KR101594981B1 - Composition for diagnosing pancreatic cancer and method for diagnosing pancreatic cancer using the same - Google Patents

Abstract

The present invention relates to a composition for the diagnosis of pancreatic cancer, a kit for diagnosing pancreatic cancer, and a method for diagnosing pancreatic cancer using the same, which can be used for judging the possibility of developing pancreatic cancer. By providing a diagnostic marker, the probability of onset, early diagnosis and disease severity of pancreatic cancer can be significantly predicted or understood, and can be utilized in the study of tumorigenesis of pancreatic cancer. In addition, the diagnostic method of the present invention can quickly diagnose pancreatic cancer from blood or the like non-invasively.

Description

TECHNICAL FIELD The present invention relates to a composition for diagnosing pancreatic cancer, and a method for diagnosing pancreatic cancer using the same. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for diagnosing pancreatic cancer,

The present invention relates to a composition and kit for diagnosing pancreatic cancer, which comprises an agent for measuring an expression level of a specific protein or an expression level of mRNA of a gene encoding the protein, which can be used for determining the onset or the probability of developing pancreatic cancer, Diagnostic method.

Among the major diseases of modern people, studies on the methods of treatment and diagnosis of cancer are progressing relatively actively, focusing on lung cancer, liver cancer, stomach cancer which have high incidence. However, studies on esophageal cancer, colorectal cancer, and pancreatic cancer which have low incidence are relatively low. In particular, pancreatic cancer initially does not feel much symptoms, and symptoms such as pain and weight loss are usually seen after systemic metastasis has occurred. Therefore, regular diagnosis is very important because the cure rate is lower. Most clinical symptoms develop slowly, appetite declines, is prone to weakness, and weight loss is the most common symptom. Pancreatic cancer is the fatal cancer with a 5-year survival rate of 1-4% and a median survival time of 5 months. It has the worst prognosis in cancer of the human body. In addition, since 80-90% of patients are found to be in an impossible state of radical resection, which is expected to be cured in diagnosis, the prognosis is poor and the treatment depends mainly on the chemotherapy. Therefore, have. To date, the efficacy of several anticancer drugs including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer, is extremely poor and the response rate to chemotherapy is only about 15% This suggests that there is an urgent need to develop more effective early diagnosis and treatment methods to improve the prognosis of pancreatic cancer patients.

The diagnosis of pancreatic cancer has been made by blood test (CA19-9), x-ray examination of the stomach, duodenum, biliary and choledochoscopic cholecystectomy, and retrograde endoscopy. Computed tomography is the most commonly used. When a lesion of a disease is found by these methods, a more accurate examination result can be obtained by performing a more precise biopsy. However, the above diagnostic method is inconvenient because the accuracy is low, Therefore, the subjects are reluctant to do so. Therefore, it has been required to develop a test method that can diagnose pancreatic cancer easily and quickly.

In this regard, Korean Patent No. 10-0819122 discloses a technique using matrilin, transthyretin and stratifin as pancreatic cancer markers, Korean Unexamined Patent Application Publication No. 2008-0082372 Discloses a technique using various pancreatic cancer markers. Korean Patent Laid-Open Publication No. 2009-0003308 discloses a method for diagnosing pancreatic cancer by detecting the expression level of REG4 protein in a blood sample of an individual. Korean Published Application No. 2012-0009781 discloses a method for diagnosing pancreatic cancer Korean Patent Application Laid-open No. 2007-0119250 discloses an assay method for measuring the expression level of XIST RNA in cancer tissues isolated from an individual in order to provide a new gene LBFL313 differentially expressed in human pancreatic cancer tissue And U.S. Patent Application Publication No. 2011/0294136 discloses a method for diagnosing pancreatic cancer using biomarkers such as keratin 8 protein. However, since the markers show a large difference in their diagnostic efficiency and accuracy, it is necessary to find out the marker with better effect.

As a result of intensive efforts to develop a marker useful for early diagnosis of pancreatic cancer, the present inventors have completed the present invention by confirming that proteins specifically raised or decreased in pancreatic cancer can be identified and used to easily diagnose pancreatic cancer .

It is therefore an object of the present invention to provide a composition for the diagnosis of pancreatic cancer, which can easily diagnose the possibility of pancreatic cancer.

Another object of the present invention is to provide a kit for diagnosing pancreatic cancer comprising the above composition.

It is still another object of the present invention to provide a method for diagnosing pancreatic cancer using the composition or kit for diagnosing pancreatic cancer.

In order to achieve the above object, the present invention provides a method for diagnosing pancreatic cancer, which comprises an agent for measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the expression level of mRNA of a gene encoding ABAT protein Lt; / RTI >

According to another aspect of the present invention, there is provided a kit for diagnosing pancreatic cancer comprising the composition.

According to another aspect of the present invention, there is provided a method for diagnosing pancreatic cancer, comprising the steps of: (a) obtaining a sample from a subject diagnosed as having pancreatic cancer; (b) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from the sample; (c) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with an expression level in a normal control sample; And (d) comparing the expression level of the ABAT protein or the mRNA expression level of the ABAT protein-encoding gene in the subject to be diagnosed with the diagnosis of pancreatic cancer to the expression level in the normal control group as a result of the comparison of the step (c) And determining that the probability of onset is high.

By providing a diagnostic marker for pancreatic cancer, the present invention can significantly predict or identify the incidence, early diagnosis, and disease severity of pancreatic cancer, and can be utilized in the study of tumorigenesis of pancreatic cancer. In addition, the diagnostic method of the present invention can quickly diagnose pancreatic cancer from blood or the like non-invasively.

FIG. 1 is a graph showing relative concentrations of ABAT in a control group and a pancreatic cancer patient group by MRM quantitative analysis. FIG.
FIG. 2 is a ROC (Receiver Operating Characteristic) graph showing ABAT's pancreatic cancer diagnostic performance (AUC).
FIG. 3 is a ROC graph showing the pancreatic cancer diagnostic performance (AUC) of CA19-9, which is known as a conventional pancreatic cancer blood marker.
FIG. 4 is a ROC graph comparing ABAT and CA19-9 with pancreatic cancer diagnostic performance (AUC).
FIG. 5 is a ROC graph showing the diagnostic performance (AUC) of pancreatic cancer when ABAT and CA19-9 were used at the same time.
FIG. 6 is a ROC graph comparing the diagnostic performance (AUC) of pancreatic cancer using ABAT and CA19-9 at the same time and the diagnostic performance of pancreatic cancer using ABAT or CA19-9, respectively.

The term "diagnosing" as used herein includes determining the susceptibility of a subject to a particular disease or disorder, determining whether a subject currently has a particular disease or disorder, Determining the prognosis of the subject afflicted with the cancer (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining the stage of the cancer, or determining the response of the cancer to treatment), or determining therametrics And monitoring the status of the object to provide information about the object. For the purposes of the present invention, the diagnosis is to ascertain whether or not the pancreatic cancer has developed or is likely to develop (risk).

The term "pancreatic cancer" in the present invention means cancer originating from pancreatic cells. There are several types of pancreatic cancer, including pancreatic adenocarcinoma, which accounts for about 90% of cases. Generally, pancreatic cancer means pancreatic adenocarcinoma. In addition, there are cystic adenocarcinoma (adenocarcinoma) and endocrine tumor. About 5% to 10% of pancreatic cancer patients have a genetic predisposition. About 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, compared with 0.6% of pancreatic cancer patients in the general population. Pancreatic cancer is a very poor prognosis with a 5 - year survival rate of less than 5%. The reason for this is that most of the cases are found after cancer has progressed, so surgical resection is possible within 20% of cases, and even if completely resected by visual examination, survival rate is not improved by micro metastasis and response to chemotherapy and radiotherapy It is low. Therefore, the most important method for improving survival rate is early detection and operation when symptomless or nonspecific.

The term "marker "," biomarker "or" diagnostic marker "used in the present invention refers to a marker capable of distinguishing a normal or pathological condition or capable of predicting a therapeutic response and measuring objectively. Particularly, in relation to pancreatic cancer in the present invention, the level of protein expression or gene expression is significantly increased or decreased in a subject having pancreatic cancer or a risk of developing pancreatic cancer (risk) compared to a normal control group (non-pancreatic cancer) Such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.

The present invention provides a composition for diagnosing pancreatic cancer comprising an agent for measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the expression level of mRNA of a gene encoding ABAT protein.

The present invention uses ABAT as a marker for diagnosing pancreatic cancer, thereby effectively diagnosing whether or not the subject has pancreatic cancer.

In the present invention, ABAT (4-aminobutyrate aminotransferase), which is used as a marker for diagnosing pancreatic cancer, is also called GABA transaminase or 4-aminobutyrate transaminase and is known to inhibit gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system It is an enzyme involved in the decomposition into succinic semialdehyde. The active enzyme is a 50 kD subunit homodimer complexed with pyridoxal-5-phosphate. Deficiency of ABAT shows mental retardation, muscle weakness, hyperreflexia, helplessness, chronic seizures and EEG abnormalities. However, the prior art disclosing that ABAT can be used as a biomarker for the diagnosis of pancreatic cancer is not known at all.

 The inventors of the present invention confirmed that using ABAT as a marker for diagnosis of pancreatic cancer can give high sensitivity and reliability to the onset or possibility of pancreatic cancer from an individual. Specifically, the present inventors analyzed plasma samples of patients with pancreatic cancer, patients with intraductal papillary mucinous neoplasm (IPMN) and patients with chronic cholecystitis, and found ABAT as a biomarker overexpressed in pancreatic cancer , And normal controls (non-pancreatic cancer) were analyzed to establish the efficacy of the biomarkers excavated.

As used herein, the term "measurement of protein expression level" refers to a process for confirming the presence and expression level of a marker (protein) for diagnosing pancreatic cancer or a gene encoding the same in a biological sample to diagnose pancreatic cancer. As a method of measuring or comparing the expression level of the protein, a protein chip analysis, an immunoassay, a ligand binding assay, a MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, a SELIF- / Ionization Time of Flight Mass Spectrometry Analysis, Radiation Immunoassay, Radiation Immunodiffusion, Oucheroton Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, 2D Electrophoresis Analysis, Liquid Chromatography-Mass Spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA).

In the composition for diagnosing pancreatic cancer according to the present invention, the agent for measuring the expression level of the ABAT protein may include an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA) or an aptamer that specifically binds to the ABAT protein have.

The term "antibody" as used herein refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein of ABAT. The antibody of the present invention includes both a polyclonal antibody, a monoclonal antibody and a recombinant antibody. The antibody can be easily prepared using techniques well known in the art. For example, a polyclonal antibody can be produced by methods well known in the art, including injecting the pancreatic cancer marker protein antigen into an animal and obtaining blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be produced from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs and the like. In addition, monoclonal antibodies can be prepared using hybridoma methods (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519) or phage antibody library techniques (Clackson et al, Nature, 352 : 624-628, 1991; Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991). The antibody prepared by the above method can be separated and purified by methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. The functional fragment of the antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

As used herein, the term "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized, DNA or RNA-like polymer, first introduced by Nielsen, Egholm, Berg and Buchardt at the University of Copenhagen, PNA has a repeated N- (2-aminoethyl) -glycine skeleton linked by peptide bonds, while DNA has a phosphate-ribose sugar backbone, which greatly increases binding capacity and stability to DNA or RNA, , Diagnostic assays and antisense therapies. PNA is described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.

In the present invention, "aptamers" are oligonucleic acid or peptide molecules and the general contents of aptamers 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).

As used herein, the term " measurement of mRNA expression level "refers to the measurement of the amount of mRNA in the biological sample to identify the presence and expression level of the mRNA encoding the protein for diagnosing pancreatic cancer in the biological sample . RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

In the composition for diagnosing pancreatic cancer according to the present invention, the agent for measuring the expression level of the mRNA of the gene encoding the ABAT protein comprises a primer, a probe or an antisense nucleotide that specifically binds to the mRNA of the gene encoding the ABAT protein. Since the information of the ABAT protein according to the present invention is known in UniProt and the like, those skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the mRNA of the gene encoding the protein.

As used herein, the term "primer" is a fragment that recognizes a target gene sequence and includes primer pairs in both forward and reverse directions, but is preferably a primer pair that provides analysis results with specificity and sensitivity. High specificity can be given 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 induce nonspecific amplification .

As used herein, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding do. The kind of probe may be a peptide nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA or a DNA, and is most preferably a peptide nucleic acid It is a PNA. More specifically, the probe is a biomolecule derived from or derived from an organism, or prepared in vitro, and includes, for example, an enzyme, a protein, an antibody, a microorganism, an animal and plant cell and an organ, RNA may include 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 the antisense oligomer is hybridized with the target sequence in the RNA by Watson-Crick base pairing, allowing the formation of the mRNA and RNA: oligomeric heterodimers in the target sequence, ≪ / RTI > sequence and the backbone between the subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence.

In one embodiment, the composition for diagnosing pancreatic cancer according to the present invention further comprises an agent for measuring an expression level of CA19-9 (Carbohydrate Antigen 19-9) protein or an expression level of mRNA of a gene encoding CA19-9 protein . The CA19-9 protein is a conventional pancreatic cancer marker (Safi F. et al., "Diagnostic importance of the tumor marker CA 19-9 in pancreatic cancer", Dtsch Med Wochenschr 109 (49): 1869-73 (1984) ; Wang FM et. al., "The significance of CA19-9 tumor antigen in the serum of patients with carcinomas ". Proc Natl Sci Council Repub. China B, Apr 9 (2): 119-25 (1985). The composition of the present invention combines the two markers, i.e. ABAT and the measured values of CA19-9, to enable more effective and more accurate diagnosis of pancreatic cancer.

The present invention also provides a pancreatic cancer diagnostic kit comprising the above composition for diagnosing pancreatic cancer. 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.

The pancreatic cancer diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method. Preferably, the diagnostic kit may further comprise essential elements necessary for performing the reverse transcription polymerase reaction. The RT-PCR kit contains a primer pair specific for the gene encoding the marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include enzymes such as test tubes or other appropriate containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC DEPC-water, sterile water, and the like.

In addition, the diagnostic kit of the present invention may contain essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, an enzyme, and the like for producing a fluorescently labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

In addition, the diagnostic kit of the present invention can include essential elements necessary for performing ELISA. The ELISA kit comprises an antibody specific for the protein. An antibody is a monoclonal antibody, polyclonal antibody or recombinant antibody, which has high specificity and affinity for the marker protein and little cross-reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

Furthermore, the present invention provides a method for diagnosing pancreatic cancer using the composition for diagnosing pancreatic cancer or the kit for diagnosing pancreatic cancer. The diagnostic method comprises the following steps:

(a) obtaining a sample from a subject to be diagnosed as having pancreatic cancer;

(b) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from the sample;

(c) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with an expression level in a normal control sample; And

(d) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with the expression level of the ABAT protein in the subject to be diagnosed as having pancreatic cancer, as a result of the comparison of the step (c) And determining that the probability is high.

In the above method, the "sample" refers to a biological sample, such as a tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine, which has a different protein expression level or gene expression level due to the onset of pancreatic cancer And preferably means blood, serum or plasma.

Since the mRNA of the ABAT protein or the gene encoding the protein is characterized in that the expression level in the pancreatic cancer patient is increased compared to the expression level in the normal control group, the expression of the ABAT protein in the subject diagnosing pancreatic cancer Level or the mRNA expression level of the gene encoding the ABAT protein is higher than the expression level of the protein or gene in the normal control group, it can be judged that the possibility of pancreatic cancer is high.

The expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein in the subject to be diagnosed as having pancreatic cancer is higher than the expression level in the normal control group means that the pancreatic cancer The expression level of the ABAT protein in the subject to be diagnosed or the mRNA expression level of the gene encoding the ABAT protein is more than 1.0, more than 1.5, more than 2, or more than 3 times the expression level in the normal control , More than 5 times, or more than 10 times.

In one embodiment, 'determining that the likelihood of developing pancreatic cancer is high' may be performed using a pancreatic cancer diagnostic function. An example of the pancreatic cancer diagnosis function is the following functional formula 1:

<Function formula 1>

는 ABAT의 신규 측정값을,

는 SVM에서의 라그랑주 승수를,

는 정상군/췌장암군의 구분자를,

는 기준 측정값을, 그리고

는 보정치를 의미한다.In this formula,

Lt; RTI ID = 0.0 &gt; ABAT &lt; / RTI &

The Lagrange multiplier in the SVM,

Is the discriminator of the normal group / pancreatic cancer group,

The reference measurement value, and

Quot; means a correction value.

The function is derived from SVM (Support Vector Machine). SVM is an algorithm that estimates a function that satisfies a given condition based on the Lagrangian optimization theory. The case of applying the classification method using the maximum margin classifier is called the support vector classification (SVC). When the MRM relative value of ABAT is substituted into the function, if the function value is 1, it is diagnosed that the possibility of pancreatic cancer is high. If the function value is -1, the diagnosis is normal.

The method of the present invention does not require the physician's clinical judgment since the possibility of pancreatic cancer can be readily determined by substituting the expression level of the ABAT protein or its mRNA into the pancreatic cancer diagnostic function and consequently the probability of developing pancreatic cancer.

In this study, SVM is constructed as a diagnostic function. However, it can be implemented by various kinds of discriminant analysis methods such as Neural Network, Random Forest, and other machine learning methods.

In the methods of the present invention, the level of protein expression can be measured and compared using an antibody that specifically binds to the protein of interest. The antibody and the protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used.

As used herein, the term "antigen-antibody complex" refers to a combination of a protein antigen and an antibody recognizing the presence or absence of the gene in the biological sample. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.

For the purpose of the present invention, protein assay, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF Immunoassay, radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blot and ELISA (enzyme linked immunosorbent assay) no.

In a specific embodiment of the present invention, the LC-MRM method was used to measure and compare the protein expression level of ABAT itself.

Specifically, the protein in the biological sample was passed through an LC analysis column with a gradient of 5% to 85% by volume in a solution of 5% distilled water, 95% acetonitrile and 0.1% formic acid for 50 minutes. Concentration gradients were performed because the resolution for specific substances may vary depending on the mixing ratio of the solutions. The range is an optimum range for simultaneously separating various proteins.

Mass spectrometry was performed by MS / MS mode multiple reaction monitoring (MRM). While SIM (Selected Ion Monitoring) is a method of using ions created by collision with the source part of the mass spectrometer, MRM once again selects a particular ion among the broken ions to make another MS source connected continuously And collide with each other and use the ions obtained in the collision. In SIM, there is a problem that the selected quantitative ions may interfere with the quantitation when the ions are also detected in the plasma. On the other hand, in the case of using MRM, when ions having the same mass are further broken, the molecular structure is different and differentiated. Therefore, when the ion is used as a quantitative ion, disturbing peaks in the background are removed, Can be obtained. Therefore, by using the MRM mode during mass spectrometry, we were able to simultaneously analyze the desired substances at better analytical sensitivity.

Through the above analysis methods, it is possible to compare the protein expression level in the normal control group with the protein expression level in the pancreatic cancer individual, judge whether the expression level of the pancreatic cancer marker gene is significantly increased or not, and diagnose the possibility of the pancreatic cancer .

In addition, in the method of the present invention, measurement or comparison of the mRNA expression level of the gene encoding the ABAT protein can be performed by a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, Or a DNA chip, but the present invention is not limited thereto. Through these measurement methods, the mRNA expression amount in a subject to be diagnosed as the amount of mRNA expression in the normal control group and the diagnosis of the development of pancreatic cancer can be confirmed, and the possibility of the development of pancreatic cancer can be diagnosed or predicted by comparing the expression levels thereof.

The present invention also provides a method for diagnosing pancreatic cancer by combining CA19-9, which is known as pancreatic cancer marker, in addition to ABAT. The method comprises the steps of:

(a) obtaining a sample from a subject to be diagnosed as having pancreatic cancer;

(b) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from the sample;

(c) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with an expression level in a normal control sample;

(d) measuring the level of expression of the CA19-9 protein or the level of the mRNA of the gene encoding the CA19-9 protein from the sample;

(e) comparing the expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the CA19-9 protein with an expression level in a normal control sample; And

(f) comparing the levels of ABAT protein expression or mRNA expression levels of the ABAT protein encoding the subject to be diagnosed with the diagnosis of pancreatic cancer to the level of expression in the normal control group as a result of the comparison between steps (c) and (e) And determining that the expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the CA19-9 protein is higher than the expression level in the normal control, that the possibility of pancreatic cancer is high.

In the above method, steps (d) and (e) can be performed in a manner similar to that described above with respect to the ABAT protein or the mRNA of the gene encoding it.

The present invention provides a method for diagnosing pancreatic cancer, comprising the steps of: (a) obtaining a sample from a subject diagnosed as having pancreatic cancer; (b) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from the sample; And (c) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with the expression level in a normal control sample.

The present invention also provides a method for diagnosing pancreatic cancer, comprising the steps of: (a) obtaining a sample from a subject to be diagnosed as having pancreatic cancer; (b) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from the sample; (c) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with an expression level in a normal control sample; (d) measuring the level of expression of the CA19-9 protein or the level of the mRNA of the gene encoding the CA19-9 protein from the sample; And (e) comparing the expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the CA19-9 protein with an expression level in a normal control sample .

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

Example : Overexpression of pancreatic cancer or Low expression  Protein discovery and performance analysis

(1) Blood sample preparation

In order to identify proteins overexpressing or underexpressing in pancreatic cancer, blood samples were collected from 50 pancreatic cancer patients, 10 patients with malignant pancreatic ductal mucinous papillary tumor, 22 patients with chronic cholecystitis, and 25 normal control patients as shown in Table 1 below Respectively. Normal control and chronic cholecystitis samples were normal, and pancreatic cancer and malignant pancreatic ductal mucinous papillary tumors were classified into pancreatic cancer.

group age gender range Average standard deviation female male sum Pancreatic cancer 44-88 65.72 ± 9.85 23 27 50 Mucinous papillary tumor in malignant pancreatic duct 49-81 63.6 ± 9.66 4 6 10 Chronic cholecystitis 43-82 56.96 ± 9.13 11 11 22 Normal control group 35-65 49.04 + - 8.92 16 9 25

40 ㎕ of each blood sample was passed through a Multiple Affinity Removal System (MARS) column (Agilent, USA) to remove 14 proteins occupying the largest proportion in the blood, followed by concentration with a 3 kDa filter and quantification of BCA Of which 200 μg of plasma was taken and denatured with 6 M urea and reduced and alkylated using 20 mM DTT and 50 mM iodoacetic acid. The protein was then treated with trypsin at a ratio of 50: 1 (protein: trypsin, w / w) at 37 ° C for 16 hours to desaturate the peptide using a C18 OASIS column (Waters, USA) And lyophilized. The lyophilized peptide was dissolved in Solution A (98% distilled water, 2% acetonitrile, 0.1% formic acid), and 5 fmol of an internal standard β-galactosidase peptide was added thereto, and MRM analysis was performed on the peptide .

(2) Transition  selection

To perform the MRM analysis, a peptide having a characteristic charge-to-mass ratio (m / z) of a specific protein was selected (Q1), and the peptide among the various fragment ions generated when the peptide was fragmented by electrical impact A fragment ion with characteristic m / z was selected (Q3). The combination of Q1 and Q3 is a combination of ions specific to a specific protein. This is called a transition. A signal obtained when ions are sequentially passed through these characteristic Q1 and Q3 in a high-resolution (triple quadrupole) And analyzed quantitatively. Based on the peptide tandem mass spectra of the National Institute of Standards and Technology (NIST) library using open software called SKYLine developed by the MacCoss research team at the University of Washington, USA, ms / ms data is present For peptides, a maximum of 10 peptides per protein were selected in order of increasing total intensity. The length of the peptide was selected from a minimum of 7 amino acids to a maximum of 24 amino acids.

However, among the whole peptides that can be generated by cleavage by trypsin, the peptides containing the amino acids or having specific motifs are excluded because they are known to have poor signal values as follows:

In the presence of methionine in the peptide, oxidation was induced by in vivo reactive oxygen species (ROS) to increase the mass value by 32 Da;

B) when histidine is present in the peptide, the charge state of the peptide is changed due to the positive charge of the R-group;

If there is an NxS / T motif in the peptide, N-saccharification occurs and the mass value shifts;

Ⓓ Missing cleavage may occur if proline is present following R or K that can be cleaved by trypsin in the peptide.

A peptide having a charge of +2 in the precursor charge was selected, and +1 ion charge was used for ion charge and y-ion was used for ion type. Protein Blast P and Skyline programs were used to select unique transitions and peptides, and the final selected transitions only used those within the retention time (RT) range. For RT prediction, 600 SIS peptide MRM assays were performed, and the calibration curve was calculated based on the hydrophobicity scale and the RT chromatogram.

(3) LC  And MRM

The LC was a Agilent 1260-capillary LC and a capillary RR 0.5 x 150 3.5 μm column was used for peptide separation. 5 μl of the sample was injected, and the flow rate was set at 20 μl / min. First, the column was equilibrated with Sol A (95% distilled shoe, 5% acetonitrile, 0.1% formic acid) for 10 minutes and then eluted with 5% to 85% solution for 50 minutes with Sol B (5% distilled water, 95% acetonitrile, 0.1% , The peptide was eluted through a concentration gradient of 85% for 5 minutes.

Transitions to selected proteins were monitored in MRM mode using Agilent's triple quadrupole 6490-QQQ instrument as a mass spectrometer. The 5 fmol beta-galactosidase peptide (GDFQFNISR [C13N15], 547.3 / 646.4) spiked into each sample was also monitored simultaneously to compensate for batch-to-batch deviations.

(4) Data quantitative analysis

(GDFQFNISR [C13N15], 547.3 / 646.4 m / z), which is an internal standard peptide, at 0.09, 0.27, 0.82, 2.5, 7.4, 22.2, 66.7 and 200 fmol After dilution, 10 [mu] g of plasma was added to the matrix in the same manner as the target peptide analysis conditions. Also, for the purpose of confirming the endogenous signal, the case where the internal standard peptide was not added was included in the analysis, and the standard curve was determined by quantifying MRM three times at all 9 concentration points.

The MRM results of each individual were generated by ion chromatography (XIC, Extraction Chromatography) of the corresponding MRM transition using the Skyline (MacCoss Lab, ver1.4.1), and the peak area of each transition was calculated Respectively. The area of each XICed peak was normalized to the XICed peak area of the internal standard beta-galactosidase peptide (GDFQFNISR [C13N15], 547.3 / 646.4 m / z), and relative quantitative analysis of each protein was performed with this value .

As a result of quantitative analysis of the MRM, it was confirmed that the expression of ABAT protein was specifically increased in the pancreatic cancer patient group as compared with the normal group as shown in FIG. 1, and the ABAT was selected as a marker for diagnosing pancreatic cancer.

(5) Data statistical analysis

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. In this case, a case where a classification method using a maximum margin classifier is used is referred to as a support vector classification Support Vector Classification (SVC). In this example, the following pancreatic cancer diagnostic functions were constructed using the SVC that maximizes the difference between the two groups (normal and cancer patient groups):

In the above equation, if the function value is 1, it is determined to be pancreatic cancer, and if it is -1, it is judged to be normal.

The function was used to determine whether pancreatic cancer was present. 0.000100328 = -1, f (0.000252726) = -1, and F (0.000100) were obtained as the results of substituting the diagnostic function for the ABAT relative measurement values of 0.000100328, 0.000252726 and 0.000301919, respectively, f (0.000301919) = -1 so that the object can be determined as normal. In addition, from the three pancreatic cancer patients, 0.087803242, 0.039893973, and 0.038009273 were obtained as the relative MRM relative measurement values of ABAT, and the result was substituted into the above diagnostic function. As a result, the function values were f (0.087803242) = 1, f (0.039893973) f (0.038009273) = 1 so that the subject could be identified as pancreatic cancer.

On the other hand, the performance of ABAT as a pancreatic cancer diagnostic marker when using the pancreatic cancer diagnosis function is shown by the AUC of the ROC graph. 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 .

ABAT according to the present invention showed an excellent AUC of 0.838 as a diagnostic marker for pancreatic cancer (Fig. 2). In particular, considering that the AUC of CA19-9, which is a commercial pancreatic cancer marker, is 0.825 (FIG. 3), ABAT of the present invention is superior to CA19-9 in diagnosing pancreatic cancer (FIG.

In addition, when ABAT was used concurrently with CA19-9, it was confirmed that the AUC was 0.972 (FIG. 5), and the ABAT or CA19-9 alone performed better than the ABAT alone (FIG. 6).

The results show that ABAT according to the present invention can be usefully used as a pancreatic cancer diagnostic marker.

Claims (12)

A composition for diagnosing pancreatic cancer comprising an expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or an expression level of mRNA of a gene encoding ABAT protein.
The method according to claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), or an aptamer that specifically binds to the protein , A composition for diagnosing pancreatic cancer.
The composition for diagnosing pancreatic cancer according to claim 1, wherein the agent for measuring the expression level of the mRNA comprises a primer, a probe or an antisense nucleotide that specifically binds to the gene.
The composition for diagnosing pancreatic cancer according to claim 1, further comprising an agent for measuring the expression level of the CA19-9 protein or the expression level of the mRNA of the gene encoding the CA19-9 protein.
A kit for the diagnosis of pancreatic cancer comprising the composition according to any one of claims 1 to 4.
The kit according to claim 5, wherein the kit is a reverse transcription polymerase chain reaction kit (RT-PCR), a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, Kit for the diagnosis of pancreatic cancer.
(a) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from blood, serum or plasma samples of a subject; And
(b) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with an expression level in a normal control sample
Wherein the method comprises the steps of:
delete The method according to claim 7, wherein the protein expression level measurement is performed using an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA) or an aptamer that specifically binds to the protein. Information delivery method.
8. The method of claim 7, wherein the protein expression level measurement or comparison is performed using protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) Laser Immunoassay, Immunoelectrophoresis, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, Two-Dimensional Electrophoresis Analysis, Liquid Chromatography - Ionization Time of Flight Mass Spectrometry Mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA). Wherein the information is provided for diagnosis of pancreatic cancer.
The method according to claim 7, wherein the mRNA expression level is measured by a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, a Northern blotting or a DNA chip , A method of providing information necessary for diagnosis of pancreatic cancer.
(a) measuring the expression level of ABAT (4-aminobutyrate aminotransferase; GenBank Accession No: NP_001120920.1) protein or the mRNA expression level of a gene encoding ABAT protein from blood, serum or plasma samples of a subject;
(b) comparing the expression level of the ABAT protein or the mRNA expression level of the gene encoding the ABAT protein with the level of expression in a normal control sample;
(c) measuring the level of expression of the CA19-9 protein or the level of mRNA of the gene encoding the CA19-9 protein from the blood, serum or plasma sample of the subject;
(d) comparing the measured expression level of the CA19-9 protein or the mRNA expression level of the gene encoding the CA19-9 protein with an expression level in a normal control sample
Wherein the method comprises the steps of:

Images