KR20240081429A - A novel biomarker for prognosis in pancreatic cancer - Google Patents

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death worldwide, but effective prognostic indicators are lacking. Methionyl-tRNA synthetase 1 (MARS1), an enzyme important in translation initiation that transfers Met to initiator tRNA, is associated with cancer development and progression. MARS1 expression was significantly increased in PDAC compared to normal pancreatic duct tissue. Additionally, high expression of MARS1 was associated with poor prognosis in PDAC patients. This suggests that MARS1 is involved in pancreatic carcinogenesis and has the potential as a new prognostic indicator for PDAC.

Description

A novel biomarker for prognosis in pancreatic cancer}

The present invention relates to a new biomarker for predicting the prognosis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among other major malignant tumors, with a 5-year survival rate of 6% after diagnosis. Because early diagnosis is difficult, only 10-20% of pancreatic cancer can be surgically resected for treatment at the time of diagnosis. As prognostic factors for unresectable pancreatic cancer, tumor factors such as CA 19-9 level and tumor stage and host factors such as performance status, serum C-reactive protein level, and neutrophil-lymphocyte ratio are reported to be related to patient survival. there is. However, predicting prognosis is not always easy. Therefore, screening new tumor biomarkers is very important for prevention, diagnosis, prognosis, and targeted treatment of PDAC. In particular, the present invention focuses on recent developments in the identification of new protein biomarkers useful for the prognosis of PDAC patients.

Human cytoplasmic methionyl-tRNA synthetase 1 (MARS1) consists of 900 amino acids and is one of the components of the multi-tRNA synthetase complex. It has been reported that the aminoacylation activity of MARS1, which is required for translation initiation, is increased in human colon cancer and that stable overexpression of the MARS1 substrate tRNAiMet can cause oncogenic transformation. Coincidentally, the 3' untranslated region (UTR) of MARS1 contains 56 base pairs of complementary sequence to the complementary sequence of the 3' UTR of the C/EBP homologous protein, which has been critically linked to the initiation of certain tumors. Additionally, overexpression of MARS1 has also been reported in several other types of cancer, such as malignant fibrous histiocytoma, sarcoma, malignant glioma, and glioblastoma. MARS1 can also control tumorigenesis indirectly through interaction with AIMP3. Any mutation in MARS1 that can affect its interaction with AIMP3 can modulate the tumor suppressor activity of AIMP3 in the nucleus. Additionally, MARS1 inhibition reduced cell transformation and tumorigenic ability of p16INK4a-negative breast cancer cell lines. This evidence suggests that MARS1 plays an important role in cancer development and progression.

To date, no study has revealed an association between MARS1 and the prognosis of PDAC. Therefore, the present invention was completed by evaluating MARS1 expression patterns and its role in PDAC through immunohistochemical analysis involving 111 clinical tissue samples.

One object of the present invention is methionyl-tRNA synthetase 1 (MARS1) or a fragment thereof; Alternatively, the present invention provides a composition for diagnosing cancer, including an agent for measuring the expression level of a gene encoding the same.

Another object of the present invention is to provide a diagnostic kit comprising the diagnostic composition.

Another object of the present invention is to detect methionyl-tRNA synthetase 1 (MARS1) in a biological sample isolated from a subject of interest; or measuring the expression level of the gene encoding the protein; To provide a method of providing information for diagnosis of cancer, including.

Another object of the present invention is to detect methionyl-tRNA synthetase 1 (MARS1) in a biological sample isolated from a subject of interest; or measuring the expression level of the gene encoding the protein; To provide a method of providing information for predicting the prognosis of cancer, including.

Another object of the present invention is to detect methionyl-tRNA synthetase 1 (MARS1) in a biological sample obtained from a subject of interest; Or a measuring unit that measures the expression level of the gene encoding the protein; and a detection unit that outputs the presence or absence of cancer based on the expression level of the gene or protein encoded by the gene measured in the measurement unit. To provide a cancer diagnostic device including.

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

According to one embodiment of the present invention, methionyl-tRNA synthetase 1 (MARS1) or a fragment thereof; Alternatively, a composition for diagnosing cancer is provided, including an agent for measuring the expression level of a gene encoding the same.

In the present invention, “methionyl-tRNA synthetase 1 (MARS1)” belongs to a family of 20 enzymes essential for protein biosynthesis. Additionally, MARS1 covalently links its cognate tRNA and methionine. Increased MARS1 catalytic activity has been reported in human colon cancer, and stable overexpression of the MARS1 substrate tRNAiMet can lead to oncogenic transformation. MARS1 also plays an important role in carcinogenesis and progression of several human malignancies, and MARS1 specifically stabilizes CDK4 by forming a complex with heat shock protein 90 and the cell division cycle, thereby reducing CDK4's proteasome dependence. Prevent decomposition. Additionally, MARS1 inhibition reduces cellular CDK4 levels, resulting in cell cycle arrest in G0/G1 phase. MARS1 competes with the tumor suppressor p16INK4a for binding to the CDK4 N-terminal domain, further enhancing its role in p16INK4a-negative cancer. Additionally, MARS1 inhibition reduces cell transformation and tumorigenic ability of p16INK4a-negative breast cancer cell lines. This evidence indicates that MARS1 plays an important role in cancer development and progression. However, its expression pattern and biological behavior in pancreatic cancer are completely unclear.

In addition, the expression level of methionyl-tRNA synthetase 1 (MARS1) may include strong expression (Strong MARS1 expression) and weak expression (Week MARS1 expression), and the two expressions are immune Distinction is possible through histochemical analysis. In the present invention, the cases of strong expression and weak expression were divided into two cases and the recurrence-free survival period and overall survival period were analyzed as prognosis of cancer.

In the present invention, the composition is intended to be applied to a biological sample isolated from an object of interest, and the biological sample includes, but is not limited to, solid tissue sample, tissue culture, liquid tissue sample, cell or cell fragment. Additionally, non-limiting examples of biological samples include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, Sputum, tears, mucus, nasal washes, nasal aspirate, 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, organ secretions, cells, cell extracts It may include one or more selected from the group consisting of cell extract and cerebrospinal fluid, but is not limited thereto.

In the present invention, the “object of interest” refers to an individual that has developed cancer or is likely to develop cancer, and may be a mammal, including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, and dogs. , may be selected from the group consisting of 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 from a biological sample isolated from an individual of interest in the present invention, the occurrence or likelihood of developing a disease can be confirmed very quickly and easily.

As used herein, “cancer” refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals. In the present invention, the cancer includes pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon 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, “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 to 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 survival rate is not improved 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. More than 90% of cancers that occur in the pancreas occur in pancreatic duct cells, and pancreatic cancer usually refers to pancreatic duct cancer or pancreatic adenocarcinoma. Therefore, preferably, pancreatic cancer in the present invention may mean pancreatic ductal (adeno)carcinoma (PDAC).

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 from benign pancreatic tumors (such as lipoma or intrapancreatic papillary mucinous neoplasm (IPMN)), or it may be to distinguish between carcinomas, 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 excessive drinking (alcohol abuse), gallstones, etc., causing the enzymes to cause autolysis of the pancreas, but it also occurs 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 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, the 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. Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function, and include 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.

Information on methionyl-tRNA synthetase 1 (MARS1) according to the present invention and the genes encoding them are known, so those skilled in the art can use this to specifically bind to the genes encoding the proteins. It will be possible to easily design primers, probes, or antisense nucleotides.

In the present invention, methionyl-tRNA synthetase 1 (MARS1) or the gene encoding it can be measured from a biological sample isolated from the subject of interest.

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 (including 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 necessary 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. 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 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 buffers (pH and magnesium concentrations 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 cancer 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 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 capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g. conjugated with antibodies) and their substrates or those capable of binding 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 chromogenic 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, it relates to a method of providing information for diagnosing cancer.

The method of the present invention includes methionyl-tRNA synthetase 1 (MARS1); Alternatively, it may include measuring the expression level of the gene encoding the protein.

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 not limited thereto.

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 method of the present invention, descriptions of 29 biomarkers, cancer, antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), aptamers, etc., and descriptions of primers, probes, etc. are duplicated as previously described and are included in the specification. To avoid excessive complexity, detailed description is omitted below.

In one embodiment of the present invention, the method includes methionyl-tRNA synthetase 1 (MARS1) measured on a biological sample of the subject of interest; Alternatively, if the expression level of the gene encoding this is higher than that of the control group, it can be predicted that the target individual has developed cancer or is 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 a healthy normal control group, or the average of the expression level of the corresponding marker protein or the gene encoding the corresponding marker protein in biological samples derived from pancreatic disease patients. It may be an intermediate value, or it may be an 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 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 subject of interest. Diseases suspected to be cancer are distinguished from other diseases, especially pancreatic diseases (e.g., pancreatitis (both acute or chronic), benign pancreatic tumors (lipoma or intraductal papillary mucinous neoplasm (IPMN), etc.)) and are classified as cancer. It includes predicting, and 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 carcinomas.

In the present invention, the cancer includes pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon 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.

According to another embodiment of the present invention, it relates to a composition for predicting the prognosis of cancer.

In the present invention, the composition for predicting prognosis includes methionyl-tRNA synthetase 1 (MARS1); Alternatively, it may include an agent that measures the expression level of the gene encoding it.

“Prognosis” in the present invention refers to the act of predicting in advance the course of a disease and the outcome of death or survival. The prognosis or prognostic diagnosis may be interpreted to mean that the course of a disease may vary depending on the patient's physiological or environmental condition, and to mean all actions that predict the course of the disease before and after treatment by comprehensively considering the patient's condition. You can. For the purposes of the present invention, the prognosis can be interpreted as the act of determining whether the survival rate after the onset of cancer is predicted to be low or the response to treatment is poor.

In addition, in the present invention, "diagnosis" means confirming the existence or characteristics of a pathological condition, and in the present invention, "prognosis" refers to the act of predicting the course of a disease and the outcome of death or survival in advance. It means that it is clearly distinguished. More specifically, cancer diagnosis means confirming whether a patient is currently suffering from a cancer disease, and cancer prognosis refers to predicting the course of the disease and the outcome of death or survival of a patient suffering from cancer disease in advance. It refers to an action, and more specifically, it refers to predicting or anticipating the recurrence-free survival period and overall survival period.

In the composition for predicting prognosis of the present invention, descriptions of methionyl-tRNA synthetase 1 (MARS1), cancer, agents for measuring the expression level of proteins or genes, etc. are duplicated as previously described in the specification. To avoid excessive complexity, detailed description is omitted below.

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

In the kit for predicting prognosis of the present invention, descriptions of prognosis, cancer, type of kit, etc. overlap with what was previously described, and to avoid excessive complexity of the specification, detailed descriptions thereof are omitted below.

According to another embodiment of the present invention, it relates to a method of providing information for predicting the prognosis of cancer.

The method of the present invention includes methionyl-tRNA synthetase 1 (MARS1); Alternatively, it may include measuring the expression level of the gene encoding the protein.

In one embodiment of the present invention, the method includes at least one protein selected from the group consisting of CNOT1, KIF11, and SLC44A1 measured on a biological sample of the subject of interest; Alternatively, if the expression level of the gene encoding this is higher than the control group, the prognosis of cancer can be predicted to be poor.

In another embodiment of the present invention, the method of the present invention includes methionyl-tRNA synthetase 1 (MARS1) measured on a biological sample of the subject of interest; Alternatively, if the expression level of the gene encoding this is lower than the control group, the prognosis of cancer can be predicted to be poor.

In the method of the present invention, descriptions of methionyl-tRNA synthetase 1 (MARS1), cancer, prognosis, subject of interest, biological sample, control group, etc. are overlapped with those described previously to avoid excessive complexity of the specification. To avoid this, the detailed description is omitted below.

According to another embodiment of the present invention, it relates to a cancer diagnostic device.

The diagnostic device of the present invention (a) methionyl-tRNA synthetase 1 (MARS1) on a biological sample obtained from a subject of interest; Or a measuring unit that measures the expression level of the gene encoding the protein; and (b) a detection unit that outputs the presence or absence of cancer based on the expression level of the gene or protein encoded by the gene measured in the measurement unit; It relates to a diagnostic device for cancer, including a.

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

In the method of the present invention, descriptions of methionyl-tRNA synthetase 1 (MARS1), cancer, prognosis, subject of interest, biological sample, control group, etc. are overlapped with those described previously to avoid excessive complexity of the specification. To avoid this, the detailed description is omitted below.

According to one embodiment of the present invention, methionyl-tRNA synthetase 1 (MARS1) or a fragment thereof; Alternatively, a composition for diagnosing cancer is provided, including an agent for measuring the expression level of a gene encoding the same.

According to another embodiment of the present invention, the agent for measuring the expression level of the protein is selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid), and aptamer that specifically binds to the protein. A diagnostic composition comprising one or more selected species is provided.

According to another embodiment of the present invention, the agent for measuring the expression level of the gene provides a diagnostic composition comprising at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene. .

According to another embodiment of the present invention, the composition provides a diagnostic composition for application to a biological sample isolated from a subject of interest.

According to another embodiment of the present invention, the biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum. ), sputum, tears, mucus, nasal washes, nasal aspirate, 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, organ secretions, cells, A diagnostic composition is provided, comprising at least one selected from the group consisting of cell extract and cerebrospinal fluid.

According to another embodiment of the present invention, the cancer is pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, and bile duct cancer. Hodgkin's lymphoma, Hodgkin's 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, and 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, a diagnostic composition is provided.

According to another embodiment of the present invention, a composition for diagnosis is provided, wherein the cancer is pancreatic cancer.

According to another embodiment of the present invention, a diagnostic composition is provided, wherein the diagnosis predicts prognosis.

According to another embodiment of the present invention, a diagnostic kit is provided, comprising the diagnostic composition of any one of claims 1 to 8.

According to another embodiment of the present invention, the kit provides a diagnostic kit, which is 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.

According to another embodiment of the present invention, in a biological sample isolated from a subject of interest, methionyl-tRNA synthetase 1 (MARS1); or measuring the expression level of the gene encoding the protein; Provides a method of providing information for diagnosis of cancer, including.

According to another embodiment of the present invention, the protein of methionyl-tRNA synthetase 1 (MARS1) measured in the biological sample; Or, encoding this includes determining that cancer has developed or is highly likely to develop if the expression level of the gene is higher than that of the control group; Provides a method of providing information for diagnosis of cancer, including.

According to another embodiment of the present invention, the biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum. ), sputum, tears, mucus, nasal washes, nasal aspirate, 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, organ secretions, cells, A method of at least one selected from the group consisting of cell extract and cerebrospinal fluid is provided.

According to another embodiment of the present invention, 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 SELDI-TOF. (Sulface Enhanced Laser Desorption/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 phase Chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), Western blotting, ELISA (enzyme linked immunosorbent assay), or multiple reaction monitoring A method based on the monitoring (MRM) method is provided.

According to another embodiment of the present invention, the expression level of the gene can be measured using reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), or real-time reverse transcription polymerase reaction (Real-time RT- Methods are provided, such as PCR), RNase protection assay (RPA), Northern blotting or DNA chip.

According to another embodiment of the present invention, the cancer is pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, and bile duct cancer. Hodgkin's lymphoma, Hodgkin's 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, and 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, provides a method of providing information.

According to another embodiment of the present invention, a method of providing information is provided, wherein the cancer is pancreatic cancer.

According to another embodiment of the present invention, a method of providing information is provided, wherein the diagnosis predicts prognosis.

According to another embodiment of the present invention, (a) methionyl-tRNA synthetase 1 (MARS1) on a biological sample obtained from a subject of interest; Or a measuring unit that measures the expression level of the gene encoding the protein; and (b) a detection unit that outputs the presence or absence of cancer based on the expression level of the gene or protein encoded by the gene measured in the measurement unit; Provides a cancer diagnostic device including.

According to another embodiment of the present invention, the cancer is stomach cancer, thyroid cancer, parathyroid 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, and bile duct cancer. Hodgkin's lymphoma, Hodgkin's 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, and 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 A diagnostic device is provided, which is at least one selected from the group consisting of pituitary adenoma.

According to another embodiment of the present invention, a diagnostic device is provided wherein the cancer is pancreatic cancer.

According to another embodiment of the present invention, a diagnostic device is provided wherein the diagnosis predicts prognosis.

In the present invention, we found that overexpression of human cytoplasmic methionyl-tRNA synthetase 1 (MARS1) is closely related to tumor size and lymph node metastasis, and these results show that MARS1 is associated with pancreatic ductal adenocarcinoma. It is suggested that it plays an important role in the proliferation and metastasis of PDAC. As a result, we further investigated the relationship between MARS1 expression and prognosis in PDAC patients, and the Kaplan-Meier survival curve revealed that patients with high MARS1 expression had shorter overall survival and recurrence-free survival than patients with low expression.

Ultimately, using MARS1 as a biomarker for predicting pancreatic cancer prognosis has the effect of accurately diagnosing pancreatic cancer, which is difficult to diagnose and predicting prognosis, and accurately predicting the prognosis of older pancreatic cancer patients.

Figure 1 shows representative microscopic features of IHC expression of human cytoplasmic methionyl-tRNA synthetase 1 (MARS1) in pancreatic ductal adenocarcinoma (PDAC). In (A) and (E), normal acinar cells (empty arrow) showed strong MARS1 expression, and normal pancreatic duct epithelium (black arrow, internal control) showed moderate to weak MARS1 expression. In (B), (F) and (C), (G), low MARS1 expression was defined as weaker than normal acinar cells, whereas in (D) and (H), high MARS1 expression was defined as equal or stronger than normal acinar cells. was defined as ((AD), H&E staining; (EH), MARS1, x400).
Figure 2 shows the microscopic results of immunohistochemistry of MARS1, which shows low expression in pancreatic ductal adenocarcinoma. (A) shows normal pancreatic tissue (above the dotted line) and pancreatic adenocarcinoma (below the dotted line) as a result of H&E staining (X20) of a representative surgical specimen. (B) shows MARS1 immunohistochemical staining of the same tissue, showing low expression of MARS1 in phloem adenocarcinoma (MARS1, x20). (C) and (D) normal acinar cells show strong MARS1 expression as an internal control. Specifically, (C) is the result of H&E staining (X400) of normal acinar cells. (D) is an internal control, showing that MARS1 is highly expressed in normal acinar cells. (E) to (H) Moderately differentiated pancreatic adenocarcinomas showed lower MARS1 expression than MARS1 expression in normal progenitor cells ((E, G), H&E staining; (F, H), MARS1, x400). It shows weakness. Specifically, (E) shows the results of H&E staining (X400) on pancreatic adenocarcinoma tissue. (F) is a result showing that MARS1 expression is weaker than MARS1 expression in normal acinar cells. (G) is the result of observing other lesions in pancreatic adenocarcinoma tissue through H&E staining (X400). (H) is a result showing that strong MARS1 expression is also weaker than MARS1 expression in normal acinar cells.
Figure 3 shows the results of immunohistochemistry (IHC) microscopic observation of MARS1, which shows high expression in pancreatic ductal adenocarcinoma (PDAC). (A) shows normal pancreatic tissue (above the dotted line) and pancreatic adenocarcinoma tissue (below the dotted line) as a result of H&E staining (X20). (B) shows MARS1 immunohistochemical staining of the same tissue, showing that MARS1 expression is high in phloem adenocarcinoma. (C) and (D) Normal acinar cells show strong MARS1 expression as an internal control. Specifically, (C) is the result of H&E staining (X400) of normal acinar cells. (D) is an internal control, showing that MARS1 is highly expressed in normal acinar cells. (E) to (H) show that moderately poorly differentiated pancreatic adenocarcinoma showed high MARS1 expression that was equal to or stronger than MARS1 expression in normal acinar cells. Specifically, (E) shows the results of H&E staining (X400) on pancreatic adenocarcinoma tissue. (F) is a result showing that strong MARS1 expression is equal to or stronger than MARS1 expression in normal acinar cells. (G) is the result of observing other lesions in pancreatic adenocarcinoma tissue through H&E staining (X400). (H) is a result showing that strong MARS1 expression is the same or stronger than MARS1 expression in normal acinar cells.
Figure 4 shows a Kaplan-Meier graph for overall survival (OS) and disease-free survival (DFS) period according to MARS1 expression. Specifically, Figure 4A shows that the median OS duration in the high MARS1 expression group was shorter than that in the low MARS1 expression group (15.2 months vs. 17.2 months, log-rank test p = 0.044). Figure 4b also shows that although the median DFS between the two groups is not statistically different, the DFS trend in the group with high MARS1 expression is also shorter than that in the group with low MARS1 expression (8.9 months vs. 11.2 months, log-rank test p = 0.067).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

[Preparation example]

[Preparation Example 1] Experimental design and sampling

In the preparation example of the present invention, tissue was collected from a patient who underwent surgical treatment for pancreatic adenocarcinoma. Because the registered patients were eligible for surgery at the time of diagnosis, neo-adjuvant chemotherapy was not administered. After surgery, adjuvant chemotherapy was administered depending on the patient's condition. IHC analysis was performed on tissue lysates prepared using surgical specimens and paraffin-embedded tissue blocks. Human cytoplasmic methionyl-tRNA synthetase 1 (MARS1) expression levels were assessed in paired lysates of normal-appearing adjacent pancreas and cancer-enriched tissue. The Ethics Committee of Gangnam Severance Hospital approved the study protocol and written consent was obtained from the enrolled patients (IRB number 3-2021-0444).

[Preparation Example 2] Immunohistochemistry (IHC) staining

Tumor tissues were fixed in 10% buffered formalin and embedded in paraffin. IHC staining was applied to 4 μm thick sections of each paraffin block. Immunohistochemical staining analysis was performed using a primary antibody against human MARS1 (1:300; 0.2 mg/mL; BicbioInc., Suwon, South Korea) and an automated IHC stainer (BenchMark XT; Ventana Medical Systems, Tucson, AZ, USA). was performed using .

[Example]

[Example 1] Interpretation of IHC Staining

IHC staining was evaluated independently by two inventors. MARS1 is strongly expressed in progenitor cells (internal control cells) of normal pancreatic tissue and weakly expressed in benign pancreatic ducts. This is because MARS1 expression is high in acinar tissue, which is present in both exocrine and endocrine organs due to high levels of protein production. MARS1 expression is low in mucus-producing epithelium, such as the pancreatic duct, where protein production is low. MARS1 expression levels in acinar cells and benign pancreatic ducts were used as reference and high MARS1 expression was defined as equal to or higher than that in normal acinar cells, as shown in Figure 1 (Figure One).

Additionally, low MARS1 expression was defined as weaker than normal acinar cells and stronger than benign pancreatic ductal epithelium. To evaluate MARS1 expression, pancreatic cancer tissue was compared with normal pancreatic tissue, such as acinar cells and pancreatic ductal epithelium. Figures 2 and 3 visualized IHC and H&E staining of the same tissue, showing tissues with low and high MARS1 expression, respectively.

IHC results were interpreted under conditions without any information about clinical data. All enrolled patients had available follow-up and clinicopathological information, including age, tumor grade, tumor size, histological type, and tumor, node, metastasis (TNM) stage.

Conclusion - MARS1, a new biomarker for predicting pancreatic cancer prognosis

1. Patients' Characteristics

Surgical samples were collected from 137 patients with pancreatic adenocarcinoma who underwent surgery between 2012 and 2022. Cancer and adjacent normal pancreatic tissue were assessed for MARS1 expression (see Table 1).

Baseline characteristics of patients with PDAC and MARS1 expression characteristic Low MARS1 expression (n=55) High MRAS1 expression (n=82) P-value Age, y (mean±SD) 65.8 ± 10.0 65.9 ± 10.3 0.543 Gender, n (M:F) 24:31 44:38 0.250 Surgery, n (%) 0.311 Pylorus-preserving pancreaticoduodenectomy (PPPD) 31 (56.4) 54 (66.9) Distal pancreatectomy 21 (38.2) 22 (26.9) Total pancreatectomy 3 (5.5) 6 (7.3) Resection margin, n (%) R0 resection 39 (70.1) 50 (61.0) 0.112 R1 resection 16 (29.9) 32 (39.0) 0.191 Tumor size, cm (mean±SD) 3.2 ± 1.2 3.1 ± 1.3 0.552 TNM stage, n (%) 0.757 IA 2 (3.6) 3 (3.7) IB 5 (9.1) 7 (8.5) IIA 7 (12.7) 9 (11.0) IIB 22 (40.1) 42 (51.2) III 19 (34.5) 21 (25.6) Degree of differentiation, n (%) 0.078 award 3 (5.5) 5 (6.1) middle 50 (90.9) 63 (76.8) under 2 (3.6) 14 (17.1) Lymph node metastases, n (%) 41 (74.5) 62 (75.6) 0.889 Lymphovascular invasion, n (%) 29 (52.7) 40 (48.9) 0.653 Perineural invasion, n (%) 45 (81.8) 62 (75.6) 0.393 CA 19-9 at adm, IU/L (mean±SD) 639.4 ± 2161.8 368.5 ± 855.2 0.112 CEA at adm, IU/L (mean±SD) 7.3 ± 19.4 4.3 ± 5.6 0.262 Adjuvant chemotherapy, n (%) 45 (81.8) 48 (58.5) 0.017

As a result, 55 and 82 of the patients had low and high MARS1 expression, respectively. The average age of the group with low MARS1 expression was 65.8 years, and the average age of the group with high MARS1 expression was 65.9 years. The male-to-female ratio in both groups was similar. There was no significant difference in surgical method (pylorus-preserving pancreaticoduodenectomy) between the two groups. The R0 resection rate was 70.1% in the group with low MARS1 expression and 61% in the group with high MARS1 expression (p=0.112). The average tumor size and TNM stage did not differ between the two groups. Compared to the group with low MARS1 expression, the group with high MARS1 expression tended to have a lower rate of moderate differentiation and a higher rate of undifferentiation, but this trend was not statistically significant. No differences were found between the two groups in terms of lymph node metastasis, lymphovascular invasion, and perineural invasion of surgical tissue. Additionally, there was no difference in the average CA 19-9 and CEA levels before surgery between the two groups. The group with low MARS1 expression had a high rate of receiving adjuvant chemotherapy after surgery.

2. Overall and Disease-Free Survival

Then, DFS and OS of 137 PDAC patients were analyzed using the Kaplan-Meier method. DFS was defined as the time from surgery to first recurrence, and OS was defined as the time from surgery to death. As shown in Figures 4A and 4B, PDAC patients with high MARS1 expression had shorter DFS and OS than patients with low MARS1 expression.

Specifically, the median OS in the group with high MARS1 expression was 15.2 months, whereas it was 17.2 months in the group with low expression (log-rank test p = 0.044). Median DFS was not significantly different between the two groups, but DFS duration tended to be shorter in the high and low MARS1 expression groups (8.9 months vs. 11.2 months, log-rank test p = 0.067). This means that MARS1 expression level affects OS and DFS of PDAC patients. Because 2-year survival rates are generally assessed in a clinically meaningful manner, z-tests were used to compare 2-year survival rates. As shown in Table 2 below, the group with low MARS1 expression had a significantly higher 2-year OS rate (p=0.0345), but there was no significant difference in DFS rate between the two groups (p=0.0624).

2-year overall survival and disease-free survival according to MARS1 expression Low MARS1 expression High MRAS1 expression P-value 2-year survival rate (se) 2-year survival rate (95% CI) full survival 0.833 (0.067) 0.662 (0.068) 0.0345 disease-free survival 0.779 (0.085) 0.608 (0.072) 0.0624

3. Risk factors for overall and relapse-free survival

In univariate analyses, tumor size >3 cm (hazard ratio [HR] = 1.546, p = 0.015), TNM stage (HR = 2.052, p = 0.009), and lymph node metastasis (HR = 2.310, p < 0.001). ), lymphovascular invasion (HR = 1.438, p = 0.041), perineural invasion (HR = 2.105, p = 0.001), and high MARS1 expression (HR = 5.663, p = 0.001, Table 3) were associated with higher OS rates in PDAC patients. It was significantly related to being unwell. In the multivariate analysis, high MARS1 expression (HR = 2.761, p = 0.022) and lymph node metastasis (HR = 8.019, p = 0.048) were independent prognostic indicators for poor OS.

Additionally, in univariate analysis, tumor size >3 cm (HR = 1.770, p = 0.009) and high MARS1 expression (HR = 4.857, p = 0.005) were significantly associated with DFS in PDAC patients (see Table 2). High MARS expression (HR = 2.774, p = 0.023) and tumor size >3 cm (HR = 1.776, p = 0.010) were independent prognostic indicators for poor DFS in multivariate analysis. Multivariate analysis identified MARS1 expression as a significant and independent prognostic factor for OS and DFS. These results show that MARS1 expression is a prognostic factor for pancreatic cancer patients.

Risk factors for overall survival and disease-free survival elements full survival disease-free survival Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR (95% CI) p-Value HR (95% CI) p-Value HR (95% CI) p-Value HR (95% CI) p-Value castle
(male vs female) 0.696
(0.492-0.985) 0.565 0.933
(0.606-1.437) 0.754 Age (y) (≤50 vs. >50) 0.789
(0.435-1.433) 0.789 0.774
(0.361-2.374)
0.872 Cancer size (cm) (≤3 vs. >3
cm)) 1.546
(1.088-2.196) 0.015 2.042
(0.945-4.410) 0.069 1.770
(1.150-2.722) 0.009 1.776
(1.147-2.717) 0.010 Differentiation (WD, MD vs. PD) 0.924
(0.328-2.601) 0.251 1.422
(0.771-2.622) 0.051 TNM stage (I vs. more II) 2.052
(1.194-3.524) 0.009 2.328
(0.993-5.457) 0.052 1.393
(0.558-3.427) 0.484 Lymph node metastasis (positive/negative) 2.310
(1.519-3.512) <.001 8.019
(1.022-62.951) 0.048 1.220
(0.715-2.084) 0.466 Lymphovascular invasion (positive/negative) 1.438
(1.015-2.036) 0.041 1.870
(0.821-4.258) 0.136 1.534
(0.992-2.374) 0.055 Perineural invasion (positive/negative) 2.105
(1.354-3.271) 0.001 1.605
(0.639-4.031) 0.314 1.440
(0.792-2.618) 0.231 R0 resection margin (positive/negative) 1.221(0.844-1.767) 0.288 1.587
(0.986-2.555) 0.057 Adjuvant chemotherapy (positive/negative) 1.132
(0.802-1.596) 0.481 1.346
(0.922-1.966) 0.124 MARS1 expression
(high vs. low) 5.663
(2.016-15.906) 0.001 2.761
(1.159-6.576) 0.022 4.857
(1.597-14.773) 0.005 2.774
(1.554-4.950) 0.023

4. Sintering

Serum levels of CA 19-9 (carbohydrate antigen 19-9) are a prognostic indicator for pancreatic ductal adenocarcinoma (PDAC). In the present invention, the ability of the expression level of human cytoplasmic methionyl-tRNA synthetase 1 (MARS1), which promotes cancer growth by regulating protein synthesis and cell cycle, is used to predict the prognosis of pancreatic adenocarcinoma. evaluated. We performed immunohistochemical (IHC) staining on pancreatic specimens obtained from patients with pancreatic adenocarcinoma undergoing surgery, and found that high MARS1 expression was defined as equal to or higher than that in normal progenitor cells, and low MARS1 expression was defined as expression in normal progenitor cells. It was defined as weaker and stronger than the pancreatic duct epithelium. In addition, univariate and multivariate analyzes were conducted on other factors related to prognosis. As a result, among 137 pancreatic adenocarcinoma patients, no significant differences in baseline characteristics were found between patients with high (n = 82) and low MARS1 expression (n = 55). However, the median overall survival of patients with high MARS1 expression was shorter than that of patients with low expression (15.2 vs. 17.2 months, log-rank test p = 0.044). The median disease-free survival (DFS) was not significantly different between the two groups, but DFS was shorter in patients with high MARS1 expression than in those with low MARS1 expression (8.9 months vs. 11.2 months, log-rank test p = 0.067). ). In multivariate analysis, lymph node metastasis and high MARS1 expression were associated with poor prognosis of pancreatic adenocarcinoma. Increased MARS1 expression detected by IHC staining is associated with poor prognosis in PDAC, showing that MARS1 functions as a prognostic indicator.

Claims (22)

methionyl-tRNA synthetase 1 (MARS1) or a fragment thereof; Or a composition for diagnosing cancer, comprising an agent for measuring the expression level of the gene encoding the same. According to paragraph 2,
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 1,
The agent for measuring the expression level of the gene is a diagnostic composition comprising at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene. According to paragraph 1,
The composition is a diagnostic composition for application to a biological sample isolated from an object of interest. According to clause 4,
The biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, and tears ( tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, Ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple Nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract and cerebrospinal fluid ( A diagnostic composition comprising at least one member selected from the group consisting of cerebrospinal fluid. According to paragraph 1,
The above cancers include pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon 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. According to clause 6,
A composition for diagnosis, wherein the cancer is pancreatic cancer. In clause 7,
A diagnostic composition, wherein the diagnosis predicts prognosis. A diagnostic kit comprising the diagnostic composition of any one of claims 1 to 8. According to clause 9,
The kit is a diagnostic kit, which is an RT-PCR kit, DNA chip kit, ELISA kit, protein chip kit, rapid kit, or MRM (multiple reaction monitoring) kit. In biological samples isolated from the subject of interest,
methionyl-tRNA synthetase 1 (MARS1); or measuring the expression level of the gene encoding the protein; Method for providing information for diagnosis of cancer, including. According to clause 11,
Methionyl-tRNA synthetase 1 (MARS1) protein measured in the biological sample; Or, encoding this includes determining that cancer has developed or is highly likely to develop if the expression level of the gene is higher than that of the control group; Method for providing information for diagnosis of cancer, including. According to clause 12,
The biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, and tears ( tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, Ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple Nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract and cerebrospinal fluid ( A method of at least one selected from the group consisting of cerebrospinal fluid. According to clause 13,
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. Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blotting, ELISA (enzyme linked immunosorbent assay), or multiple reaction monitoring (MRM) methods, method. According to clause 13,
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), Northern blotting or DNA chip method. According to claim 14 or 15,
The above cancers include pancreatic cancer, thyroid cancer, parathyroid cancer, stomach cancer, ovarian cancer, colon 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, How to Provide Information. According to clause 16,
A method of providing information, wherein the cancer is pancreatic cancer. According to clause 17,
A method of providing information, wherein the diagnosis predicts prognosis. (a) methionyl-tRNA synthetase 1 (MARS1) on a biological sample obtained from the subject of interest; Or a measuring unit that measures the expression level of the gene encoding the protein; and
(b) a detection unit that outputs the presence or absence of cancer based on the expression level of the gene or protein encoded by the gene measured in the measurement unit; A diagnostic device for cancer, including a. According to clause 19,
The above cancers include stomach cancer, thyroid cancer, parathyroid 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, and pituitary adenoma. At least one, diagnostic device. According to clause 20,
A diagnostic device wherein the cancer is pancreatic cancer. According to clause 21,
The diagnosis is a diagnostic device that predicts prognosis.