer.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention is a commercial pancreatic cancer diagnostic marker.
  • pancreatic cancer The early stage diagnosis of pancreatic cancer was superior to that of CA19-9 alone.
  • the MRM-MS method of Example 2 the diagnostic performance for the cancer and pancreatic cancer classification of the combination markers of CA19-9, LRG1 and KLKB1 was analyzed.
  • the CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention has an AUC of 0.8924, and Sn
  • Sp 0.9 was 0.8625, indicating a very good performance as a diagnostic marker for cancer and pancreatic cancer.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention was confirmed that the cancer / pancreatic cancer discrimination performance is superior to when using a commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention has an AUC of 0.8349, and Sn
  • Sp 0 9 was 0.6207, indicating a very good performance as a diagnostic marker for pancreatic cancer.
  • the combination of CA19-9, LRG1 and KLKB1 according to the present invention was confirmed that the pancreatic cancer diagnostic performance is better than when using a single pancreatic cancer diagnostic marker CA19-9 alone.
  • SVM Small Vector Machine
  • SVM is an algorithm that estimates a function that satisfies a given condition based on the Lagrangian optimization theory. Among them, the SVM uses a classification method using the maximum margin classifier. Support Vector Classification (SVC). In this embodiment, using one sample group of two sample groups, the SVC that maximizes the difference between two groups (normal group and cancer patient group) within the sample group is derived through machine learning to construct a pancreatic cancer diagnosis function as follows. It was.
  • X is a measure of expression level of pancreatic cancer diagnostic markers
  • cti is the Lagrange multiplier in SVM
  • yi is the delimiter of normal group / pancreatic cancer group
  • a value of 1 is determined to be pancreatic cancer, and a value of -1 is normal. remind Pancreatic cancer was determined using the function.
  • pancreatic cancer was determined by using Formula 1 based on SVM (Support Vector Machine).
  • pancreatic cancer was determined using Function 1 based on SVM (Support Vector Machine).
  • SVM Serial Vector Machine
  • MRM quantitative values of LRG1 and CLU respectively (7.4, 1.451, 3.3803), (6.3, 1.0718, 3.1325) And (26.1, 1.2053, 2.8642)
  • f (7.4, 1.451, 3.3803) -1
  • f (6.3, 1.0718, 3.1325) -1
  • f ( 26.1, 1.2053, 2.8642) -1 to determine that the subject was normal.
  • pancreatic cancer was determined using Function 1 based on SVM (Support Vector Machine).
  • SVM Serial Vector Machine
  • MRM quantitative values of L G1 and KLKB1 respectively (7.4, 1.451, 1.2801).
  • f (7.4, 1.451, 1.2801) -1
  • f (6.3, 1.0718, 0.961) -1
  • f (26.1, 1.2053, 1.5657) -1 was able to determine that the subject is normal.
  • the patient was diagnosed with early stage pancreatic cancer.
  • Example 14 Diagnosis of Pancreatic Cancer from Other Cancers-Data Statistical Analysis
  • the initial stage of the pancreatic cancer was determined using the functional formula 1 based on SVM (Support Vector Machine).
  • the test group was divided into high risk groups.
  • Test 5 consisted of high grade dysplasia and invasive types of high risk (malignant) subtype IPMN experimental group, low dysplasia IPMN, intermediate dysplasia IPMN, normal group and patients with gallstones as benign inflammatory diseases.
  • Test 6 screens for high and low risk groups according to MMS analysis To determine whether the high grade dysplasia and invasive types of the high risk (malignant) subtype IPMN test group consisted of low dysplasia IPMN and intermediate dysplasia IPMN and the test group for screening detection.
  • Test 7 screens for the detection of high-risk and low-gastric groups according to the ELISA assay.
  • the invasive type-i-Jl " 3 ⁇ 4 (malignant) subtype IPMN test group consisted of a low dysplasia IPMN and an intermediate dysplasia IPMN test group for screening detection.
  • the CA19-9 protein is Roche Diagnostics By chemiluminescent enzyme immunoassay (CLEIA) using a COB AS Elecsys CA 19-9 instrument, LRG1 and TTR proteins were measured by MRM quantitative analysis.
  • CLIA chemiluminescent enzyme immunoassay
  • the ROC graph is a representation of the relationship between sensitivity and specificity on a two-dimensional plane. The larger the area under the ROC graph (AUC; 0 ⁇ AUC ⁇ 1), the more accurate it is. . Sn
  • Sp 0.9
  • Sp 0 .
  • the measurement result of 9 is shown in Table 18 and FIGS. 39 to 63.
  • the markers according to the present invention or a combination of two or more markers are diagnostic markers for selectively detecting high-risk IPMNs, distinct from the control group. Its performance was shown to be very good. In particular, the combination markers according to the present invention were able to confirm that the high-risk IPMN detection performance is superior to that of using commercial pancreatic cancer diagnostic marker CA19-9 alone.
  • Example 17 Screening Detection of Low Risk IPMN and High Risk IPMN by MRM-MS Method The sample test of Example 15 for the differential detection of low risk IPMN and high risk IPMN. For 6, the tests shown in Table 33 were performed in substantially the same manner as in the MMS assay of Example 16. The AUC and Sn
  • the measurement result of Sp o.9 is shown in Table 19 and FIGS. 64 to 89.
  • the marker according to the present invention or a combination of two or more markers is a diagnostic marker for selectively detecting high-risk IPMN ol, distinguished from low-risk IPMN. Its performance was shown to be very good.
  • the combinatorial markers according to the present invention may be used for the pancreatic cancer diagnostic marker CA19-9.
  • Example 18 Screening Detection of Low-Risk IPMN and High-Risk IPMN by ELISA Method
  • the diagnostic performance of the marker pancreatic cancer was analyzed using the MRM-MS method.
  • the marker combination of Table 20 was confirmed to be excellent in distinguishing diagnostic performance of high-risk IPMN.
  • the marker according to the present invention was shown to have a very good performance as a diagnostic marker for selectively detecting a high risk group IPMN to be distinguished from a low risk group IPMN.
  • the markers according to the present invention were confirmed to be superior to the high-risk IPMN detection performance than when using commercial pancreatic cancer diagnostic marker CA19-9 alone.

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Abstract

La présente invention concerne : une composition pour le diagnostic de cancer du pancréas, contenant une préparation pour mesurer le niveau d'expression d'une protéine ou d'un gène de cette dernière, et capable d'être utilisée pour déterminer s'il y a un risque de cancer du pancréas ; un kit ; et un procédé pour le diagnostic de cancer du pancréas à l'aide de cette dernière. La présente invention peut significativement prédire ou identifier le risque de cancer du pancréas ou d'une lésion précancéreuse de cancer du pancréas, le diagnostic précoce de ce dernier, et l'étendue de maladies de ce dernier par fourniture d'un marqueur de diagnostic de cancer du pancréas, et peut être utilisée dans la recherche d'oncogenèse de cancer du pancréas. De plus, le procédé de diagnostic de la présente invention peut fournir un diagnostic simple et précoce de cancer du pancréas à partir du sang et analogue d'une manière non-effractive.
PCT/KR2015/009827 2014-10-17 2015-09-18 Composition pour le diagnostic de cancer du pancréas, et procédé pour le diagnostic de cancer du pancréas à l'aide de cette dernière Ceased WO2016060382A1 (fr)

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EP15850998.4A EP3208614B1 (fr) 2014-10-17 2015-09-18 Composition pour le diagnostic de cancer du pancréas, et procédé pour le diagnostic de cancer du pancréas à l'aide de cette dernière
CN201811471602.XA CN109576368B (zh) 2014-10-17 2015-09-18 用于诊断胰腺癌的组合物以及使用其诊断胰腺癌的方法
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