WO2006080597A1 - Markers for the diagnosis of lung cancer - Google Patents

Markers for the diagnosis of lung cancer Download PDF

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Publication number
WO2006080597A1
WO2006080597A1 PCT/KR2005/000279 KR2005000279W WO2006080597A1 WO 2006080597 A1 WO2006080597 A1 WO 2006080597A1 KR 2005000279 W KR2005000279 W KR 2005000279W WO 2006080597 A1 WO2006080597 A1 WO 2006080597A1
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WIPO (PCT)
Prior art keywords
lung cancer
genes
antibody
pkpl
abcc5
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PCT/KR2005/000279
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French (fr)
Inventor
Jeong Ho Yoon
Se Nyun Kim
Jong Ho Park
Ja-Eun Kim
Young-Hwa Song
Sung Han Kim
Dong Yoon Park
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Digital Genomics Inc.
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Application filed by Digital Genomics Inc. filed Critical Digital Genomics Inc.
Priority to US11/815,198 priority Critical patent/US7871774B2/en
Priority to PCT/KR2005/000279 priority patent/WO2006080597A1/en
Priority to JP2007553017A priority patent/JP2008528024A/en
Priority to KR1020050015045A priority patent/KR100690250B1/en
Publication of WO2006080597A1 publication Critical patent/WO2006080597A1/en

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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
    • 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 for lung 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 lung cancer using the composition or kit .
  • Lung cancer is a leading cause of cancer death worldwide. Lung cancer is responsible for about one-sixth of all cancer deaths .
  • lung cancer There are two major types of lung cancer: small cell lung cancer and non-small cell lung cancer .
  • Non-small cell lung cancer is the representative lung cancer, which accounts for about 80% of all lung cancer .
  • Adenocarcinoma, squamous cell carcinoma and large cell carcinoma are three types of non-small cell lung cancer . Since there are differences in histological properties as well as prognosis and therapy according to the type of lung cancer, accurate diagnosis is important .
  • ten-year survival rates of patients with non-small cell lung cancer are 10% or even less .
  • the present inventors in order to develop biomarkers capable of simply and accurately diagnosing lung cancer, performed primary screening for genes overexpressed only in lung cancer using a DNA chip, and identified highly significant markers by performing RT-PCR.
  • PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 genes were identified .
  • the genes were practically applied to lung cancer samples, they were found to accurately diagnose lung cancer, thereby leading to the present invention.
  • a diagnostic marker of lung cancer which is at least one selected from among PKPl (plakophilin 1) , ABCC5
  • KRT15 keratin 15
  • KRT14 keratin 14
  • TRIM29 tripartite motif-containing 29
  • SERPINB5 serine (or cysteine) proteinase inhibitor
  • clade B ovalbumin
  • TKl thymidine kinase 1, soluble
  • GPX2 glutthione peroxidase
  • MMPl matrix metalloproteinase 1
  • ITGB4 integrated protein, beta 4
  • compositions for detecting a diagnostic marker of lung cancer comprising a pair of primers specific for one or more genes selected from among PKPl, ABCC5, KRT15, KRT14 ,
  • FIG . 1 shows the clustering of 57 tumorous and 40 normal lung tissues using gene expression data .
  • Tumorous lung tissues are represented by "Tumor (black rectangular shape) "
  • normal lung tissues are represented by "Normal (white rectangular shape) " .
  • the left white bar indicates a group in which most of the normal lung tissues cluster together
  • the right black bar indicates another group in which the tumor lung tissues cluster together .
  • FIG . 2 shows a 3-D visualization of gene expression patterns by multidimensional scaling .
  • the black circle indicates tumorous lung tissues
  • the gray circle indicates normal lung tissues .
  • Spots placed near each other in three dimensional space indicate that they have similar gene expression patterns .
  • Tumorous lung tissues form one cluster
  • normal lung tissues form another cluster, indicating that each of the tumorous and normal lung tissues has a specific gene expression pattern .
  • FIG . 3 shows the results of RT-PCR for confirming the difference in expression levels between tumorous and normal lung tissues .
  • Ten genes are expressed not in normal lung tissues but in tumorous lung tissues .
  • the present invention relates to a lung cancer diagnostic marker selected from PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
  • diagnosis refers to evaluation of the presence or properties of pathological states . With respect to the obj ects of the present invention, the diagnosis is to determine the incidence of lung cancer .
  • lung cancer refers to malignant tumor occurring in the lung, and includes both small cell lung cancer and non-small cell lung cancer including adenocarcinoma, squamous cell carcinoma and large cell carcinoma .
  • marker for diagnosing is intended to indicate a substance capable of diagnosing lung cancer by distinguishing lung cancer cells from normal cells, and includes organic biological molecules, quantities of which are increased or decreased in lung cancer cells relative to normal cells , such as polypeptides or nucleic acids (e . g . , mRNA, etc . ) , lipids , glycolipids, glycoproteins and sugars
  • lung cancer diagnostic markers are PKPl , ABCC5 , KRT15 , KRT14 ,
  • TRIM29 SERPINB5
  • TKl TKl
  • GPX2 GPX2
  • MMPl MMPl
  • ITGB4 ITGB4
  • 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 lung cancer diagnostic markers of the present invention which are genes whose expression always increases by direct or indirect factors when lung cancer occurs , 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 .
  • the PKPl , ABCC5 , KRT15, KRT14 , TRIM29, SERPINB5, TKl , GPX2 , MMPl and ITGB4 genes of the present invention may be used as diagnostic markers of lung cancer because they are expressed at high levels specifically in lung cancer cells compared to cells of normal lung tissue . All of the genes are useful as diagnostic markers of lung cancer . However, with respect to the present obj ect of providing rapid, simple and accurate markers, it is preferable to detect a limited number of markers make a medical decision . This is economical in terms of preventing waste of time and resources . Since PKPl and ABCC5 are expressed specifically in lung cancer cells, they have very high reliability allowing diagnosis of lung cancer even when used alone . Therefore, the diagnosis of lung cancer is preferably carried out using either PKPl or ABCC5 alone, or both of them, as markers .
  • the diagnosis of lung cancer may be carried out using one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 , as markers .
  • genes expressed at almost the same levels in cells of normal lung tissue and lung cancer cells for example, DCK (deoxycytidine kinase) and SEP15 ( selenoprotein, 15-KD) , may be used as quantitative controls .
  • the present invention relates to a kit for detecting a diagnostic marker of lung cancer, comprising an agent measuring mRNA or protein levels of one or two genes selected from PKPl and ABCC5.
  • the kit may further include an agent measuring mRNA or protein levels of one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
  • Gene expression levels of biological samples may be determined by measuring mRNA or protein levels .
  • measurement of mRNA expression levels is a process of assessing the presence and expression levels of mRNA of lung cancer marker genes in biological samples for diagnosing lung 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”, as used herein, is a process of assessing the presence and expression levels of proteins expressed from lung cancer marker genes in biological samples for diagnosing lung 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, enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis , immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip assay .
  • the detection kit of the present invention is composed of a composition, solution or apparatus , which includes one or more kinds of different constituents suitable for analysis methods .
  • the present invention relates to a kit for detecting a diagnostic marker, which is 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 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 .
  • dNTPs deoxynucleotides
  • enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor, DEPC- treated water, and sterile water.
  • the present invention relates to a diagnostic kit, which is 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 a control gene or fragments thereof, such as cDNA or oligonucleotides .
  • the present invention relates to a diagnostic kit, which is 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 . , conj ugated with an antibody) and their substrates, or other substances capable of binding to the antibodies .
  • An RT-PCR kit for detecting lung cancer markers includes a pair of primers specific to one to two genes selected from PKPl and ABCC5. Also, the RT-PCR kit may include a pair of primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5 , TKl, GPX2 , MMPl and ITGB4 genes .
  • a DNA chip kit for detecting lung cancer markers includes a base plate onto which cDNA corresponding to one to two genes selected from PKPl and ABCC5, or fragments thereof, is attached . Also, cDNA corresponding to one to eight genes selected from among KRT15, KRT14 , TRIM29,
  • SERPINB5 TKl , GPX2 , MMPl and ITGB4 genes , or fragments thereof, may be attached and immobilized onto a base plate .
  • An ELISA kit for detecting lung cancer markers includes an antibody specific to one or two proteins selected from PKPl and ABCC5. Also, the ELISA kit may include an antibody specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
  • the present invention relates to a composition for detecting a diagnostic marker of lung cancer, comprising primers specific to one or two genes selected from PKPl and ABCC5.
  • composition may further include primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
  • 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 nucleosides 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 conj ugates (e . g . , methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc . ) or charged conj ugates (e . g . , phosphorothioate, phosphorodithioate, etc . ) .
  • uncharged conj ugates e . g . , methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc .
  • charged conj ugates e . g . , phospho
  • Nucleic acids may contain one or more additionally covalent-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 .
  • the nucleic acid sequences of the present invention may also be altered using a label capable of directly or indirectly supplying a detectable signal . Examples of the label include radioisotopes , fluorescent molecules and biotin .
  • the composition for detecting a diagnostic marker of lung cancer includes a pair of primers specific to one to two genes selected from PKPl and ABCC5.
  • Primers for amplifying PKPl SEQ ID NO . 1
  • Primers for amplifying ABCC5 are preferably represented by SEQ ID NOS . 2 and 3
  • compositions may further include a pair of primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 genes .
  • Primers for amplifying KRT15 (SEQ ID NO . 7 ) are preferably represented by SEQ ID NOS . 8 and 9.
  • Primers for amplifying KRT14 ( SEQ ID NO . 10 ) are preferably represented by SEQ ID NOS . 11 and 12.
  • Primers for amplifying TRIM29 (SEQ ID NO . 13 ) are preferably represented by SEQ ID NOS . 14 and 15.
  • Primers for amplifying SERPINB5 are preferably represented by SEQ ID NOS . 17 and 18.
  • Primers for amplifying TKl are preferably represented by SEQ ID NOS . 20 and 21.
  • Primers for amplifying GPX2 are preferably represented by SEQ ID NOS . 23 and 24.
  • Primers for amplifying MMPl are preferably represented by SEQ ID NOS . 26 and 27.
  • Primers for amplifying ITGB4 (SEQ ID NO . 28 ) are preferably represented by SEQ ID NOS . 29 and 30.
  • the present invention relates to a composition for detecting a diagnostic marker of lung cancer, comprising an antibody specific to one or two proteins selected from PKPl and ABCC5.
  • the above composition may further include an antibody specific to one to eight proteins selected from among KRT15, KRT14 , TRIM29, SERPINB5 , TKl , GPX2 , MMPl and ITGB4.
  • an antibody refers to a specific protein molecule that indicates an antigenic region .
  • an antibody binds specifically to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies .
  • Antibody production using the lung 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 inj ecting the lung 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 ) .
  • the hybridoma method employs cells from an immunologically suitable host animal injected with a diagnostic marker protein of lung cancer as an antigen, such as mice, and a cancer or myeloma cell line as another group .
  • a diagnostic marker protein of lung cancer as an antigen
  • a cancer or myeloma cell line as another group .
  • Cells of the two groups are fused with each other by a method widely known in the art, for example, using polyethylene glycol , and antibody-producing cells are proliferated by a standard tissue culture method . After uniform cell colonies are obtained by subcloning using a limited dilution technique, hybridomas capable of producing an antibody specific for the diagnostic marker protein of lung cancer are cultivated in large scale in vitro or in vivo according to a standard technique .
  • Monoclonal antibodies produced by the hybridomas may be used in an unpurified form, but are preferably used after being highly purified by a method widely known in the art so as to obtain best results .
  • the phage antibody library method includes constructing a phage antibody library in vitro by obtaining genes for antibodies ( single-chain fragment variable ( scFv) ) to a variety of intracellular lung cancer markers and expressing them in a fusion protein form on the surface of phages, and isolating monoclonal antibodies binding to lung cancer-specific proteins from the library .
  • scFv single-chain fragment variable
  • 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 and the like.
  • the composition for detecting a lung cancer marker includes an antibody specific for one to two proteins selected from PKPl and ABCC5. Also, the composition may further include an antibody specific to one to eight proteins selected from among KRTl5, KRTl4 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
  • the present invention relates to a method of diagnosing lung cancer, comprising measuring mRNA levels in a biological sample from a patient with suspected lung cancer using primers specific to one or two genes selected from PKPl and ABCC5, 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 above method may further include diagnosing lung cancer using primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
  • 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, but is not limited to, samples displaying a difference in expression levels of a lung cancer marker gene, such as tissues , cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine .
  • Analysis methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, realtime RT-PCR, RNase protection assay (RPA) , Northern blotting and DNA chip assay.
  • a patient with suspected lung cancer is compared with a normal control for mRNA expression levels of a lung cancer marker gene, and the patient' s suspected lung cancer is diagnosed by determining whether expression levels of mRNA from the lung cancer marker gene have significantly increased .
  • mRNA expression levels are preferably measured by RT- PCR using primers specific to a gene as a lung cancer marker .
  • RT-PCR is a method that was introduced to analyze RNA by P . Seeburg (Cold Spring Harb Symp Quant Biol 1986, Pt 1 : 669-677 ) , with which cDNA is synthesized from mRNA by reverse transcription and amplified by PCR.
  • a pair of primers prepared in a fashion specific to a diagnostic marker of lung cancer is used .
  • 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 lung cancer while comparing the mRNA expression and levels with those of a control, thereby simply diagnosing the incidence of lung cancer .
  • mRNA expression levels are measured using a DNA chip in which the lung cancer marker genes or nucleic acid 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 lung cancer .
  • the present invention relates to a method of diagnosing lung cancer, comprising measuring protein levels by contacting an antibody specific to one or two genes selected from PKPl and ABCC5 with a biological sample from a patient with suspected lung 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 .
  • the above method may further include diagnosing lung cancer using an antibody specific to one to eight proteins selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
  • 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 (RIA) , radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS , and protein chip assay .
  • a patient with suspected lung cancer is compared with a normal control for the amount of formed antigen-antibody complexes, and the patient' s suspected lung cancer is diagnosed by evaluating a significant increase in expression levels of a protein from the lung cancer marker gene .
  • the term "antigen-antibody complexes", as used herein, refers to binding products of a lung cancer marker protein to an antibody specific thereto .
  • the amount of formed antigen-antibody complexes may be quantitatively determined by measuring the signal size of a detection label .
  • Such 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 .
  • Examples of the fluorescent substances include, but are not limited to, fluorescin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamin.
  • Examples of the ligands include, but are not limited to, biotin derivatives .
  • Examples of luminescent substances include, but are not limited to, acridinium esters, luciferin and luciferase .
  • Examples of the microparticles 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, K4W (CN) 8 , [Os (bpy) 3 ] 2+ , [RU (bp ⁇ ) 3 ] 2+ and [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 a labeled antibody recognizing an antigen bound to a antibody immobilized on a solid support; and indirect sandwich ELISA, in which a captured antigen bound to an antibody immobilized on a solid support is detected by first adding an antigen-specific antibody, and then a secondary labeled antibody which binds the antigen-specific antibody.
  • 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 lung cancer may be diagnosed by measuring the degree of complex formation of a lung cancer marker protein and an antibody thereto .
  • the protein expression levels are preferably measured using a protein chip in which one or more antibodies to the lung cancer markers are arrayed and immobilized at predetermined positions of a base plate at high density.
  • proteins are isolated from the sample and hybridized with the protein chip to form antigen-antibody complexes .
  • the protein chip is then read to determine the presence or expression levels of the proteins, thereby- diagnosing the incidence of lung cancer .
  • the measurement of protein expression levels is preferably achieved using Western blotting using one or more antibodies to the lung cancer makers .
  • 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 antigen-antibody complexes produced using a labeled antibody, thereby diagnosing the incidence of lung cancer .
  • the detection methods are composed of methods of assessing expression levels of maker genes in a control and cells in which lung 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 .
  • EXAMPLE 1 Analysis of gene expression in squamous cell carcinoma cells of the lung and normal lung tissue
  • the gene expression patterns of the patient specimens were assessed using an 8K human cDNA microarray (GenePlorerTMTwinChipTM human-8K setl) , which was purchased from Digital Genomics Inc . , Korea .
  • the microarray contained 8 , 170 different cDNA probes that were repeated twice, and the related information is available from a web site, http: //annotation. digital-genomics , co. kr/excel/human8 2ksetl .xls .
  • To compare 97 samples with each other for gene expression patterns the gene expression of each specimen was compared with that of a common reference sample .
  • the common reference sample was prepared by mixing the equal amounts of RNA isolated from eight lung-derived cell lines . Cell lines used were NCI-H23, NCI-H1299, NCI-H596, A-549, NCI- H358 , NCI-H128 , SK-LU-I, and Malme-3M.
  • Samples for hybridization were prepared as follows .
  • RNA was reverse transcribed in the presence of aminoallyl-modified dUTP and coupled to a fluorescent dye by a chemical method.
  • Samples extracted from lung cancer patients were labeled with a Cy5 fluorescent dye, and the common reference sample RNA was labeled with a Cy3 fluorescent dye .
  • the two samples labeled with two different fluorescent dyes were mixed and hybridized to the microarray.
  • the DNA chip was washed with a washing solution containing SSC to eliminate non-specific hybridizations .
  • the washed DNA chip was scanned using a confocal laser scanner ( Perkin Elmer, Scanarray Lite) , and the obtained fluorescent data present at each spot were saved as TIFF images .
  • a confocal laser scanner Perkin Elmer, Scanarray Lite
  • the TIFF images were quantified with GenePix 3.0 (Axon Instruments ) to quantify the fluorescence intensity at each spot .
  • Quantitative results obtained from GenePix 3.0 were normalized using the ⁇ lowess' function supplied by the S-plus statistical package (Insightful) according to a method suggested by Yang et al . (Nucleic Acids Res 2002 , 30 : el5 ) .
  • Gene expression patterns in squamous cell carcinomas of the lung and normal lung tissues were analyzed through analysis of the cDNA (complementary DNA) microarray, which contained over 8 , 000 probes .
  • the whole gene expression patterns of squamous cell carcinomas of the lung were evaluated using clustering analysis and multidimensional scaling .
  • Clustering analysis revealed that the squamous cell carcinoma and normal lung tissues were separated into two large distinct clusters (FIG. 1) .
  • Multidimensional scaling confirmed the obvious difference between the squamous cell carcinoma and normal lung tissues in gene expression patterns ( FIG. 2 ) .
  • EXAMPLE 2 Selection of genes expressed at different levels in non-small cell lung cancer and normal lung tissue ⁇ 2-l> Gene selection by t-test
  • RT-PCR RT-PCR
  • tumorous lung tissues were collected from eight patients with squamous cell carcinoma of the lung, and normal tissues were also prepared.
  • RT-PCR was carried out as follows . 5 ⁇ g of RNA was reverse transcribed in a 20 ⁇ l reaction volume, and was diluted with distilled water to 100 ⁇ l . Using 2 ⁇ l of the diluted RT-PCR product as a template, a 25-cycle PCR was carried out with a pair of primers specific to each gene in a 25- ⁇ l reaction volume . 8 ⁇ l of the PCR product was electrophoresed on a 2% agarose gel containing 0.5 ⁇ g/ml of EtBr, and DNA bands were visualized under UV light .
  • RT-PCR revealed that 39 of the 49 genes were expressed in tumorous lung tissues at higher levels than in normal tissues, indicating that 78% of the microarray results are consistent with the RT-PCR results .
  • the 10 genes specifically expressed in tumorous lung tissues can be used as diagnostic markers for diagnosing lung cancer.
  • the lung cancer markers of the present invention allow the simple accurate diagnosis of lung cancer through detection of their mRNA or protein expression levels .

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Abstract

Disclosed is a diagnostic marker specific for lung 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 lung cancer using the composition or kit.

Description

MARKERS FOR THE DIAGNOSIS OF LUNG CANCER
Technical Field
The present invention relates to a diagnostic marker specific for lung 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 lung cancer using the composition or kit .
Background Art
Lung cancer is a leading cause of cancer death worldwide. Lung cancer is responsible for about one-sixth of all cancer deaths . There are two major types of lung cancer: small cell lung cancer and non-small cell lung cancer . Non-small cell lung cancer is the representative lung cancer, which accounts for about 80% of all lung cancer . Adenocarcinoma, squamous cell carcinoma and large cell carcinoma are three types of non-small cell lung cancer . Since there are differences in histological properties as well as prognosis and therapy according to the type of lung cancer, accurate diagnosis is important . Despite the recent advances in cancer therapy, ten-year survival rates of patients with non-small cell lung cancer are 10% or even less . This is because non-small cell lung cancer is generally difficult to diagnose until the disease is relatively advanced . Under present situations, early diagnosis is the most effective method for increasing survival rates of the patients . A variety of attempts have been made to diagnose lung cancer using markers . Some research reported diagnostic markers of lung cancer through expression of a limited number of target genes and proteins (Hibi et al . , Am. J. Pathol . 1999, 155 : 711-715; Brechot et al . Eur . J. Cancer 1997, 33 : 385-391; Pastor et al . , Eur . Respir . J. 1997, 10 : 603-609; Morita et al . , Int . J. Cancer 1998 , 78 : 286-292 ) . Also, some reports described the finding of lung cancer marker genes using microarray techniques (Oncogenel9_1519; Oncogene23_7734 ; Oncogene21_7749) . However, there is no report involving the possibility of early diagnosis of lung cancer using the presently identified diagnostic markers of lung cancer .
Based on this background, the present inventors, in order to develop biomarkers capable of simply and accurately diagnosing lung cancer, performed primary screening for genes overexpressed only in lung cancer using a DNA chip, and identified highly significant markers by performing RT-PCR. As a result, PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 genes were identified . When the genes were practically applied to lung cancer samples, they were found to accurately diagnose lung cancer, thereby leading to the present invention.
Disclosure of the Invention
It is therefore an obj ect of the present invention to provide a diagnostic marker of lung cancer, which is at least one selected from among PKPl (plakophilin 1) , ABCC5
(ATP-binding cassette, subfamily C (CFTR/MRP) , member 5) ,
KRT15 ( keratin 15) , KRT14 ( keratin 14 ) , TRIM29 (tripartite motif-containing 29) , SERPINB5 (serine (or cysteine) proteinase inhibitor, clade B (ovalbumin) , member 5 ) , TKl (thymidine kinase 1, soluble) , GPX2 (glutathione peroxidase
2 ) , MMPl (matrix metalloproteinase 1 ) and ITGB4 (integrin, beta 4 ) .
It is another object of the present invention to provide a kit for detecting a diagnostic marker of lung cancer, comprising an agent measuring mRNA or protein levels of one or more genes selected from among PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
It is a further obj ect of the present invention to provide a composition for detecting a diagnostic marker of lung cancer, comprising a pair of primers specific for one or more genes selected from among PKPl, ABCC5, KRT15, KRT14 ,
TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
It is yet another obj ect of the present invention to provide a composition for detecting a diagnostic marker of lung cancer, comprising an antibody specific for one or more proteins selected from among PKPl, ABCC5, KRT15, KRT14, TRIM29, SERPINB5, TKl , GPX2 , MMPl and ITGB4. It is still another obj ect of the present invention to provide a method of diagnosing lung cancer using primers specific for one or more genes selected from among PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4. It is still another obj ect of the present invention to provide a method of diagnosing lung cancer using an antibody specific for one or more proteins selected from among PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
Brief Description of the Drawings
FIG . 1 shows the clustering of 57 tumorous and 40 normal lung tissues using gene expression data . Tumorous lung tissues are represented by "Tumor (black rectangular shape) " , and normal lung tissues are represented by "Normal (white rectangular shape) " . The left white bar indicates a group in which most of the normal lung tissues cluster together, and the right black bar indicates another group in which the tumor lung tissues cluster together .
FIG . 2 shows a 3-D visualization of gene expression patterns by multidimensional scaling . The black circle indicates tumorous lung tissues , and the gray circle indicates normal lung tissues . Spots placed near each other in three dimensional space indicate that they have similar gene expression patterns . Tumorous lung tissues form one cluster, and normal lung tissues form another cluster, indicating that each of the tumorous and normal lung tissues has a specific gene expression pattern .
FIG . 3 shows the results of RT-PCR for confirming the difference in expression levels between tumorous and normal lung tissues . Ten genes are expressed not in normal lung tissues but in tumorous lung tissues .
Best Mode for Carrying Out the Invention
In one aspect, the present invention relates to a lung cancer diagnostic marker selected from PKPl, ABCC5, KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
The term "diagnosis", as used herein, refers to evaluation of the presence or properties of pathological states . With respect to the obj ects of the present invention, the diagnosis is to determine the incidence of lung cancer .
The term "lung cancer", as used herein, refers to malignant tumor occurring in the lung, and includes both small cell lung cancer and non-small cell lung cancer including adenocarcinoma, squamous cell carcinoma and large cell carcinoma .
The term "marker for diagnosing, marker for diagnosis or diagnostic marker", as used herein, is intended to indicate a substance capable of diagnosing lung cancer by distinguishing lung cancer cells from normal cells, and includes organic biological molecules, quantities of which are increased or decreased in lung cancer cells relative to normal cells , such as polypeptides or nucleic acids (e . g . , mRNA, etc . ) , lipids , glycolipids, glycoproteins and sugars
(monosaccharides , disaccharides , oligosaccharides, etc . ) .
With respect to the obj ects of the present invention, lung cancer diagnostic markers are PKPl , ABCC5 , KRT15 , KRT14 ,
TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 , which are genes whose expression is increased in lung 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 lung cancer diagnostic markers of the present invention, which are genes whose expression always increases by direct or indirect factors when lung cancer occurs , 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 . The PKPl , ABCC5 , KRT15, KRT14 , TRIM29, SERPINB5, TKl , GPX2 , MMPl and ITGB4 genes of the present invention may be used as diagnostic markers of lung cancer because they are expressed at high levels specifically in lung cancer cells compared to cells of normal lung tissue . All of the genes are useful as diagnostic markers of lung cancer . However, with respect to the present obj ect of providing rapid, simple and accurate markers, it is preferable to detect a limited number of markers make a medical decision . This is economical in terms of preventing waste of time and resources . Since PKPl and ABCC5 are expressed specifically in lung cancer cells, they have very high reliability allowing diagnosis of lung cancer even when used alone . Therefore, the diagnosis of lung cancer is preferably carried out using either PKPl or ABCC5 alone, or both of them, as markers .
In addition, the diagnosis of lung cancer may be carried out using one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 , as markers . Herein, genes expressed at almost the same levels in cells of normal lung tissue and lung cancer cells, for example, DCK (deoxycytidine kinase) and SEP15 ( selenoprotein, 15-KD) , may be used as quantitative controls .
In another aspect, the present invention relates to a kit for detecting a diagnostic marker of lung cancer, comprising an agent measuring mRNA or protein levels of one or two genes selected from PKPl and ABCC5.
The kit may further include an agent measuring mRNA or protein levels of one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
Gene expression levels of 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 lung cancer marker genes in biological samples for diagnosing lung 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", as used herein, is a process of assessing the presence and expression levels of proteins expressed from lung cancer marker genes in biological samples for diagnosing lung 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, enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis , immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip assay .
The detection kit of the present invention is composed of a composition, solution or apparatus , which includes one or more kinds of different constituents suitable for analysis methods .
Preferably, the present invention relates to a kit for detecting a diagnostic marker, which is 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 . Also, the RT-PCR kit may include primers specific to a nucleic acid sequence of a control gene . The RT-PCR 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 . In addition, preferably, the present invention relates to a diagnostic kit, which is 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 . Also, the base plate may include a control gene or fragments thereof, such as cDNA or oligonucleotides . Further, preferably, the present invention relates to a diagnostic kit, which is 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 . Also, 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 . , conj ugated with an antibody) and their substrates, or other substances capable of binding to the antibodies .
An RT-PCR kit for detecting lung cancer markers includes a pair of primers specific to one to two genes selected from PKPl and ABCC5. Also, the RT-PCR kit may include a pair of primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5 , TKl, GPX2 , MMPl and ITGB4 genes .
A DNA chip kit for detecting lung cancer markers includes a base plate onto which cDNA corresponding to one to two genes selected from PKPl and ABCC5, or fragments thereof, is attached . Also, cDNA corresponding to one to eight genes selected from among KRT15, KRT14 , TRIM29,
SERPINB5, TKl , GPX2 , MMPl and ITGB4 genes , or fragments thereof, may be attached and immobilized onto a base plate .
An ELISA kit for detecting lung cancer markers includes an antibody specific to one or two proteins selected from PKPl and ABCC5. Also, the ELISA kit may include an antibody specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
In a further aspect, the present invention relates to a composition for detecting a diagnostic marker of lung cancer, comprising primers specific to one or two genes selected from PKPl and ABCC5.
The above composition may further include primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
As used herein, "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 nucleosides 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 conj ugates (e . g . , methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc . ) or charged conj ugates (e . g . , phosphorothioate, phosphorodithioate, etc . ) . Nucleic acids may contain one or more additionally covalent-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 . The nucleic acid sequences of the present invention may also be altered using a label capable of directly or indirectly supplying a detectable signal . Examples of the label include radioisotopes , fluorescent molecules and biotin .
The composition for detecting a diagnostic marker of lung cancer includes a pair of primers specific to one to two genes selected from PKPl and ABCC5. Primers for amplifying PKPl ( SEQ ID NO . 1 ) are preferably represented by SEQ ID NOS . 2 and 3 , and primers for amplifying ABCC5
( SEQ ID NO . 4 ) are preferably represented by SEQ ID NOS . 5 and 6. Also, the composition may further include a pair of primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4 genes . Primers for amplifying KRT15 (SEQ ID NO . 7 ) are preferably represented by SEQ ID NOS . 8 and 9. Primers for amplifying KRT14 ( SEQ ID NO . 10 ) are preferably represented by SEQ ID NOS . 11 and 12. Primers for amplifying TRIM29 (SEQ ID NO . 13 ) are preferably represented by SEQ ID NOS . 14 and 15. Primers for amplifying SERPINB5 (SEQ ID NO . 16) are preferably represented by SEQ ID NOS . 17 and 18. Primers for amplifying TKl (SEQ ID NO . 19) are preferably represented by SEQ ID NOS . 20 and 21. Primers for amplifying GPX2 (SEQ ID NO. 22 ) are preferably represented by SEQ ID NOS . 23 and 24. Primers for amplifying MMPl (SEQ ID NO . 25 ) are preferably represented by SEQ ID NOS . 26 and 27. Primers for amplifying ITGB4 (SEQ ID NO . 28 ) are preferably represented by SEQ ID NOS . 29 and 30. In yet another aspect, the present invention relates to a composition for detecting a diagnostic marker of lung cancer, comprising an antibody specific to one or two proteins selected from PKPl and ABCC5.
The above composition may further include an antibody specific to one to eight proteins selected from among KRT15, KRT14 , TRIM29, SERPINB5 , TKl , GPX2 , MMPl and ITGB4.
The term "antibody", as used herein, refers to a specific protein molecule that indicates an antigenic region . With respect to the obj ects of the present invention, an antibody binds specifically to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies .
Antibody production using the lung 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 inj ecting the lung 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 ) .
The hybridoma method employs cells from an immunologically suitable host animal injected with a diagnostic marker protein of lung cancer as an antigen, such as mice, and a cancer or myeloma cell line as another group . Cells of the two groups are fused with each other by a method widely known in the art, for example, using polyethylene glycol , and antibody-producing cells are proliferated by a standard tissue culture method . After uniform cell colonies are obtained by subcloning using a limited dilution technique, hybridomas capable of producing an antibody specific for the diagnostic marker protein of lung cancer are cultivated in large scale in vitro or in vivo according to a standard technique . Monoclonal antibodies produced by the hybridomas may be used in an unpurified form, but are preferably used after being highly purified by a method widely known in the art so as to obtain best results . The phage antibody library method includes constructing a phage antibody library in vitro by obtaining genes for antibodies ( single-chain fragment variable ( scFv) ) to a variety of intracellular lung cancer markers and expressing them in a fusion protein form on the surface of phages, and isolating monoclonal antibodies binding to lung cancer-specific proteins from the library .
Antibodies prepared by the above methods are isolated using gel electrophoresis , dialysis, salting out, ion exchange chromatography, affinity chromatography, and the like . In addition, 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 and the like.
The composition for detecting a lung cancer marker includes an antibody specific for one to two proteins selected from PKPl and ABCC5. Also, the composition may further include an antibody specific to one to eight proteins selected from among KRTl5, KRTl4 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
In still another aspect, the present invention relates to a method of diagnosing lung cancer, comprising measuring mRNA levels in a biological sample from a patient with suspected lung cancer using primers specific to one or two genes selected from PKPl and ABCC5, 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 above method may further include diagnosing lung cancer using primers specific to one to eight genes selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
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 .
The term "biological sample", as used herein, includes, but is not limited to, samples displaying a difference in expression levels of a lung cancer marker gene, such as tissues , cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine .
Analysis methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, realtime RT-PCR, RNase protection assay (RPA) , Northern blotting and DNA chip assay.
With the detection methods , a patient with suspected lung cancer is compared with a normal control for mRNA expression levels of a lung cancer marker gene, and the patient' s suspected lung cancer is diagnosed by determining whether expression levels of mRNA from the lung cancer marker gene have significantly increased . mRNA expression levels are preferably measured by RT- PCR using primers specific to a gene as a lung cancer marker .
RT-PCR is a method that was introduced to analyze RNA by P . Seeburg (Cold Spring Harb Symp Quant Biol 1986, Pt 1 : 669-677 ) , with which cDNA is synthesized from mRNA by reverse transcription and amplified by PCR. At the amplification step, a pair of primers prepared in a fashion specific to a diagnostic marker of lung cancer is used . 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 lung cancer while comparing the mRNA expression and levels with those of a control, thereby simply diagnosing the incidence of lung cancer .
Alternatively, mRNA expression levels are measured using a DNA chip in which the lung cancer marker genes or nucleic acid 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 lung cancer . In still another aspect, the present invention relates to a method of diagnosing lung cancer, comprising measuring protein levels by contacting an antibody specific to one or two genes selected from PKPl and ABCC5 with a biological sample from a patient with suspected lung 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 .
The above method may further include diagnosing lung cancer using an antibody specific to one to eight proteins selected from among KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
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 (RIA) , radioimmunodiffusion, ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, immunoprecipitation assay, complement fixation assay, FACS , and protein chip assay .
With the analysis methods, a patient with suspected lung cancer is compared with a normal control for the amount of formed antigen-antibody complexes, and the patient' s suspected lung cancer is diagnosed by evaluating a significant increase in expression levels of a protein from the lung cancer marker gene . The term "antigen-antibody complexes", as used herein, refers to binding products of a lung cancer marker protein to an antibody specific thereto . The amount of formed antigen-antibody complexes may be quantitatively determined by measuring the signal size of a detection label .
Such 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 . Examples of enzymes available as detection labels include, but are not limited to, β- glucuronidase, β-D-glucosidase, β-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 β-latamase . Examples of the fluorescent substances include, but are not limited to, fluorescin, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamin. Examples of the ligands include, but are not limited to, biotin derivatives . Examples of luminescent substances include, but are not limited to, acridinium esters, luciferin and luciferase . Examples of the microparticles include, but are not limited to, colloidal gold and colored latex . Examples of the redox molecules include, but are not limited to, ferrocene, ruthenium complexes , viologen, quinone, Ti ions, Cs ions, diimide, 1, 4- benzoquinone, hydroquinone, K4W (CN) 8, [Os (bpy) 3] 2+ , [RU (bpγ) 3] 2+ and [MO (CN) 8] 4" . Examples of the radioactive isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I and 186Re .
Preferably, the protein expression levels are measured by ELISA. Examples of 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 a labeled antibody recognizing an antigen bound to a antibody immobilized on a solid support; and indirect sandwich ELISA, in which a captured antigen bound to an antibody immobilized on a solid support is detected by first adding an antigen-specific antibody, and then a secondary labeled antibody which binds the antigen-specific antibody. More preferably, 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 lung cancer may be diagnosed by measuring the degree of complex formation of a lung cancer marker protein and an antibody thereto . In addition, the protein expression levels are preferably measured using a protein chip in which one or more antibodies to the lung cancer markers are arrayed and immobilized at predetermined positions of a base plate at high density. By a method of analyzing a sample using a protein chip, proteins are isolated from the sample and hybridized with the protein chip to form antigen-antibody complexes . The protein chip is then read to determine the presence or expression levels of the proteins, thereby- diagnosing the incidence of lung cancer . Further, the measurement of protein expression levels is preferably achieved using Western blotting using one or more antibodies to the lung cancer makers . 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 antigen-antibody complexes produced using a labeled antibody, thereby diagnosing the incidence of lung cancer . The detection methods are composed of methods of assessing expression levels of maker genes in a control and cells in which lung cancer occurs . mRNA or protein levels may be expressed as an absolute (e . g . , μg/ml) or relative (e . g. , relative intensity of signals) difference in the amount of marker proteins . A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
EXAMPLE 1 : Analysis of gene expression in squamous cell carcinoma cells of the lung and normal lung tissue
<1-1> RNA isolation from patient specimens
Patient specimens were obtained from Korean Cancer Center Hospital . RNA was isolated from the specimens from patients with squamous cell carcinoma of the lung using a TriZol reagent (InVitrogen) according to the manufacturer' s protocol . 10 ml of the TriZol reagent was used per patient specimen, which was cut into a size of 1 cm3. The concentration of the isolated RNA was determined using a spectrophotometer .
<l-2> Microarray analysis
The gene expression patterns of the patient specimens were assessed using an 8K human cDNA microarray (GenePlorer™TwinChip™ human-8K setl) , which was purchased from Digital Genomics Inc . , Korea . The microarray contained 8 , 170 different cDNA probes that were repeated twice, and the related information is available from a web site, http: //annotation. digital-genomics , co. kr/excel/human8 2ksetl .xls . To compare 97 samples with each other for gene expression patterns, the gene expression of each specimen was compared with that of a common reference sample . The common reference sample was prepared by mixing the equal amounts of RNA isolated from eight lung-derived cell lines . Cell lines used were NCI-H23, NCI-H1299, NCI-H596, A-549, NCI- H358 , NCI-H128 , SK-LU-I, and Malme-3M.
Samples for hybridization were prepared as follows .
20 μg of RNA was reverse transcribed in the presence of aminoallyl-modified dUTP and coupled to a fluorescent dye by a chemical method. Samples extracted from lung cancer patients were labeled with a Cy5 fluorescent dye, and the common reference sample RNA was labeled with a Cy3 fluorescent dye . The two samples labeled with two different fluorescent dyes were mixed and hybridized to the microarray. Then, the DNA chip was washed with a washing solution containing SSC to eliminate non-specific hybridizations . The washed DNA chip was scanned using a confocal laser scanner ( Perkin Elmer, Scanarray Lite) , and the obtained fluorescent data present at each spot were saved as TIFF images . The TIFF images were quantified with GenePix 3.0 (Axon Instruments ) to quantify the fluorescence intensity at each spot . Quantitative results obtained from GenePix 3.0 were normalized using the λlowess' function supplied by the S-plus statistical package (Insightful) according to a method suggested by Yang et al . (Nucleic Acids Res 2002 , 30 : el5 ) .
<l-3> Overall evaluation of microarray results data
Gene expression patterns in squamous cell carcinomas of the lung and normal lung tissues were analyzed through analysis of the cDNA (complementary DNA) microarray, which contained over 8 , 000 probes . The whole gene expression patterns of squamous cell carcinomas of the lung were evaluated using clustering analysis and multidimensional scaling . Clustering analysis revealed that the squamous cell carcinoma and normal lung tissues were separated into two large distinct clusters (FIG. 1) . Multidimensional scaling confirmed the obvious difference between the squamous cell carcinoma and normal lung tissues in gene expression patterns ( FIG. 2 ) . These results indicate that gene expression results obtained through microarray analysis are useful data for selection of marker genes capable of diagnosing squamous cell carcinoma of the lung .
EXAMPLE 2 : Selection of genes expressed at different levels in non-small cell lung cancer and normal lung tissue <2-l> Gene selection by t-test
A t-test was conducted in a significance level of p=10"δ so as to select genes whose expression is significantly different between squamous cell carcinoma and normal lung tissues . Since gene selection in the significance level is expected to generate only one false- positive gene in one million tests, all genes thus selected are genes practically different in expression levels . The gene selection by the t-test resulted in the selection of 832 genes exhibiting a significant difference in expression levels . Among the selected genes, 319 genes were expressed at higher levels in squamous cell carcinoma lung tissues, and 513 genes were expressed at higher levels in normal lung tissues . Since genes displaying high-level expression in tumorous lung tissues are required for diagnosing lung cancer, diagnostic markers were selected from among the 319 genes highly expressed in lung cancer.
<2-2> Identification of diagnostic markers using RT-PCR
Expression levels of the selected genes were estimated by RT-PCR in order to identify genes highly useful as diagnostic markers for the early diagnosis of lung cancer. For RT-PCR, tumorous lung tissues were collected from eight patients with squamous cell carcinoma of the lung, and normal tissues were also prepared. RT-PCR was carried out as follows . 5 μg of RNA was reverse transcribed in a 20 μl reaction volume, and was diluted with distilled water to 100 μl . Using 2 μl of the diluted RT-PCR product as a template, a 25-cycle PCR was carried out with a pair of primers specific to each gene in a 25-μl reaction volume . 8 μl of the PCR product was electrophoresed on a 2% agarose gel containing 0.5 μg/ml of EtBr, and DNA bands were visualized under UV light .
Among the 319 genes highly expressed in squamous cell carcinoma of the lung, 39 genes exerting a two-fold difference in expression levels were selected (Table 1) , and gene expression levels were confirmed by RT-PCR.
TABLE 1
Genes expressed in tumorous lung tissues more than twice as high as in normal tissues among the genes selected by the t-test
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Among the 39 selected genes, 10 genes were found to be expressed not in normal lung tissues but in tumorous lung tissues (FIG. 3) . The rest 29 genes were expressed in tumorous lung tissues at higher levels than in normal tissues, and 10 genes did not display any difference in expression levels between tumorous and normal lung tissues . RT-PCR revealed that 39 of the 49 genes were expressed in tumorous lung tissues at higher levels than in normal tissues, indicating that 78% of the microarray results are consistent with the RT-PCR results . In particular, the 10 genes specifically expressed in tumorous lung tissues can be used as diagnostic markers for diagnosing lung cancer.
Industrial Applicability
The lung cancer markers of the present invention allow the simple accurate diagnosis of lung cancer through detection of their mRNA or protein expression levels .

Claims

Claims
1. A kit for detecting a diagnostic marker of lung cancer, comprising an agent measuring mRNA or protein levels of one or two genes selected from PKPl and ABCC5.
2. The kit according to claim 1, which further comprises an agent measuring mRNA or protein levels of one to eight genes selected from KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
3. The kit according to claim 1, which is used for RT-PCR, DNA chip assay or ELISA.
4. A composition for detecting a diagnostic marker of lung cancer, comprising a pair of primers specific to one or two genes selected from PKPl and ABCC5.
5. The composition according to claim 4, which further comprises a pair of primers specific to one to eight genes selected from KRT15, KRT14, TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
6. A composition for detecting a diagnostic marker of lung cancer, comprising an antibody specific to one or two proteins selected from PKPl and ABCC5.
7. The composition according to claim 6, which further comprises an antibody specific to one to eight proteins selected from KRT15, KRT14, TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
8. A method of diagnosing lung cancer, comprising: measuring mRNA levels in a biological sample from a patient with suspected lung cancer using primers specific to one or two genes selected from PKPl and ABCC5; and comparing mRNA levels of the sample from the patient with those of a normal control sample to determine an increase in mRNA levels .
9. The method according to claim 8 , which further comprises using primers specific to one to eight genes selected from KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2, MMPl and ITGB4.
10. A method of diagnosing lung cancer, comprising : measuring protein levels by contacting an antibody specific to one or two proteins selected from PKPl and ABCC5 with a biological sample from a patient with suspected lung 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 .
11. The method according to claim 10, which further comprises using an antibody specific to one to eight proteins selected from KRT15, KRT14 , TRIM29, SERPINB5, TKl, GPX2 , MMPl and ITGB4.
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