KR101994790B1 - Composition for diagnosing cancer - Google Patents

Abstract

The present invention relates to a kit and composition which can be used to predict cancer, furthermore the stage, grade, survival rate and therapeutic responsiveness of ovarian cancer, and a method of providing information for the prediction thereof, by measuring the expression level of autotaxin (ATX) or the gene encoding the same for biological samples of the subject of interest, preferably pelvic fluids.

Description

[Composition for diagnosing cancer]

The present invention relates to a composition, a kit, and a method for providing information for the prediction, which can be used for predicting the likelihood of developing cancer, and further, the stage, differentiation grade and therapeutic response of ovarian cancer.

Ovarian cancer is the fourth most common cause of female cancer, and it is one of the leading cause of death among gynecologic malignancies. Although ovarian cancer is rarely found, it is located in the deep pelvis, so it is mostly found in the advanced stage because ovarian cancer does not cause any symptoms until it spreads widely and spreads, and despite 5 years of survival rate It is one of the very serious diseases of less than 1%. Early detection of ovarian cancer is crucial to improve the cure rate. The 5-year survival rate is 85% when ovarian cancer is diagnosed within 2 days, while the 5-year survival rate is lower than 50% when the 3-stage diagnosis is diagnosed. Therefore, for effective cancer treatment, early diagnosis using an appropriate diagnostic marker (labeling substance) is essential.

Tumor biomarker measurement is one of the clinically useful methods for the diagnosis of malignant tumor, evaluation and follow - up of treatment outcome. Ideal tumor biomarkers should be produced in tumor cells and should be readily available in body fluids, have high specificity to distinguish malignant from benign, and should be helpful in early detection. It should also be useful in population testing and should reflect the progression of the tumor and the outcome of the treatment.

To date, various biomarkers have been proposed to diagnose ovarian cancer, and studies have been conducted through various technology platforms and data analysis. CA-125, CA19-9, and carcinoembryonic antigen (CEA) are commonly used biomarkers for the diagnosis of ovarian cancer. CA-125 is widely used for screening, diagnosis, monitoring and follow-up . However, the use of such a single biomarker is limited in the diagnosis of ovarian cancer because sensitivity and specificity for malignant tumor cells are low, and an alternative to this is needed. In this connection, Korean Patent Laid-Open No. 10-2008-0044801 (titled "biomarker for endometrial cancer and endometrial cancer: heptidine, hereinafter referred to as prior art 1") was prepared by using hepcidin as a biomarker Lt; RTI ID = 0.0 > endometrial cancer. ≪ / RTI > However, using the biomarker having specificity for both ovarian cancer and endometrial cancer, the above-described prior art 1 has a limitation in increasing the accuracy of diagnosis because sensitivity and specificity for diagnosis of ovarian cancer are decreased.

It is an object of the present invention to provide a composition capable of diagnosing cancer susceptibility, cancer stage, differentiation degree, or therapeutic response to cancer patients with high accuracy.

Another object of the present invention is to provide a kit capable of diagnosing the likelihood of cancer development, cancer stage, degree of differentiation, or therapeutic response of a cancer patient with high accuracy.

Another object of the present invention is to provide a method for providing information for diagnosing cancer possibility, cancer stage, differentiation degree, cancer survival rate or therapeutic response with high accuracy.

It is another object of the present invention to provide a method for screening a drug for treating the cancer.

According to one embodiment of the present invention, there is provided a diagnostic composition for cancer comprising an agent for measuring the expression level of autotaxin (ATX) or a gene encoding the same.

In the present invention, the term "autotaxin (ATX)" is also referred to as 'accession nucleotide pyrophosphatase / phosphodiesterase family member 2 (ENPP2)', and the secreted lipofusporphase D (lysophospholipase D, lysoPLD). It cleaves choline from lysophosphatidylcholine (LPC) to form lysophosphatidic acid (LPA), a lipid signaling molecule.

In the present invention, the expression level of the autotaxin or the gene encoding the same may be measured from a biological sample of the desired individual. In the present invention, the term "biological sample" refers to any substance, biological fluid, tissue or cell obtained from or derived from an individual, including, for example, whole blood, leukocytes, Blood, sputum, tears, mucus, nasal washes (including peripheral blood mononuclear cells, buffy coat, plasma and serum) ), Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, Bronchial aspirate, synovial fluid, joint aspirate, organ secretions, It may include a cell, a cell extract, or a cerebrospinal fluid. However, it is preferable that a hollow needle or the like is inserted into a living body without cutting the skin of a patient having a high possibility of developing it, It may be a liquid biopsy (for example, a tissue, a cell, blood, serum, plasma, saliva, sputum or pelvic fluids, etc.) (pelvic fluids) can increase the accuracy of diagnosis.

The present invention further includes an agent for measuring the expression level of cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the same, thereby improving the accuracy of the cancer diagnosis.

In the present invention, the term " cancer antigen 125 (CA125) "is also referred to as 'mucin 16' or 'MUC16' and corresponds to a protein encoded by the MUC16 gene. MUC16 belongs to the mucin family glycoprotein.

In the present invention, the agent for measuring the expression level of auto-Thaksin or cancer antigen 125 is not particularly limited, but preferably an antibody, oligopeptide, ligand, PNA ( peptide nucleic acid, and aptamer.

In the present invention, "measurement of protein expression level" refers to a process of confirming the presence and expression level of the protein of autotaxin, which is a diagnostic marker for cancer, in a biological sample in order to diagnose cancer. As a method of measuring or comparing the expression level of the above auto-Thaksin, there may be mentioned protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) Desorption / Ionization Time of Flight Mass Spectrometry Analysis, Radiation Immunoassay, Radiation Immunodiffusion, Ouchteroni Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complement Fixation Analysis, Two Dimensional Electrophoresis Analysis, Liquid Chromatography-Mass But are not limited to, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA)

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

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

In the present invention, the term " aptamer "is an oligonucleotide or peptide molecule, and the general contents of the aptamer 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 diagnostic composition according to the present invention, the agent for measuring the expression level of the gene encoding ototoxin or cancer antigen 125 is selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the above or the gene encoding cancer antigen 125 And the like. Since the information on auto-Thaksin and cancer antigen 125 according to the present invention is known, a person skilled in the art will be able to easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein.

The expression "measurement of gene expression level" in the present invention means measuring the amount of a gene by confirming the presence and expression level of a gene encoding autotaxin in a biological sample to diagnose cancer. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

In the present invention, the term "primer" is a primer pair that includes a primer pair in both forward and reverse directions, but preferably provides specific and sensitive analysis results. High specificity can be given when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that only the target gene sequence containing the complementary primer binding site is amplified and does not induce nonspecific amplification .

In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in the sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding. The kind of probe may be a peptide nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA or a DNA, and is most preferably a peptide nucleic acid Can be a PNA. More specifically, the probe is a biomolecule derived from or derived from an organism, or prepared in vitro, and includes, for example, an enzyme, a protein, an antibody, a microorganism, an animal and plant cell and an organ, RNA may include cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the term "LNA (Locked nucleic acids) " means a nucleic acid analogue including 2'-O, 4'-C methylene bridges [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502 ]. LNA nucleosides include the common nucleotide bases of DNA and RNA, and can form base pairs according to the Watson-Crick base pair rule. However, due to the 'locking' of the molecule due to the methylene bridge, the LNA fails to form an ideal shape in the Watson-Crick bond. When LNAs are incorporated into DNA or RNA oligonucleotides, LNAs can pair with complementary nucleotide chains more rapidly to enhance the stability of the double strand. In the present invention, the term "antisense" means that the antisense oligomer is hybridized with the target sequence in the RNA by Watson-Crick base pairing to allow the formation of the mRNA and RNA: oligomer heterodimers in the target sequence, And an oligomer having a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence.

In the present invention, the term "diagnosis" is intended to include determining the susceptibility of a subject to a particular disease or disorder, determining whether a subject currently has a particular disease or disorder, Determining the prognosis of the subject (e.g., identifying a pre-metastatic or metastatic cancerous condition, determining the stage of the cancer or determining the response of the cancer to treatment), or determining therametrics (e.g., And monitoring the status of the object to provide information). For the purpose of the present invention, the diagnosis is to confirm the incidence or risk of developing the disease, the stage or differentiation of the cancer, or the survival rate or therapeutic response of the cancer patient.

In the present invention, the term " stage "refers to a degree of progression of cancer cells, and the international classification according to the progress of cancer generally follows the classification of the TNM stage. Here, 'T (Tumor Size)' is classified according to the size of the primary tumor, 'N (Lymph Node)' is classified according to the degree of lymph node metastasis, 'M (Metastasis) . The detailed classification of T, N, and M is shown in Table 1 below, and the cancer stage classification is shown in Table 2 below.

TNM armory Justice Size of primary tumor
(T arm)
Size of the primary tumor (T stage) T0 Tumor cells are seen in malignant tumors but are confined to the mucosa or epithelium but not yet invade the basement membrane. T1 There is limited lesion in primary organs, tumor is moving smoothly and there is no involvement in adjacent and surrounding tissues T2 The size of tumor is 2 ~ 5cm T3 Tumor size greater than T2 but limited to organs T4 Adherence and infiltration with surrounding tissue Lymph node metastasis
(N armory)
Lymph node status (N stage) N0 No evidence of lymph node lesion N1 Involvement in one lymph node (1 to 2 cm or more, usually up to 3 cm) that is palpable, mobile, and limited to the first position N2 A palpable, partially mobilized or hard or firm lymph node, microscopically evidence of invasion, clinically intertwined and appearing on the opposite or bilateral side (3-5 cm) N3 What about? It is fixed and passes through the capsule and is completely fixed to bones, large blood vessels, skin, Whether remote transfer
(M armory)
Distant metastasis
(M stage) M0 No remote metastasis M1 Have remote metastasis

Stage classification T1 T2 T3 T4 N0 First Second N1 Third N2 N3 M1 Four

In the present invention, the term "grade of cancer" refers to the degree of maturation or differentiation of cancer cells, and can be classified into Grade 1, Grade 2 and Grade 3 according to the degree of differentiation, 3 has a poor prognosis after treatment because of its poor metastasis, poor therapeutic effect, and poor prognosis as compared with Grade 2 or 1 highly metastatic or metastatic cancer (Histopathology 2002 Sep ; 41 (3A): 154-61, Nat Genet. 2008 May; 40 (5): 499-507).

Classification Degree of differentiation Degree of differentiation Grade 1 Low grade Well-differentiated stages (well differentiated) Grade 2 The degree of differentiation (Ingermediate grade) Moderately differentiated, Grade 3 High grade Poorly differentiated (Poorly differentiated)

As a disease to be diagnosed in the present invention, the term "cancer" refers to or indicates a physiological condition characterized by cell growth that is not typically regulated in mammals. The cancer to be diagnosed in the present invention may be selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Hodgkin lymphoma, hematologic cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectum cancer, brain tumor, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, Cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, urothelial cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, It may be an adenoma, but it may preferably be an ovarian cancer.

According to another embodiment of the present invention, there is provided a diagnostic kit for cancer comprising the diagnostic composition for cancer of the present invention.

In the present invention, the diagnosis kit can be used to diagnose the incidence of cancer disease, the possibility of developing cancer, the degree of differentiation or stage of cancer.

In the present invention, the cancer is selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Cancer of the uterus, cancer of the uterus, cancer of the uterus, cancer of the brain, cancer of the anus, cancer of the fallopian tubes, endometrial carcinoma, vaginal cancer, vulvar carcinoma, endometrial carcinoma, endometrioid carcinoma, Renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brain stem nerve, neuroendocrine carcinoma, endometrioid carcinoma, small intestine cancer, soft tissue sarcoma, Glioma or pituitary adenoma, but may be preferably an ovarian cancer.

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

The cancer diagnostic kit of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

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

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

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

According to another embodiment of the present invention, there is provided a composition for predicting the therapeutic response of cancer, comprising an agent for measuring the expression level of autotaxin (ATX) or a gene encoding the same.

In the present invention, the expression level of the autotaxin or the gene encoding the same may be measured from a biological sample of the desired individual.

In the present invention, the blood, which contains the biological whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma and serum, (eg, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal cavity, The use of peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph (lymph) fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, , Cell extract or cerebrospinal fluid, but is preferably administered to a patient with a high likelihood of developing (Eg, tissue, cells, blood, serum, plasma, saliva, sputum or fluid in the pelvis) of a patient who has not been excised, pelvic fluids, etc.), more preferably pelvic fluids, can improve the accuracy in predicting the therapeutic response of cancer.

In the present invention, it is possible to further improve the accuracy in predicting the therapeutic response of the cancer by further comprising an agent for measuring the expression level of cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the same.

In the present invention, the agent for measuring the expression level of auto-Thaksin or cancer antigen 125 is not particularly limited, but preferably an antibody, oligopeptide, ligand, PNA ( peptide nucleic acid, and aptamer.

In the diagnostic composition according to the present invention, the agent for measuring the expression level of the gene encoding ototoxin or cancer antigen 125 is selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the above or the gene encoding cancer antigen 125 And the like. Since the information on auto-Thaksin and cancer antigen 125 according to the present invention is known, a person skilled in the art will be able to easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein.

In the present invention, the cancer is selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Endometrioid carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine carcinoma, endometrioid carcinoma, endometrial carcinoma, Pancreatic cancer, pituitary adenoma, soft tissue sarcoma, urethral cancer, penile cancer, urothelial carcinoma, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, , Preferably an ovarian cancer.

According to another embodiment of the present invention, there is provided a kit for predicting the therapeutic response of a cancer comprising a composition for predicting therapeutic response of a cancer of the present invention.

In the present invention, the susceptibility of the cancer patient to the standard treatment method can be predicted using the kit for predicting the therapeutic response of the cancer, and the prognosis can be predicted after performing the standard treatment for the cancer patient.

In the present invention, the cancer is selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Endometrioid carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine carcinoma, endometrioid carcinoma, endometrial carcinoma, , Pancreatic cancer, soft tissue sarcoma, urethral cancer, penile cancer, urothelial carcinoma, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma But can preferably be an ovarian cancer.

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

The cancer diagnostic kit of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

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

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

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

According to another embodiment of the present invention, there is provided a method for providing information for diagnosis of cancer, comprising the step of measuring the expression level of autotaxin or a gene encoding the same in a biological sample separated from a desired individual.

In the present invention, the above-mentioned "object of interest" means an individual having an uncertain onset of the cancer, and having a high possibility of developing the cancer.

In the present invention, the term "biological sample" refers to any substance, biological fluid, tissue or cell obtained from or derived from an individual, including, for example, whole blood, leukocytes, Blood, sputum, tears, mucus, nasal washes (including peripheral blood mononuclear cells, buffy coat, plasma and serum) ), Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, Bronchial aspirate, synovial fluid, joint aspirate, organ secretions, It may include a cell, a cell extract, or a cerebrospinal fluid. However, it is preferable that a hollow needle or the like is inserted into a living body without cutting the skin of a patient having a high possibility of developing it, It may be a liquid biopsy (for example, a tissue, a cell, blood, serum, plasma, saliva, sputum or pelvic fluids, etc.) (pelvic fluids) can increase the accuracy of diagnosis.

The present invention may include a step of measuring the expression level of autotaxin or a gene encoding the same in the biological sample as described above.

In addition, the present invention further includes the step of measuring the expression level of the cancer antigen 125 (CA125) or a gene encoding the cancer antigen 125 in the separated biological sample, thereby improving the accuracy of the cancer diagnosis.

In the present invention, the agent for measuring the expression level of autotaxin or cancer antigen 125 is not particularly limited, but preferably an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA) binding specifically to the protein of autotaxin, And aptamers. The term " aptamer "

As a method for measuring or comparing the expression level of the above-mentioned auto-thaxin, there may be mentioned protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) Immunoassay, radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA) It is not.

In the present invention, the agent for measuring the expression level of the gene encoding Ottashine or cancer antigen 125 is selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding Ottashine or cancer antigen 125 And may include one or more species.

In the present invention, the presence or absence of a gene encoding autoantagonase or cancer antigen 125 and the degree of expression of the gene may be determined by RT-PCR, competitive reverse transcription polymerase chain reaction (RT-PCR) But are not limited to, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, and DNA chip. .

The present invention may include a step of predicting that the cancer is likely to occur when the level of expression of the auto-thapsin or the gene encoding the same is higher than that of the normal control in the biological sample of the subject.

In the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is 250 ng / ml, 260 ng / ml, 270 ng / ml, 280 ng / ml, 290 ng / ml , 299 ng / ml or more, or 300 ng / ml or more, preferably 299 ng / ml or more.

In the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is 80 to 200 ng / ml, 85 to 190 ng / ml, 95 to 180 ng / ml, 100 to 180 ng / ml, 160 ng / ml, it can be predicted that the degree of differentiation of the cancer is Grade 1.

In the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is 300 to 420 ng / ml, 310 to 415 ng / ml, 320 to 410 ng / ml, 330 to 400 ng / To 390 < RTI ID = 0.0 > ng / ml, < / RTI >

In the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is 310 to 420 ng / ml, 320 to 415 ng / ml, 330 to 410 ng / ml, 340 to 400 ng / To 390 ng / ml, the degree of differentiation of the cancer can be predicted to be Grade 3.

In the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is in the range of 150 to 250 ng / ml, 160 to 240 ng / ml, 170 to 230 ng / ml, 180 to 220 ng / ml, 210 ng / ml, it can be predicted that the stage of cancer is the first stage.

Also, in the present invention, the expression level of autotaxin measured in the biological sample of the subject individual is 280 to 390 ng / ml, 290 to 380 ng / ml, 300 to 370 ng / ml, 310 to 360 ng / ml, or In the case of 320 to 350 ng / ml, it can be predicted that the stage of cancer is stage 2.

In the present invention, the expression level of autotaxin measured in the biological sample of the subject individual is 430 to 530 ng / ml, 440 to 520 ng / ml, 450 to 510 ng / ml, 460 to 500 ng / ml, or In the case of 470 to 490 ng / ml, it can be predicted that the stage of cancer is three stages.

Also, in the present invention, the expression level of autotaxin measured in the biological sample of the desired individual is in the range of 480 to 600 ng / ml, 490 to 590 ng / ml, 500 to 580 ng / ml, 510 to 570 ng / To 560 ng / ml, or 520 to 550 ng / ml, the stage of cancer can be predicted to be four stages.

In the present invention, the cancer is selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Endometrioid carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine carcinoma, endometrioid carcinoma, endometrial carcinoma, Pancreatic cancer, pituitary adenoma, soft tissue sarcoma, urethral cancer, penile cancer, urothelial carcinoma, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, , Preferably an ovarian cancer.

According to another embodiment of the present invention, there is provided a method for providing information for predicting therapeutic response of a cancer, which comprises measuring the expression level of autotaxin or a gene encoding the same in a biological sample separated from a desired individual .

In the present invention, the biological sample includes blood (including 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, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymphatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells cell extract, cell extract, or cerebrospinal fluid, but preferably is a highly likely pathogen (Eg, tissue, cells, blood, serum, plasma, saliva, sputum or fluid in the pelvis) of a patient who has received a liquid needle biopsy Pelvic fluids, etc.), and more preferably pelvic fluids.

The present invention may include a step of measuring the expression level of autotaxin or a gene encoding the same in the biological sample as described above.

In addition, the present invention further includes a step of measuring the expression level of cancer antigen 125 (CA125) or a gene encoding the cancer antigen 125 in the biological sample as described above, thereby improving accuracy in predicting the therapeutic response of the cancer have.

In the present invention, the agent for measuring the expression level of autotaxin or cancer antigen 125 is not particularly limited, but preferably an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA) binding specifically to the protein of autotaxin, And aptamers. The term " aptamer "

As a method for measuring or comparing the expression level of the above-mentioned auto-thaxin, there may be mentioned protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) Immunoassay, radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA) It is not.

In the present invention, the agent for measuring the expression level of the gene encoding Ottashine or cancer antigen 125 is selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding Ottashine or cancer antigen 125 And may include one or more species.

In the present invention, the presence or absence of a gene encoding autoantagonase or cancer antigen 125 and the degree of expression of the gene may be determined by RT-PCR, competitive reverse transcription polymerase chain reaction (RT-PCR) But are not limited to, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, and DNA chip. .

In the present invention, the expression level of autotaxin measured in a biological sample of the desired individual is at least 250 ng / ml, at least 260 ng / ml, at least 270 ng / ml, at least 280 ng / ml, at least 290 ng / at least 320 ng / ml, at least 330 ng / ml, at least 340 ng / ml, at least 350 ng / ml, at least 360 ng / ml, at least 370 ng / ml, at least 310 ng / ml, at least 314 ng / ml or more, or 372 ng / ml or more, preferably 270 ng / ml or more, 280 ng / ml or more, 290 ng / ml or more, 300 ng / ml or more, 310 or more or 314 ng / ml or more, more than 330 ng / ml, more than 340 ng / ml, more than 350 ng / ml, more than 360 ng / ml, more than 370 ng / ml, more preferably more than 374 ng / Or more; The level of expression of the cancer antigen 125 is at least 20 ng / ml, at least 22 ng / ml, at least 24 ng / ml, at least 26 ng / ml, or at least 26.8 ng / ml, preferably at least 26 ng / ml or more, more preferably 26.8 ng / ml or more; It is predicted that treatment response to standard chemotherapy is low, recurrence rate is high, and prognosis is bad.

In the present invention, the cancer is selected from the group consisting of ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, Endometrioid carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine carcinoma, endometrioid carcinoma, endometrial carcinoma, Pancreatic cancer, pituitary adenoma, soft tissue sarcoma, urethral cancer, penile cancer, urothelial carcinoma, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, , Preferably an ovarian cancer.

In the present invention, by measuring the expression level of auto-thaksin in a biological sample of an objective individual, preferably pelvic fluids, it is possible to determine whether or not the cancer, preferably ovarian cancer, The stage or degree of differentiation and the therapeutic response to standard chemotherapy can be predicted with high accuracy.

FIG. 1 shows the expression level of autotaxin in ovarian cancer tissues in Example 1 of the present invention.
FIG. 2 is a graph showing the amount of autotaxy secreted in the pelvic fluid of patients with ovarian cancer and benign tumors using ELISA in Example 2 of the present invention.
FIG. 3 is a value obtained by measuring the amount of autotaxin secreted from the serum of the ovarian cancer patient and the normal person by ELISA in Example 2 of the present invention.
FIG. 4 is a graph showing the amount of autotaxin secreted in plasma of an ovarian cancer patient and a normal person using ELISA in Example 2 of the present invention.
FIG. 5 shows the ROC curve using the level of autotaxy expression in the pelvic fluid of patients with ovarian cancer and benign tumors in Example 2 of the present invention.
FIG. 6 is a graph showing an analysis of the expression level of autotaxin according to ovarian cancer differentiation grade in fluid of the pelvis of an ovarian cancer patient in Example 3 of the present invention. FIG.
FIG. 7 is a graph showing an analysis of the expression level of autotaxin according to the stage of ovarian cancer in the fluid of the pelvis of an ovarian cancer patient in Example 4 of the present invention. FIG.
FIG. 8 is a graph showing the results of the CA125 alone evaluation in the fluid of the pelvis of the ovarian cancer patient in Example 5 and the predictive value of the diagnosis of ovarian cancer when CA125 and ototaxin are combined with a double marker.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

[Example 1] Overexpression of autotaxin (Fig. 1) in ovarian cancer tissues [

For this immunohistochemical staining, formalin-fixed paraffin embedded tissue was approved by the IRB of the Ministry of Health, Gangnam Severance Hospital, received. The obtained TMA (tissue microarray) confirmed the expression of autotaxin using a monoclonal antibody against autotaxin using an EnVision-HRP detection system (Dakocytomation, Carpinteria, Calif.) For immunotol- ogy to autotaxin And the expression level of autotaxin was measured using the VisioPharm software. The numerical values were maximum "300" and minimum "0".

182 cases of ovarian cancer tissue and 48 cases of non-tumor tissue surrounding ovarian cancer were used for immunohistochemical staining to confirm the expression pattern of autotaxin. Analysis of the expression level of autotaxin using immunohistochemical staining revealed that the expression of autotaxin was significantly increased in ovarian cancer (OC) compared to non-tumor tissue (Fig. 1). Statistical significance was assessed using the mann whitney test. The p-value was less than 0.0001.

[Example 2] Use of ototaxin as a diagnostic marker for ovarian cancer by fluid in the pelvis of ovarian cancer patients (Figs. 2 to 5)

In order to select patient samples for the confirmation of the expression of autotaxin, an autotaxin ELISA kit of R & D was used using a fluid (Fig. 2), serum (Fig. 3) and plasma Over-expression of autotaxin was observed. As a result, the expression level of autotaxin in serum (FIG. 3) and plasma (FIG. 4) of ovarian cancer patients was not different from that of normal persons, but the expression level of autotaxin in the pelvic fluid (FIG. 2) Compared to the control group. Thus, the fluid in the pelvis is more suitable for the diagnosis of ovarian cancer than the serum and plasma of ovarian cancer patients. Statistical significance was assessed using the mann whitney test. The p-value was less than 0.05.

Therefore, the area under the curve (AUC) was 0.648 as shown in the ROC graph for use in diagnosing and predicting autotaxin in fluid in the pelvis.

[Example 3]. Increased levels of autotaxin expression in pelvic fluid following ovarian cancer differentiation (Figure 6)

Ovarian cancer and fluid in the pelvis originating from benign tumors were distributed through the Kangnam Severance Hospital Human Dental Bank (Korea Maternity Cancer Bank) with the approval of the Ethics Committee for Clinical Research (IRB). The obtained samples were assayed for the level of autotaxin expression using an Otothoxin ELISA kit and the measured values were analyzed by the GraphPad Prism 7 statistical program (FIG. 6). As a result, it was confirmed that the level of autotaxin expression was increased as the degree of ovarian cancer differentiation increased. In addition, statistical significance between Grade 1 and Grade 2, and Grade 1 and Grade 3 of differentiation was confirmed (p <0.05). The categories of the expression levels of autotaxin according to ovarian cancer differentiation degree are shown in Table 4 below.

Degree of differentiation The level of expression level of autotaxin (ng / ml) Mean value (ng / ml) Grade 1 10.37 to 456.04 138.93 + 132.7155 Grade 2 6.10-1174.01 363.55 + 357.31 Grade 3 4.14-1121.92 366.72 + 335.03

[Example 4] Increase in the level of autotaxin expression in the pelvic fluid according to ovarian cancer stage (Fig. 7)

Ovarian cancer and fluid in the pelvis originating from benign tumors were distributed through the Kangnam Severance Hospital Human Dental Bank (Korea Maternity Cancer Bank) with the approval of the Ethics Committee for Clinical Research (IRB). The obtained samples were assayed for the level of autotaxin expression using an Otothar's ELISA kit, and the measured values were analyzed by the GraphPad Prism 7 statistical program (FIG. 7). As a result, it was confirmed that the expression level of autotaxin increased as the stage of ovarian cancer increased. Especially, it was confirmed that autotaxin was expressed at a high concentration in ovarian cancer stage 3 and stage 4, which had worse prognosis than stage 1. The categories of the expression levels of autotaxin according to the stage of ovarian cancer are shown in Table 5 below. +; mean value; -; median value.

ordnance The level of expression level of autotaxin (ng / ml) Mean value (ng / ml) Stage 1 4.15 to 979.74 198.232 + 237.36 Stage 2 23.6 to 786.78 333.57 + 401.25 Stage 3 25.44 to 1221.92 485.25 + 329.34 Stage 4 8.16 to 931.54 538.97 + 232.95

[Example 5] CA125, which is effective for diagnosing ovarian cancer and discriminating therapeutic response from fluid in the pelvis derived from ovarian cancer,

Ovarian cancer and fluid in the pelvis originating from benign tumors were distributed through the Kangnam Severance Hospital Human Dental Bank (Korea Maternity Cancer Bank) with the approval of the Ethics Committee for Clinical Research (IRB). The obtained samples were assayed for the level of autotaxin expression using an Otothoxin ELISA kit, and the measured values were subjected to a SAS version 9.3 statistical program to generate ROC curves (FIG. 8). As a result, the sensitivity of ovarian cancer diagnosis was improved from 0.737 to 0.801 when CA125 and Ototaxin were used in combination with CA125 alone in the diagnosis of ovarian cancer. The predictive power of ovarian cancer according to the cut-off value of CA125 and ototoxin is shown in Tables 6 to 8 below.

variable CA125 cut-off value
AUC (CA125 cut-off value + ATX cut-off value) AUC ((CA125 cut-off value + ATX cut-off value) - CA125 cut-off value)
AUC p-value Cut-off value of ATX ≥ 246.45201742, <246.45201742 0.737 (0.674-0.799) 0.770 (0.686-0.855) 0.034 (-0.020-0.087) 0.2171 ≥274.03594175, <274.03594175 0.737 (0.674-0.799) 0.802 (0.736-0.868) 0.065 (0.018-0.112) 0.0072 ≥299.766730025, <299.766730025 0.737 (0.674-0.799) 0.796 (0.734-0.859) 0.060 (0.017-0.102) 0.0057 ≥314.22390002, <314.22390002 0.737 (0.674-0.799) 0.801 (0.742-0.860) 0.064 (0.028-0.100) 0.0005 ≥372.304646465, <372.304646465 0.737 (0.674-0.799) 0.776 (0.717-0.836) 0.040 (0.006-0.073) 0.0195

Benign tumors _ fluid in pelvis vs. ovary cancer _ fluid in pelvis The cut-off value (ng / ml) responsiveness
(95% CI) Specificity
(95% CI) PPV
(95% CI) ATX cut-off value ≥299.766730025, 299.766730025 0.513
(0.433-0.593) 0.917
(0.806-1.027) 0.975
(0.94-1.009)

variable CA125 cut-off value
AUC (CA125 cut-off + ATX cut-off) AUC p-value Cut-off value of ATX ≥274.03594175, <274.03594175 0.737 (0.674-0.799) 0.802 (0.736-0.868) 0.0072 ≥299.766730025, <299.766730025 0.737 (0.674-0.799) 0.796 (0.734-0.859) 0.0057 ≥314.22390002, <314.22390002 0.737 (0.674-0.799) 0.801 (0.742-0.860) 0.0005 ≥372.304646465, <372.304646465 0.737 (0.674-0.799) 0.776 (0.717-0.836) 0.0195

Claims (32)

A diagnostic composition for cancer, comprising an agent that measures the level of expression of autotaxin (ATX) or a gene encoding the same.
The composition is intended for pelvic fluids of the desired individual,
The cancer is an ovarian cancer,
Wherein the expression level of the autotaxin or the gene encoding the same is higher in the fluid of the pelvis of the subject than in the normal control group. delete delete The method according to claim 1,
Wherein the composition further comprises an agent for measuring the expression level of cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the same. The method according to claim 1,
The agent for measuring the expression level of autotaxin comprises at least one selected from the group consisting of an antibody specifically binding to autotaxin, an oligopeptide, a ligand, a peptide nucleic acid (PNA) and an aptamer , &Lt; / RTI &gt; The method according to claim 1,
The agent for measuring the expression level of the gene encoding ototoxin includes at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene encoding the ototoxin. . delete The method according to claim 1,
Wherein said composition is for predicting the grade or stage of cancer. A composition for predicting the therapeutic response of a cancer, comprising an agent for measuring the expression level of autotaxin (ATX) or a gene encoding the same.
The composition further comprises an agent for measuring the expression level of cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the cancer antigen 125,
The composition is intended for pelvic fluids of the desired individual,
Wherein the cancer is ovarian cancer. delete delete delete delete A cancer diagnostic kit comprising the composition of any one of claims 1, 4, 6, and 8,
Wherein the cancer is ovarian cancer. A kit for predicting the therapeutic response of a cancer comprising the composition of claim 9,
Wherein said cancer is ovarian cancer. Measuring the expression level of autotaxin (ATX) or a gene encoding the same in a biological sample separated from the desired individual; And
And predicting that cancer is likely to occur when the expression level of the auto-thapsin or the gene encoding the same is increased as compared to that of a normal control, as measured in a biological sample of the subject individual, As a method of providing,
Wherein the biological sample is pelvic fluids,
Wherein the cancer is ovarian cancer. delete 17. The method of claim 16,
Wherein the information providing method further comprises the step of measuring an expression level of a cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the same. 17. The method of claim 16,
The method for providing information for diagnosis of cancer wherein the expression level of autotaxin measured in the biological sample of the target individual is 290 ng / ml or more, which is predicted to have a high possibility of the cancer. 17. The method of claim 16,
The method for providing information for diagnosis of cancer, wherein the expression level of autotaxin is 299 ng / ml or more as measured in a biological sample of the subject, and the predicted probability of the cancer is high. 17. The method of claim 16,
Wherein the degree of differentiation of the cancer is predicted to be Grade 1 when the expression level of autotaxin measured in the biological sample of the desired individual is 80 to 200 ng / ml. 17. The method of claim 16,
Wherein the degree of differentiation of the cancer is predicted to be Grade 2 when the expression level of autotaxin measured in the biological sample of the subject is 300 to 420 ng / ml. 17. The method of claim 16,
Wherein the degree of differentiation of the cancer is predicted to be grade 3 when the expression level of autotaxin is 310 to 420 ng / ml. 17. The method of claim 16,
Wherein the cancer stage is predicted to be stage 1 when the expression level of autotaxin measured in the biological sample of the subject is 150 to 250 ng / ml. 17. The method of claim 16,
Wherein the cancer stage is predicted to be stage 2 when the expression level of autotaxin measured in the biological sample of the subject is 280 to 390 ng / ml. 17. The method of claim 16,
Wherein the cancer stage is predicted to be in the third stage when the expression level of autotaxin measured in the biological sample of the subject individual is 430 to 530 ng / ml. 17. The method of claim 16,
Wherein the cancer stage is predicted to be in the fourth stage when the expression level of autotaxin measured in the biological sample of the subject is 480 to 600 ng / ml. delete 1. A method for providing information for predicting the therapeutic response of a cancer, comprising the step of measuring the expression level of autotaxin (ATX) or a gene encoding the same in a biological sample separated from a desired individual,
The method of providing information further comprises the step of measuring the expression level of cancer antigen 125 (cancer antigen 125, CA125) or a gene encoding the same,
The biological sample is pelvic fluids,
Wherein the cancer is ovarian cancer. delete delete 30. The method of claim 29,
When the expression level of autotaxin measured in the biological sample of the desired individual is 314 ng / ml or more; And
When the expression level of cancer antigen 125 measured in the biological sample of the desired individual is 26.8 ng / ml or more; The predicted therapeutic response to standard chemotherapy is low. &Lt; Desc / Clms Page number 13 &gt;

Images