KR20230160425A - Composition for diagnosing pancreatic cancer - Google Patents

Abstract

The present invention relates to a composition, a kit, and a method of providing information for diagnosis that can diagnose pancreatic cancer and distinguish it from other pancreatic diseases or other carcinomas.

Description

Composition for diagnosing pancreatic cancer}

The present invention relates to a composition for diagnosing pancreatic cancer, a kit, and a method for providing information for diagnosis.

Among the major diseases of modern people, research on cancer treatment and diagnosis methods is being conducted relatively actively, focusing on lung cancer, liver cancer, and stomach cancer, which have high incidences. However, research on esophageal cancer, colon cancer, and pancreatic cancer, which have low incidence, is relatively poor.

In particular, pancreatic cancer does not show many symptoms in the early stages, and symptoms such as pain and weight loss usually appear after systemic metastases have already occurred, making the cure rate even lower, so regular diagnosis is very important. Most clinical symptoms develop slowly, are prone to weakness, 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 time of 5 months, and has the poorest prognosis among human cancers. In addition, since 80-90% of patients are found at the time of diagnosis in a state where curative resection with the expectation of cure is impossible, the prognosis is poor and treatment mainly relies on chemotherapy, so the development of early diagnosis methods is more urgently needed than for any other human cancer. there is.

To date, the therapeutic effectiveness of several anticancer drugs including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer, is extremely low, and the response rate to anticancer treatment is only around 15%. This fact suggests that the development of more effective early diagnosis and treatment methods is urgently needed to improve the prognosis of pancreatic cancer patients. Appropriate diagnosis and treatment of pancreatic cancer precursor lesions, which are stages before pancreatic cancer progresses to fatal pancreatic cancer, are very important in improving pancreatic cancer treatment outcomes.

Diagnosis of pancreatic cancer or pancreatic cancer precursor lesions includes blood tests (CA19-9), X-ray contrast examination of the stomach and duodenum, cholangiography through the skin and liver, and retrograde endoscopic cholangiography. These methods have been used to detect disease lesions, but recently ultrasonography and computed tomography are most commonly used. Relatively accurate test results can be obtained by performing a more precise biopsy. However, the diagnostic method is very inconvenient due to low accuracy or pain to the patient, so subjects are reluctant to use it. Therefore, there has been a demand for the development of a test method that can easily and quickly diagnose pancreatic cancer or pancreatic cancer precursor lesions.

Republic of Korea Patent No. 10-0819122 and Korea Patent Publication No. 2012-0082372 disclose technology using various pancreatic cancer markers including matrilin, transthyretin, stratifin, etc. , Since each marker shows a large difference in diagnostic efficiency and accuracy, there is a need to discover markers with better effectiveness and develop a diagnostic method using them.

One object of the present invention is to provide a composition, kit, or method of providing information for diagnosing the occurrence or possibility of developing pancreatic cancer.

Another object of the present invention is to provide a composition, kit, or information provision method for diagnosing and distinguishing whether a lesion occurring in an individual is pancreatic cancer, another inflammatory disease, or a benign pancreatic tumor.

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

One. Biomarkers for diagnosis of cancer

According to one embodiment of the present invention, the present invention relates to a biomarker composition for diagnosing cancer, including at least one protein of leukocyte immunoglobulin like receptor B5 (LILRB5) and cathelicidin antimicrobial peptide (CAMP) or a gene encoding the protein.

In the present invention, the "LILRB5 (leukocyte immunoglobulin like receptor B5)" belongs to the leukocyte immunoglobulin-like receptor (LIR) family, in particular, has two or four extracellular immunoglobulin domains and a membrane-transmitting It belongs to the subfamily B class of LIR receptors, containing a domain and 2 to 4 cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Several other LIR subfamily B receptors are expressed on immune cells where they bind to MHC class I molecules on antigen-presenting cells and inhibit stimulation of the immune response. The LILRB5 may be composed of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, the “CAMP (cathelicidin antimicrobial peptide)” is a polysaccharide mainly stored in the lysosomes of macrophages and polymorphonuclear leukocytes, and corresponds to an amphipathic substance. Members of this family react with pathogens by disrupting, damaging, or puncturing cell membranes. Accordingly, the CAMP is known to play an important role in innate immune defense against bacterial infection in mammals. The CAMP may be composed of the amino acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

In one example of the present invention, the biomarker composition may include LILRB5 protein or a gene encoding it.

In another example of the present invention, the biomarker composition includes the LILRB5 protein or the gene encoding it, and may further include the CAMP protein or the gene encoding it.

In the present invention, the LILRB5 or CAMP is expressed in a biological sample isolated from the subject of interest, preferably in a liquid biopsy, for example, in cells or exosomes isolated from blood, serum, or plasma. level, etc. can be measured.

In the present invention, the “object of interest” refers to an individual who has developed or is likely to develop cancer, especially pancreatic cancer, and may be a mammal, including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, It may be selected from the group consisting of monkeys, dogs, cats, cows, horses, pigs, sheep, and goats, and preferably may be humans, but is not limited thereto.

When measuring the expression level of a marker according to the present invention in a liquid biopsy isolated from an object of interest in the present invention, or cells or exosomes isolated from the liquid biopsy, the patient is opened and tissue (e.g., pancreatic tissue) is measured. There is no need for an invasive process such as separating tissue cells from the tissue, it takes approximately 5 minutes to obtain the biopsy, and it takes approximately 2 hours to measure the expression levels of various disease biomarkers according to the present invention from the biopsy. It takes less than 10 minutes, so you can very quickly and easily check whether or not the disease has developed or is likely to develop the disease.

In the present invention, the term “cancer” refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals. The above cancers include thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, and blood cancer. , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer. , soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, preferably It may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

In the present invention, “pancreatic cancer” refers to cancer originating from pancreatic cells. There are several types of pancreatic cancer, and pancreatic adenocarcinoma, which arises from pancreatic duct cells, accounts for about 90%, so pancreatic cancer generally refers to pancreatic ductal adenocarcinoma. In addition, there are cystic cancer (cystadenocarcinoma) and endocrine tumors. Approximately 5-10% of pancreatic cancer patients have a genetic predisposition, and approximately 7.8% of pancreatic cancer patients have a family history of pancreatic cancer, which is higher than the 0.6% incidence of pancreatic cancer in the general population. Pancreatic cancer is a cancer with a very poor prognosis, with a 5-year survival rate of less than 5%. This is because most cancers are discovered after they have progressed, so surgical resection is possible in less than 20% of cases at the time of discovery, and even if the cancer is completely resected with the naked eye, the improvement in survival rate is small due to micrometastases, and the response to anticancer drugs and radiation therapy is low. Because it is low. Therefore, the most important way to improve survival rate is to detect early and perform surgery when there are no symptoms or when symptoms are non-specific.

In the present invention, “diagnosis” means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis may be to predict the possibility of the onset, growth, progression or metastasis of cancer, or may refer to cancer as another disease, such as a pancreatic disease, especially pancreatitis (both acute and chronic). , it may be to distinguish pancreatic benign tumors (such as lipoma or intrapancreatic papillary mucinous neoplasm (IPMN)), or it may be to distinguish carcinoma, especially pancreatic cancer from other carcinomas.

In the present invention, “pancreatitis” refers to a disease caused by inflammation of the pancreas and includes acute pancreatitis and chronic pancreatitis. Pancreatic juice contains digestive enzymes such as amylase (which hydrolyzes carbohydrates), trypsin (which hydrolyzes proteins), and lipase (which hydrolyzes fats). Pancreatitis not only occurs when the pancreatic juice does not flow smoothly due to alcohol abuse, gallstones, etc., causing the enzymes to cause autolysis of the pancreas, but also due to various causes such as metabolic disorders, drugs, and abdominal injuries. Pancreatitis is an inflammatory disease of the pancreas that causes damage to pancreatic glandular cells, extensive interstitial edema, hemorrhage, and migration of neutrophilic granulocytes to the site of injury. Pancreatitis can be broadly divided into two types: mild type, in which interstitial edema and peripancreatic fat necrosis are found, peripancreatic and intrapancreatic (peripancreatic) pancreatitis. There is severe type of pancreatitis accompanied by extensive intrapancreatic fatty necrosis, pancreatic parenchymal necrosis, and hemorrhage.

In the present invention, the biological sample isolated from the subject of interest essentially contains at least one protein of LILRB5 and CAMP, preferably LILRB5, and optionally further contains CAMP, or expression of genes encoding these proteins. By measuring the level, it is possible to predict the possibility of onset, growth, progression, or metastasis of cancer, especially pancreatic cancer, and distinguish pancreatic cancer from other pancreatic diseases, such as pancreatitis or benign pancreatic tumors, or other carcinomas. Or, the likelihood of an outbreak can be predicted.

According to another embodiment of the present invention, it relates to a composition for diagnosing cancer comprising as an active ingredient an agent for measuring the expression level of at least one protein of LILRB5 and CAMP or the gene encoding the same.

In one example of the present invention, the diagnostic composition may include an agent for measuring the expression level of the LILRB5 protein or the gene encoding it.

In another example of the present invention, the diagnostic composition includes an agent for measuring the expression level of the LILRB5 protein or the gene encoding it, and may further include an agent for measuring the expression level of the CAMP protein or the gene encoding it.

In the present invention, the agent for measuring the expression level of the protein includes at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. It can be done, but is not limited to this.

In the present invention, the “antibody” refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, antibody refers to an antibody that specifically binds to the protein. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibody can be easily produced using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, including the process of injecting the protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. These polyclonal antibodies can be produced from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, etc. In addition, monoclonal antibodies can be prepared using the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), which is well known in the art, or phage antibody library technology (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 separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Additionally, antibodies of the invention include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

In the present invention, the “oligopeptide” is a peptide composed of 2 to 20 amino acids and may include dipeptide, tripeptide, tetrapeptide, and pentapeptide, but is not limited thereto.

In the present invention, "Peptide Nucleic Acid (PNA)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt at the University of Copenhagen, Denmark in 1991. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases its binding force and stability to DNA or RNA, making it useful in molecular biology. , is used in diagnostic analysis and antisense therapy. PNA was 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] is disclosed in detail.

In the present invention, the “aptamer” is an oligonucleic acid or peptide molecule, and general details 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)].

In the present invention, the agent for measuring the expression level of the gene encoding the protein may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the protein. It is not limited.

In the present invention, the “primer” is a fragment that recognizes the target gene sequence and includes forward and reverse primer pairs, but is preferably a primer pair that provides analysis results with specificity and sensitivity. High specificity can be granted 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 the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .

In the present invention, the “probe” refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of the target substance in the sample through the binding. The type of probe is not limited as it is a material commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and is most preferred. It is PNA. More specifically, the probe is a biomaterial that is derived from or similar to living organisms or includes those manufactured in vitro, such as enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

In the present invention, “LNA (Locked nucleic acids)” refers to nucleic acid analogs containing 2'-O, 4'-C methylene bridges [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502 ]. LNA nucleosides contain the common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rules. However, due to the 'locking' of the molecule due to the methylene bridge, LNA does not form the ideal shape in Watson-Crick bonding. When LNA is included in a DNA or RNA oligonucleotide, the LNA can pair with the complementary nucleotide chain more quickly and increase the stability of the double helix.

In the present invention, the "antisense" refers to a sequence of nucleotide bases in which an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, allowing the formation of an oligomeric heteroduplex, typically mRNA and RNA, within the target sequence. and an oligomer having an intersubunit backbone. Oligomers may have exact or approximate sequence complementarity to the target sequence.

Since information about the LILRB5 and CAMP proteins according to the present invention and the genes encoding them are known, those skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the genes encoding the proteins based on this information. There will be.

In the present invention, the LILRB5 or CAMP protein, or the gene encoding it, is a biological sample isolated from the subject of interest, preferably a liquid biopsy, for example, cells or exosomes isolated from blood, serum, or plasma. It can be measured from a little bit.

In the present invention, the cancer includes thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, and Hodgkin lymphoma. , blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, and endocrine cancer. , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma. , Preferably, it may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

In the present invention, the diagnostic composition can be used to predict the possibility of cancer onset, growth, progression, or metastasis, or can be used to treat cancer as another disease, such as pancreatic disease, especially pancreatitis (both acute and chronic). , It can be used to distinguish pancreatic benign tumors (lipoma or intrapancreatic papillary mucinous neoplasm (IPMN), etc.), or it can be used to differentiate carcinoma, especially pancreatic cancer, from other carcinomas. .

According to another embodiment of the present invention, it relates to a kit for diagnosing cancer comprising the diagnostic composition of the present invention.

In the present invention, the “kit” refers to a tool that can evaluate the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody by reaction with a substrate, but also indirect labeling in which a label that develops color through reactivity with another directly labeled reagent is conjugated. It may include a chromogenic substrate solution, a washing solution, and other solutions that will undergo a color reaction with the label, and may be prepared including reagent components to be used. In the present invention, the kit may be a kit containing the essential elements required to perform RT-PCR, including a test tube, reaction buffer, deoxynucleotides (dNTPs), and Taq-polymerization, in addition to each primer pair specific for the marker gene. It may contain enzymes, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. Additionally, the kit may be a kit for detecting genes for cancer diagnosis that includes essential elements needed to perform a DNA chip. The DNA chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

In the present invention, the kit may be an RT-PCR kit, DNA chip kit, ELISA kit, protein chip kit, rapid kit, or MRM (multiple reaction monitoring) kit.

The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, in the present invention, the kit may further include essential elements required to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains a pair of primers specific for the gene encoding the marker protein. Primers are nucleotides 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 include primers specific to the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, and the RNase inhibitor DEPC. -Can include DEPC-water, sterilized water, etc.

Additionally, the kit for diagnosing cancer of the present invention may include essential elements required to perform DNA chip testing. A DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. The substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

Additionally, the cancer diagnostic kit of the present invention may include essential elements necessary to perform ELISA. ELISA kits contain antibodies specific for these proteins. Antibodies are antibodies that have high specificity and affinity for a marker protein and almost no cross-reactivity to other proteins, and may be monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or those that can bind to antibodies. It may contain other substances, etc.

In the present invention, the fixture for the antigen-antibody binding reaction includes a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, and a glass slide glass. It may be used, but is not limited thereto.

In addition, in the present invention, the label for the secondary antibody is preferably a conventional coloring agent that produces a color reaction, such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, and FITC (poly L Labels such as fluorescein and dye, such as -lysine-fluorecein isothiocyanate) and RITC (rhodamine-B-isothiocyanate), may be used, but are not limited thereto.

In addition, the chromogenic substrate for inducing color development in the present invention is preferably used according to the label that produces a color reaction, such as TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. At this time, it is more preferable that the chromogenic substrate is provided dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker for the secondary antibody conjugate to produce a chromogenic deposit, and the presence or absence of the marker proteins is detected by visually checking the degree of deposition of the chromogenic deposit.

In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) consisting of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferable. do. After the antigen-antibody binding reaction, the washing solution reacts with the secondary antibody to the antigen-antibody conjugate, then adds an appropriate amount to the fixative and washes 3 to 6 times. The reaction stopping solution may preferably be a sulfuric acid solution (H ₂ SO ₄ ).

According to another embodiment of the present invention, for the diagnosis of cancer, comprising measuring the expression level of at least one protein of LILRB5 and CAMP or a gene encoding the protein in a biological sample isolated from a subject of interest. It's about how to provide information.

In the present invention, the object of interest is an individual that has developed or is likely to develop cancer and may be a mammal, including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, and cats. , may be selected from the group consisting of cattle, horses, pigs, sheep, and goats, and preferably may be humans, but is not limited thereto.

In the present invention, the biological sample refers to any material, biological fluid, tissue, or cell obtained from or derived from an individual, such as whole blood, leukocytes, and peripheral blood mononuclear cells. ), white blood cell buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, respiration (breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid) , glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate. It may be one or more selected from the group consisting of joint aspirate, organ secretions, cells, cell extract, and cerebrospinal fluid, but is preferably liquid biopsy. For example, it may be cells or exosomes isolated from blood, serum or plasma.

In one example of the present invention, the method of providing information may include measuring the expression level of the LILRB5 protein or the gene encoding it in a biological sample isolated from the subject of interest.

In another example of the present invention, the method for providing information includes measuring the expression level of the LILRB5 protein or the gene encoding the same in a biological sample isolated from the subject of interest, and the expression level of the CAMP protein or the gene encoding the same. An additional step of measuring may be further included.

In the present invention, the agent for measuring the expression level of the protein includes at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. can do.

In the present invention, the expression level of the protein can be measured using protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, and SELDI-TOF (Sulface Enhanced Laser Desorption/SELDI-TOF). Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry ( It can be performed by liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay).

Additionally, in the present invention, the expression level of the protein can be measured by a multiple reaction monitoring (MRM) method.

In the present invention, in the multiple reaction monitoring method, the internal standard material may be a synthetic peptide or Escherichia coli beta galactosidase in which a specific amino acid constituting the target peptide is isotopically substituted.

In the present invention, the agent for measuring the expression level of the gene encoding the protein may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the protein.

In the present invention, the expression level of the gene encoding the protein can be measured using reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), and real-time reverse transcription polymerase reaction (Real-time RT-PCR). , RNase protection assay (RPA), Northern blotting, or DNA chip.

In the information provision method of the present invention, descriptions of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), aptamers, etc., and descriptions of primers, probes, etc. overlap with what was previously described, and to avoid excessive complexity of the specification, they are provided below. The detailed description is omitted.

In the present invention, if the expression level of the LILRB5 protein or the gene encoding it measured in the biological sample of the subject of interest is higher than that of the control group, it can be predicted that the subject of interest has developed or is highly likely to develop cancer.

In the present invention, if the expression level of the CAMP protein or the gene encoding it measured in the biological sample of the subject of interest is lower than that of the control group, it can be predicted that the subject of interest has developed or is highly likely to develop cancer.

In the present invention, “control group” refers to the expression level of the corresponding biomarker protein or the gene encoding the protein in healthy normal controls, or the average value of the expression level of the corresponding marker protein or the gene encoding the corresponding marker protein in biological samples derived from patients with pancreatic disease. Alternatively, it may be the median value, or it may be the average or median value of the expression level of the marker protein or the gene encoding the marker protein in a biological sample derived from a cancer patient, preferably a patient with another cancer type other than pancreatic cancer, but is not limited thereto.

In the information provision method of the present invention, predicting that the cancer has developed or is highly likely to develop includes not only predicting the possibility of the onset, growth, progression, or metastasis of the cancer, but also predicting that the cancer has developed or is likely to develop in the target individual. Diseases suspected of having cancer are distinguished from other diseases, especially pancreatic diseases (e.g., pancreatitis (both acute or chronic), benign pancreatic tumors (lipoma or intrapancreatic papillary mucinous tumor (IPMN), etc.)) and are classified as cancer. In addition, it may include predicting that the cancer that has developed or is suspected to have developed in the subject of interest is pancreatic cancer by distinguishing it from other cancer types.

In the present invention, the cancer includes thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, and Hodgkin lymphoma. , blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, and endocrine cancer. , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma. , Preferably, it may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

2. Biomarkers to distinguish cancer from inflammatory or benign tumors

According to one embodiment of the present invention, the present invention relates to a biomarker composition for distinguishing between cancer and inflammatory or benign tumors, including at least one protein of LILRB5 and CAMP or a gene encoding the same.

In one example of the present invention, the biomarker composition may include LILRB5 protein or a gene encoding it.

In another example of the present invention, the biomarker composition may include CAMP protein or a gene encoding it.

In another example of the present invention, the biomarker composition may include the LILRB5 protein or the gene encoding it, and may include the CAMP protein or the gene encoding it.

In the present invention, the LILRB5 or CAMP is expressed in a biological sample isolated from the subject of interest, preferably in a liquid biopsy, for example, in cells or exosomes isolated from blood, serum, or plasma. level, etc. can be measured.

In the present invention, the object of interest may be a mammal, including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It may be selected from the group consisting of, and preferably may be a human, but is not limited thereto.

In the present invention, the cancer includes thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, and Hodgkin lymphoma. , blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, and endocrine cancer. , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma. , Preferably, it may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

In the present invention, the “inflammation” refers to the formation of an abnormal inflammatory response in the tissue or organ in which cancer suspected of developing in the subject of interest has occurred. In particular, for the purpose of the present invention, it may be acute or chronic pancreatitis. It is not limited to this.

In the present invention, the “benign tumor” is also called a “benign nodule” or “nodule,” and is a lump formed by excessive proliferation of cells, and not only does it grow slowly, but it does not grow beyond a certain size. There is a characteristic that it does not exist. Additionally, the benign tumor does not metastasize to other tissues like cancer and can be easily cured. The benign tumor has little harm to the human body, but can be problematic if it puts pressure on major organs. For the purpose of the present invention, the benign tumor may be a pancreatic lipoma, a pancreatic mass, or an intrapancreatic papillary mucinous tumor (IPMN), but is not limited thereto.

In the present invention, cancer, especially pancreatic cancer, can be distinguished from pancreatitis or benign pancreatic tumor by measuring the expression level of at least one protein of LILRB5 and CAMP or the gene encoding it for biological samples isolated from the subject of interest. Can be predicted or diagnosed.

According to another embodiment of the present invention, a diagnostic composition for distinguishing cancer from inflammation or benign tumor, comprising as an active ingredient an agent for measuring the expression level of at least one protein of LILRB5 and CAMP or the gene encoding the same. will be.

In one example of the present invention, the diagnostic composition may include an agent for measuring the expression level of the LILRB5 protein or the gene encoding it.

In another example of the present invention, the diagnostic composition may include an agent for measuring the expression level of CAMP protein or the gene encoding it.

In another example of the present invention, the diagnostic composition includes an agent for measuring the expression level of the LILRB5 protein or the gene encoding it, and may include an agent for measuring the expression level of the CAMP protein or the gene encoding it.

In the present invention, the agent for measuring the expression level of the protein includes at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. It can be done, but is not limited to this.

In the present invention, the agent for measuring the expression level of the gene encoding the protein may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the protein. It is not limited.

Since information about the LILRB5 and CAMP proteins according to the present invention and the genes encoding them are known, those skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the genes encoding the proteins based on this information. There will be.

In the present invention, the LILRB5 or CAMP protein, or the gene encoding it, is a biological sample isolated from the subject of interest, preferably a liquid biopsy, for example, cells or exosomes isolated from blood, serum, or plasma. It can be measured from a little bit.

In the present invention, the cancer includes thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, and Hodgkin lymphoma. , blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, and endocrine cancer. , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma. , Preferably, it may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

In the present invention, the inflammation may be acute or chronic pancreatitis, but is not limited thereto.

In the present invention, the benign tumor may be a pancreatic lipoma, a pancreatic mass, or an intrapancreatic papillary mucinous tumor (IPMN), but is not limited thereto.

In the present invention, the composition for differentiation is used to distinguish cancer, especially pancreatic cancer, from other pancreatic diseases such as pancreatitis (including both acute and chronic) and benign pancreatic tumors (lipoma or intrapancreatic papillary mucinous neoplasm (IPMN), etc.) It can be used to predict or diagnose its onset.

According to another embodiment of the present invention, it relates to a diagnostic kit for distinguishing between cancer and inflammatory or benign tumor, comprising the diagnostic composition of the present invention.

In the present invention, the kit may be an RT-PCR kit, DNA chip kit, ELISA kit, protein chip kit, rapid kit, or MRM (multiple reaction monitoring) kit.

The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, in the present invention, the kit may further include essential elements required to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains a pair of primers specific for the gene encoding the marker protein. Primers are nucleotides 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 include primers specific to the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, and the RNase inhibitor DEPC. -Can include DEPC-water, sterilized water, etc.

Additionally, the diagnostic kit of the present invention may include essential elements required to perform DNA chip testing. A DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. The substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

Additionally, the diagnostic kit of the present invention may include essential elements required to perform ELISA. ELISA kits contain antibodies specific for these proteins. Antibodies are antibodies that have high specificity and affinity for a marker protein and almost no cross-reactivity to other proteins, and may be monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or those that can bind to antibodies. It may contain other substances, etc.

In the present invention, the fixture for the antigen-antibody binding reaction includes a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, and a glass slide glass. It may be used, but is not limited thereto.

In addition, in the present invention, the label for the secondary antibody is preferably a conventional coloring agent that produces a color reaction, such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, and FITC (poly L Labels such as fluorescein and dye, such as -lysine-fluorecein isothiocyanate) and RITC (rhodamine-B-isothiocyanate), may be used, but are not limited thereto.

In addition, the chromogenic substrate for inducing color development in the present invention is preferably used according to the label that produces a color reaction, such as TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. At this time, it is more preferable that the chromogenic substrate is provided dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker for the secondary antibody conjugate to produce a chromogenic deposit, and the presence or absence of the marker proteins is detected by visually checking the degree of deposition of the chromogenic deposit.

In the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) consisting of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferable. do. After the antigen-antibody binding reaction, the washing solution reacts with the secondary antibody to the antigen-antibody conjugate, then adds an appropriate amount to the fixative and washes 3 to 6 times. The reaction stopping solution may preferably be a sulfuric acid solution (H ₂ SO ₄ ).

According to another embodiment of the present invention, comprising measuring the expression level of at least one protein of LILRB5 and CAMP or a gene encoding the protein in a biological sample isolated from a subject of interest, including cancer, inflammation, or benign It concerns a method of providing information to distinguish tumors.

In the present invention, the subject of interest refers to an subject whose cancer is uncertain, meaning an subject with a high probability of developing the cancer. Preferably, an entity in which a lesion has occurred may refer to an entity in which it is unclear whether the lesion or tumor is inflammatory, a benign tumor, or cancer.

In the present invention, the object of interest may be a mammal, including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It may be selected from the group consisting of, and preferably may be a human, but is not limited thereto.

In the present invention, the biological sample refers to any material, biological fluid, tissue, or cell obtained from or derived from an individual, such as whole blood, leukocytes, and peripheral blood mononuclear cells. ), white blood cell buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, respiration (breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid) , glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate. It may be one or more selected from the group consisting of joint aspirate, organ secretions, cells, cell extract, and cerebrospinal fluid, but is preferably liquid biopsy. For example, it may be cells or exosomes isolated from blood, serum or plasma.

In one example of the present invention, the method of providing information may include measuring the expression level of the LILRB5 protein or the gene encoding it in a biological sample isolated from the subject of interest.

In another example of the present invention, the method of providing information may include measuring the expression level of CAMP protein or the gene encoding it in a biological sample isolated from the subject of interest.

In another example of the present invention, the method for providing information includes measuring the expression level of the LILRB5 protein or the gene encoding it in a biological sample isolated from the object of interest, and measuring the expression level of the CAMP protein or the gene encoding it. It can include all measurement steps.

In the present invention, the agent for measuring the expression level of the protein includes at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. can do.

In the present invention, the expression level of the protein can be measured using protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, and SELDI-TOF (Sulface Enhanced Laser Desorption/SELDI-TOF). Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry ( It can be performed by liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blotting, or ELISA (enzyme linked immunosorbent assay).

Additionally, in the present invention, the expression level of the protein can be measured by a multiple reaction monitoring (MRM) method.

In the present invention, in the multiple reaction monitoring method, the internal standard material may be a synthetic peptide or Escherichia coli beta galactosidase in which a specific amino acid constituting the target peptide is isotopically substituted.

In the present invention, the agent for measuring the expression level of the gene encoding the protein may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the protein.

In the present invention, the expression level of the gene encoding the protein can be measured using reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), and real-time reverse transcription polymerase reaction (Real-time RT-PCR). , RNase protection assay (RPA), Northern blotting, or DNA chip.

In the information provision method of the present invention, descriptions of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), aptamers, etc., and descriptions of primers, probes, etc. overlap with what was previously described, and to avoid excessive complexity of the specification, they are provided below. The detailed description is omitted.

In the present invention, if the expression level of the LILRB5 protein or the gene encoding it measured in the biological sample of the object of interest is higher than that of the control group, the object of interest has developed cancer or is likely to develop cancer, that is, in the individual. It can be predicted that the lesion or tumor that has occurred has a high probability of being cancer.

In the present invention, if the expression level of CAMP protein or the gene encoding it measured in the biological sample of the object of interest is lower than that of the control group, the object of interest has developed cancer or is highly likely to develop cancer, that is, in the individual. It can be predicted that the lesion or tumor that has occurred has a high probability of being cancer.

In the present invention, if the expression level of the LILRB5 protein or the gene encoding it measured in the biological sample of the subject of interest is lower than that of the control group, the likelihood of developing cancer in the subject of interest is low, that is, the lesion occurring in the subject. Alternatively, it can be predicted that the tumor is likely to be pancreatitis or a benign pancreatic tumor rather than cancer.

In the present invention, if the expression level of CAMP protein or the gene encoding it measured in the biological sample of the object of interest is higher than that of the control group, the possibility of developing cancer in the object of interest is low, that is, the lesion occurring in the object. Alternatively, it can be predicted that the tumor is likely to be pancreatitis or a benign pancreatic tumor rather than cancer.

In the present invention, the control group is the average or median value of the expression level of the corresponding marker protein or the gene encoding it in biological samples derived from patients with pancreatic diseases, or the corresponding marker in biological samples derived from patients with pancreatic diseases other than pancreatic cancer among pancreatic diseases. It may be the average or median value of the expression level of the protein or the gene encoding it, or the average or median value of the expression level of the marker protein or the gene encoding it in biological samples derived from patients with pancreatitis or benign pancreatic tumor. It is not limited.

In the present invention, the cancer includes thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, and Hodgkin lymphoma. , blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, and endocrine cancer. , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma. , Preferably, it may be pancreatic cancer, but is not limited thereto as long as it is a type of cancer in which cancer progression, such as tumor differentiation and/or proliferation, is dependent on the cancer cells and/or cancer stem cells described in the present invention.

By measuring the expression level of the biomarker protein of the present invention or the gene encoding it, the occurrence or likelihood of developing cancer, especially pancreatic cancer, can be accurately predicted or diagnosed.

In addition, the present invention enables diagnosis by distinguishing between pancreatic cancer and other pancreatic diseases, which were previously difficult to distinguish through CT/MRI, etc.

Moreover, in the present invention, the onset of the disease can be predicted or diagnosed by obtaining a liquid biopsy from the individual at the time of the above diagnosis and measuring the expression level of the biomarker, so it is simple and quick in a non-invasive way compared to the conventional method. Cancer can be diagnosed very accurately.

Figure 1 shows the mRNA expression level of LILRB5 in samples derived from the pancreatic cancer patient group, normal people, high-risk group (pancreatic cyst, chronic pancreatitis, etc.), and benign pancreatic disease patient group (acute pancreatitis, pancreatic lipoma) in Experimental Example 2 by qPCR. It shows one result.
Figure 2 shows the results of an ROC curve for diagnosing pancreatic cancer based on the results of analyzing the mRNA expression level of LILRB5 in samples derived from the pancreatic cancer patient group and the pancreatic cyst patient group in Experimental Example 2.
Figure 3 shows the results of analyzing the mRNA expression level of CAMP in samples derived from the pancreatic cancer patient group, acute pancreatitis patient group, and pancreatic benign tumor patient group in Experimental Example 2 by qPCR.
Figure 4 shows the results showing the ROC curve for diagnosing pancreatic cancer based on the results of analyzing the mRNA expression level of CAMP in samples derived from the pancreatic cancer patient group and the acute pancreatitis patient group in Experimental Example 2.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example

[Experimental Example 1] Selection of pancreatic cancer diagnostic markers

If you have a chronic or acute pancreatic disease or a pancreatic tumor, the process of differentiating it from pancreatic cancer is very complicated, and the testing process is invasive. Therefore, in the following experiment, non-invasive marker discovery was performed for the purpose of differentiating malignant tumors (pancreatic cancer) from benign lesions in patient groups that can show pancreatic masses on CT/MRI (pancreatic cancer, chronic pancreatitis, pancreatic mass, etc.). The experimental group was a pancreatic cancer (PDAC) patient group (n=15), and the control group was a normal group (n=7), acute pancreatitis patient group (n=5), chronic pancreatitis patient group (n=7), and pancreatic cyst patient group (n=8). The following mRNA-seq analysis (transcriptome sequencing) was performed to identify genes whose expression levels were significantly increased or decreased in peripheral blood mononuclear cells (PBMC) obtained from a total of 42 individuals. was carried out. As an experimental method, total RNA was isolated from the sample, DNA contamination was removed using DNase, RNA was purified using the TruSeq Stranded mRNA LT Sample Prep kit, and then randomly fragmented for sequencing with short reads ( fragmentation). cDNA was synthesized through reverse transcription of the finely cleaved RNA fragment, and then different adapters were attached to both ends and connected. The length of the read was sequenced from both ends of the cDNA fragment. After going through a preprocessing process to reduce deviation from the raw reads obtained through this sequencing, they were mapped to the reference genome using the HISAT2 program that takes splice into account, and then aligned reads ( aligned reads) were created. Transfer assembly was performed using the StringTie program using reference-based alignment read information. The expression level obtained through transcript quantification of each sample is calculated as FPKM (Fragments Per Kilobase of transcript per Million mapped reads) or RPKM (Reads Per Million), which is a normalized value that takes into account rear count, transcript length, and depth of coverage. The expression profile was extracted with Kilobase of transcript per Million mapped reads). Genes or transcripts differentially expressed were selected through statistical hypothesis testing of the expression values of two or more groups under different conditions. When known genetic information was available, functional annotation and gene-set enrichment analysis of differentially expressed genes was performed based on GO and KEGG databases.

As a result of the above experiment, as shown in Tables 1 and 2 below, it was confirmed that the expression level of LILRB5 mRNA in the pancreatic cancer patient group was significantly increased compared to the healthy control group, acute pancreatitis patient group, chronic pancreatitis patient group, and pancreatic cyst patient group. I was able to.

Gene_ID Transcript_ID Gene_Symbol Description 10990 NM_001081442,NM_001081443,NM_001304457,NM_006840 LILRB5 leukocyte immunoglobulin like receptor B5

LILRB5 Test/Control.fc Test/Control.raw.pval Test/Control.bh.pval PDAC/Healthy Control 4.777884 1.15165E-05 0.001284 PDAC/Acute Pancreatitis 4.610999 0.000141 0.010693 PDAC/Chronic Pancreatitis 4.368164 3.47871E-05 0.003952 PDAC/IPMN 4.890152 3.09082E-06 0.000668

In addition, as shown in Tables 3 and 4 below, it was confirmed that the expression level of CAMP mRNA in the pancreatic cancer patient group was significantly reduced compared to the acute pancreatitis patient group or the pancreatic cyst patient group.

Gene_ID Transcript_ID Gene_Symbol Description 820 NM_004345 CAMP cathelicidin antimicrobial peptide

CAMP Test/Control.fc Test/Control.raw.pval Test/Control.bh.pval PDAC/Acute Pancreatitis -9.419506 0.00012093 0.00999674 PDAC/IPMN -6.819309 0.00010433 0.01059615

[Experimental Example 2]

Peripheral blood mononuclear cells (PBMC) were obtained from the blood of the control group (normal group, high-risk group (pancreatic cyst, chronic pancreatitis, etc.), benign pancreatic disease patient group (acute pancreatitis, pancreatic lipoma), and pancreatic cancer patient group. After isolating RNA (Qiagen, USA), cDNA was prepared using PrimeScript RT Master MIX (Perfect Real Time, Takara #RR036A), and QuantStudio 6 Flex Real-Time PCR System (in vitro number 15-758) from Applied biosystems. ) was used to perform PCR to measure the expression levels of LILRB5 and CAMP mRNA in each group, and the results are shown in Figures 1 to 4. The primer sequences used at this time are as shown in Table 5 below. However, in the following analysis Sensitivity indicates the probability that a person with the disease will obtain a positive result in diagnosis, specificity indicates the probability that a person without the disease will obtain a negative result in diagnosis, and AUC (Area) Under the ROC Curve) is the area under the ROC curve and is a scale for evaluating the performance of predictive factors. Sensitivity and specificity are measured at the LILRB5 marker according to the cutoff values shown in Table 6 below and are shown in Table 6. Sensitivity and specificity were measured for the CAMP marker according to the cutoff values shown in Table 7 below and are shown in Table 7.

gene form Sequence (5' -> 3') LILRB5 (Ref. NM_001081442) forward primer TCG AAC CCC AGT GAC CTC reverse primer GTC ATA GCC GAC ATC AGA GC probe CCA GGC GTG TCT AGG AAG CCC CAMP (Ref. NM_004345) forward primer ACC AGA GGA TTG TGA CTT CAA G reverse primer GCA GGG CAA ATC TCT TGT TAT C probe TCC CCA TAC ACC GCT TCA CCA G

cut-off responsiveness(%) 95% CI Specificity (%) 95% CI < 10.5 80 59.5% to 90.8% 95 82.3% to 99.0%

cut-off responsiveness(%) 95% CI Specificity (%) 95% CI > 6.02 88 65.7% to 97.9% 75 40.9% to 95.6%

As shown in Figures 1 and 2 and Table 6, the mRNA expression level of LILRB5 was measured in samples from the control group (normal group, high-risk group (pancreatic cyst, chronic pancreatitis, etc.), benign pancreatic disease patient group (acute pancreatitis, pancreatic lipoma), and pancreatic cancer patient group. As a result of confirmation and analysis by qPCR, it was found that LILRB5 showed a lower ΔCt value in the pancreatic cancer patient group compared to the control group, that is, the mRNA expression level was significantly increased.

As shown in Figures 3 and 4 and Table 7, CAMP showed a higher ΔCt value in the pancreatic cancer patient group compared to the control group of patients with acute pancreatitis and benign pancreatic tumor, that is, the mRNA expression level was significantly reduced. I was able to.

Through this, it was found that in the present invention, pancreatic cancer can be diagnosed and distinguished from other diseases using single markers of LILRB5 and CAMP or a combination thereof.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> ACURASYSBIO Co., Ltd. <120> Composition for diagnosing pancreatic cancer <130> DP2202204 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 590 <212> PRT <213> Homo sapiens <400> 1 Met Thr Leu Thr Leu Ser Val Leu Ile Cys Leu Gly Leu Ser Val Gly 1 5 10 15 Pro Arg Thr Cys Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Ala Ser Val Ile Ala Arg Gly Lys Pro Val Thr Leu Trp 35 40 45 Cys Gln Gly Pro Leu Glu Thr Glu Glu Tyr Arg Leu Asp Lys Glu Gly 50 55 60 Leu Pro Trp Ala Arg Lys Arg Gln Asn Pro Leu Glu Pro Gly Ala Lys 65 70 75 80 Ala Lys Phe His Ile Pro Ser Thr Val Tyr Asp Ser Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Glu Thr Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro 100 105 110 Leu Glu Leu Val Ala Thr Gly Phe Tyr Ala Glu Pro Thr Leu Leu Ala 115 120 125 Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Val Thr Leu Gln Cys 130 135 140 Asp Thr Leu Asp Gly Leu Leu Thr Phe Val Leu Val Glu Glu Glu Gln 145 150 155 160 Lys Leu Pro Arg Thr Leu Tyr Ser Gln Lys Leu Pro Lys Gly Pro Ser 165 170 175 Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser Cys Arg Trp Arg 180 185 190 Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn Pro Gln Val Trp Ser Asn 195 200 205 Pro Ser Asp Leu Leu Glu Ile Leu Val Pro Gly Val Ser Arg Lys Pro 210 215 220 Ser Leu Leu Ile Pro Gln Gly Ser Val Val Ala Arg Gly Gly Ser Leu 225 230 235 240 Thr Leu Gln Cys Arg Ser Asp Val Gly Tyr Asp Ile Phe Val Leu Tyr 245 250 255 Lys Glu Gly Glu His Asp Leu Val Gln Gly Ser Gly Gln Gln Pro Gln 260 265 270 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser 275 280 285 His Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser Pro Arg 290 295 300 Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly Leu Ile 305 310 315 320 Pro Asp Ile Pro Ala Leu Ser Val Gln Pro Gly Pro Lys Val Ala Ser 325 330 335 Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp His Gln Ile Asp Thr 340 345 350 Phe Phe Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Cys Leu Lys 355 360 365 Ser Lys Tyr Gln Ser Tyr Arg His Gln Ala Glu Phe Ser Met Ser Pro 370 375 380 Val Thr Ser Ala Gln Gly Gly Thr Tyr Arg Cys Tyr Ser Ala Ile Arg 385 390 395 400 Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser Tyr Pro Gln Glu Leu Val 405 410 415 Val Ser Gly Pro Ser Gly Asp Pro Ser Leu Ser Pro Thr Gly Ser Thr 420 425 430 Pro Thr Pro Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Leu Asp 435 440 445 Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Thr Gly Val Ser 450 455 460 Val Ala Phe Val Leu Leu Leu Phe Leu Leu Leu Phe Leu Leu Leu Arg 465 470 475 480 His Arg His Gln Ser Lys His Arg Thr Ser Ala His Phe Tyr Arg Pro 485 490 495 Ala Gly Ala Ala Gly Pro Glu Pro Lys Asp Gln Gly Leu Gln Lys Arg 500 505 510 Ala Ser Pro Val Ala Asp Ile Gln Glu Glu Ile Leu Asn Ala Ala Val 515 520 525 Lys Asp Thr Gln Pro Lys Asp Gly Val Glu Met Asp Ala Arg Ala Ala 530 535 540 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 545 550 555 560 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Pro Ser Gln Glu Arg Glu 565 570 575 Pro Pro Ala Glu Pro Ser Ile Tyr Ala Pro Leu Ala Ile His 580 585 590 <210> 2 <211> 170 <212> PRT <213> Homo sapiens <400> 2 Met Lys Thr Gln Arg Asp Gly His Ser Leu Gly Arg Trp Ser Leu Val 1 5 10 15 Leu Leu Leu Leu Gly Leu Val Met Pro Leu Ala Ile Ile Ala Gln Val 20 25 30 Leu Ser Tyr Lys Glu Ala Val Leu Arg Ala Ile Asp Gly Ile Asn Gln 35 40 45 Arg Ser Ser Asp Ala Asn Leu Tyr Arg Leu Leu Asp Leu Asp Pro Arg 50 55 60 Pro Thr Met Asp Gly Asp Pro Asp Thr Pro Lys Pro Val Ser Phe Thr 65 70 75 80 Val Lys Glu Thr Val Cys Pro Arg Thr Thr Gln Gln Ser Pro Glu Asp 85 90 95 Cys Asp Phe Lys Lys Asp Gly Leu Val Lys Arg Cys Met Gly Thr Val 100 105 110 Thr Leu Asn Gln Ala Arg Gly Ser Phe Asp Ile Ser Cys Asp Lys Asp 115 120 125 Asn Lys Arg Phe Ala Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu 130 135 140 Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe 145 150 155 160 Leu Arg Asn Leu Val Pro Arg Thr Glu Ser 165 170

Claims (20)

A diagnostic biomarker composition for pancreatic cancer comprising the LILRB5 (leukocyte immunoglobulin like receptor B5) protein or the gene encoding it. According to paragraph 1,
The biomarker composition further includes a CAMP (cathelicidin antimicrobial peptide) protein or a gene encoding it. A diagnostic composition for pancreatic cancer comprising, as an active ingredient, an agent that measures the expression level of the LILRB5 (leukocyte immunoglobulin like receptor B5) protein or the gene encoding it. According to paragraph 3,
The diagnostic composition further includes an agent for measuring the expression level of CAMP (cathelicidin antimicrobial peptide) protein or the gene encoding it. According to paragraph 3,
The agent for measuring the expression level of the protein is a diagnostic agent comprising at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. Composition. According to paragraph 3,
A diagnostic composition wherein the agent for measuring the expression level of the gene includes at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene. A diagnostic kit for pancreatic cancer comprising the diagnostic composition of any one of claims 3 to 6. A method of providing information for the diagnosis of pancreatic cancer, comprising measuring the expression level of LILRB5 (leukocyte immunoglobulin like receptor B5) protein or the gene encoding it in a biological sample isolated from a subject of interest. According to clause 8,
The method of providing information further includes the step of measuring the expression level of CAMP (cathelicidin antimicrobial peptide) protein or the gene encoding it in a biological sample isolated from the object of interest. According to clause 8,
The agent for measuring the expression level of the protein includes at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acids), and aptamers that specifically bind to the protein. How to provide. According to clause 8,
The expression level of the protein can be measured using protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and Surface Enhanced Laser Desorption/Ionization Time of SELDI-TOF (SELDI-TOF). Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry. Performed by Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), multiple reaction monitoring (MRM), Western blotting, or ELISA (enzyme linked immunosorbent assay), How to provide information. According to clause 8,
The agent for measuring the expression level of the gene includes at least one selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene. According to clause 8,
The expression level of the gene can be measured using reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), and RNase protection assay (RPA). RNase protection assay), an informative method performed by Northern blotting or DNA chip. According to clause 8,
A method of providing information that predicts that the target individual has developed or is likely to develop pancreatic cancer when the expression level of the LILRB5 protein or the gene encoding it measured in the biological sample of the target individual is higher than that of the control group. A biomarker composition for distinguishing pancreatic cancer from pancreatic inflammation or pancreatic benign tumor, comprising at least one protein of leukocyte immunoglobulin like receptor B5 (LILRB5) and cathelicidin antimicrobial peptide (CAMP) or a gene encoding the same. Distinguishing pancreatic cancer from pancreatic inflammation or benign pancreatic tumor, which contains as an active ingredient an agent that measures the expression level of at least one protein of LILRB5 (leukocyte immunoglobulin like receptor B5) and CAMP (cathelicidin antimicrobial peptide) or the gene encoding it. Diagnostic composition for. A diagnostic kit for distinguishing pancreatic cancer from pancreatic inflammation or benign pancreatic tumor, comprising the diagnostic composition of claim 16. Pancreatic cancer is diagnosed by pancreatic inflammation or How to provide information to differentiate pancreatic tumors from benign ones. According to clause 18,
A method of providing information that predicts that the target individual has developed or is likely to develop pancreatic cancer when the expression level of the LILRB5 protein or the gene encoding it measured in the biological sample of the target individual is higher than that of the control group. According to clause 18,
A method of providing information that predicts that the target individual has a high possibility of developing pancreatic cancer when the expression level of the CAMP protein or the gene encoding it measured in the biological sample of the target individual is lower than that of the control group.