WO2016060382A1 - Composition for diagnosing pancreatic cancer and method for diagnosing pancreatic cancer by using same - Google Patents

Abstract

The present invention relates to: a composition for diagnosing pancreatic cancer, containing a preparation for measuring the expression level of a protein or a gene thereof, and capable of being used for determining whether there is a risk of pancreatic cancer; a kit; and a method for diagnosing pancreatic cancer by using the same. The present invention can significantly predict or identify the risk of pancreatic cancer or a precancerous lesion of pancreatic cancer, the early diagnosis thereof and the extent of diseases thereof by providing a diagnostic marker of pancreatic cancer, and can be utilized in the research of pancreatic cancer oncogenesis. In addition, the diagnostic method of the present invention can provide simple and early diagnosis of pancreatic cancer from the blood and the like in a non-invasive manner.

Description

 【Specification】

 [Name of invention]

 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

It is about a diagnosis method.

[Technique to become background of invention]

 Among the major diseases of modern people, researches on the treatment and diagnosis of cancer are relatively active, mainly in lung cancer, liver cancer, gastric cancer, and the like. However, studies on esophageal cancer, colorectal cancer, and pancreatic cancer with low incidence incidence are relatively low.

 In particular, 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.

 5-fluorouracil, known to date for 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

Malignant lesions of the pancreas are frequently found due to many imaging tests such as abdominal ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI). Lesions showing cystic lesions in the diagnosis were precancerous from benign lesions.

It is clinically important because it contains a variety of diseases ranging from premalignant and malignant lesions.

 However, despite the development of imaging tests such as CT and MRI, it is still difficult to accurately differentiate the cystic lesions of the pancreas with various pathologic findings from benign lesions to precancerous and cystic lesions. Therefore, it is important to clinically distinguish whether cystic lesions of the pancreas are malignant tumors, and if so, whether they are still in the precancerous stage or already have malignant lesions.

 Republic of Korea Patent No. 10-0819122 and Republic of Korea Patent 1

In 2012-0082372, matrilin, transthyretin,

Techniques have been disclosed using various pancreatic cancer markers, including stratifin and the like. In addition, 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, and 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. However, since 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.

[Content of invention]

 Problem to be solved

 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.

 It is a further object of the present invention to provide a method for determining the presence or absence of malignancy of pancreatic cancer in a subject to be detected using the diagnostic composition or kit.

 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.

[Measures of problem]

In order to achieve the above object, 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. In another embodiment of the present invention, 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 /.

Specifically, the 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. Known pancreatic cancer markers, such as pancreatic cancer with a CA19-9 of less than 37 U / ml, and 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

 (a) carbohydrate antigen 19-9 (CA 19-9);

 (b) Leucine-rich alpha-2-glycoprotein 1, LRG1; And

 (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).

 It includes markers for diagnosing pancreatic cancer.

 In order to achieve the above another object, the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition.

 In order to achieve the above another object, the present invention comprises the steps of obtaining a sample from a subject to diagnose whether the pancreatic cancer;

As a combination of at least three kinds or more marker genes from the sample, specifically, (a) CA19- ^9; (b) LRGl; And (c) measuring the expression level or mRNA expression level of the protein of pancreatic cancer markers including any one or more marker genes selected from the group consisting of TTR, C 1 R, CLU and KLKB 1, respectively; To compare the expression levels of marker genes obtained from the subjects with expression levels in normal control samples, respectively, to diagnose the onset of 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.

 In the determining step, as a result of comparing the expression level of the marker gene, 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, or 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. For example, 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.

 Further embodiments of the present invention, CLU (Clusterin preproprotein); And

Leucine-rich alpha-2-glycoprotein 1 (LRG1), carbohydrate antigen 19-9 (CA 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. For example, 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,

 Comparing the measured expression level of the marker with the expression level of the control marker, and

 Using the comparison result of the marker expression levels to determine whether the subject is at high risk of IPMN,

 A method for diagnosing high risk group IPMN. 【Effects of the Invention】

 By providing a diagnostic marker for pancreatic cancer, 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. In addition, the diagnostic method of the present invention can non-invasively diagnose early pancreatic cancer simply from blood or the like.

[Brief Description of Drawings]

 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).

 2 is a control and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis

It is a graph showing the expression level of L G1 protein.

 3 is a control and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis

It is a graph which shows the expression amount of TTR protein.

 4 is a control and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis

It is a graph showing the expression level of C 1 R protein.

5 is a control and pancreatic catheter adenocarcinoma patient group by MRM quantitative analysis It is a graph showing the expression level of CLU protein.

 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.

 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.

 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.

 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.

 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.

 11 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 chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by MRM quantitative analysis.

 12 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer when using CA19-9, LRG1 and TTR 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 TTR proteins were measured by MRM quantitative analysis.

 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. ᅳ

 15 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 chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by ELISA method.

 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.

 FIG. Π 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

By assay (CLEIA), 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.

20 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 is By chemiluminescent enzyme immunoassay (CLEIA), LRG1 protein was measured by ELISA method, TTR protein was measured by immunobidification method.

 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

By assay (CLEIA), LRG1 protein was determined by ELISA method and TTR protein was determined by immunobidification method.

23 compares pancreatic cancer diagnostic performance (AUC and detection sensitivity) 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. will be. The 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.

 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.

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

By Assay (CLEIA), LRG1 and C1R proteins were measured by MRM quantitative analysis.

 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. will be. The CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.

 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.

 29 shows diagnostic performance for distinguishing pancreatic cancer from other cancers 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. And detection sensitivity). The CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and CLU proteins were measured by MRM quantitative analysis.

 FIG. 30 illustrates an experimental group having a CA19-9 value of 37 U / or less.

Comparison of 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

By assay (CLEIA), LRG1 and CLU proteins were measured by MRM quantitative analysis.

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. will be. 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. By the CA19-9 protein ^ chemiluminescent enzyme immunoassay (CLEIA), LRG1 and CLU proteins were measured by ELISA method.

 33 shows 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

By assay (CLEIA), 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.

 36 shows early stage diagnostic performance (AUC and detection sensitivity) of pancreatic cancer 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. It is a comparison. The CA19-9 protein was determined by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis.

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

By Assay (CLEIA), LRG1 and KLKB 1 proteins were measured by MRM quantitative analysis.

 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.

 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

By Assay (CLEIA), LRG1, TTR, CLU, C1R and KLKB1 proteins were measured by MRM quantitative analysis.

 45-48 show (L G 1 + C A 19-9 + TTR), (LRG 1 + C A 19-9 + CLU),

Diagnostic performance (AUC and detection sensitivity) of high-risk IPMN when three combinations of (LRG1 + CA19-9 + C 1R) and (LRG1 + CA19-9 + KLKB1) were used simultaneously and CA19-9 protein alone Is a comparison. The CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1, TTR, CLU, C1 R and KLKBl proteins were measured by MRM quantitative analysis.

 49-54 show (LRG1 + TTR + CLU), (LRG1 + TTR + C1R),

(LRG1 + TTR + KL B 1), (LRG1 + CLU + C1R), (LRG1 + CLU + KLKB 1), and

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.

 58-63 show (CLU + CA19-9 + TTR), (CLU + CA19-9 + ClR),

(CLU + CA 19-9 + KLKB 1), (CLU + TTR + C 1 R), (CLU + TTR + KLKB 1), and

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 comparison of diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs with 9 protein alone compared to low-risk IPMNs. 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.

Figure 70 to Fig. 73 (CA19-9 + LRGl + TTR), (CA19- 9 + LRGl + ClR),

 Diagnostic performance of high-risk IPMNs distinguished from low-risk IPMNs when using three combination marker proteins (CA19-9 + LRG1 + CLU), and (CA19-9 + LRG1 + KLKB1) and CA19-9 protein alone ( AUC and detection sensitivity). The CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1, TTR, CLU, C1R and KLKB1 proteins were measured by MRM quantitative analysis.

 74-79 show (LRG1 + TTR + CLU), (LRG1 + TTR + C1R),

(LRG1 + TTR + KLKB1), (LRG1 + CLU + C1R), (LRG1 + CLU + KLKB1) and

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

It was measured by MRM quantitative analysis.

 80 to 83 show two combination marker proteins of (CLU + CA19-9), (CLU + TTR), (CLU + C1R), and (CLU + KLKB1) and when using CA19-9 protein alone. Comparison of diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs with low-risk IPMNs. The CA19-9 protein is chemiluminescent enzyme immunity

By Assay (CLEIA), LRG1, TTR, CLU, C1R and KLKB1 proteins were measured by MRM quantitative analysis.

 84-89 show (CLU + CA19-9 + TTR), (CLU + CA19-9 + C1R),

(CLU + CA19-9 + KLKB1), (CLU + TTR + C1R), (CLU + TTR + KLKB1) and

 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

It was measured by MRM quantitative analysis.

 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.

 91-93 show (LRG1 + CA19-9), (LRG1 + TTR) and (LRG1 + CLU)

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.

 94-97 show (LRG1 + CA19-9 + TTR), (LRG1 + CA19-9 + CLU),

 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 diagnostic performance (AUC and detection sensitivity) of high-risk IPMNs distinguished from low-risk IPMNs when using a combination marker protein of (CLU + CA19-9 + TTR) versus CA19-9 protein alone. The CA19-9 protein is chemiluminescent enzyme immunity

By Assay (CLEIA), LRG1, TTR, CLU, C1R and KLKB1 proteins were measured by ELISA assay.

[Specific contents to carry out invention]

 In the present invention, the term "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. This includes malignant tumors, including benign tumors, malignant tumors, and benign or malignant cases, for example, pancreatic adenocarcinoma (Pancreatic ductal adenocarcinoma) PD AC) 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. Standing sheet, and the like liquid cystic tumor, mucinous cystic tumor, pancreatic duct in papillary mucinous tumors (Intraductal papillary mucinous neoplasm, IPMN), solid tumor pseudogenes heads.

 Preferably, the pancreatic cancer may be pancreatic adenocarcinoma or IPMN, and 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.

 WHO's tumor classification defines 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. In the pancreatic duct It is characterized by papillary growth of mucus-secreting cells and has a broad spectrum from benign to malignant. 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.

 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.

 Therefore, 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).

 In the present invention, 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. Provided are methods, compositions, and kits that differentiate these malignancies. The pancreatic cancer-related markers according to the present invention can select high risk group IPMN by distinguishing it from low risk group IPMN.

 In addition, the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition. Preferably, 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.

 As used herein, the term "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). Preferably, the diagnosis in the present specification is to determine whether pancreatic cancer develops or the possibility (danger).

 As used herein, the terms "marker biomarker" or "diagnostic marker" can distinguish between normal or pathological conditions or predict treatment response.

It means a marker that can be measured objectively. In particular, with respect to pancreatic cancer in the present invention, 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). Refers to 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

protein expression level of the 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.

 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.

Another pancreatic cancer diagnostic marker used in the present invention 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

It may be at least one selected from the group consisting of KLKB1. TTR protein is related to the subunit consisting of 127 amino acid residues, etc. As one of the plasma proteins to form a four-polymer in ^blood, and all brain functions to transport the thyroid hormone, Alzheimer's disease, breast cancer, ovarian cancer, stomach cancer. 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.

 As a specific example, TTR may be NCBI Accession # NP # 000362 · l, ClR may be NCBI Accession # NP_001724.3, CLU may be NCBI Accession # NP_001822.3, and KLKB1 may be NCBI Accession #NP — 000883.2.

 We analyzed plasma samples from patients with pancreatic ductal adenocarcinoma (PD AC), patients with intraductal papillary mucinous neoplasm (IPMN), and patients with chronic cholecystitis. By using a combination of three types of marker genes consisting of CA19-9 and LRG1 and one of TTR, C1R, CLU, and KLKB1 in the same manner as described above, the biomarker was analyzed by analyzing a plasma sample of a normal control subject without the disease. The validity of the combination was established.

 Table 1

 Course Method Count

 Data mining

 Pancreatic cancer diagnostic marker candidate 1,000

 Microarray analysis

 MRM-MS Applicable Candidate MRM-MS 246 Excellent diagnostic performance Excellent candidate Single marker criteria AUC> 0.6 143

SIS Applicable Candidate MRM-MS 54

 1〜 among 3 protein combination marker candidates

 Finalists 4

본 발명에 사용된 용어, "단백질의 발현 수준 측정"이란 췌장암을 진단하기 위하여 생물학적 시료에서 췌장암 진단용 마커 (단백질) 또는 이를 암호화하는 유전자의 존재 여부와 발현 정도를 확인하는 과정을 의미한다. Performance under a variety of conditions

As used herein, 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). 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

 Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blotting, and enzyme—linked immunosorbentassay (ELISA). It is not.

 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).

As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and results in an antigen-antibody reaction. For the purposes of the present invention, 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. Such polyclonal antibodies can be prepared from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like. In addition, 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. In addition, 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.

 As used herein, the term "Peptide Nucleic Acid" is artificially synthesized,

It refers to a polymer similar to DNA or RNA, and was first introduced in 1991 by professors Nielsen, Egholm, Berg and Buchardt at the University of Copenhagen, Denmark. DNA has a phosphate-ribose sugar backbone, whereas ΡΝΑ has a repeating Ν- (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. ΡΝΑ is described in Nielsen ΡΕ, Egholm Μ, Berg RH, Buchardt 0 (December 1991). "Sequence-selective recognition of DNA by strand displacement with a

thymine-substituted polyamide ". Science 254 (5037): 1497-50 is disclosed in detail.

 "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.

78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

 The term "measurement of expression level of mRNA" used in the present invention 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. .

Analytical methods for this include reverse transcriptase (RT-PCR), competitive reverse transcriptase Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips It doesn't happen.

 In the composition for diagnosing pancreatic cancer according to the present invention, 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

Nucleotides. CA19-9, LRG1, TTR, C1R, CLU and according to the present invention

The information of KL B 1 protein is known from UniProt et al.

Based on the mRNA of the gene encoding the protein

Primers, probes or antisense nucleotides that bind will be readily designed. .

 As used herein, the term “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. .

 As used herein, the term “probe” refers to a substance that can specifically bind to a target substance to be detected in a sample.

Specifically, 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,

It 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 八, 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. . As used herein, the term “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.

 In addition, the present invention provides a pancreatic cancer diagnostic kit comprising the pancreatic cancer diagnostic composition. For example, 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.

 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.

 For example, 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

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,

DNase, RNase inhibitor DEPC-water, sterile water, and the like.

In addition, the diagnostic kit of the present invention may include the necessary elements necessary to perform the DNA chip. 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.

 In addition, 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.

 In addition, the 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.

 Accordingly, 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

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

To provide a composition.

 In addition, the 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. Accordingly, 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 Provided are compositions for distinguishing pancreatic cancer from other cancers, including agents that measure the expression level of one or more markers.

 Furthermore, 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:

Obtaining a sample from a subject to diagnose whether pancreatic cancer develops; (A) CA19-9, (b) LRG1 and (C) TTR, C1R, CLU and

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

Comparing the expression levels of 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. For example, 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. In one embodiment, the subject may mean a mammal, and in another embodiment, a human.

 Determining whether the target pancreatic cancer using the expression level comparison results of the marker, specifically, the expression level of the CA19-9 protein or mRNA of the gene encoding the CA19-9 protein in the subject to diagnose whether the pancreatic cancer 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.

Higher than the expression level in the control group, 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.

In the above method, 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.

 For example, 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 When the expression level in pancreatic cancer patients decreases as compared with the expression level in the normal control group, 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.

In addition, 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.

 In one embodiment, the 'determining the high likelihood of developing pancreatic cancer' may be performed using a pancreatic cancer diagnostic function. Examples of the pancreatic cancer diagnostic function include the following formula (1):

<Function 1>

 Where

 X is a measure of expression level of pancreatic cancer diagnostic markers,

 αι is the Lagrange multiplier in SVM,

 y, is the separator of normal group / pancreatic cancer group ，

 X i is the reference measurement, and

 b means a correction value.

 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). When M19 relative values of any one of CA19-9 and LRG1 and TTR, C 1 R, CLU and KLKB1 were substituted into the function, a value of 1 diagnosed as having a high probability of developing pancreatic cancer, and a value of- If 1, the diagnosis is normal.

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.

In this study, 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. In addition, (a) obtaining a sample from a subject to diagnose whether or not pancreatic cancer; (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 from the sample; (c) measuring the expression level of any one protein selected from the group consisting of TTR, C1R, CLU and KLKB1 from the sample, or the expression level of mRNA of a gene encoding the protein; And (d) substituting the numerical values measured in (b) and (c) into the following Formula 1 to determine the possibility of developing pancreatic cancer.

 <Function 1>

상기 식에서, X는 CA19-9 및 LRG1 , TTR, C1R, CLU 및 KLKB1 중 어느 하나의 단백질의 신규 측정값을, oti는 SVM에서의 라그랑주 승수를, yi는 n

Wherein 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. In the method of the present invention, 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.

As used herein, the term “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.

 For the purposes of the present invention, the protein expression level measurement or comparative analysis

Methods include protein chip analysis, immunoassays, ligand binding assays,

Matrix Assisted Laser Desorption / Ionization Time of Flight Mass (MALDI-TOF)

 Spectrometry analysis, Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry (SELDI-TOF) analysis, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D Electrophoresis, Liquid Chromatography-Mass

Spectrometry, LC-MS), LC -MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western block ¾ ^"and ELISA (but include enzyme linked immunosorbent assay) are not limited thereto.

 In the present invention, 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.

Specifically, a biological protein in the sample by volume _^0/0 95% based on distillation shoe,

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. First equilibrate the column with Sol A (95% distilled water, 5% acetonitrile, 0.1% formic acid) for 10 minutes, then use Sol B (5% distilled water, 95% acetonitrile, 0.1% formic acid) for 5 minutes to 85%. Until 5 minutes, eluted the tempide through a concentration gradient of 85%.

In mass spectrometry, quantitation can be performed by MRM (Multiple Reaction Monitoring), an MS / MS mode. Whereas Selected Ion Monitoring (SIM) is a method that uses ions generated by collisions once in the source section of a mass spectrometer, 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. In 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. On the other hand, when using MRM, If ions are broken once more, the molecular structure is different and tends to be differentiated. Therefore, when used as a quantitative ion, a disturbing peak in the background can be removed to obtain a cleaner baseline. In addition, by synthesizing and measuring the stable isotope standard (SIS) peptide and comparing it with the measurement of the target peptide, it is possible to simultaneously analyze the desired substances with better analytical sensitivity.

 Through the above analysis methods, it is possible to compare the protein expression level in the normal control group and the protein expression level in the pancreatic cancer individual, and to determine whether there is a significant increase or decrease in the expression level of the pancreatic cancer marker gene to the protein to diagnose the possibility of pancreatic cancer. Can be.

 In addition, in the method of the present invention, 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. Through the above measurement methods, it is possible to confirm the mRNA expression level of the subject to be diagnosed with the occurrence of pancreatic cancer and the expression level of the normal control group, and to compare the degree of expression of the pancreatic cancer can be diagnosed or predicted.

 On the other hand, 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

Measuring the expression level of any one protein selected from the group consisting of TTR, C1R, CLU, and KLKB1 from the sample, or the expression level of mRNA of a gene encoding the protein; (d) the expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the CA19-9 protein, and the expression level of the LRG1 protein or the mNRA expression level of the gene encoding the LRG1 protein, respectively, in the normal control sample. Comparing with expression level; And (e) the expression level of any one protein selected from the group consisting of TTR, C1R, CLU and KLKB1, or the expression level of mRNA of the gene encoding the protein, respectively, as a normal control group. Provided is a method of detecting pancreatic cancer markers, the method comprising comparing with expression levels in a sample. In the markers, compositions, kits, and diagnostic methods for diagnosing pancreatic cancer of the present invention, 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.

 In a further embodiment of the invention, 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. For example, 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.

 Another example of this is CLU (Clusterin preproprotein); And

 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) and 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.

 Another example of the present invention, the sample of the subject, the IPMN marker

Measuring the expression level of the proteins or _m RNA expression level of the gene encoding the protein, respectively, Comparing the measured expression level of the marker with the expression level of the control marker, and

 And 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.

 Prior to measuring the expression level of the marker, 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)

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. In addition, 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.

 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. In addition, when 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.

 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.

In addition, the IPMN marker, the expression level measurement, and determining the steps are as described above. Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited by these examples. Example 1 Preparation of Samples for Experiment

 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.

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).

 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.

 Table 2

 Pancreatic cancer

 Category Training Set * Exam 1 Exam 2 Exam 3 Exam 4

 (1/2/3/4)

 396 316 80 50 80 29 Total

 (20/228/31/1 17) (16/182/25/93) (4/46/6/24) (4/46/0/0) (4/46/6/24) (3/21 / 1/4)

75 60 15 15 15 10

AMC

 (10/63/1/1) (8/50/1/1) (2/13/0/0) (2/13/0/0) (2/13/0/0) (2/8 / 0/0)

128 102 26 6 26 7

NCC

 (3/27/25/73) (2/22/20/58) (1/5/5/15) (1/5/0/0) (1/5/5/15) (1/3 / 1/2)

96 76 19 10 19 5

SMC

 (1/50/2/43) (1/40/1/34) (0/10/1/9) (0/10/0/0) (0/10/1/9) (0/3 / 0/2)

50 40 10 10 10 1

SNUH

(3/45/2/0) (2/36/2/0) (1/9/0/0) (1/9/0/0) (1/9/0/0) (0/1 / 0/0) 47 38 9 9 9 6

YMC

 (3/43/1/0) (3/34/1/0) (0/9/0/0) (0/9/0/0) (0/9/0/0) (0/6 / 0/0)

Table 3

-AMC: Asan Medical Center (Seoul Asan Hospital)

 -NCC: National Cancer Center

 -SMC: Samsung Medical Center (Samsung Seoul Hospital)

 -SNUH: Seoul National University Hospital (Seoul National University Hospital)

 -YMC: Yonsei Medical Center (Severance Hospital)

 * Perform all tests with classifier derived from training set Example 2: Discovery and performance analysis of pancreatic cancer diagnostic marker combination by MRM-MS method

 <2-1> Blood Sample Pretreatment

. 40 / of each blood sample is passed through a depletion column to delete the seven proteins that make up the largest proportion in the blood, concentrated with a 3 kDa filter, and then subjected to BCA quantification to obtain 200 equivalents of plasma and 6 M urea. (urea) and reduced and alkylated with 20 mM DTT and 50 mM iodoacetic acid. Herein, trypsin was treated at 37 ^° C. for 16 hours at a ratio of 50: 1 (protein: trypsin, w / w) to make the protein as a peptide, and then the peptide was C18 OASIS. Desalted and lyophilized using a column (Waters, USA). The lyophilized peptide was dissolved in Solution A (98% distilled water, 2% acetonitrile, 0.1% formic acid), and MRM analysis was performed. <2-2> Transition Selection

 To perform MRM analysis, the characteristic charge versus specific protein

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

For 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.

 However, peptides containing the following amino acids or having specific motifs were excluded from all peptides that could be produced by cleavage by trypsin because the signal values are known to be poor as follows:

 When methionine is present in the peptide, oxidation occurs by reactive oxygen species (ROS) in vivo, increasing the mass value by 32 Da;

 When histidine is present in the peptide, the charge state of the peptide changes due to the positive charge of the R-group;

 When NxS / T motifs are present in the peptide, Ν-glycosylation occurs and the mass value shifts;

 @ 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. In order to predict the RT, 600 SIS peptide RM analyzes were performed, and a calibration curve based on the hydrophobicity scale and the RT chromatogram was calculated.

<2-3> LC and MRM

 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. First equilibrate the column with Sol A (95% distilled shoe, 5% acetonitrile, 0.1% formic acid by volume) for 10 minutes, then Sol A and Sol B (5% distilled water, 95% acetonitrile, 0.1% formic acid by volume). ) For 50 minutes at 95: 5

Peptides were added to a concentration gradient of 15:85 at 15:85 for 5 minutes.

Eluted.

 As a mass spectrometer, Agilent's triple quadrupole 6490-QQQ instrument is used for MRM mode for transitions to selected proteins.

Monitored. Each sample to compensate for deviations between batches.

The spiked 5 fmol beta-galactosidase peptide (GDFQFNISR [C 13 N15], 547.3 / 646.4) was also monitored simultaneously.

<2-4> Data Quantitative Analysis

 To confirm quantitation, 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. After dilution, 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. In addition, 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.

^'Expression of pancreatic cancer in the patient population of the protein contained in the MRM quantitative analysis of four markers a combination by using the result obtained by the method of the Table 1 are as follows. As shown in Figures 1 and 2 it was confirmed that the expression of CA19-9 and LRG1 protein is specifically increased in the pancreatic cancer patient group compared to the normal group, the CA19-9 and LRG1 was selected as a diagnostic marker for pancreatic cancer.

 As shown in Figures 3 to 6, it was confirmed that the expression of TTR, CLU and KLKB1 protein was specifically decreased in the pancreatic cancer patient group compared to the normal group, and the expression of the C1R protein was specifically increased in the pancreatic cancer patient group compared to the normal group. Could confirm. Example 3: discovery and performance analysis of pancreatic cancer diagnostic protein combination by ELISA method

 The expression levels of LRG1, TTR and CLU proteins were measured in the pancreatic cancer patients and normal groups by ELISA. In the case of LRG1, IBL's hLRGl ELISA kit was used, and in the case of TTR, AssayPro's prialbumin ELISA kit was used, and in the case of CLU, R & D Systems' Human Clusterin Quantikine kit was used, and each ELISA measurement was performed according to the manufacturer's protocol. .

 Specifically, 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:

2,091 times). 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 540πηι or 570ηπι, and the results were summarized by calculating concentration values.

 As a result of the ELISA quantitative analysis, as shown in FIGS. 7 to 9, the expression of LRG1 protein was specifically increased in the pancreatic cancer patient group compared to the normal group, and the expression of TTR protein was specifically decreased in the pancreatic cancer patient group compared to the normal group. , The expression of CLU protein was confirmed to be specifically reduced in the pancreatic cancer patient group compared to the normal group, which was the same as the MRM-MS results of Example 2. Example 4 Identification and Performance Analysis of Pancreatic Cancer Diagnosis Markers by Immunoturbidimetr Assay

 The expression level of TTR protein in pancreatic cancer patients and normal patients

It was measured by an immunonubibimetric assay. The measurement using the R _oc he Diagnostics Inc. COBAS INTEGRA 800 Prealbumin was performed according to the manufacturer's protocol.

 Specifically, 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.

 As a result of the quantitative analysis by the immunobinding method, as shown in FIG. 10, it was confirmed that the expression of 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

<5-l> Diagnostic Performance of CA19-9, LRG1 and TTR Combination Markers for PDAC Classification Using MRM-MS method of Example 2, Diagnostic Performance of Pancreatic Cancer Classification of Combination Markers of CA19-9, LRG1 and TTR Was analyzed. The CA19-9 protein is

By Chemiluminescent Enzyme Immunoassay (CLEIA) using Roche Diagnostics COBAS Elecsys CA 19-9 instrument, LRG1 and TTR proteins were analyzed by MRM quantitative analysis. Measured.

When pancreatic cancer diagnostic function was used, the performance as a pancreatic cancer diagnostic marker of the combination of CA19-9, LRG1 and TTR was compared with AUC and Sn | _{Sp = 0.9} . The ROC graph represents the relationship between sensitivity and specificity on a two-dimensional plane. The larger the area under the ROC graph (八 1; 0≤ 八;。 <1), the more accurate it is. can do. Sn | _Sp = _0.9 is a sensitivity value when the specificity is 0.9, which is a numerical value representing the detection sensitivity, and it can be determined that the larger the value is, the more accurate it is.

The measurement results of the AUC and Sni _{Sp = 0.9} are shown in Table 4 and FIG. 1.

 Table 4

상기 표 4 및 도 11에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 AUC가 0.9312이고, | =₀.₉가 0.8250으로서 췌장암 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히, 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 CA19— 9, LRG1 및 TTR을 각각 단독 사용 또는 2개씩 조합하여 사용할 때보다 췌장암 진단 성능이 더 우수함을 확인할 수 있었다.

As shown in Table 4 and FIG. 11, 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. In particular, 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.

<5-2> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for Early Stage Identification of PDAC

Using the MRM-MS method of 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. The AUC and Sn | Table 5 shows the measurement results of _Sp = _0.9 And shown in FIG. 12.

 Table 5

상기 표 5 및 도 12에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 AUC가 0.9070이고, Sn|_Sp=_{0 9}가 으7600으로서 췌장암 초기 병기 구분을 위한 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히, 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 상용 췌장암 진단 마커인 CA19-9를 단독으로 사용했을 때보다 췌장암 초기 병기 진단 성능이 더 우수함을 확인할 수 있었다.

As shown in Table 5 and Figure 12, 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. In particular, 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.

<5-3> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for Cancer / Pancreatic Cancer

 Using 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 TTR was analyzed. The CA19-9 protein is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is

Measured by MRM quantitative analysis. The AUC and Sn | The measurement result of _Sp = _0.9 is shown in Table 5 and FIG.

As shown in Table 5 and Figure 13, 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. In particular, 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.

<5-4> PDAC Classification Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers in Experimental Groups with CA19-9 <37 U / m £

 Using the MRM-MS method of Example 2, 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 £. In clinical practice, 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

Chemiluminescent Enzyme Immunization with Diagnostics COBAS Elecsys CA 19-9 Instrument

By Assay (CLEIA), LRG1 and TTR Proteins by MRM Quantitative Assay

Measured.

When pancreatic cancer diagnostic function was used, the performance as a pancreatic cancer diagnostic marker of the combination of CA19-9, LRG1 and TTR was compared with AUC and Sn | _Sp = o. ₉ . ROC

The graph is a representation of the relationship between sensitivity and specificity on a two-dimensional plane. The larger the area under the ROC graph (ᅀ 1 :; 0 <八 1 ^ <1), the more accurate it is. can do. Sn | _{Sp = 0 9} is a sensitivity value when the specificity is 0.9, and is a numerical value indicating the detection sensitivity, and it can be determined that the larger the value is, the more accurate it is. The AUC and Sn | The measurement result of _Sp = _0.9 is shown in Table 5 and FIG.

As shown in Table 5 and Figure 14, 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

The performance as a marker was found to be very good. In particular, 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 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. In the ELISA method, the combination of CA19-9, LRG1, and TTR was confirmed to be excellent in pancreatic cancer diagnosis.

<6-l> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for PDAC Classification Using the ELISA method of Example 3, the diagnostic performance of the CA19-9, LRG1, and TTR combination markers for pancreatic cancer classification was analyzed. It was. The CA19-9 protein was measured by chemiluminescent enzyme immunoassay (CLEIA), and the LRG1 and TTR proteins were measured by ELISA method.

The AUC and Sn | _Sp = ₀ . The measurement result of ₉ is shown in Table 6 and FIG. 15.

 Table 6

상기 표 6 및 도 15에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 AUC가 0.9315이고, |^_{=0 9}가 0.8250으로서 췌장암 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히， 본 발명에 따른 CA19-9, LRG1 및 TTR의 조합은 상용 췌장암 진단 마커인 CA19-9를 단독으로 사용했을 때보다 췌장암 진단 성능이 더 우수함을 확인할 수 있었다. <6-2> PDAC 초기 병기 구분을 위한 CA19-9, LRG1 및 TTR조합 마커의 진단 성능

As shown in Table 6 and FIG. 15, the combination of CA19-9, LRG1, and TTR according to the present invention showed that AUC is 0.9315 and | ^ _{= 0 9} is 0.8250, which is very excellent as a pancreatic cancer diagnostic marker. In particular, 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. <6-2> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for Early Stage Identification of PDAC

 Using the ELISA method of Example 3, the diagnostic performance of the early stages of pancreatic cancer classification of the combination markers of CA19-9, LRG1 and TTR was analyzed. The CA19-9 protein is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is ELISA

It measured by the method. The AUC and Sn | The measurement result of _Sp = _o.9 is shown in Table 6 and FIG. 16.

As shown in Table 6 and Figure 16, 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. In particular, 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

<6-3> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for Cancer / Pancreatic Cancer.

 Using the ELISA method of Example 3, 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 is chemiluminescent enzyme immunoassay (CLEIA), LRG1 and TTR protein is ELISA

It measured by the method. The AUC and Sn | The measurement result of _{Sp = 9} is shown in Table 6 and FIG.

As shown in Table 6 and Figure 17, 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. In particular, 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.

<6-4> PDAC Classification Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers in Experimental Groups with CA19-9 <37 U / m

Using the ELISA method of Example 3, a combination marker of CA19-9, LRG1, and TTR The diagnostic performance of pancreatic cancer was analyzed in the experimental group with CA19-9 <37 U / ^. In clinical practice, pancreatic cancer is 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 AUC and Sn | The measurement result of _Sp = _{0 9} is shown in Table 6 and FIG. As shown in Table 6 and Figure 18, the combination of CA19-9, LRG1 and TTR according to the present invention has an AUC of 0.8287, Sn | Diagnosis of pancreatic cancer with _Sp = _0.9 = 0.5172

 The performance as a marker was found to be very good. In particular, 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.

<7-l> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for PDAC Separation Using the immunobinding method of Example 4, the diagnostic performance of the combination markers of CA19-9, LRG1, and TTR for pancreatic cancer was determined. 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 ^'AUC and Sn | The measurement result of _Sp = _0.9 is shown in Table 7 and FIG. 19.

CA19-9 0.8259 0.7250

CA19-9 0.8259 0.7250

 CA19-9 + LRG1 + TTR 0.9146 0.7600

7-2 CA19-9 + TTR 0.8678 0.6800

 CA19-9 0.7924 0.6400

CA19-9 + LRG1 + TTR 0.8965 0.8250

7-3 CA19-9 + TTR 0.8367 0.7500

 CA19-9 0.7964 0.6875

CA19-9 + LRG1 + TTR 0.8439 0.5172

7-4 CA19-9 + TTR 0.7255 0.3103

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.

<7-2> Diagnostic Performance of CA19-9, LRG1, and TTR Combination Markers for Early Stage Identification of PDAC

Using the immunobinding method of 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 AUC and Sn | The measurement result of _Sp = _0.9 is shown in Table 7 and FIG. 20.

As 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. In particular, 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. <7-3> Diagnostic Performance of CA19-9JLRG1 and TTR Combination Markers for Cancer / Pancreatic Cancer

Using the immunobinding method of 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 AUC and Sn | The measurement result of _Sp = _{0 9} is shown in Table 7 and FIG. 21.

As shown in Table 7 and Figure 21, 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. In particular, 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.

<7-4> Diagnosis of PDAC Classification of CA19-9, LRG1, and TTR Combination Markers in CA19-9 <37 U / n Experimental Groups

 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 / ^.

Analyzed. In clinical practice, 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. The AUC and Sn | _Sp = ₀ . The measurement result of ₉ is shown in Table 7 and FIG. 22.

As shown in Table 7 and Figure 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 performance as a marker was found to be very good. In particular, 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 8 Diagnosis of CA19-9, LRG1 and C1R Combination Markers by MRM-MS Method Performance

<8-l> Diagnostic Performance of CA19-9, LRG1 and C1R Combination Markers for PDAC Classification Using MRM-MS method of Example 2, Diagnostic Performance of Pancreatic Cancer Classification of 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 C 1R proteins were determined by MRM quantitative analysis. The AUC and Sn | The measurement result of _{Sp = 09} is shown in Table 8 and FIG.

 Table 8

상기 표 8 및 도 23에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 C1R의 조합은 AUC가 0.9296이고, Sn|_Sp=₀.₉가 으8375로서 췌장암 진단

As shown in Table 8 and FIG. 23, 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. In particular, 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.

<8-2> Diagnostic Performance of CA19-9, LRG1, and C1R Combination Markers for Early Stage Identification of PDAC

Using the MRM-MS method of 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 AUC and Sn | The measurement result of _{Sp = 0 9} is shown in Table 9 and FIG.

 Table 9

. AUC Sn s _p = o.9

CA19-9 + LRG1 + C1R 0.9097 0.7800 CA19-9 + LRG1 0.9094 0.7400

 CA19-9 + C1R 0.8658 0.6600

 LRG1 + C 1R 0.7297 0.3800

CA19-9 0.7924 0.6400

 LRG1 0.6362 0.3800

CIR 0.5137 0.1400 As shown in Table 9 and Figure 24 above, 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. In particular, 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.

<8-3> 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. The AUC and Sn | _Sp = o. The measurement result of ₉ is shown in Table 10 and FIG. 25.

 Table 10

상기 표 10 및 도 25에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 C1R의 조합은 AUC가 0.9088이고, Sn|_Sp=_{0 9}가 0.8375로서 암과 췌장암 구분을 위한 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히, 본 발명에 따른 CA19-9, LRG1 및 C1R의 조합은 상용 췌장암 마커인 CA19-9을 단독으로 사용할 때보다 암 /췌장암 구분 성능이 더 우수함을 확인할 수 있었다.

As shown in Table 10 and Figure 25, CA19-9, LRG1 according to the present invention And C1R have a AUC of 0.9088 and Sn | _Sp = _{0 9} was 0.8375, which shows a very good performance as a diagnostic marker for cancer and pancreatic cancer. In particular, the combination of CA19-9, LRG1 and C1R according to the present invention was confirmed that the cancer / pancreatic cancer discrimination performance is better than when using a commercial pancreatic cancer marker CA19-9 alone.

<8-4>. 八 19-9 CA19-9 for the experimental group <37 11/111 £. PDAC-sensitive Diagnostic Performance of LRG1 and C1R Combination Markers

Using the MRM-MS method of Example 2, 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 AUC and Sn | The measurement result of _Sp = _{0 9} is shown in Table 10 and FIG. 26.

As shown in Table 10 and Figure 26, 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 performance as a marker was found to be very good. In particular, 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 9 Diagnostic Performance of CA19-9 LRG1 and CLU Combination Markers by MRM-MS Method

<9-l> Diagnostic Performance of CA19-9, LRG1 and CLU Combination Markers for Separating PDACs Using the MRM-MS method of Example 2, the diagnostic performance of CA19-9, LRG1 and CLU combination markers for pancreatic cancer 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 AUC and Sn | The measurement result of _Sp = _{0 9} is shown in Table 11 and FIG.

CA19-9+LRG1 0.9305 0.8125 [Table 1 1]

CA19-9 + LRG1 0.9305 0.8125

 CA19-9 + CLU 0.8662 0.7250

 LRG1 + CLU 0.8004 0.6125

 CA19-9 0.8259 0.7250

LRG1 0.7104 0.4375

CLU ᅳ 0.6456 0.2250 As shown in Table 1 1 and FIG. 27, the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.9338, and Sn | _Sp = _a9 was 0.8500, indicating a very good performance as a diagnostic marker for pancreatic cancer. In particular, 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 CA19-9JLRG1 and CLU alone or in combination of two.

<9-2> Diagnostic Performance of CA19-9, LRG1, and CLU Combination Markers for Early Stage Identification of PDAC

Using 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 ο MRM quantitative analysis. The AUC and Sn | The measurement result of _{Sp = 0.9} is shown in Table 12 and FIG.

 Table 12

 Test Marker Combination AUC

 CA19-9 + LRG1 + CLU 0.9027 0.7800

9-2 CA19-9 + TTR 0.8440 0.6400

 CA19-9 0.7924 0.6400

CA19-9 + LRG1 + CLU 0.9093 0.8500

9-3 CA19-9 + TTR 0.8494 0.7125

 CA19-9 0.7964 0.6875

CA19-9 + LRG1 + CLU 0.8384 0.5862

9-4 CA19-9 + TTR 0.6753 0.2069

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. In particular, 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.

<9-3> Diagnosis of CA19-9JLRG1 and CLU Combination Markers for Cancer / Pancreatic Cancer

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. The AUC and Sn | _Sp = ₀ . The measurement result of ₉ is shown in Table 12 and FIG. 29.

As shown in Table 12 and 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. In particular, 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.

<9-4> CA19-9 for an experimental group with CA19-9 <37 U /. PDAC-sensitive Diagnosis Performance of LRG1 and CLU Combination Markers

Using the MRM-MS method of 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 AUC and Sn | The measurement result of _{Sp = 0.9} is shown in Table 12 and FIG.

As shown in Table 12 and Figure 30, 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. In particular, 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 In 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.

<10-1> Diagnostic Performance of CA19-9, LRG1 and CLU Combination Markers for PDAC Classification Using the ELISA method of Example 3, the diagnostic performance of the CA19-9, LRG1 and CLU combination markers for pancreatic cancer 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 AUC and Sn | The measurement result of _Sp = _o.9 is shown in Table 13 and FIG. 31.

 Table 13

상기 표 13 및 도 31에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 CLU의 조합은 AUC가 0.9399이고, Sn|_Sp=0.9가 0.8000으로서 췌장암 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히, 본 발명에 따른 CA19-9_: LRG1 및 CLU의 조합은 CA19-9, LRG1 및 CLU를 각각 단독 또는 2개씩 조합하여 사용할 때보다 췌장암 진단 성능이 더 우수함을 확인할 수 있었다.

As shown in Table 13 and Figure 31, 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. In particular, 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.

<10-2> Diagnostic Performance of CA19-9, LRG1, and CLU Combination Markers for Early Stage Identification of PDAC

Using the ELISA method of 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 AUC and Sn | The measurement result of _Sp = _0.9 is shown in Table 14 and FIG.

 Table 14

상기 표 14 및 도 32에서 보는 바와 같이, 본 발명에 따른 CA19-9, LRG1 및 CLU의 조합은 AUC가 0.9193이고， Sn|_Sp=0.9가 0.6400으로서 췌장암 초기 병기 구분을 위한 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히 본, 발명에 따른 CA19-9, LRG1 및 CLU의 조합은 상용 췌장암 진단 마커인 CA19-9을 단독으로 사용할 때보다 췌장암 초기 병기를 진단하는 성능이 더 우수함을 확인할 수 있었다. <10-3> 암 /췌장암 구분을 위한 CA19-9, LRG1 및 CLU 조합 마커의 진단 실시예 3의 ELISA 방법을 이용하여, CA19-9, LRG1 및 CLU의 조합 마커의 암 및 췌장암 구분에 대한 진단 성능을 분석하였다. 상기 CA19-9 단백질은 화학발광 효소 면역 분석법 (CLEIA)에 의해, LRG1 및 CLU 단백질은 ELISA 정량 분석에 의해 측정하였다ᅳ 상기 AUC 및 Sn|_Sp=_a9의 측정 결과를 표 14 및 도 33에 나타내었다.

As shown in Table 14 and Figure 32, 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. In particular, 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. <10-3> Diagnosis of CA19-9, LRG1 and CLU Combination Markers for Cancer / Pancreatic Cancer Using the ELISA method of Example 3, the diagnostic performance for the cancer and pancreatic cancer classification of the combination markers of CA19-9, LRG1 and CLU was analyzed. The CA19-9 protein was determined by chemiluminescence enzyme immunoassay (CLEIA) and the LRG1 and CLU proteins were determined by ELISA quantitative analysis. The AUC and Sn | The measurement result of _Sp = _a9 is shown in Table 14 and FIG. 33.

As shown in Table 14 and Figure 33, 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. In particular, 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.

<10-4> CA19-9 <19 U / ^ CA19-9 for the experimental group. PDAC-sensitive Diagnosis Performance of LRG1 and CLU Combination Markers

Using the ELISA method of 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. The AUC and Sn | _Sp = o. The measurement result of ₉ is shown in Table 14 and FIG. 34.

As shown in Table 14 and Figure 34, the combination of CA19-9, LRG1 and CLU according to the present invention has an AUC of 0.8435, Sn | Pan = cancer diagnosis as _Sp = _{0 9} is 0.4828

The performance as a marker was found to be very good. In particular, the combination of CA19-9, L G1 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 11 Diagnostic Performance of CA19-9, LRG1 and KLKB1 Combination Markers by MRM-MS Method

<1 1-1> Diagnostic Performance of CA19-9, LRG1, and KLKB1 Combination Markers for PDAC Classification

Combination of CA19-9, LRG1 and KLKB1 using the MRM-MS method of Example 2 The diagnostic performance of markers for pancreatic cancer 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 AUC and Sn | _Sp = ₀ . The measurement result of ₉ is shown in Table 15 and FIG. 35.

 Table 15

, As 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. In particular, 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.

<11-2> Diagnostic Performance of CA19-9, LRG1 and KLKB1 Combination Markers for Early Stage Identification of PDAC

Using 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. The AUC and Sn | The measurement result of _Sp = o.9 is shown in Table 16 and FIG. 36.

 Table 16

Test Marker Combination AUC

11-2 CA19-9 + LRG1 + KLKB1 0.9148 U 8000 CA19-9 + TTR 0.8440 0.6400

 CA19-9 0.7924 0.6400

CA19-9 + LRG1 + KLKB1 0.8924 0.8625

1 1 -3 CA19-9 + TTR 0.8494 0.7125

 CA19-9 0.7924 0.6400

CA19-9 + LRG1 + KLKB1 0.8349 0.6207

1 1 -4 CA19-9 + TTR 0.6753 0.2069

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. In particular, the combination of CA19-9, LRG1 and KLKB1 according to the present invention is a commercial pancreatic cancer diagnostic marker.

 The early stage diagnosis of pancreatic cancer was superior to that of CA19-9 alone.

<1 1 -3> Diagnostic Capability of CA19-9, LRG1, and KLKB1 Combination Markers for Cancer / Pancreatic Cancer

Using 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 AUC and Sn | The measurement result of _Sp = _o.9 is shown in Table 16 and FIG. 37.

As shown in Table 16 and FIG. 37, 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. In particular, 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.

<1 1-4> PDAC Classification Diagnostic Performance of CA19-9, LRG1, and KLKB1 Confusion Markers in CA19-9 <37 U / ^ Using the MRM-MS method of Example 2, the diagnostic performance for the pancreatic cancer classification of the combination marker of CA19-9, LRG1 and KLKB1 was analyzed. The CA19-9 protein was determined by chemiluminescence enzyme immunoassay (CLEIA), and the LRG1 and KLKB1 proteins were determined by MRM quantitative analysis. The AUC and Sn | The measurement result of _Sp = _a9 is shown in Table 16 and FIG. 38.

As shown in Table 16 and FIG. 38, 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. In particular, 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. Example 12 Pancreatic Cancer Diagnosis-Data Statistical Analysis

<12-1> CA19-9 + LRG1 + TTR

 SVM (Support Vector Machine) was used for statistical analysis. 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.

 n

f (x) = sgn ^^ ^ y ^ <x, x _t > + b)

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,

 X i is the reference measurement, and

 b means a correction value.

In the above formula, 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.

 Specifically, the CA19-9 measurement, TTR and LRG1 MRM quantitative values (7.4, 1.451, 3.2359), (6.3, 1.0718, 2.136) and (26.1, 1.2053, 3.1797) were obtained from three normal controls, respectively. As a result of assigning to the diagnostic function, the function value is calculated as f (7.4, 1.451 3.2359) = -l, f (6.3, 1.0718,2.136) = -l, f (26.1, 1.2053, 3.1797) = -1 Could be determined to be normal.

Also, from the three patients with pancreatic cancer each MRM as a quantitative measurement value of CA19-9, TTR and ^{LRG1 (4 5318, 4 .898,} 1. 2 514), (1 45, 2. 46 08, 1.6616) and ⁽⁸⁸⁹ , 2. ⁵ i53, 1.5474) the obtained result of substituting the diagnostic function the purpose, the function value f (45318, 4.898, 1.2514, respectively) = l, f (145, 2.4608, 1.6616) = l, f (889, 2.5153, 1.5474) = 1, the subject could be identified as pancreatic cancer.

<12-2> CA19-9 + LRG1 + C1R

 For statistical analysis, pancreatic cancer was determined by using Formula 1 based on SVM (Support Vector Machine).

 (7.4, 1.451, 1.0748), (6.3, 1.0718, 0.4531) and (26.1, 1.2053, 1.0929) were obtained as CA19-9 measurements, MRG quantitative values of LRG1 and C1R from three normal controls, respectively, As a result of the substitution, the function value is calculated as (7.4, 1.451, 1.0748) = -1, f (6.3, 1.0718, 0.4531) = -l, f (26.1, 1.2053, 1.0929) = ᅳ 1 to return the subject to normal. Could be determined.

In addition, CA19-9 measurements from three pancreatic cancer patients, LRG1 _. And each of a MRM quantitative value of ^{C1R (4 5318, 4 .89 3} , 1.37 42), (1 4 5, 2. 4 608, 1. 4 83) and (889, ^{2.5 15} 3, ^1.47 4), and as a result of substituting it into the diagnostic function, the function value is f (45318, 4.893, 1.3742) = l, f (145, 2.4608, 1.483) = l, f (889, 2.5153, 1.474) = 1 The subject was determined to be pancreatic cancer.

<12-3> CA19-9 + LRG1 + CLU

For statistical analysis, pancreatic cancer was determined using Function 1 based on SVM (Support Vector Machine). CA19-9 measurements from three normal controls, 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), and as a result of substituting it into the diagnostic function, the value of the function is 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.

 In addition, CA19-9 measurements, MRM quantitative values of LRG1 and CLU from three pancreatic cancer patients (45318, 4.893, 1.6821), (145, 2.4608, 2.545) and (889,2.5153,

 1.5101), and as a result of substituting it into the diagnostic function, f (45318, 4.893, 1.6821) = 1, f (145, 2.4608, 2.545) = 1, f (889, 2.5153, 1.5101) = 1 The subject was determined to be pancreatic cancer. <12-4> CA19-9 + L G1 + KLKB1

 For statistical analysis, pancreatic cancer was determined using Function 1 based on SVM (Support Vector Machine). CA19-9 measurements from three normal controls, as MRM quantitative values of L G1 and KLKB1, respectively (7.4, 1.451, 1.2801). , (6.3, 1.0718, 0.961) and (26.1, 1.2053, 1.5657), and as a result of substituting it into the diagnostic function, the function values are respectively 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.

 In addition, CA19-9 measurements from three pancreatic cancer patients, as MRM quantitative values of LRG1 and KLKB1 (45318, 4.893, 0.555), (145,2.4608, 0.5347) and (889,2.5153,

0.8084), and as a result of substituting it into the diagnostic function, the function value is f (45318, 4.893, 0.555) = l, f (145, 2.4608,0.5347) = l, f (889, 2.5153, 0.8084) = 1 The subject was determined to be pancreatic cancer. Example 13 Early Stage Pancreatic Cancer Diagnosis-Statistical Analysis of Data For the statistical analysis, the initial stage of pancreatic cancer was determined by using Equation 1 based on SVM (Support Vector Machine).

As CA19-9 measurements, MRM quantitative values of LRG1 and KLKB1, (7.4, 1.451, 1.2801), (6.3, 1.0718,0.961) and (26.1, 1.2053, 1.5657), respectively, were obtained. The values were calculated as f (7.4, 1.451, 1.2801) = -l, f (6.3, 1.0718, 0.961) = -1, f (26.1, 1.2053, 1.5657) = -1, respectively, to determine the subject as normal. . In addition, three patients with early stage pancreatic cancer obtained CA19-9 measurements, MRM quantitative values of LRG1 and KLKB1 (154.52, 4.0994, 1.2722), (190.16, 4.5008, 0.7645) and (1052.8, 3.5696, 0.6775), respectively. As a result of substituting the diagnostic function, the function value is calculated as f (154.52, 4.0994, 1.2722) = 1, f (190.16, 4.5008, 0.7645) = 1, f (1052.8, 3.5696, 0.6775) = 1, respectively. The patient was diagnosed with early stage pancreatic cancer. Example 14 Diagnosis of Pancreatic Cancer from Other Cancers-Data Statistical Analysis For the purpose of statistical analysis, the initial stage of the pancreatic cancer was determined using the functional formula 1 based on SVM (Support Vector Machine). (8, 1.3985, 0.7085), (10.68, 0.9864, 0.776) and (7.32, 1.1431, 0.9214) were obtained as CA19-9 measurements, MRM quantitative values of LRG1 and KLKB1, respectively, from three other cancer patients, and this diagnostic function As a result, the function values are calculated as f (8, 1.3985, 0.7085) = -1, f (10.68, 0.9864, 0.776) = -1, f (7.32, 1.1431, 0.9214) = -1, respectively. Other cancer patients could be identified. In addition, three pancreatic cancer patients obtained CA19-9 measurements, MRM quantitative values of LRG1 and KLKB1 (280.72, 4.0849, 0.9165), (4000, 5.7558, 0.7216) and (120.32, 6.2917, 0.555), respectively. As a result of assigning to the function, the function value was calculated as f (154.52 _> 4.0994, 1.2722) = 1, f (190.16, 4.5008, 0.7645) = 1, f (1052.8, 3.5696, 0.6775) = 1 Could be determined. Example 15 Preparation of Samples for Experiments To effectively detect IPMNs of malignant subtypes, the following table:

As shown in 17, with the consent of the patients of Seoul National University Hospital,

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.

invasive type-i- Jl위 ^" ¾ (malignant) subtype IPMN 실험군을 low dysplasia IPMN 및 intermediate dysplasia IPMN과 선별 탐지를 위한 실험군으로 구성되었다. Test 7 screens for the detection of high-risk and low-gastric groups according to the ELISA assay.

The invasive type-i-Jl ^" ¾ (malignant) subtype IPMN test group consisted of a low dysplasia IPMN and an intermediate dysplasia IPMN test group for screening detection.

 Table 17

실시예 16: MRM-MS 방법에 의한고위험 IPMN의 탐지

Example 16: Detection of High Risk IPMN by MRM-MS Method

Using the MRM-MS method of Example 2, the diagnostic performance of the markers of Table 32 below for high risk IPMN was analyzed. 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.

When the diagnostic function was used in the same way as the pancreatic cancer diagnosis function, the performance as a diagnostic marker of the combination of CA19-9, LRG1, and TTR was compared to AUC and Sn | _Sp = ₉ . 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

It is a sensitivity value when specificity is 0.9, and it is a numerical value which shows detection sensitivity, and it can be judged that the larger the value is, the more accurate.

The AUC and Sn | _Sp = ₀ . The measurement result of ₉ is shown in Table 18 and FIGS. 39 to 63.

 Table 18

 Marker AUC Sn | Sp = 0.9 Results

CA19-9 0.6948 0.2000 **

 LRG1 0.7375 0.4000 39

LRG1 + CA19-9 0.8438 0.5333 40

 LRG1 + TTR 0.9479 0.7333 41

 LRG1 + CLU 0.8 146 0.6000 42

 LRG1 + C1R 0.8448 0.7333 43

 LRG1 + KLKB1 0.8969 0.7333 44

 LRG1 + CA19-9 + TTR 0.9500 0.9333 45

 LRG1 + CA19-9 + CLU 0.9104 0.8667 46

 LRG1 + CA19-9 + C1R 0.8708 0.6667 47

 LRG1 + CA19-9 + KLKB1 0.8938 0.8667 48

 LRG1 + TTR + CLU 0.9653 0.8667 49

 LRG1 + TTR + C1R 0.9542 0.7333 50

 LRG1 + TTR + KLKB1 0.9010 0.8000 51

 LRG1 + CLU + C1R 0.9542 0.8000 52

 LRG1 + CLU + LKB1 0.9031 0.8667 53

 LRG1 + C1R + KLKB1 0.8500 0.7333 54

CLU + CA19-9 0.8479 0.7333 55 CLU + TTR 0.8885 0.6000 56

CLU + KLKB1 0.8656 0.7333 57

CLU + CA19-9 + TTR 0.9365 0.6667 58

CLU + CA19-9 + C 1R 0.8229 0.6000 59

CLU + CA19-9 + KLKB 1 0.8969 0.7333 60

 CLU + TTR + C 1 R 0.9062 0.6667 61

CLU + TTR + KLKB 1 0.8604 0.6667 62

CLU + C1R + KL B 1 0.8917 0.8000 63 As 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.

 Table 19

150 AUC Sn s _P = o.9 Result Drawing

CA19-9 0.6296 0.2000 **

LRG1 0.7148 0.2667 64

LRG1 + CA19-9 0.8333 0.4667 65

 LRG1 + TTR 0.8704 0.4000 66

LRG1 + CLU 0.8222 0.4667 67

LRG1 + C1R 0.8593 0.4000 68

L G1 + KLKB1 0.8630 0.4000 69 CA19-9 + LRG1 + TTR 0.9074 0.6000 70

 CA19-9 + LRG1 + C1R 0.9000 0.6000 71

 CA19-9 + LRG1 + CLU 0.8667 0.6000 72

 CA19-9 + LRG1 + LKB1 0.9037 0.8000 73

 LRG1 + TTR + CLU 0.8407 0.4667 74

 LRG1 + TTR + C1R 0.8185 0.4667 75

 LRG1 + TTR + KLKB1 0.8815 0.3333 76

 LRG1 + CLU + C1 R 0.8963 0.6000 77

 LRG1 + CLU + KLKB1 0.8889 0.4000 78

 LRG1 + C1R + KL B1 0.9074 0.6000 79

 CLU + CA19-9 0.7407 0.4000 80

 CLU + TTR 0.7333 0.3333 81

 CLU + C1R 0.7778 0.6000 82

 CLU + KLKB1 0.81 1 1 0.3333 83

 CLU + CA19-9 + TTR 0.8741 0.6000 84

 CLU + CA19-9 + C1R 0.8185 0.7333 85

 CLU + CA19-9 + KLKB1 0.8741 0.6000 86

 CLU + TTR + C1 R 0.8481 0.3333 87

 CLU + TTR + KL B1 0.8333 0.4000 88

 CLU + C1R + KLKB1 0.8889 0.6000 89 As 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. In particular, the combinatorial markers according to the present invention may be used for the pancreatic cancer diagnostic marker CA19-9.

It can be seen that the high-risk IPMN detection performance is better than the single use.

Example 18 Screening Detection of Low-Risk IPMN and High-Risk IPMN by ELISA Method In Example 17, the diagnostic performance of the marker pancreatic cancer was analyzed using the MRM-MS method. In the ELISA method, the marker combination of Table 20 was confirmed to be excellent in distinguishing diagnostic performance of high-risk IPMN.

Specifically, in order to distinguish between low risk and high risk IPMNs, the test shown in Table 20 below was carried out on Test 7 of Example 15 in substantially the same manner as the ELISA assay of Example 6. The AUC and Sn | The measurement result of _{Sp = 0.9} is shown in Table 34 and FIGS. 90 to 100.

 Table 20

상기 표 20 및 도 90 내지 도 100에서 보는 바와 같이, 본 발명에 따른 마커 또는 2이상의 마커 조합은 저위험군 IPMN와 구별되게 고위험군 IPMN을 선별적으로 탐지하기 위한 진단 마커로서의 성능이 매우 우수한 것으로 나타났다. 특히, 본 발명에 따른 마커들은 상용 췌장암 진단 마커인 CA19-9를 단독으로 사용했을 때보다 고위험군 IPMN 탐지 성능이 더 우수함을 확인할 수 있었다.

As shown in Table 20 and FIGS. 90 to 100, the marker according to the present invention, or a combination of two or more markers, 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. In particular, 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.

Claims

 [Patent Claims]  [Claim 1]  (a) carbohydrate antigen 19-9, (b) Leucine-rich alpha-2-glycoprotein 1, LRG, and (c) Transthyretin, ATTR, Prealbumin, TBPA; Preparation agent for measuring expression level of mRNA of pancreatic cancer diagnostic marker protein or gene encoding the same, including one or more markers selected from the group consisting of ClR (Complement Clr subcomponent precursor), CLU (Clusterin preproprotein) and KLKB1 (Plasma Kallikrein protein;) Containing, pancreatic cancer diagnostic composition.  [Claim 2] The pancreatic cancer diagnosis according to claim 1, wherein the pancreatic cancer diagnosis is performed by detecting pancreatic cancer selectively from a normal group, selectively detecting pancreatic cancer among various cancers, or pancreatic cancer having a CAI 9-9 value of less than 37 U / m £ ^'. To detect the composition.  [Claim 3]  According to claim 1, wherein the pancreatic cancer is pancreatic adenocarcinoma or high-risk intra pancreatic mucous papilloma (Intraductal papillary mucinous neoplasm, IPMN) composition.  [Claim 4] The composition of claim 3, wherein the pancreatic adenocarcinoma is stage 1 or 2 pancreatic adenocarcinoma. ^'  [Claim 5]  4. The composition of claim 3, wherein the pancreatic adenocarcinoma is not IPMN derived. [Claim 6] The method of claim 1, wherein the protein is LRG1 NCPI Accession No: NP_443204.1, and comprises the amino acid sequence of the TTR proteins NCPI Accession No: comprises the amino acid sequence of the NP-00036 ² .1, and the C1R proteins NCPI Accession No: NP_001724.3 comprises the amino acid sequence of, and the CLU protein NCPI Accession No: NPJX), and comprises the amino acid sequence of the 1822.3 KLKB1 proteins NCPI Accession No: 3. NP_000 ⁸⁸ comprises a ^second amino acid sequence Phosphorus composition.  [Claim 7] According to claim 1, The agent for measuring the expression level of the marker protein is an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) that specifically binds to the protein acid) or aptamer (aptamer), characterized in that, pancreatic cancer diagnostic composition.  [Claim 8]  The pancreatic cancer diagnostic composition according to claim 1, wherein the agent for measuring the expression level of the marker mRNA comprises a primer, a probe or an antisense nucleotide specifically binding to the gene.  [Claim 9]  9. A kit for diagnosing pancreatic cancer, comprising the composition of claim 1.  [Claim 10]  10. The method of claim 9, wherein the kit is a reverse transcription polymerase chain reaction (RT-PCR) key H, DNA ¾ kit, Enzyme linked immunosorbent assay (ELISA) kit, protein chip kit, rapid kit, or multiple reaction monitoring. ) Kit for pancreatic cancer, characterized in that the kit. - [Claim 1 11]  Measuring the expression level of at least three or more pancreatic cancer marker proteins or a mRNA expression level of a gene encoding the same in a sample of a subject,  Comparing the measured expression levels of each marker with the expression levels of the markers obtained from the normal control samples, respectively, and  Determining a risk of developing pancreatic cancer in the subject using the comparison result of the expression levels,  The at least three or more pancreatic cancer markers include (a) CA19-9 (carbohydrate antigen 19-9), (b) Leucine-rich alpha-2-glycoprotein 1, LRG, and (c) TTR (Transthyretin, ATTR, Prealbumin). , TBPA); Complement Clr (component Clr subcomponent precursor), CLU (Clusterin preproprotein) and KLKBK Plasma Kallikrein protein;) pancreatic cancer diagnostic markers comprising one or more markers selected from the group consisting of. [Claim 12] The method of claim 1 1, wherein the level of expression than in the determining step, the result of comparing the expression level of the marker in the sample to be measured _(a) CAl ⁹ -9 and (b) are respectively top LRGl expression level control High, and (c) TTR, CLU, and If the KLKB1 expression level is lower than the expression level in the normal control group, or the expression level of C1R in the sample to be measured is higher than the expression level in the normal control group, it is determined as pancreatic cancer.  【Claim 13】  The method for diagnosing pancreatic cancer according to claim 1, wherein the determining of the pancreatic cancer comprises determining the possibility of developing pancreatic cancer by substituting the expression level of the markers into the following Formula 1.  <Function 1>  n  fi ^) = s ^ n (^ <x, χ> + h)  X is a measure of the expression level of any one of the pancreatic cancer markers,  oti is the Lagrange multiplier in SVM (Support Vector Machine),  yi is the delimiter of normal group / pancreatic cancer group,  X i is the reference measurement, and  b means a correction value.  [Claim 14]  The method of diagnosing pancreatic cancer according to claim U, wherein the sample is blood, serum or plasma.  [Claim 15]  The method for diagnosing pancreatic cancer according to claim 11, wherein the expression level measurement of the marker is performed using an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), or an aptamer that specifically binds to the corresponding protein. .  [Claim 16]  The method of claim 1, wherein the measurement or comparison of the expression level of the marker is performed by protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass) Spectrometry analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis Selected from the group consisting of analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), western blot and enzyme linked immunosorbentassay (ELISA) It is carried out using the method of diagnosing pancreatic cancer.  [Claim 17]  The method of claim 11, wherein the measurement of mRNA expression level is performed by reverse transcriptase polymerase reaction, competitive transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. Phosphorus, pancreatic cancer diagnostic method.  [Claim 18]  The method of claim 1, wherein the pancreatic cancer is a subject with pancreatic adenocarcinoma (PDAC) likelihood. ,  [Claim 19]  19. The composition of claim 18, wherein the pancreatic adenocarcinoma is IPMN derived pancreatic adenocarcinoma.  [Claim 20]  The pancreatic cancer diagnosis according to claim 1, wherein the pancreatic cancer is differentiated from the pancreas-related diseases other than the normal group and cancer, or the pancreatic cancer is selectively detected among various cancers, or the level of CA19-9 is increased. Detecting pancreatic cancer less than.  [Claim 21]  The method of claim 20, wherein the pancreatic cancer diagnosis is to selectively detect pancreatic cancer from at least one pancreatic related disease selected from the group consisting of pancreatitis and cholecystitis.  [Claim 22]  The method of claim 11, wherein the pancreatic cancer is pancreatic adenocarcinoma or high-risk pancreatic ductal intramucosal papillary mucinous neoplasm (IPMN).  [Claim 23] Intraductal papillary mucinous neoplasm (IPMN) marker protein comprising LRGl (Leucine-rich alpha-2-glycoprotein 1, LRG1) or It includes an agent for measuring the expression level of mRNA of the gene encoding,  Composition for screening diagnosis of high risk group IPMN.  [Claim 24]  24. The composition of claim 23, wherein the composition selects high risk group IPMNs in distinction from low risk group IPMNs.  [Claim 25]  The composition of claim 23, wherein the IPMN marker further comprises one or more markers selected from the group consisting of carbohydrate antigen 19-9 (CA19-9), TTR, C1R, CLU, and KLKB1.  [Claim 26]  The composition of claim 25, wherein the IPMN markers are markers comprising LRG1 and one marker selected from the group consisting of TTR, C1R, CLU, and KLKB1.  [Claim 27]  The composition of claim 25, wherein the IPMN marker is a combination marker comprising LRG1 and two markers selected from the group consisting of TTR, C1R, CLU and KLKB1. [Claim 28]  Cluster preproprotein (CLU); And  LRG 1 (Leucine-rich alpha-2-glycoprotein 1, LRGl), CA 19-9 (carbohydrate antigen 19-9), TTR (Transthyretin, ATTR, Prealbumin, TBPA), ClR (Complement Clr subcomponent precursor) and KLKB1 (Plasma) Kallikrein protein;) IPMN marker protein comprising one or more markers selected from the group consisting of or a formulation for measuring the expression level of mRNA of the gene encoding the same, composition for screening diagnosis of high-risk group IPMN.  [Claim 29]  The method of claim 28, wherein the IPMN marker is LRGl, CA19-9, TTR, C1R and The composition is a combination marker comprising a CLU and one marker selected from the group consisting of KLKB1.  [Claim 30]  The method of claim 28, wherein the IPMN marker is LRGl, CA19-9, TTR, C1R and Combination comprising LRG1 and two markers selected from the group consisting of KLKB1 A composition that is a marker.  [Claim 31]  31. The method according to any one of claims 26 to 30, wherein the LRG1 protein comprises an amino acid sequence of NCPI Accession No: NP_443204.1, the TTR protein comprises an amino acid sequence of NCPI Accession No: NP_000362.1, The C1R protein comprises the amino acid sequence of NCPI Accession No: NP_001724.3, the CLU protein comprises the amino acid sequence of NCPI Accession No: NP_001822.3, and the KLKB1 protein comprises the NCPI Accession No: NP_000883.2 amino acid sequence. A composition comprising. [Claim 32]  29. The method according to claim 23 or 28, wherein the method for measuring the expression level of the protein comprises an antibody, oligopeptide, ligand, peptide nucleic acid (PNA) or aptamer that specifically binds to the protein. Composition.  [Claim 33]  29. The composition of claim 23 or 28, wherein the agent for measuring the expression level of said mRNA comprises primers, probes or antisense nucleotides that specifically bind said gene.  [Claim 34]  31. A kit for screening diagnosis of high risk group IPMN, comprising the composition of any one of claims 26-30.  [Claim 35]  35. The kit of claim 34, wherein the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit or a multiple reaction monitoring (MRM). Kit for screening diagnosis of high risk group IPMN.  [Claim 36]  The expression level of the intraductal papillary mucinous neoplasm (IPMN) marker proteins or the gene encoding the same in the pancreatic duct according to the composition for screening diagnosis of the high risk group IPMN according to any one of claims 26 to 30, for a sample of the subject. measuring each mRNA expression level, The expression level of the measured marker is compared with that of the control marker. Steps, and  Determining whether the subject is at high risk of IPMN using the comparison result of the marker expression levels;  How to diagnose high risk IPMNs.  [Claim 37]  The method of claim 36, further comprising determining whether the subject has IPMN prior to measuring the expression level of the marker. [Claim 38]  38. The method of claim 37, wherein the step of identifying is performed by imaging, histology or genetic markers.  [Claim 39]  The method of claim 36, wherein the subject is a patient whose IPMN tissue has been removed by surgical resection.  [Claim 40]  37. The method of claim 36, wherein the control group is a normal group, a patient with pancreatic disease except for IPMN and pancreatic adenocarcinoma, or a subject with low risk IPMN.  [Claim 41]  The method of claim 36, wherein the high risk IPMN comprises high grade dysplasia and invasive type IPMN.  [Claim 42]  37. The method of claim 36, wherein the method further comprises performing a surgical step of surgical resection or drug administration when determined to be a high risk IPMN of the subject.  [Claim 43]  The method of claim 38, wherein the method further comprises performing drug administration or prognostic monitoring when determined to be a low risk IPMN of the subject. [Claim 44]  37. The method of claim 36, wherein determining the high risk IPMN is As a result of comparing the expression levels of the markers, (a) CA19-9 and (b) LRGl expression levels in the sample to be measured were higher than those in the control group. (C) TTR, CLU, and KLKB1 expression levels in the sample are lower than the expression level in the normal control group, or C1R expression levels in the sample to be measured are higher than the expression level in the normal control group. How to do.  [Claim 45] ^'37. The method of claim 36, wherein said high risk IPMN comprises IPMN line derived from pancreatic jongam.  [Claim 46]  37. The method of claim 36, wherein the sample is blood, serum or plasma.  [Claim 47]  The method of claim 36, wherein the expression level measurement of the marker is characterized in that using an antibody, loli peptide, ligand, peptide nucleic acid (PNA) or aptamer (binmer) that specifically binds to the protein.  [Claim 48]  The method of claim 36, wherein the measuring or comparing the expression level of the marker,  Protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass) Spectrometry analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-Mass Spectrometry , LC-MS), LC-MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blot and ELISA (enzyme linked imimmosorbentassay).  [Claim 49]  37. The method of claim 36, wherein said mRNA expression level measurement is performed by reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting or DNA chip. Way.