WO2009057849A1 - Marqueurs de diagnostic du cancer du colon utilisant des gènes régulés de façon positive - Google Patents

Marqueurs de diagnostic du cancer du colon utilisant des gènes régulés de façon positive Download PDF

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WO2009057849A1
WO2009057849A1 PCT/KR2007/006108 KR2007006108W WO2009057849A1 WO 2009057849 A1 WO2009057849 A1 WO 2009057849A1 KR 2007006108 W KR2007006108 W KR 2007006108W WO 2009057849 A1 WO2009057849 A1 WO 2009057849A1
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colon cancer
kit
sppl
ifitml
cks2
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PCT/KR2007/006108
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English (en)
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Hee Gu Lee
Eun Young Song
Young Il Yeom
Seon-Young Kim
Young Ho Kim
Ho Kyung Chun
Kyung-Sook Chung
Misun Won
Jae Wha Kim
Do Young Yoon
Jong-Seok Lim
Min-A Kang
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Korea Research Institute Of Bioscience And Biotechnology
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Publication of WO2009057849A1 publication Critical patent/WO2009057849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a diagnostic marker specific to colon cancer. Also, the present invention relates to a composition and a kit, comprising an agent measuring the presence of the marker, and a method of diagnosing colon cancer using the same.
  • the large intestine is a part of the digestive system where foods taken into the mouth are digested and absorbed, and waste products and indigestible food matter stay. Another function is to reabsorb the water; as the water is removed, the wastes become feces that contain various bacteria .
  • Human large intestine is about 2 m long, and consists of the colon, rectum, and anus. Cancer may occur in any part of mucosa lining the large intestine, and commonly develops in the sigmoid colon and rectum. Recently, the incidence of colon cancer has increased remarkably in Korea . Colon cancer is the fourth most common cancer in men after cancer of the stomach, lung, and liver, and the case for women is similar.
  • Colon cancer has an incidence of 5%-10% among people in their thirties, usually amongpeople witha family history.
  • Environmental factors, rather than hereditary factors, are thought to contribute considerably to colon cancer.
  • a western diet, in particular, high intake of animal fat or protein, has been regarded as the most important environmental influence on colon cancer.
  • hereditary factors are responsible for about 5% of all cases of colon cancer.
  • patients at increased risk for colon cancer include those with (1) a previous adenomatous polyp, (2) a family history of colon cancer, (3) a history of chronic ulcerative colitis, and (4) a chronic anal fistula.
  • Colon cancer can be completely cured by surgery alone or endoscopic resection without further treatment, when it is detected at an early stage. Even if the cancer has spread to distant organs, liver or lung (distant metastasis) at an operable stage, it is curable by a surgical operation. Thus, surgery is still the most effective treatment modality for colon cancer. However, when colon cancer is detected at an advanced stage, in which metastasis occurs to organs such as lung, liver, lymph node and peritoneum, surgical operation can not be performed. Thus, for the effective treatment of colon cancer, early diagnosis and treatment are essential.
  • recurrence After surgical operation, recurrence is often detected, and thus regular follow-up examination should be performed every 3-4 months.
  • the liver, lung, and peritoneum are organs susceptible to tumor recurrence, and local recurrence is also detected in the resected area .
  • recurrence of colon cancer is developed for a relatively short period of time, and the recurrent lesion can be completely cured by resection. Since 80% or more of recurrence commonly occurs within 3 years after surgery, no recurrence within 5 years is generally regarded as a complete cure.
  • CEA is only used as a clinical marker for the management of colon cancer, and CEA is not a tumor marker for accurate early diagnosis .
  • the present inventors manufactured DNA chips for the identification of the putative, colon cancer related-genes, and they examined the expression levels thereof in stomach cancer, breast cancer, prostate cancer, and liver cancer, as well as in colon cancer. As a result, the present inventors could screen genes that are specifically up-regulated only in colon cancer tissue, and then finally select the genes that are useful as potential diagnostic markers . They found that the selected genes can be used for accurate early diagnosis of colon cancer, thereby completing the present invention.
  • the present invention provides diagnostic markers for detecting metastasis and prognosis of colon cancer, thereby providing useful information for the treatment and management of colon cancer.
  • diagnostic markers can be used for the development of colon cancer-specific anticancer agents.
  • FIG. 1 is the result of microarray analysis by using 48K human microarray chip (Illumina), which shows the expression levels of 2,230 genes in normal colon tissue and colon cancer tissue (p value ⁇ 0.05), DF: Disease-free, RC: Recurrent);
  • FIG. 2 is a graph showing genes that are specifically up-regulated in colon cancer tissue, based on the result of microarray analysis by using 48K human microarray chip (Illumina) ;
  • FIGs. 3 to 6 are the results of microarray data mining to compare various known microarray data with the result of microarray analysis of colon cancer-specific genes, in which FIG. 3 is the result of microarray data mining for KLK6 gene, FIG. 4 is the result of microarray data mining for CKS2 gene, FIG.
  • FIG. 5 is the result of microarray data mining for IFITMl gene
  • FIG. 6 is the result of microarray data mining for SPPl gene
  • FIG. 7 is the result of DNA chip analysis to compare expression levels of the same genes in colon cancer, stomach cancer, breast cancer, prostate cancer, and liver cancer;
  • FIG. 8 is the result of electrophoresis to analyze the expression levels of CKS2, IFITMl, KLK6, and SPPl diagnostic markers in normal tissue and colon cancer tissue by RT-PCR (odd number lanes: normal tissue, even number lanes: cancer tissue) ;
  • FIG. 9 is the result of electrophoresis to analyze the expression levels of CEACAM6, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, and CTHRCl diagnostic markers in normal tissue and colon cancer tissue by RT-PCR (odd number lanes: normal tissue, even number lanes: cancer tissue);
  • FIG. 10 is the result of electrophoresis to analyze the expression levels of HS6ST2, EGFL ⁇ , LCN2, MMPl, and CA9 diagnostic markers in normal tissue and colon cancer tissue by RT-PCR (odd number lanes: normal tissue, even number lanes: cancer tissue); FIG.
  • 11 is the result of electrophoresis to analyze the expression levels of ANLN, CEACAM ⁇ , CSTl, CTHRCl, DPEPl, KLK ⁇ , and MELK diagnostic markers in 10 types of colon cancer cell lines (Lane 1, DLD-I; Lane 2, HT29; Lane 3, HCTll ⁇ ; Lane 4, colo205; Lane 5, SW480; Lane 6, SW620; Lane 7, SNU Cl; Lane 8, SNU C2A; Lane 9,KM 12C; Lane 10, KM 12SM) by RT-PCR;
  • FIG. 12 is the result of Western blotting showing the expression of KLK ⁇ in the sera from healthy subjects and colon cancer patients; and FIGs. 13 to 17 are photographs showing the protein expression in normal mucosa (left) and colon cancer tissue (right) by immunohistostaining, in which FIG. 13, 14, 15, and 16 are the results of immunohistostaining by using CEACAM6, SPPl, IFITMI 55-1-8, CKS2, and KLK ⁇ antibodies, respectively.
  • the present invention relates to one or more diagnostic markers for colon cancer, selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9.
  • diagnostic markers for colon cancer selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9.
  • diagnosis refers to evaluation of the presence or properties of pathological states. With respect to the objects of the present invention, the diagnosis is to determine the incidence of colon cancer.
  • colon cancer refers to malignancy that arises from the mucosa, inner lining of the colon, and includes cancerous growths in the colon, rectum and an
  • diagnosis marker is intended to indicate a substance capable of diagnosing colon cancer by distinguishing colon cancer cells from normal cells, and includes organic biomolecules, of which quantities are increased in colon cancer cells relative to normal cells, such as polypeptides, nucleic acids (mRNA, etc. ) , lipids, glycolipids, glycoproteins, and sugars (monosaccharide, disaccharide, oligosaccharides, etc.) .
  • organic biomolecules of which quantities are increased in colon cancer cells relative to normal cells, such as polypeptides, nucleic acids (mRNA, etc. ) , lipids, glycolipids, glycoproteins, and sugars (monosaccharide, disaccharide, oligosaccharides, etc.) .
  • the diagnostic markers for colon cancer are KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM6, MMPl, LCN2, HS6ST2, EGFL ⁇ , or CA9, which are up-regulated in colon cancer cells.
  • the selection and application of significant diagnostic markers determine the reliability of diagnosis results.
  • a significant diagnostic marker means a marker that has high validity, giving accurate diagnosis results, and high reliability, supplying constant results upon repeated measurement.
  • the diagnostic markers for colon cancer of the present invention display the same results upon repeated tests, and have high reliability due to a great difference in expression levels compared to a control, thus having a very low possibility of giving false results. Therefore, diagnosis based on the results obtained by measuring the expression levels of the significant diagnostic markers of the present invention is valid and reliable.
  • diagnosis based on the results obtained by measuring the expression levels of the significant diagnostic markers of the present invention is valid and reliable.
  • the genes that are up-regulated by 2-9 fold in colon cancer tissues were selected.
  • the present invention relates to a diagnostic composition for colon cancer, comprising an agent measuring expression or protein levels of one or more genes selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM6, MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9.
  • Gene expression levels in biological samples may be determined by measuring mRNA or protein levels.
  • the term "measurement of mRNA expression levels”, as used herein, is a process of assessing the presence and expression levels of mRNA of colon cancer marker genes in biological samples for diagnosing colon cancer, in which the amount of mRNA is measured. Analysis methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) , Northern blotting and DNA chip assay.
  • the "measurement of protein expression levels” is a process of assessing the presence and expression levels of proteins expressed from colon cancer marker genes in biological samples for diagnosing colon cancer, in which the amount of protein products of the marker genes is measured using antibodies specifically binding to the proteins.
  • Analysis methods for measuring protein levels include, but are not limited to, Western blotting, ELISA (enzyme linked immunosorbent assay) , radioimmunoassay (RIA) , radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS) , and protein chip assay.
  • the present invention provides a diagnostic composition for colon cancer, comprising a primer sequence specific to one or more genes selected from KLK6, CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9.
  • primer means a short nucleic acid sequence having a free 3' hydroxyl group, which is able to form base-pairing interaction with a complementary template and serves as a starting point for replication of the template strand.
  • a primer is able to initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at suitable buffers and temperature.
  • the primers of the present invention specific to each of the marker genes, are sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides.
  • the primer may have additional properties that do not change the nature of the primer to serve as a starting point for DNA synthesis.
  • the primers of the present invention may be chemically synthesized using a phosphoramidite solid support method or other widely known methods. These nucleic acid sequences may also be modified using many means known in the art . Non-limiting examples of such modifications include methylation, capsulation, replacement of one or more native nucleotides with analogues thereof, and inter-nucleotide modifications, for example, modifications to uncharged conjugates (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged conjugates (e.g., phosphorothioate, phosphorodithioate, etc.) .
  • uncharged conjugates e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.
  • charged conjugates e.g., phosphorothioate, phosphorodithioate, etc.
  • Nucleic acids may contain one or more additionally covalently-bonded residues, which are exemplified by proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralene, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidative metals, etc.) , and alkylating agents.
  • proteins e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • intercalating agents e.g., acridine, psoralene, etc.
  • chelating agents e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • alkylating agents e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • the composition for detecting a diagnostic marker for colon cancer includes a pair of primers specific to one or more genes selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM6, MMPl, LCN2, HS6ST2, EGFL ⁇ , andCA9, inparticular, primers represented by SEQ ID NOs. 1 to 36.
  • the present invention provides a diagnostic composition for colon cancer, comprising an antibody specific to one or more proteins selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl,
  • antibody refers to a specific proteinmolecule that indicates an antigenic region. With respect to the objects of the present invention, an antibody specifically binds to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies.
  • Antibody production using the colon cancer marker proteins identified as described above may be easily carried out using techniques widely known in the art.
  • Polyclonal antibodies may be produced by a method widely known in the art, which includes injecting the colon cancer marker protein antigen into an animal and collecting blood samples from the animal to obtain serum containing antibodies .
  • Such polyclonal antibodies may be prepared from a certain animal host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows and dogs.
  • Monoclonal antibodies may be prepared by a method widely known in the art, such as a hybridoma method (see, Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or a phage antibody library technique (Clackson et al . , Nature, 352: 624-628, 1991; Marks et al, J . MoI. Biol., 222: 58, 1-597, 1991) .
  • Antibodies prepared by the above methods are isolated using gel electrophoresis, dialysis, salting out, ion exchange chromatography, affinity chromatography, and the like.
  • the antibodies of the present invention include complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules.
  • the functional fragments of antibody molecules refer to fragments retaining at least an antigen-binding function, and include Fab, F(ab'), F(ab')2, Fv or the like.
  • the present invention provides a diagnostic kit for colon cancer, comprising the diagnostic composition for colon cancer according to the present invention.
  • the diagnostic kit of the present invention is preferably composed of a composition, solution or apparatus, which includes one or more kinds of different constituents suitable for analysis methods.
  • the diagnostic kit may be a diagnostic kit characterized by including essential elements required for performing RT-PCR.
  • An RT-PCR kit includes a pair of primers specific for each marker gene.
  • the primers are nucleotides having sequences specific to a nucleic acid sequence of each marker gene, and are about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length.
  • the RT-PCR kit may include primers specific to a nucleic acid sequence of a control gene.
  • the RT-PCR kit may further include test tubes or other suitable containers, reaction buffers (varying in pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor, DEPC-treated water, and sterile water.
  • reaction buffers varying in pH and magnesium concentrations
  • dNTPs deoxynucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase
  • DNAse DNAse
  • RNAse inhibitor RNAse inhibitor
  • DEPC-treated water DEPC-treated water
  • sterile water sterile water
  • the present invention relates to a diagnostic kit, characterized by including essential elements required for performing a DNA chip assay.
  • a DNA chip kit may include a base plate onto which genes or fragments thereof, cDNA or oligonucleotides, are attached, and reagents, agents and enzymes for preparing fluorescent probes.
  • the base plate may include cDNA or oligonucleotides corresponding to a control gene or fragments thereof.
  • the present invention relates to a diagnostic kit, characterized by including essential elements required for performing ELISA.
  • An ELISA kit includes antibodies specific to marker proteins.
  • the antibodies are monoclonal, polyclonal or recombinant antibodies, which have high specificity and affinity to each marker protein and rarely have cross-reactivity to other proteins.
  • the ELISA kit may include an antibody specific to a control protein.
  • the ELISA kit may further include reagents capable of detecting bound antibodies, for example, a labeled secondary antibody, chromophores, enzymes (e.g., conjugated with an antibody) and their substrates, or other substances capable of binding to the antibodies .
  • the present invention provides a method of diagnosing colon cancer, using the diagnostic composition or kit for colon cancer.
  • the present invention provides a method of diagnosing colon cancer, comprising the steps of measuring mRNA levels in a biological sample from a patient with suspected colon cancer using primers specific to one or more genes selected from KLK ⁇ , CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9; and comparing mRNA levels of the sample from the patient with those of a normal control sample to determine an increase in mRNA levels.
  • the isolation of mRNA from a biological sample may be achieved using a known process, and mRNA levels may be measured by a variety of methods.
  • biological sample includes samples displaying a difference in expression levels of a colon cancer marker gene, such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, but is not limited thereto.
  • Analysis methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR,
  • RNase protection assay RPA
  • Northern blotting RNase protection assay
  • the mRNA expression level of a colon cancer marker gene in a patient with suspected colon cancer is compared with that in a normal control, and the patient's suspected colon cancer is diagnosed by determining whether expression levels of mRNA from the colon cancer marker gene have significantly increased.
  • mRNA expression levels are preferably measured by RT-PCR or DNA chip assay using primers specific to a gene as a colon cancer marker.
  • RT-PCR products are electrophoresed, and patterns and thicknesses of bands are analyzed to determine the expression and levels of mRNA from a gene used as a diagnostic marker of colon cancer while comparing the mRNA expression and levels with those of a control, thereby simply diagnosing the incidence of colon cancer (FIGs.8 to 11) .
  • mRNA expression levels are measured using a DNA chip in which the colon cancer marker genes or fragments thereof are anchored at high density to a glass-like base plate.
  • a cDNA probe labeled with a fluorescent substance at its end or internal region is prepared using mRNA isolated from a sample, and is hybridized with the DNA chip. The DNA chip is then read to determine the presence or expression levels of the gene, thereby diagnosing the incidence of colon cancer .
  • the present invention provides a method of diagnosing colon cancer, comprising the steps of measuring protein levels by contacting an antibody specific to one or more proteins selected from KLK6, CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9 with a biological sample from a patient with suspected colon cancer to form antigen-antibody complexes, and comparing protein levels of the sample from the patient with those of a normal control sample to determine an increase in protein levels.
  • an antibody specific to one or more proteins selected from KLK6, CKS2, IFITMl, SPPl, DPEPl, CSTl, CDH3, ANLN, CXCLl, MELK, CDCAl, CTHRCl, CEACAM ⁇ , MMPl, LCN2, HS6ST2, EGFL ⁇ , and CA9
  • the isolation of proteins from a biological sample may be achieved using a known process, and protein levels may be measured by a variety of methods.
  • Analysis methods for measuring protein levels include, but are not limited to, Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS , and protein chip assay.
  • a patient with suspected colon cancer is compared with a normal control for the amount of formed antigen-antibody complexes, and the patient's suspected colon cancer is diagnosed by evaluating a significant increase in expression levels of a protein from the colon cancer marker gene.
  • antigen-antibody complexes refers to binding products of a colon cancer marker protein to an antibody specific thereto.
  • the amount of formed antigen-antibody complexes may be quantitatively determined by measuring the signal intensity of a detection label.
  • a detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, but the present invention is not limited to the examples.
  • enzymes available as detection labels include, but are not limited to, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urase, peroxidase or alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphenolpyruvate decarboxylase, and ⁇ -latamase.
  • fluorescent substances include, but are not limited to, fluorescin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamin.
  • ligands include, but are not limited to, biotin derivatives.
  • luminescent substances include, but are not limited to, acridinium esters, luciferin and luciferase .
  • micropartides include, but are not limited to, colloidal gold and colored latex.
  • redox molecules examples include, but are not limited to, ferrocene, ruthenium complexes, viologen, quinone, Ti ions, Cs ions, diimide, 1, 4-benzoquinone, hydroquinone, K 4 W (CN) 8 , [Os (bpy) 3 ] 2+ , [RU (bpy) 3 ] 2+ , [MO(CN) 8 ] 4" .
  • the radioactive isotopes include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • the protein expression levels are measured by ELISA.
  • ELISA include direct ELISA using a labeled antibody recognizing an antigen immobilized on a solid support, indirect ELISA using a labeled antibody recognizing a capture antibody forming complexes with an antigen immobilized on a solid support, direct sandwich ELISA using another labeled antibody recognizing an antigen in an antigen-antibody complex immobilized on a solid support, and indirect sandwich ELISA, in which another labeled antibody recognizing an antigen in an antigen-antibody complex immobilized on a solid support is reacted, and then a secondary labeled antibody recognizing the another labeled antibody is used.
  • the protein expression levels are detected by sandwich ELISA, where a sample reacts with an antibody immobilized on a solid support, and the resulting antigen-antibody complexes are detected by adding a labeled antibody specific for the antigen, followed by enzymatic development, or by first adding an antigen-specific antibody and then a secondary labeled antibody which binds to the antigen-specific antibody, followed by enzymatic development.
  • the incidence of colon cancer may be diagnosed by measuring the degree of complex formation of a colon cancer marker protein and an antibody thereto.
  • the protein expression levels are preferably measured by Western blotting using one or more antibodies to the colon cancer markers .
  • Total proteins are isolated from a sample, electrophoresed to be separated according to size, transferred onto a nitrocellulose membrane, and reacted with an antibody.
  • the amount of proteins produced by gene expression is determined by measuring the amount of produced antigen-antibody complexes using a labeled antibody, thereby diagnosing the incidence of colon cancer.
  • the detection methods are composed of methods of assessing expression levels of marker genes in a control and cells in which colon cancer occurs.
  • mRNA or protein levels may be expressed as an absolute (e.g., ⁇ g/ml) or relative (e.g., relative intensity of signals) difference in the amount of marker proteins (FIG. 12) .
  • the protein expression levels are preferably measured by immunohistostaining using one or more antibodies to the colon cancer markers.
  • Normal colon epithelial tissues and cancer tissues were collected and fixed, and then paraffin-embedded blocks were prepared according to a widely known method. The blocks were cut into small sections (several urn in thickness), and attached to glass slides to be reacted with one selected from the antibodies according to a known method. Subsequently, the unreacted antibodies were washed, and the reacted antibodies were labeled with one selected from the above mentioned detection labels, and then observed under a microscope (FIGs. 13 to 17) .
  • a DNA chip 48Khumanmicroarray, Illumina was used to analyze the expression levels of 2,230 genes.
  • RNAs were extracted from the normal colon epithelial and colon cancer cells. Extraction of the total RNA was performed using an RNeasy Mini Kit (QIAGEN) , and quantified using an Experion RNA StdSens (Bio-Rad) chip. For hybridization, the extracted total RNA was labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion) . cDNA was prepared using a T7 Oligo (dT) primer, and in vitro transcription was performed using a biotin-UTP to prepare a biotin-labeled cRNA. The prepared cRNA was quantified using NanoDrop.
  • cRNAs that were generated from the normal colon epithelial and colon cancer cells were hybridized to a Human- ⁇ V2 (Illumina) chip. Then, to remove non-specific hybridization, the DNA chip was washed with an Illumina Gene Expression System Wash Buffer (Illumina) , and the washed DNA chip was labeled with streptavidin-Cy3 (Amersham) . The fluorescent-labeled DNA chip was scanned using a confocal laser scanner (Illumina), and fluorescent data present in each spot were saved with TIFF image files. TIFF image files were analyzed by Illumina BeadStudio version 3 (Illumina) to assess spot fluorescent intensities. Differences in spot intensities were normalized using ⁇ quantile' with Avadis Prophetic version 3.3 program (Strand Genomics) .
  • genes showing the expression level of 2 fold or more were observed in 60% of patients (Table 4), in which high and low expressions are 281 and 605, respectively.
  • the expression levels of the same genes were also examined in stomach cancer, breast cancer, and pancreatic cancer (FIG. 7) to finally screen the genes that are specifically expressed in colon cancer (FIGs. 3 to 6) .
  • RNA samples normal colon epithelial tissues and colon cancer tissues were taken from 20 patients with colon cancer, and mRNAs were isolated from the total 40 tissues.
  • the tissues were immediately washed with sterile phosphate buffered saline to remove blood, and frozen in liquid nitrogen.
  • total RNA was isolated by a guanidinium method, and single-step RNAisolation was performed. The isolated total RNA was quantified using a spectrophotometer, and then stored at a -70 0 C freezer.
  • Total 10 colon cell lines (DLD-I, HT29, HCT116, colo205, SW480, SW620, SNU Cl, SNU C2A, KM 12C, KM 12SM) were selected and obtained from Korean Cell Line Bank (KCLB: 28 Yongon-dong, Chongno-gu, Seoul, Korea) .
  • Each cell line was cultured in a suitable medium, DMEM (Invitrogen) or RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclon) and Penicillin/Streptomycin (1 mg/ml, Sigma) for 5-6 days, and then total RNA was isolated by a guanidiniummethod, as described above, to perform the single-step RNA isolation.
  • the isolated RNA was quantified using a spectrophotometer, and then stored at a -70 0 C freezer until use.
  • IFITM 1 Forward ATG TCG CTCT GGT CCC TGT TC 224bp
  • CEACAM 6 Forward CGC ATA CAG TGG TCG AGA GA 318bp
  • Reverse TCT CAT CTG CAC AAG GAA CG cDNA was constructed by RT-PCR using mRNA that was extracted from the tissues and cell lines in Example 2.
  • the cDNA construction was performed using an AccuScript High Fidelity 1st Stand cDNA Synthesis Kit (STRATAGENE) , and the resulting cDNA and primers in Table 5 were used for RT-PCR.
  • FIGs. 8 to 10 differences in the expression levels between normal and colon cancer cells were observed, and their expressions were also observed in colon cancer cell lines, as shown in FIG. 11.
  • samples were isolated from the blood of colon cancer patients and healthy subjects, and boiled in the same volume of sample buffer solution (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% BME) for 5 min. Then, proteins were separated using a 12% SDS-PAGE. The SDS-PAGE to separate the proteins according to their size was contacted with a nitrocellulose membrane, and an electric current was supplied to transfer the proteins to the nitrocellulose membrane.
  • sample buffer solution 125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% BME
  • KLK ⁇ antibodies (ABNOVA, 1:2000) that were reacted in a TBST solution (10 mM Tris, 100 mM sodium chloride, 0.05% Tween 20) containing 3% fetal bovine serum albumin for 30 min were added thereto, and stirred at 4 0 C for 2 hrs . Then, the membrane was washed with TBST to remove residual antibodies, and HRP-conjugated secondary antibodies (ABCAM, Rabbit polyclonal to Mouse IgG) was added thereto, followed by stirring at 4°C for 1 hr.
  • a TBST solution 10 mM Tris, 100 mM sodium chloride, 0.05% Tween 20
  • HRP-conjugated secondary antibodies (ABCAM, Rabbit polyclonal to Mouse IgG) was added thereto, followed by stirring at 4°C for 1 hr.
  • the nitrocellulose membrane was stirred in a mixture of solution A (containing Luminol and enhancer) and Solution B (containing Hydrogen peroxide) in the same amount for 1 min, and water was removed therefrom.
  • the nitrocellulose membrane was placed in a film cassette, and the film was developed in a dark room.
  • the KLK ⁇ protein was not detected in healthy subjects (Lanes 2, 3, and 4), but the KLK ⁇ protein was detected in colon cancer patients (Lanes ⁇ , 7, and 8) .
  • tissue slides were subjected to immunostaining.
  • thenormal colon epithelial tissues and colon cancer tissues were taken from colon cancer patients by surgical resection, and paraffin embedded blocks were prepared. The blocks were sectioned using a microtome in a thickness of 5 urn, and attached to glass slides to prepare tissue slides. The tissue slides were stained with a known immunostaining method, and the expression of proteins and their location in the tissues were observed under a microscope.
  • CEACAM 6 (ABCAM, 1:5000), SPPl (ABNOVA, 1:2000), IFITM l 55-1-8 (SANTA CRUZ, 1:1000), and CKS 2 (ZYMED Laboratories, 1:2000) .
  • SPPl ABNOVA, 1:2000
  • IFITM l 55-1-8 SANTA CRUZ, 1:1000
  • CKS 2 ZYMED Laboratories, 1:2000
  • FIG. 16 was observed in the nucleus
  • KLK ⁇ expression (FIG. 17) was observed in cytoplasm and cell membrane. In addition, they were detected in the blood of colon cancer patient, suggesting that they were secreted into the blood. All five types of antibodies were more highly detected in the tumor than in the normal colon mucosa.
  • CEACAM6 (FIG. 13) and SPPl (FIG. 14) were thought to be secreted into the blood, and the possibility that IFITMl
  • FIG. 15 CEACAM ⁇ and SPPl showed a positive reaction in cytoplasm, cell membrane, and lumen of glandular structures, suggesting high possibility of secretion.
  • IFITMl and KLK ⁇ expressions were not observed in glandular structures, suggesting no possibility of secretion. However, their structure implies the possibility of secretion, and in immunohistochemical staining, low levels of expression were observed in cytoplasm or cell membrane, which also indicates the possibility.

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Abstract

La présente invention concerne des marqueurs de diagnostic spécifiques du cancer du colon. De plus, la présente invention concerne une composition ou un kit comportant un agent mesurant la présence des marqueurs, et un procédé de diagnostic du cancer du colon l'utilisant.
PCT/KR2007/006108 2007-10-30 2007-11-29 Marqueurs de diagnostic du cancer du colon utilisant des gènes régulés de façon positive WO2009057849A1 (fr)

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WO2011100453A2 (fr) * 2010-02-10 2011-08-18 Maine Medical Center Compositions et procédés pour le traitement d'une maladie cardiaque et vasculaire
WO2012046064A3 (fr) * 2010-10-07 2012-09-27 The University Of York Différenciation cellulaire
WO2014150720A1 (fr) * 2013-03-15 2014-09-25 The Regents Of The University Of Michigan Compositions et procédés associés à l'inhibition de la croissance et/ou de la prolifération de cellules cancéreuses
EP2751561A4 (fr) * 2011-08-31 2015-08-12 Oncocyte Corp Méthodes et compositions pour le traitement et le diagnostic du cancer colorectal
CN106526203A (zh) * 2016-11-22 2017-03-22 郎秀玲 一种用于左半结肠癌检测的试剂盒
WO2017184059A1 (fr) * 2016-04-20 2017-10-26 Hiloprobe Ab Gènes marqueurs pour la classification du cancer colorectal, procédé d'évaluation de métastase des ganglions lymphatiques pour le pronostic du cancer colorectal et kit associé
US20180313846A1 (en) * 2017-04-03 2018-11-01 Maine Medical Center Blood test to predict endurance athletic performance
CN109402227A (zh) * 2017-08-08 2019-03-01 安徽普元生物科技股份有限公司 一步法实时荧光定量逆转录聚合酶链反应检测人基质金属蛋白酶1的试剂盒
US11578121B2 (en) 2017-10-01 2023-02-14 Taipei Medical University Anti-EGF like domain multiple 6 (EGFL6) antibodies

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US9718878B2 (en) 2010-02-10 2017-08-01 Maine Medical Center Kit for detecting Cthrc1 in a sample
WO2011100453A3 (fr) * 2010-02-10 2011-12-08 Maine Medical Center Compositions et procédés pour le traitement d'une maladie cardiaque et vasculaire
WO2011100453A2 (fr) * 2010-02-10 2011-08-18 Maine Medical Center Compositions et procédés pour le traitement d'une maladie cardiaque et vasculaire
WO2012046064A3 (fr) * 2010-10-07 2012-09-27 The University Of York Différenciation cellulaire
EP2751561A4 (fr) * 2011-08-31 2015-08-12 Oncocyte Corp Méthodes et compositions pour le traitement et le diagnostic du cancer colorectal
WO2014150720A1 (fr) * 2013-03-15 2014-09-25 The Regents Of The University Of Michigan Compositions et procédés associés à l'inhibition de la croissance et/ou de la prolifération de cellules cancéreuses
US9850300B2 (en) 2013-03-15 2017-12-26 The Regents Of The University Of Michigan Compositions and methods relating to inhibiting cancer cell growth and/or proliferation
WO2017184059A1 (fr) * 2016-04-20 2017-10-26 Hiloprobe Ab Gènes marqueurs pour la classification du cancer colorectal, procédé d'évaluation de métastase des ganglions lymphatiques pour le pronostic du cancer colorectal et kit associé
US10988811B2 (en) 2016-04-20 2021-04-27 Hiloprobe Ab Marker genes for colorectal cancer classification, method for judging lymph node metastasis for prognosis of colorectal cancer and kit therefor
CN106526203A (zh) * 2016-11-22 2017-03-22 郎秀玲 一种用于左半结肠癌检测的试剂盒
US20180313846A1 (en) * 2017-04-03 2018-11-01 Maine Medical Center Blood test to predict endurance athletic performance
CN109402227A (zh) * 2017-08-08 2019-03-01 安徽普元生物科技股份有限公司 一步法实时荧光定量逆转录聚合酶链反应检测人基质金属蛋白酶1的试剂盒
US11578121B2 (en) 2017-10-01 2023-02-14 Taipei Medical University Anti-EGF like domain multiple 6 (EGFL6) antibodies

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