WO2015023110A1 - Biomarker for diagnosing tumor disease and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing a human tumor disease and a use thereof. According to the present invention, when a biological sample includes single nucleotide polymorphism (SNP) nucleotide (e.g., 'A/A', 'A/C', 'A/c', or 'C/a') in the 50^th nucleotide of the Ras homolog gene family, member A (RhoA) gene of SEQ ID NO: 1, the biological sample is at a very high risk of having a tumor disease (specifically, lymphoma or leukemia). Therefore, a kit and method of the present invention can be very effectively and easily used to detect/diagnose a human tumor disease (e.g., lymphoma or leukemia).

Description

 【Specification】

 [Name of invention]

 Biomarkers for Tumor Disease Diagnosis and Uses thereof [Technical Field]

 The present invention relates to a biomarker for diagnosing tumor disease and its use.

[Background technology]

 Oncogenesis includes chromosomal instability, aneuploidy and various genetic aberrations of genes important for cell growth and survival. For example, balanced translocations and gene fusions have often been observed in blood and mesenchymal tumors including sarcomas, leukemias and lymphomas (Rabbits, TH, Nature 372 (6502): 143- 149 (1994)), such as carcinomas Less has been observed in epithelial tumors. Recently, tumorigenic gene fusions have been developed in the prostate gland (Tomlins, SA, et al., Science 310 (5748): 644-648 (2005)), thyroid gland (Bongar zone, I., et al., Cancer Res. 54 (11). ): 2979-2985 (1994); Kroll, TG, et al., Science 289 (5483): 1357-1360 (2000)), and lungs (Soda, M., et al., Nature 448 (7153): 561) 566 (2007)), which shows that the oncogenic gene fusions occur very frequently in carcinomas. For example, recurrent chromosomal translocations are found characteristically in many leukemias, lymphomas and sarcomas (eg mesenchymal tumors). In addition, the single-gene may be used as a tumor biomarker if the single-gene variation affects the entire pathogenesis process.

On the other hand, lymphomas, which can be classified as non-Hodgk in 's lymphomas (NHL) and Hodgkin's lymphomas (HL), are immune that not only protects the body from infection and disease, divides faster than normal cells, but also helps them survive longer. A type of blood tumor that occurs in B or T lymphocytes, which are part of the system's white blood cells, making up about 4% of all tumors. Lymphoma can occur in the lymphatic vessels, spleen, bone marrow, blood, or other organs and occurs more often in men than in women. Typically, lymphomas Represents a solid tumor of lymphoid cells. Treatment is mostly chemotherapy but in some cases radiation therapy and / or bone marrow transplantation. In general, lymphomas are treatable according to history, type and stage. The malignant cells often originate in the lymphatic vessels and may affect other organs. Lymphomas occurring in other organs are referred to as extraranodal lymphomas. Extralymphatic sites include the skin, brain, intestine, and bone. Lymphoma is closely related to lymphoid leukemi as, which typically occurs not only in lymphocytes but also in the circulating blood and bone marrow. Thus, lymphomas exist in many different types and make up part of a broad group of diseases called hemato logical neoplasms.

 Because lymphomas have a tendency to relapse as they progress to later stages, it is very important to diagnose lymphoma at an early stage. In general, the diagnosis of lymphoma is carried out through histopathological analysis of biopsy samples obtained from lymphatic vessels. The type of lymphoma can be identified through microscopic morphology analysis of cancer cells or detection of molecules (biomarkers) specific for lymphoma. The identification of new biomarkers is urgently needed in the art because the method of diagnosing lymphoma using biomarkers is more effective and faster than biopsy using a microscope. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Detailed Description of the Invention]

 [Technical problem]

We have developed biomarkers for rapid identification and / or diagnosis of tumor diseases (eg, hematologi cal neoplasms). Efforts have been made to earnest research. As a result, we investigated the mutant gene profile through whole exome sequencing and RNA-sequencing on biological samples obtained from patients with lymphoma, so that the 50th nucleotide of the RhoA gene is a single nucleotide polymorphism (single). The present invention has been completed by discovering that RhoA G17V mutations that are mutated to nucleotide polymorphi sm (SNP) nucleotides can be effectively used for the diagnosis of lymphoma.

 Accordingly, it is an object of the present invention to provide a kit for diagnosing tumor disease.

 Another object of the present invention is to provide a method for detecting a tumor disease. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

Technical Solution

 According to one aspect of the invention, the invention is 10-100 of the T allele comprising the substitution of G to T of the 50th nucleotide of the RhoA (Ras homo log gene fami ly, member A) gene of SEQ ID NO: 1 Provided are a kit for diagnosing a tumor disease comprising a primer or probe specifically binding to two consecutive nucleotide sequences.

 According to another aspect of the present invention, the present invention provides a method for detecting a tumor disease, comprising the steps of: a) separating a nucleic acid molecule from a biological sample of a subject; And (b) identifying the 50th nucleotide of the Rho Ras homo log gene fami ly, member A) gene of SEQ ID NO: 1 in the nucleic acid molecule of step (a), wherein the 50th nucleotide is from G to T. If substituted, the subject is determined to have a tumor disease.

The inventors have made extensive research efforts to develop biomarkers for rapid identification and / or diagnosis of tumor diseases (eg, blood tumors). As a result, the inventors have found that biological samples obtained from lymphoma patients Examination of the mutant gene profile through total exome sequencing and RNA-sequencing revealed that RhoA G17V mutations in which the 50th nucleotide of the RhoA gene was mutated to a single nucleotide polymorphic nucleotide could be effectively used to diagnose lymphoma.

 The Rho protein is a well-known member of the p21 Ras superfamily of small GTPases and exhibits intrinsic GTPase activity through a conformational change between an inactive GDP-binding state and an active GTP-binding state. RhoA proteins regulate signal transduction from cell surface receptors to intracellular target molecules and include a variety of cell types, motility, cytokinesis, smooth muscle contraction, and tumor progression. Included in biological processes (Yoshioka, K., et al., Cancer Research, 59: 2004-2010 (1999)). It has been reported that the expression level of RhoA in tumor tissues is significantly higher compared to surrounding normal tissues, and increases positively with the stage of colorectal cancer (Nakamori, S., et al., Rec. Adv. Gastroenterol) Carcinogenesis, 1: 901904 (1996). In particular, the RhoA and RhoC genes were expressed at relatively much higher levels at metastasis sites (Suwa, H., et al., Br. J. Cancer, 77: 147152 (1998)). Thus, RhoA functions positively for the migration of tumor cells. In addition, RhoA has been reported to regulate transcriptional activation by serum response factor (SRF) and to be involved in oncogenes is and intracellular transformation (Perona R., et al., Oncogene , 8 (5): 1285-1292 (1993); Hill CS, et al., Cell, 81 (7): 1159-1170 (1995).

According to the present invention, the RhoA G17V mutation of the present invention was found with high frequency in AITL lymphoma and T-cell-derived lymphoma (see Table 1). Kits of the invention are primers that specifically bind to 10-100 contiguous nucleotide sequences, including the G to T substitution of the 50th nucleotide of the Rho Ras homolog gene family, member A) gene, which is the first sequence Comprising a probe, Complementary expression of the T nucleotides are A nucleotides and can be used to identify and / or diagnose tumor disease in a subject. Therefore, the "50th time" of step (b) of the present invention Nucleotide substituted from G to T "means" the 50th nucleotide is a single nucleotide polymorphism (SNP) nucleotide 'Α / Α', 'A / C', Ά / c * or X / a ' ，， can be expressed as As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990).

 The present invention relates to an allele wherein the 50th nucleotide of the RhoA (Ras homo log gene family, member A) gene of SEQ ID NO: 1 is "T", but the allele nucleotide is a double stranded gDNA (genomic DNA) If found, it may be interpreted to include a nucleotide sequence that is complementary to the above nucleotide sequence. In other words, 'T', a single nucleotide polymorphism (SNPs), becomes 'Α' in the complementary nucleotide sequence. In this respect, all sequences herein are based on sequences in the sense strand in gDNA, unless otherwise noted.

As used herein, the term "single nucleotide polymorphisms (SNPs)" refers to a single base (A, T, C, or G) in the genome between members of a species or between a pair of individuals. Refers to the diversity of DNA sequences that occur in different cases between chromosomes, eg, DNA fragments of different individuals (eg TGTG [G / T] AAAG, the complementary base of G / T in the SNP of the present invention). As indicated, when the difference includes a single base, it is called two alleles (G or T), and in general, almost all SNPs have two alleles, and within a population, SNPs are minor alleles. Minor allele frequency (MAF), which is the lowest allele frequency found in a locus found in a particular population .. Variations exist within the human population, geological or ethnic One SNP allele common in the group is very regressive. Single bases can be altered (replaced), removed (deleted) or added (inserted) to the polynucleotide sequence. SNPs can cause changes in translation frames (in-frame shi ft).

 Single nucleotide polymorphisms can be included in coding sequences of genes, non-coding regions of genes or intergeni c regions between genes. SNPs in the coding sequence of a gene do not necessarily cause a change in the amino acid sequence of the target protein due to the codon degeneracy of the genetic code. SNPs that form the same polypeptide sequence are called synonymous (sometimes called silent mutations), and non-synonymous for SNPs that form other polypeptide sequences. Non-synonymous SNPs can be missense or nonsense, where the missense change results in another amino acid, while the nonsense change forms a nonmature termination codon. SNPs that are not at the protein-coding site can cause gene silencing, transcription factor binding or non-coding RNA sequences.

 Variability in human DNA sequences can affect the onset of disease and how humans react to pathogens, chemicals, drugs, vaccines, and other reagents. SNPs are also thought to be a key enabler for the realization of customized medicine concepts. Above all, SNPs, which are being actively developed as markers in recent years, are very important in biomedical research to diagnose disease by comparing genomic regions between groups with or without disease. SNPs are the most mutant of the human genome and are believed to exist at one SNP ratio per 1.9 kb (Sachidanandam et al., 2001). SNPs are very stable genetic markers, sometimes directly affecting the phenotype, and are well suited for automated genotyping systems (Landegren et al., 1998; Isaksson et al., 2000). SNPs research is also important in grain and livestock raising programs.

According to the present invention, the kit of the present invention comprises a primer pair designed to amplify a polynucleotide comprising a single nucleotide polymorphism (SNP) nucleotide (S T nucleotide) of the 50th nucleotide in SEQ ID NO: 1 Tumor disease diagnosis kit. Specifically, Of these primer pairs, the forward primer is a nucleotide sequence capable of annealing with a sequence adjacent to a tumor disease (eg, lymphoma or leukemia) -associated single nucleotide polymorphism (SNP) nucleotide corresponding to the 50th nucleotide in SEQ ID NO: 1 Wherein the 3 ′ ′ end of the forward primer has a base complementary to the SNP base.

 According to certain embodiments of the invention, the kits and methods of the invention may be gene amplification or microarrays. More specifically, the amplification of the present invention is carried out according to polymerase chain react ion (PCR). According to certain embodiments of the present invention, the primers of the present invention may be used for amplification react ions.

The term "amplification reaction" as described herein means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800, 159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al. , Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (200D), Miller, H. I. (W0 89/06700) and Davey, C. et al. (EP 329,822), Ligase chain reaction (Hgase) chain reaction; LCR (17, 18), Gap-LCR (W0 90/01069), repair chain reaction (EP 439,182), transcription ion-mediated amplification (TMA) (19) ( W0 88/10315) self sustained sequence repl icat ion (20) (W0 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) ), Consensus sequence primed polymerase chain react ion (CP—PCR) (US Pat. Nos. 4, 437, 975), arbitrarily ly primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861, 245), nucleic acid bases Nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130,238, Crab 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification ) And ring-mediated isothermal amplification; LAMP), but is not limited thereto. available Other amplification methods are described in US Pat. Nos. 5, 242, 794, 5,494,810, 4,988,617, and US Pat. Nos. 09/854, 317, the teachings of which are incorporated herein by reference. do.

 PCR is the most well-known method of nucleic acid amplification, and many modifications and uses have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse PCRGnverse polymerase chain reaction (IPCR) Vectorette PCR and TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Spr inger-Ver lag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference. When the diagnostic kit of the present invention is performed using a primer, gene amplification reaction is performed to analyze the nucleotide sequence of the biomarker of the present invention. Since the present invention detects the nucleotide sequence of the biomarker of the present invention, the presence or absence of a tumor disease can be determined by determining the nucleotide sequence of the biomarker of the present invention in a sample to be analyzed (for example, genomic DNA).

 According to some embodiments of the invention, a biological sample that can be used in the kit or method of the present invention is a biological sample isolated from human, blood, plasma, serum, tissue, cells, lymph, bone marrow, saliva, ocular fluid , Semen, brain extract, spinal fluid, joint fluid, thymus fluid, ascites fluid, amniotic fluid, cell tissue fluid and cell culture fluid, and more specifically blood, plasma, serum, tissue, cell, lymph, bone marrow fluid, cell tissue fluid and Cell culture fluid, more specifically blood, plasma, serum, tissue, cells, lymph fluid and cell culture fluid, and even more specifically blood, plasma, serum, tissue and lymph fluid, most specific Examples include blood and tissues.

In some embodiments of the invention, the amplification process of the invention is US patent No. 4,683,195 claim, disclosed to 4, 683,202 and No. 4,800, 159 call can be carried out according to PCR (polymerase chain react ion ^•).

As used herein, the term "primer" refers to a lygonucleotide, the conditions under which the synthesis of a primer extension product complementary to the nucleic acid chain (template) is induced, i.e. the presence of a polymer such as nucleotide and DNA polymerase, and it can function at a suitable temperature and condition of _P H to the starting point of the synthesis. Specifically, the primer is deoxyribonucleotide and single chain. Primers used in the present invention may include, but are not limited to, natural ly occurring dVP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides, non-natural nucleotides and ribonucleotides.

 As used herein, the term "extension primer" refers to a primer that is annealed to a target nucleic acid (biomarker) to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase. This extension primer extends to the position where the immobilized probe is annealed to occupy the site where the probe is annealed.

 Extension primers used in the present invention include nucleotide sequences that can be complementarily localized to the first position of the target nucleic acid. As used herein, the term "complementary" means that the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under certain annealing or hybridization conditions, and is referred to as "substant i al." ly complementary 'and' perfect ly complementary 'are all encompassing meanings. Specifically, it means' fully complementary'. As used herein, the term "substantially complementary sequence", as used in connection with a primer sequence, is not only a completely identical sequence, but also within the scope of annealing to a specific sequence to serve as a primer, and in part with the sequence to be compared. It is also meant to include inconsistent sequences.

The primer should be long enough to prime the synthesis of the extension product in the presence of the polymerizer. Suitable lengths of primers are typically polynucleotides of 15-30 bp, depending on a number of factors, such as temperature, application and source of the primer. Short primer molecules are used to form a complex sufficiently stable with the template Generally requires lower temperatures. As used herein, the term “annealing” or “priming” means that an oligodioxynucleotide or nucleic acid is applied to a template nucleic acid, wherein the polymerase polymerizes the nucleotides to allow the template nucleic acid or its To form nucleic acid molecules that are complementary to a portion.

 The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of being unique to the primer by being common with the template. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the above-described nucleotide sequence as a template, and is sufficient if it has a sufficient complementarity within a range capable of acting as a primer by the gene sequence. The design of such a primer can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, for example, by using a primer design program (eg, PRIMER 3 program).

 The nucleotide sequence of the target of the present invention to be referred to in the preparation of the primer can be found in GenBank. For example, the GenBank accession number of the oA gene, which is the first sequence of the biomarker of the present invention, is AF498970. Primers can be designed with reference to the above sequences and the single nucleotide polymorphic nucleotides of the invention. Specifically, among the primer pairs, the forward primer includes a nucleotide sequence capable of annealing with a sequence adjacent to a human lymphoma-associated single nucleotide polymorphism (SNP) base corresponding to the 50th nucleotide in SEQ ID NO: 1; The 3'-end of the primer has a base complementary to the SNP base.

 As used herein, the term "nucleic acid molecule" has the meaning encompassing DNA (gDNA and cDNA) and RNA molecules, and the nucleotides, which are the basic structural units in nucleic acid molecules, are not only natural nucleotides but also sugar or base sites. Modified analogues (Schei t, Nucleotide Analogs, John Wiey, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

If the starting material in the kit of the invention is gDNA, isolation of gDNA can be carried out according to conventional methods known in the art (see Rogers & Bendich (1994)).

 If the starting material is mRNA, total RNA is isolated and performed by conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press ( 2001); Tesniere, C. et al., Plant Mo J. Biol.Rep., 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wi 1 ley & Sons (1987); And Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized into cDNA using reverse transcriptase. Since the total RNA is isolated from humans (e.g., lymphoma patients), it has a poly-A tail at the end of the mRNA, and can easily synthesize cDNA using dL primer and reverse transcriptase using this sequence characteristic. (PNAS USA, 85: 8998 (1988); Libert F, et al., Science, 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001).

 In the kit of the present invention, identification / identification of the specific sequence can be carried out by using various methods known in the art. For example, techniques applicable to the present invention include fluorescence in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-strand conformation analysis (SSCA, Orita). et al., PNAS, USA 86: 2776 (1989)), RNase protection assays (Finkelstein et al., Genomics, 7: 167 (1990)), dot blot analysis, denaturation gradient gel electrophoresis (DGGE, Wartell et al., Nuc I. Acids Res., 18: 2699 (1990), methods using proteins that recognize nucleotide mismatches (eg, mutS proteins of E. ra //) (Modrich, Ann. Rev. Genet., 25: 229-253 (1991)) and allelic-specific PCR.

 Sequence changes result in differences in base bonds within single-stranded molecules, leading to the appearance of bands with different mobility, which SSCA detects. DGGE analysis uses denaturation gradient gels to detect sequences that exhibit mobility different from wild-type sequences.

Other techniques generally employ probes or primers that are complementary to sequences comprising the SNPs of the invention. For example, RNase protection In the analysis, riboprobes complementary to the sequences comprising the SNPs of the invention are used. DNA or mRNA isolated from the riboprobe and human is then hybridized and then cleaved with an RNase A enzyme capable of detecting mismatches. If there is a mismatch and RNase A recognizes, a smaller band is observed.

 In assays using the shake signal, probes complementary to the sequences comprising the SNPs of the invention are used. In this technique, the presence of tumor disease is determined directly by detecting the signal of the probe and target sequence. As used herein, the term “probe” refers to a linear oligomer having naturally occurring or modified monomers or bonds, including deoxyribonucleotides and ribonucleotides that can be localized to specific nucleotide sequences. Specifically, the probes are single stranded for maximum efficiency in localization, more specifically deoxyribonucleotides.

 As the probe used in the present invention, a sequence that is perfectly ly complementary to the sequence including the SNP may be used, but is substantially complementary within a range that does not prevent specific localization. Can also be used. Specifically, the probe used in the present invention is localized to a sequence comprising 10-100 contiguous nucleotide residues (more specifically, 10-30 contiguous nucleotide residues) containing 50 nucleotides in sequence 1 of the sequence sequence system. Sequences that can be included. More specifically, the 3'-end or 5'-end of the probe has a base complementary to the SNP base. In general, probes with bases complementary to the SNP base at the 3 '-terminus or 5'-terminus tend to be determined by the conformity of the terminal sequence, as the stability of the duplex formed by the hybridization If the terminal portion is not localized, these duplexes may disintegrate under stringent conditions.

Conditions suitable for isomerization are described by Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, B. D., et al. , Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. Decisions may be made with reference to those disclosed in (1985). The stringent condit ions used for the hybridization can be determined by adjusting the temperature, ionic strength (concentrate concentration) and the presence of compounds such as organic solvents. Such stringent conditions can be determined differently depending on the sequence to be shaken.

 According to some embodiments of the invention, humans with the T allele of the invention have a very high risk of tumor disease.

 According to certain embodiments of the invention, the T allele of the invention comprises a TT homozygote or TG genotype. According to the present invention, when a single nucleotide polymorphism in a sample detected by the kit of the present invention comprises T nucleotides (eg 'Α / Α', Ά / C, Ά / c 'or' C / a ') 37% or more (see FIG. 2 and Table 1).

 According to certain embodiments of the invention, the subject with the T allele of the invention is at least 37% in blood tumors.

 As used herein, the term “diagnosis” refers to determining the suscept ibiity of an object for a particular disease or condition, or determining whether an object currently has a particular disease or condition (eg To determine the prognosis of a subject with a particular disease or condition, and to monitor the condition of the subject to provide information about therapeutic efficacy (eg, treatment efficacy). More specifically, to determine whether a subject currently has a particular disease or condition.

According to certain embodiments of the present invention, tumor diseases that can be diagnosed / detected through the kits or methods of the present invention are lymphoma, leukemia, myeloma, hemangiosarcoma, endothelial cell sarcoma (endothel iosarcoma), lymphatic sarcoma, lymphangiovascular endothelial cells. Sarcoma (lymphangioendothel iosarcoma), EMC (extraskeletal myxoid chondrosarcoma), cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, colorectal cancer, prostate carcinoma, colon cancer, breast cancer, ovarian cancer, prostate Cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (adenocarcinoma renal cell carcinoma, hepatocellular carcinoma, biliary tract cancer, fibrosarcoma, myxedema, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, synovial sarcoma, mesothelioma Ewing tumor s tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, Cystic carcinoma, soft thyroid carcinoma, bronchial carcinoma, choriocarcinoma, testicular, embryonal carcinoma, wale tumor, astrocytoma, Kaposi's sarcoma, medulloblastoma, craniocytoma, epithelial cell carcinoma, pineal carcinoma, hemangioblastoma, auditory neuroma , Dendritic glioma, meningioblastoma and retinoblastoma, more specifically hematological tumors, and more specifically T-cell derived lymphoma and leukemia.

 According to certain embodiments of the present invention, T-cell derived lymphoma or leukemia that can be diagnosed / detected through the kit or method of the present invention is AITLCangioimmunoblast ic T-ce 11 lymphoma (PTL), pannicul it is ike l ike T cel l lymphoma, NK-T cel l lymphoma, lymphoblast ic lymphomas, peripheral T-cell lymphoma, cutaneous T cel l lymphoma, T-cell type anaplastic large cell lymphoma, T1 cel l type (ALCL), long stool T_ cell lymphoma, HTLV-1 (Hiiman T-lymphotropic virus) — 1) One associated ATL (Adult T—cel l 1 eukem ia / 1 ymphoma), T-cel prolymphocytic leukemia, T-cell l lymphocytes (T-cel l granular) lymphocyt ic leukemia) and aggressive N-cell leukemia. The.

 According to another aspect of the invention, the invention is SEQ ID NO: 2

It provides a kit for diagnosing tumor disease comprising an antibody or aptamer that specifically binds a variant amino acid sequence of Gly to Val substituted amino acid sequence of the amino acid sequence of RhoA (Ras homo log gene fami ly, member A) . Since the tumor disease to be diagnosed in the present invention has already been described above, the description thereof is omitted to avoid excessive duplication.

According to the invention, monobasic polymorphisms on the RhoA gene of the invention result in substitution of the coding amino acid. G17V is the amino acid sequence of the variant protein encoded by the above-described variant nucleotides found by the inventors. Accordingly, the kit of the present invention detects a mutant protein encoded by the mutant nucleotide of the present invention according to an immunoassay method using an antigen-antibody reaction to diagnose a tumor disease. Can be used.

Such immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art. For example, when the method of the present invention is carried out according to a radioimmunoassay, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) detects a mutant Lp-PLA ₂ protein. It can be used to.

 The antibody to the variant protein used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

 Antibodies to variant proteins can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. 4,816,567) or phage antibody library method (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mo J. Biol., 222: 58, 1-597 (1991) It can be prepared by). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wi ley / Greene, NY, 1991. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting the above-described variant protein antigens into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

 By analyzing the intensity of the final signal by the above-described immunoassay process, oxidative stress-related diseases can be diagnosed. That is, when a signal for a mutant protein is stronger in a human sample than a normal sample, it is diagnosed as having a high risk of oxidative stress-related diseases.

Kits of the present invention can use aptamers that bind specifically to the above-described variant RhoA protein instead of the antibody. As used herein, the term "aptamer" refers to a single-stranded nucleic acid (RNA or DNA) molecule. Or as a peptide molecule, means that it binds to a specific target material with high affinity and specificity. General contents of aptamers are described in Bock LC et al. , Nature 355 (6360): 564-6 (1992); Hoppe-Seyler F, Butz K "Peptide a tamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 426-30 (2000); Cohen BA, Colas P, Brent R. "An artificial cell ᅳ cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 14272-7 (1998).

 According to a specific embodiment of the present invention, the kit of the present invention is applied to Asians.

 As used herein, the term "Asia" refers to the Far East region where Mongolian races, including Korea, China and Japan, reside. "Asian" means a population in which the ancestor is Asian, preferably a population in which at least 10 or more ancestors are Asian. More specifically, the Asian of the present invention is a Korean. According to another aspect of the present invention, the present invention provides a variant nucleotide sequence in which the 50th nucleotide of the RhoA (Ras homo log gene family, member A) gene, which is SEQ ID NO: 1, is substituted for G to T. According to another embodiment of the present invention, the present invention provides a variant amino acid sequence in which the 17th amino acid of the amino acid sequence of the RhoA (Ras homo log gene family, member A) protein of SEQ ID NO: 2 is substituted with Gly to Val. do.

Advantageous Effects

 The features and advantages of the present invention are summarized as follows:

 (a) The present invention relates to a biomarker for diagnosing human tumor disease and use thereof.

(b) According to the present invention, the biological sample is the 50th of the RhoA (Ras homo log gene family, member A) gene of SEQ ID NO: 1 If the nucleotide contains a single nucleotide polymorphism (SNP) nucleotide (eg, 'A / A', Ά / C, Ά / c 'or' C / a '), the biological sample is a tumor disease (Specifically, lymphoma or leukemia).

 (C) Thus, the kits and methods of the present invention can be used to detect / diagnose human tumor diseases (eg, lymphoma or leukemia) very effectively and easily.

[Brief Description of Drawings]

 1 is a result showing the mutant gene profile identified through total exome sequencing and RNA-siemens analysis of AITL patient samples. The top number refers to the sample number of the patient tissue deposited in the hospital.

 Figure 2 shows the results showing RhoA mutations (G17V) identified through Sanger sequencing in AITL patient samples.

 ᅳ Figure 3 is a result of measuring GTPase activity according to the RhoA mutation position (Fig. 3a) and a graph quantitatively showing the result (Fig. 3b). Wild-type RhoA and RhoA G14V mutations retained GTPase activity, while RhoA G17V mutations did not show activity. RhoA T19N mutation was used as a positive control without GTPase activity.

 4 shows the effect of G17V mutant RhoA on cell proliferation. G17V and T19N mutants without RhoA activity showed increased cell proliferation rates than wild type and G14V mutants.

 5 is a result showing the association between G17V mutant RhoA and cell invasion (invas ion). G17V and T19N mutants without RhoA activity showed significantly increased cell invasiveness than wild type and G14V mutants.

[Form for implementation of invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . EXAMPLES Experimental Methods Axome Sequencing Data Processing

 Paired-end 101-bp reads were obtained using the IUumina HiSeq 2000 platform in lymphoma and normal samples, respectively, under the same conditions. Axle data. The analysis was processed in the following pipeline. First, perform a basic QCXquality control process to clean up the affine sequences from the read information and use fastx (http://hannonlab.cshl.edu/fastx_toolkit) to detect bad read results and sequence errors (artifacts). Removed. Thereafter, the above-described quality check procedures of the data using FastQC (http://www.bioinformatics.bbsrc.ac.uk/pro.iects/fastqc)-ir may be used to perform problems at a later stage. Excluded. Next, alignment with the human reference genome hgl9 was performed using Burrows-Wheel Aligner v0.5.9 (Li and Durbin 2010). For more accurate variant detection using Genome Analysis ToolKit vl.6-5 (Mc enna, Hanna et al. 2010), Pi cardOttp: // pi card, sourceforge.net), local realignment and base quality recal Various 'clean up' processes were performed, including the removal of duplicates using ibrat ions. Through the above process, completely sorted and ordered night files (bam files) were obtained.

The night files were used to identify somatic varients. Somatic single-nucleotide variations (SNV), and small insertions and deletions were detected using VarScan v2.3.3 (Koboldt, Zhang et al. 2012). The variants may have a VarScan (minimum Filtration was carried out using 1-value (> 0.05) provided by the minimum coverage,>10; and the minimum normal depth,> 2). Finally, we annotated various predicted biological effects using a list of non-synonymous mutations at the coding site, which included functional impact, cancer type in COSMIC and Mutation assessor. (Reva, Antipin et al. 2007), including known functions of genes in cancer. The outputs described above were further filtered to a segmental duplication score (<0.5) and 1000 genome project allele frequency value = 0 using ANN0VAR.

RNA sequence data processing

 Paired-end 101-bp reads were obtained using Illumina HiSeq 2000 Sequencer in each lymphoma sample. Basic QC processes were performed on RNA-Seq data as described in Exome Sequential Data Processing. After alignment with the human reference genome hgl9 using tophat2 (Trapnell, Pachter et al. 2009), duplication was removed using Picard (http://picard.sourceforge.net). Using completely sorted and ordered bam files, we determined whether somatic mutations were expressed. Genomic DNA Sequencing

 After exome sequencer, candidate mutations were identified by Sanger sequencer. Genomes obtained from formaHn fixed paraffin embedded (FFPE) samples using I-StarTaq-Maxime PCR PreMix kit (iNtRON) and suitable primer pairs to detect genomic mutations in RhoA genes in samples of lymphoma patients deposited at Samsung Medical Center. PCR analysis was performed on DNA. PCR products were electrophoresed on agarose gels and sequenced using nested primers.

RhoA Activity Assay The amount of GTP-loaded RhoA was measured with a pull down based RhoA activation assay kit 0 ^ 031 1 011, Denver, CO) according to the manufacturer's instructions. Jurkat cells were transfected and maintained for 48 hours and then cultured in RPMI 1640 medium containing 10% FBS. The cells were then washed with ice cold PBS and resuspended in lysate complete layer. The cell lysates were transferred to 1.5 ml refrigerated centrifuge tubes and then centrifuged at 10,000 xg for 1 minute at 4 ° C. Protein concentration was determined using a protein assay kit (Precision Red advance protein assay (Cytoskeleton, Denver, CO)), and about 200-300 proteins were used for the RhoA activation assay per prescript ion. GTP-binding RhoA protein in cell lysates was purified by reaction at 4 ^° C. on rhotekin-Rho binding domain (RBD) glutathione affinity beads and rotator. The active GTP-bound RhoA protein in the cell lysate binds to the beads, while the inactive GDP-bound RhoA protein was removed through a wash step. Bead pellets were obtained by precipitating and resuspending in Laemml i buffer, followed by SDS-PAGE and immunoblotting using anti-RhoA and anti-HA antibodies. Cell proliferation assay

48 hours after transfection, cells were aliquoted in triplicate in 100 μl RPMI 1640 medium containing 10% FBS and antibiotics at a density of 5 × 10 ³ cells / well in 96-well plates. After plating, cells were incubated at 37 ^° C. in a humidity-maintained chamber. Cell proliferation was examined by cell counting kit -8 (cel l Counting Ki t-8; Doj indo) every 24 hours according to the manufacturer's instructions, and the absorbance value of each well was determined by a microplate reader ( Spectra Max 180, Molecular Devices) was measured at 450 nm. Invas ion assays

Cell invasion assays were bio-cocelcel invasion pre-coated with basement membrane matrigel after re-hydration according to the manufacturer's instructions. Chambers s (Bee t on D i ck i nson; 8 μηι pupil size) were used. Transfected Jurkat cells were loaded into each insert (upper chamber) at a density of 3 × 10 ⁵ cells / ml in serum-deficient media. The lower compartments were filled with 750 μΐ medium containing 10% FBS. After 24 hours of incubation, the infiltrated cells were quantified by counting with a hemocytometer. Each experiment was repeated three times. Infiltration was determined by cell counting and calculated using the following formula: Cell Infiltration (%) = Lower N / (Lower N + Upper N) X 100, where N represents the mean value of the number of cells in each compartment.

Experimental results: Whole exome sequencing and RNA-sequence analysis using angioi imoblastic T-cell lymphoma (AITL) sample

 In order to elucidate the carcinogenic mechanism of lymphoma (AITL) and develop anti-cancer drug resistance treatment targets, we conducted mutant gene profiles by performing whole axome sequencing and RNA-sequencing using samples from AITL patients. Blood samples obtained from lymphoma patients and controls (e.g., normal people) were used as templates for exome + RNA-sequence (5), axome sising (2) and RNA-sequence (4). Gene mutations were analyzed via (FIG. 1). As can be seen in Figure 1, mutations in which the base is substituted in genes such as RhoA, TET2, CD28, etc. have been found. Among these, the mutation position of the RhoA gene is a mutation in which the 50th nucleotide, G, is replaced with T in the RhoA CDS (coding sequence; SEQ ID NO: GenBank Accession Number: AF498970. 1), and the RhoA amino acid sequence of SEQ ID NO: 2 In GenBank Accession Number: M21117.1, the 17th glycine (GGA) was substituted with valine (GTA). Detection of RhoA Mutations in Lymphoma Patient Samples

RhoA mutations found through the axome and RNA sequencing described above were identified using a paraffin block patient sample collected at Samsung Medical Center. Sanger after amplifying genome c DNA by PCR method Sequencing was used to confirm the mutation location of RhoA. Table 1

 RhoA G17V mutation detected in 27 AITL patients.

상기 표 1에서 확인할 수'있듯이， 27명의 AITL 환자 시료를

As can be seen in Table 1 above, 27 AITL patient samples were collected.

PCR with the template detected RhoA G17V mutations in 10 patient samples (37.0%). Ten patient samples for which RhoA G17V mutations were detected were not only completely reversed nucleotides, but also partially mutated and detected with wild type (FIG. 2). Detection of RhoA Mutations in Different Lymphoma Samples

 To investigate whether the RhoA G17V mutation is specific for AITL, we have identified the mutation in other types of lymphomas as well.

Table 2

Identification of RhoA G17V Mutations in Patients with Different Types of Lymphoma.

상기 표 2에 나타난 바와 같이， PCTL 시료에서 13개 중 1개 (7.6%) , NK/T 시료에서 20개 중 3개 ( 15.0%)에서 동일한 위치에서의 RhoA 돌연변이 검출되었는데， 이는 RhoA G17V 돌연변이가 T-세포-유래된 림프종에서 발견된다는 것을 의미한다.

As shown in Table 2, one out of 13 (7.6%) in PCTL samples and three out of 20 (15.0%) in NK / T samples detected RhoA mutations at the same position, indicating that the RhoA G17V mutation It is found in T-cell-derived lymphoma.

Changes in RhoA Activity by RhoA Gi Mutations

 RhoA is a member of the Ras family that has GTPase activity and is involved in cell proliferation. Accordingly, the inventors examined the GTPase activity of G17V mutated RhoA by the RhoA activity assay (RTK pull-down assay) (FIG. 3). RhoA G14V mutant, a wild type RhoA and a continuously active form, showed high GTPase activity, but RhoA G17V mutant showed no GTPase activity. Thus, the RhoA G17V mutation is a mutation that inhibits GTPase activity. Effect of RhoA G17V Mutation on Cell Proliferation

 To investigate the effects of RhoA G17V mutations on cell proliferation, we used RhoA s iRNA to eliminate endogenous RhoA and transfect wild-type RhoA or RhoA G17V mutants to Jurkat cells, respectively. Was compared. G17V and T19N mutants without RhoA activity significantly increased cell proliferation compared to wild type and G14V mutants (FIG. 4). Therefore, it was confirmed that the lower the RhoA activity, the higher the cell proliferation rate and the RhoA G17V mutant induces cell proliferation. Effect of RhoA G17V Mutation on Cell Infiltration

To investigate the effect of RhoA G17V mutations on cell invasion, we used RhoA s iRNA to eliminate endogenous RhoA and transfect wild-type RhoA or RhoA G17V mutants to Jurkat cells, respectively. (invas iveness) was compared (FIG. 5). G17V and T19N mutants without RhoA activity had very high cell invasion compared to wild-type and G14V mutants. This means that the invasiveness is increased and the RhoA G17V mutation causes an increase in cellular invasiveness. The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that these specific technologies are merely certain embodiments, and thus the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. Reference

 1. Koboldt, D. C., et al. (2012). "VarScan 2: somat ic mutat ion and copy number alterat ion discovery in cancer by exome sequencing." Genome research 22 (3): 568-576.

 2. Li, H. and R. Durbin (2010). Fast and accurate long- re ad alignment with BurrowsWheeler transform. Bioinformatics. 26: 589-595.

 3. McKenna, A., et al. (2010). The Genome Analysis Toolkit ： a MapReduce framework for analyzing next -generation DNA sequencing data. Genome research. 20: 1297-1303.

 4. Reva, B., et al. (2007). Determinants of protein function revealed by combinatorial entropy optimization. Genome biology. 8: 1-

15.

 5. Trapnel 1, C., et al. (2009). Top Hat: discovering splice junct ions with RNA-Seq. Bioinformatics. 25: 1105-1111.

 6. Wang, K., et al. (2010). ANN0VAR: functional annotation of genet ic variants from high-throughput sequencing data. Nucleic acids research. 38: el64-el64.

Claims

 [Patent Claims]  [Claim 11  Contains primers or probes that specifically bind to 10-100 consecutive nucleotide sequences, including G to T substitutions of the 50th nucleotide of the RhoA (Ras homo log gene family, member A) gene, SEQ ID NO: 1 Tumor disease diagnostic kit. [Claim 2]  The method of claim 1, wherein the subject having a substitution of G to T of the 50th nucleotide of the RhoA gene, which is the first sequence of the sequence listing, has a risk of hematologic tumor of 37% or more. [Claim 3]  The kit for diagnosing tumor disease according to claim 1 or 2, wherein the tumor disease is hematological tumors. [Claim 4]  4. The kit for diagnosing tumor disease according to claim 3, wherein the blood tumor is T-cell derived lymphoma or leukemia. [Claim 5] The method of claim 4, wherein the T-cell-derived lymphoma or leukemia is an anioimmunoblast ic T-cel 1 lymphoma (AITL), pannicul itis ^to l ike T cel l lymphoma (PTC), NK / T (NK-T cel l). lymphoma, lymphoblast ic lymphomas, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, T-cell type anaplastic large cell lymphoma lymphoma, T-cel 1 type (ALCL), enteropathy-type T-cell lymphoma, HTLV-1 (Hiiman T-lymphotropic virus-l) -associated ATL (Adul t T-cel 1) leukemia / lymphoma, T-cell 1 pro-lymphocytic leukemia, T-cell Kit for diagnosing tumor disease, characterized in that the T-cell granular lymphocytic leukemia or aggressive NK-cell leukemia. [Claim 6]  Tumor disease comprising an antibody or ¾ timer specifically binding to the variant amino acid sequence of Gly to Val substituted amino acid sequence of the amino acid sequence of RhoA (Ras homo log gene family, member A) of SEQ ID NO: 2 Diagnostic kit. [Claim 7]  7. The kit for diagnosing tumor disease according to claim 6, wherein the subject having the variant amino acid sequence has a risk of blood tumor of 37% or more. [Claim 8]  8. The kit for diagnosing tumor disease according to claim 6 or 7, wherein the tumor disease is hematological tumors. [Claim 9]  9. The kit for diagnosing tumor disease according to claim 8, wherein the blood tumor is T-cell derived lymphoma or leukemia. [Claim 10] The method of claim 9, wherein the T-cell-derived lymphoma or leukemia is AITLCangioimmunoblast ic T-cell lymphoma, PTCLCpannicul it is-1 ike T-cell lymphoma, NK / T (NK-T cell lymphoma), lymphoblastic lymphoma (lymphoblastic lymphomas), peripheral T-cell lymphoma, cutaneous T cell lymphoma, T-cell type anaplastic large cell 1 lymphoma, T-cel 1 type; ALCL ), Long side T- Cellular lymphoma (enteropathy-type T lymphocyte), HTLV-K human T-lymphotropic virus (1), associated ATL (Adult T-cel 1 1 eukem ia / 1 ymphoma), and T-cell prelymphocytic leukemia (T- cel 1 pro lymphocyt ic leukemia, T-cell granular lymphocyt ic leukemia, or aggressive NK-cell leukemia. [Claim 111]  A method for detecting a tumor disease comprising the following steps:  (a) isolating nucleic acid molecules from a biological sample of the subject; And  (b) identifying the 50th nucleotide of the RhoA (Ras homo log gene family, member A) gene of SEQ ID NO: 1 in the nucleic acid molecule of step (a), wherein the 50th nucleotide is G to T If substituted, the subject is determined to have a tumor disease. [Claim 12]  The biological sample of claim 11, wherein the biological sample is blood, plasma, serum, tissue, cells, lymph, bone marrow, saliva, ocular fluid, semen, brain extract, spinal fluid, joint fluid, thymus fluid, ascites fluid, amniotic fluid, It is a cell tissue fluid or cell culture fluid, The detection method of the tumor disease characterized by the above-mentioned. [Claim 13]  The method of claim 11, wherein the tumor disease is a blood tumor. [Claim 14] 12. The method of claim 11, wherein the detecting frequency of the single nucleotide polymorphic nucleotide in step (b) is at least 37%. [Claim 15]  The method of claim 11, wherein the step (b) is performed through gene amplification or microarray. [Claim 16]  A variant nucleotide sequence in which the 50th nucleotide of the RhoA (Ras homo log gene fami ly, member A) gene, SEQ ID NO: 1, is substituted with G to T. [Claim 17]  A variant amino acid sequence in which the 17th amino acid of the amino acid sequence of the RhoA (Ras homo log gene fami ly, member A) protein of SEQ ID NO: 2 is replaced with Val in Gly.