KR101744169B1 - Biomarkers for Diagnosing a Tumor Disorder and Uses Thereof - Google Patents

Abstract

The present invention relates to biomarkers for the diagnosis of human tumor diseases and uses thereof. According to the present invention, a biological sample is a single nucleotide polymorphism (SNP) nucleotide (e.g., 'A / A', or a polynucleotide) at the 50th nucleotide of the Rho homolog gene family member A gene of Sequence Listing 1. The biological sample is highly at risk for a tumor disease (specifically, lymphoma or leukemia) when it comprises an A / C, an A / c or a C / a. Therefore, the kit and method of the present invention can be used to detect / diagnose a human tumor disease (for example, lymphoma or leukemia) very effectively and easily.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for diagnosing a tumor disease,

The present invention relates to biomarkers for the diagnosis of tumor diseases and uses thereof.

Oncogenesis involves 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 sarcoma, leukemia and lymphoma (Rabbits, TH, Nature 372 (6502): 143-149 (1994) But less in epithelial tumors. In recent years, tumorigenic fusions have been reported to be associated with prostate (Tomlins, SA, et al. , Science 310 (5748): 644-648 (2005)), thyroid (Bongarzone, I., et al. , Cancer Res. : 2979-2985 (1994); Kroll, TG, et al, Science 289 (5483):. 1357-1360 (2000)), and lung (. Soda, M., et al , Nature 448 (7153): 561- 566 (2007)), which suggests that the tumorigenic fusants occur very frequently in carcinomas. For example, recurrent chromosomal translocation is characteristically found in many leukemias, lymphomas, and sarcomas (e.g., mesenchymal tumors). In addition, the single-gene can be used as a tumor biomarker if the single-gene mutation affects the overall pathogenesis process.

On the other hand, lymphomas that can be classified as non-Hodgkin's lymphomas (NHL) and Hodgkin's lymphomas (HL) are part of the immune system that protects against infection and disease and helps them to survive longer as well as divide faster than normal cells It is a type of hematologic tumor that occurs in leukocyte B or T lymphocyte, accounting for about 4% of total tumor. Lymphomas can occur in the lymphatic, spleen, bone marrow, blood or other organs and are more common in males than females. Typically, lymphoma represents a solid tumor of lymphoid cells. Most treatment methods use chemotherapy, but in some cases, radiation therapy and / or bone marrow transplantation is used. In general, lymphoma can be treated according to history, type and stage. The malignant cells often originate in the lymphatic canal and can also affect other organs. Lymphomas from other organs are referred to as extranodal lymphomas. Non-lymph node locations include skin, brain, intestine and bone. Lymphomas are closely related to lymphoid leukemias, which typically occur not only in lymphocytes but also in circulating blood and bone marrow. Thus, lymphoma is present in many types and is a part of a broad group of diseases called hematological neoplasms.

Because lymphoma tends to recur as it progresses to later stages, it is very important to diagnose lymphoma at an early stage. In general, the diagnosis of lymphoma is made by histopathologic analysis of biopsy samples obtained from lymphatic vessels. The type of lymphoma can be identified through morphological analysis of cancer cells using a microscope or detection of a lymphoma-specific molecule (biomarker). Since the method of diagnosing lymphoma using a biomarker is more effective and quicker than a biopsy using a microscope, it is urgently required in the art to identify a novel biomarker.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop biomarkers for the rapid identification and / or diagnosis of tumor diseases (e.g., hematological neoplasms). As a result, we examined the mutant gene profile through whole exome sequencing and RNA-sequencing of biological samples obtained from patients with lymphoma and found that the 50th nucleotide of the RhoA gene is a single nucleotide polymorphism polymorphism, SNP) nucleotide mutation can be effectively used for the diagnosis of lymphomas, thereby completing the present invention.

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

It is another object of the present invention to provide a method for detecting a tumor disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a recombinant vector comprising 10-100 consecutive sequences of the T allele comprising substitution of G to T of the 50 < th > nucleotide of Rho homologue gene family member (RhoA) Provided is a kit for diagnosing tumor diseases, which comprises a primer or a probe that specifically binds to a nucleotide sequence.

According to another aspect of the present invention, the present invention provides a method of 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 homologue gene family member (RhoA) gene of the first sequence of the sequence in the nucleic acid molecule of step (a), wherein the 50th nucleotide is substituted from G to T The subject is judged to have a tumor disease.

The present inventors have made extensive efforts to develop biomarkers for the rapid identification and / or diagnosis of tumor diseases (for example, blood tumors). As a result, we examined the mutant gene profile through total exome sequencing and RNA-sequencing of biological samples obtained from patients with lymphoma and found that the RhoA G17V mutation, in which the 50th nucleotide of the RhoA gene was mutated into a single nucleotide polymorphic nucleotide, And can be effectively used.

The Rho protein is a well-known member of the p21 Ras superfamily of small GTPases, exhibiting intrinsic GTPase activity through conformational changes between an inactive GDP-binding state and an active GTP-binding state. The RhoA protein regulates signal transduction from cell surface receptors to intracellular target molecules and is useful for a variety of cellular functions including cell shape, motility, cytokinesis, smooth muscle contraction, and tumor progression Are 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 than that of surrounding normal tissues, and positively increases 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 higher levels at the metastatic site (Suwa, H., et al. , Br. J. Cancer , 77: 147152 (1998)). Thus, RhoA positively functions in transmigration of tumor cells. In addition, RhoA has been reported to regulate transcriptional activation by serum response factors (SRF) and to be involved in oncogenesis 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 mutant of the present invention was found at high frequency in AITL lymphoma and T-cell-derived lymphoma (see Table 1). The kit of the present invention comprises a primer which specifically binds to 10-100 consecutive nucleotide sequences comprising substitution of G to T of the 50th nucleotide of Rho homologue gene family member A gene (RhoA) Wherein the T nucleotide is complementary to A nucleotide and can be used to identify and / or diagnose a tumor disease in a subject. Thus, in the case of the step (b) of the present invention, "50th nucleotide is substituted by G to T" means that the 50th nucleotide is single nucleotide polymorphism (SNP) nucleotides 'A / A'',' A / c ', or' C / a '.

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 in which the 50th nucleotide of the Rho homologue gene family member (RhoA) gene of SEQ ID No. 1 is "T ", but such an allele nucleotide is found in double stranded gDNA (genomic DNA) , It can be interpreted that it also includes a nucleotide sequence complementary to the aforementioned nucleotide sequence. That is, single nucleotide polymorphisms (SNPs) 'T' in a complementary nucleotide sequence become 'A'. In this regard, 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 the nucleotide polymorphisms of a single nucleotide (A, T, C or G) in the genome, DNA sequence diversity occurring in different cases in the liver. For example, DNA fragments of different individuals (e.g., (G or T) when it comprises a difference in a single base, such as a TGTG [ G / T ] AAAG, designated as the complementary base of G / T in the SNP of the present invention ) SNPs have two alleles. Within a population, SNPs can be assigned to a minor allele frequency (MAF; the lowest allele frequency in locus found in a particular population). There are variations in the human population, and one common SNP allele in the geological or ethnic group is very rare. Single bases can be changed (substituted), removed (deleted), or added (inserted) to the polynucleotide sequence. SNPs can cause in-frame shifts in translation frames.

A single nucleotide polymorphism can be included in the coding sequence of a gene, in a non-coding region of a gene, or in intergenic regions between genes. SNPs in the coding sequence of a gene do not necessarily cause changes 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 referred to as silent mutations) and are referred to as non-synonymous for SNPs that form other polypeptide sequences. Non-consensual SNPs can be missense or nonsense, and mismatch changes produce other amino acids while nonsense changes form non-mature termination codons. SNPs that are not in the protein-coding region can induce gene silencing, transcription factor binding, or non-coding RNA sequences.

Human DNA sequence variability can affect disease outbreaks and how humans respond to pathogens, chemicals, drugs, vaccines and other reagents. In addition, SNPs are thought to be key enablers for realizing the concept of customized medicines. Above all, SNPs that are actively being developed as markers are very important in biomedical research to diagnose disease by comparing genomic sites between groups with or without disease. SNPs are the most abundant variants of the human genome and are presumed to exist at a SNP ratio of 1.9 kb (Sachidanandam et al., 2001). SNPs are highly stable genetic markers, sometimes directly affecting phenotypes, and are well suited for automated genotyping systems (Landegren et al., 1998; Isaksson et al., 2000). In addition, SNPs studies are also important in grain and livestock breeding programs.

According to the present invention, the kit of the present invention comprises a pair of primers designed to amplify a polynucleotide comprising a single nucleotide polymorphism (SNP) nucleotide (e.g., T nucleotide) of the 50 th nucleotide in the first sequence of the sequence listing It is a kit for the diagnosis of tumor diseases. Specifically, an omnidirectional primer among the primer pairs anneals to a sequence adjacent to a tumor disease (e.g., lymphoma or leukemia) -related single nucleotide polymorphism (SNP) nucleotide corresponding to the 50 th nucleotide in the first sequence of the sequence listing , And the 3'-terminal of the forward primer has a base complementary to the SNP base.

According to some 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 a polymerase chain reaction (PCR). According to some embodiments of the present invention, the primers of the present invention can be used for amplification reactions.

The term "amplification reaction" as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual , 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 19 (WO 88/10315) (US Ser. No. 6,410, 276), self-sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence priming polymerase chain (CP-PCR) (U.S. Patent No. 4,437,975), random (US Pat. Nos. 5,413,909 and 5,861, 245), nucleic acid sequence based amplification (NASBA) (US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification. LAMP), but is not limited thereto. Other amplification methods that may be used are described in U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Pat. No. 09 / 854,317, the teachings of which are incorporated herein by reference.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof 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 polymerase chain reaction (PCR) IPCR), vectorette PCR and TAIL-PCR (thermal asymmetric interlaced PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is carried out using a primer, a gene amplification reaction is carried out to analyze and diagnose 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 nucleotide sequence of the biomarker of the present invention can be determined from a sample (for example, genomic DNA) to be analyzed and the presence or absence of a tumor disease can be determined.

According to some embodiments of the present invention, the biological sample that may be used in the kit or method of the present invention is a biological sample isolated from a human and is a blood sample, blood, plasma, serum, tissue, cells, lymph, bone marrow, saliva, Blood serum, plasma, serum, tissue, cells, lymph fluid, bone marrow fluid, cell tissue fluid and the like, and more particularly, the blood, plasma, serum, And more specifically, blood, plasma, serum, tissue, and lymph, and more specifically, blood, plasma, serum, tissue, cells, lymphatic fluid and cell culture liquid, Include blood and tissue.

In some embodiments of the invention, the amplification process of the present invention may be carried out according to the polymerase chain reaction (PCR) set forth in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159.

As used herein, the term "primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include, but are not limited to, naturally occurring dNMPs (i.e. dAMP, dGMP, dCMP and dTMP), modified nucleotides, non-natural nucleotides and ribonucleotides.

As used herein, the term " extension primer "means 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. The extension primer extends to a position where the immobilization probe is annealed to occupy a portion where the probe is annealed.

The extension primer used in the present invention includes a nucleotide sequence that can be complementarily hybridized to the first position of the target nucleic acid. As used herein, the term "complementary" means that under certain annealing or hybridization conditions the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence, and the terms " substantially complementary & Perfectly complementary ', and specifically, it is' completely complementary'. As used herein, the term "substantially complementary sequence" as used in connection with a primer sequence is intended to encompass a complete sequence as well as a sequence that is comparable to that of the sequence to be compared, Inconsistent sequences are also included.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer, but is typically a polynucleotide of 15-30 bp. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The term "annealing" or "priming ", as used herein, refers to oligodeoxynucleotides or nucleic acids apposition to a template nucleic acid, wherein the polymerase is capable of polymerizing a nucleotide to form a template nucleic acid, To form nucleic acid molecules.

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

The nucleotide sequence of the present invention target to be referred to in the production of the primer can be confirmed in GenBank. For example, the genBank accession number of the RhoA gene, the first sequence of the sequence listing of the present invention, which is a biomarker, is AF498970.1, and a primer can be designed with reference to the above sequence and the single nucleotide polymorphic nucleotide of the present invention. Specifically, the forward primer among the primer pairs comprises a nucleotide sequence capable of annealing with a sequence adjacent to a human lymphoma-related single nucleotide polymorphism (SNP) base corresponding to the 50 th nucleotide in the first sequence of the sequence listing, The 3'-end of the primer has a base complementary to the SNP base.

The term "nucleic acid molecule" referred to in the present specification has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and the nucleotide which is a basic constituent unit in the nucleic acid molecule is not only a natural nucleotide, Also included are modified analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

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

When the starting material is mRNA, the total RNA is isolated by a conventional method 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 Mol Biol Rep, 9:..... 242 (1991); Ausubel, FM et al, Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized by cDNA using reverse transcriptase. Since the total RNA is isolated from a human (for example, a patient with lymphoma), it has a poly-A tail at the end of mRNA, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptase there (see: 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, the identification / identification of the specific sequence can be carried out by applying various methods known in the art. For example, techniques that can be applied to the present invention, fluorescence in situ hybridization (fluorescence in situ hybridization, FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-strand keonpeo decimation analysis (SSCA, Orita et . al, PNAS, USA 86: 2776 (1989)), RNase protection assay (Finkelstein et al, Genomics, 7 :. 167 (1990)), datteu blot (dot blot) analysis, a modified gradient gel electrophoresis (DGGE, Wartell et al, Nucl.Acids Res, 18: .. using the method of E. coli mutS protein) (Modrich, Ann Rev. Genet, 25: 2699 (1990)), protein (e.g., to recognize nucleotide mismatches.: 229-253 (1991)) and allele-specific PCR.

Sequence changes result in differences in base-linkage within the single-stranded molecule, leading to the appearance of different bands of mobility, and SSCA detects this band. DGGE analysis uses a denaturing gradient gel to detect sequences that represent wild type sequences and other mobility.

Other techniques generally use probes or primers complementary to sequences comprising the SNPs of the invention. For example, in an RNase protection assay, a riboprobe complementary to a sequence comprising a SNP of the present invention is used. The riboprobe is hybridized with DNA or mRNA isolated from human, and then cleaved with an RNase A enzyme capable of detecting a mismatch. If there is a mismatch and RNase A recognizes, a smaller band is observed.

In the analysis using a hybridization signal, a probe complementary to the sequence containing the SNP of the present invention is used. In this technique, hybridization signals of the probe and the target sequence are detected to determine the presence or absence of the tumor disease directly.

The term "probe" as used herein refers to a linear oligomer having a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a particular nucleotide sequence. Specifically, the probe is a single strand for maximum efficiency in hybridization, and more specifically, a deoxyribonucleotide.

As the probe used in the present invention, a sequence complementary to the sequence including the SNP may be used, but a sequence substantially complementary to the sequence that does not interfere with the specific hybridization is used It is possible. Specifically, a probe used in the present invention may hybridize to a sequence comprising 10-100 consecutive nucleotide residues (more specifically, 10-30 consecutive nucleotide residues) comprising 50 nucleotides in the first sequence of the sequence listing . More specifically, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If the part is not hybridized, such a duplex can be disassembled under stringent conditions.

Suitable conditions for hybridization are described by Joseph Sambrook, et al. (1985), published by Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington, DC (1985), and Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY And can be determined by referring to The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

According to some embodiments of the present invention, a human having the T allele of the present invention has a very high risk of tumor disease.

According to some embodiments of the invention, the T allele of the invention comprises a TT homozygote or a TG genotype. According to the present invention, when a single nucleotide polymorphism in a sample detected by the kit of the present invention comprises a T nucleotide (e.g., 'A / A', 'A / C', 'A / ') Was more than 37% (Note: Fig. 2 and Table 1).

According to some embodiments of the invention, the subject with the T allele of the invention is greater than 37% in hematologic tumors.

The term "diagnosing" as used herein includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder (e.g., Determination of the prognosis of an object that has suffered a particular disease or disorder, and therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy) And more specifically, to determine whether an object currently has a particular disease or disorder.

According to some embodiments of the present invention, tumor diseases that can be diagnosed / detected through the kit or method of the invention include lymphoma, leukemia, myeloma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic vessel endothelial cell sarcoma cancer, prostate cancer, breast cancer, ovarian cancer, prostate cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, lymphangioendotheliosarcoma, extraskeletal myxoid chondrosarcoma, cervical cancer, testicular tumor, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, tumor of the thyroid gland, tumor of the thyroid, adenocarcinoma of the papillary, adenocarcinoma of the thyroid, tumor of the thyroid, bronchial carcinoma, choriocarcinoma, testicular, embryonal carc The present invention relates to a method for the treatment of hematologic malignancies such as malignant neoplasms, tumors, adenocarcinomas, adenocarcinomas, adenocarcinomas, adenoma, Wilms tumor, astrocytoma, Kaposi sarcoma, hematoblastoma, craniopharyngioma, ependymoma, pineal gland, angioblastoma, hematological tumors, 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 selected from the group consisting of angiimmunoblastic T-cell lymphoma (AITL), pancreatitis-like T-cell lymphoma ), NK / T (NK-T cell lymphoma), lymphoblastic lymphomas, peripheral T-cell lymphoma, cutaneous T cell lymphoma, T- (AMLL), enteropathy-type T cell lymphoma, HTLV-1 (T-lymphotropic virus-1) -related ATL (Adult T-cell leukemia / lymphoma, T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, and aggressive NK-cell leukemia. However, the present invention is not limited thereto.

According to another aspect of the present invention, the present invention provides a mutant amino acid sequence in which the 17th amino acid sequence of the amino acid sequence of Rho homologue gene family member (RhoA), the second sequence of the sequence listing, A kit for diagnosing tumor diseases, which comprises an antibody or an aptamer which binds to a tumor.

Since the tumor diseases to be diagnosed in the present invention have already been described above, the description thereof is omitted in order to avoid excessive duplication.

According to the present invention, the single base polymorphism on the RhoA gene of the invention results in the substitution of the coding amino acid. G17V is the amino acid sequence of a mutant protein encoded by the above-described mutant nucleotide found by the present inventors. Accordingly, the kit of the present invention can be used for diagnosing a tumor disease by detecting a mutant protein encoded by the mutant nucleotide of the present invention according to an immunoassay method using an antigen-antibody reaction.

Such immunoassay can be performed according to various immunoassay or immunostaining protocols developed conventionally. For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e. G., C ^14, I ^125, P ³² and S ³⁵⁾ The antibody detects a variation Lp-PLA ₂ protein labeled with . ≪ / RTI >

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

Antibodies to the variant protein can be obtained by methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (U.S. Patent No. 4,816,567 (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , 222: 58, 1-597 (1991)) have. 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 , Wiley / Greene, NY, 1991. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. A polyclonal antibody can be obtained by injecting a mutant protein antigen described above into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

By analyzing the intensity of the final signal by the above-described immunoassay, it is possible to diagnose an oxidative stress-related disease. In other words, if a signal for a mutant protein in a human sample is stronger than a normal sample, the risk of the oxidative stress-related disease is high.

The kit of the present invention can use an aptamer that specifically binds to the mutated RhoA protein described above in place of the antibody. As used herein, the term " aptamer " refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. The general contents of the Aptamer are described in Bock LC et al., Nature 355 (6360): 564-6 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 426-30 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-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 reside, including Korea, China, and Japan. "Asian" refers to a population whose ancestors are Asian, preferably a population of at least 10 ancestors of Asian populations. More specifically, the Asians of the present invention are Korean.

According to another aspect of the present invention, the present invention provides a mutant nucleotide sequence in which the 50 th nucleotide of the Rho homologue gene family member (RhoA) gene sequence of SEQ ID No. 1 is substituted with G to T nucleotide.

According to another embodiment of the present invention, the present invention provides a mutant amino acid sequence in which the 17th amino acid sequence of the amino acid sequence of RhoA (Ras homolog gene family, member A) .

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

(a) The present invention relates to a biomarker for diagnosis of human tumor disease and its use.

(b) According to the present invention, the biological sample is a single nucleotide polymorphism (SNP) nucleotide (e.g., 'A / A') at the 50th nucleotide of the Rho homologue gene family member (RhoA) A ',' A / C ',' A / c 'or' C / a '), the biological sample has a very high risk of tumor disease (specifically lymphoma or leukemia).

(c) Therefore, the kit and method of the present invention can be used to detect / diagnose a human tumor disease (for example, lymphoma or leukemia) very effectively and easily.

Figure 1 shows the results of a mutant gene profile identified through total exome sequencing and RNA-sequence analysis of AITL patient samples. The number at the top indicates the sample number of the patient tissue deposited at the hospital.
Figure 2 shows the RhoA mutation (G17V) identified through Sanger sequencing in AITL patient samples.
FIG. 3 is a graph (FIG. 3 a) showing GTPase activity according to the RhoA mutation position and FIG. 3 b showing quantitatively the results. The wild-type RhoA and RhoA G14V mutations maintained GTPase activity, but the RhoA G17V mutation showed no activity. The RhoA T19N mutation was used as a positive control without GTPase activity.
Figure 4 is a plot showing the effect of G17V mutant RhoA on cell proliferation. G17V and T19N mutants without RhoA activity showed increased cell proliferation rates compared to wild type and G14V mutants.
Figure 5 shows the relationship between G17V mutant RhoA and cellular invasion. G17V and T19N mutants without RhoA activity showed significantly increased invasiveness than wild-type and G14V mutants.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Exome sequencing data processing

The paired-end 101-bp readings were obtained using the Illumina HiSeq 2000 platform on lymphoma and normal samples under the same conditions, respectively. Exom data analysis was processed with the following pipeline. First, a basic QC (quality control) process is performed to organize the adapter sequences from the read information and to use the fastx (http://hannonlab.cshl.edu/fastx_toolkit) to generate bad read results and sequence errors, . Thereafter, the above-described quality check procedures are performed using FastQC ( http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc ), thereby eliminating problems that may occur at a later stage . Next, alignment was performed with the human reference genome hg19 using Burrows-Wheel Aligner v0.5.9 (Li and Durbin 2010). Picard (http://picard.sourceforge.net), local realignment and base quality recalibration for more accurate mutant detection using the Genome Analysis Toolkit v1.6-5 (McKenna, Hanna et al. 2010) And a process of removing the duplicates by using a variety of 'clean up' processes. Through the above procedures, we obtained perfectly sorted and ordered bam files.

Somatic variants were identified using the night files. Somatic cell single-nucleotide variations (SNV), and small insertions and deletions were detected using VarScan v2.3.3 (Koboldt, Zhang et al. 2012). The variants were filtered using p-values (> 0.05) provided by VarScan (minimum coverage,>10; and minimum normal variant depth,> 2). Finally, a list of non-synonymous mutations at the coding site was used to annotate several predicted biological effects, including functional impact, cancer type in COSMIC, and mutation assessor (Reva, Antipin et al. 2007). The above-described outputs were further filtered with a segmental duplication score (< 0.5) and a 1000 genome project allele frequency value = 0 using ANNOVAR.

RNA sequencing data processing

Paired-end 101-bp readings were obtained using an Illumina HiSeq 2000 sequencer in each lymphoma sample. A basic QC process for RNA-Seq data was performed, as described in Exome Sequencing Data Processing. After alignment with the human reference genome hg19 was performed using tophat2 (Trapnell, Pachter et al. 2009), duplicate removal was performed using Picard (http://picard.sourceforge.net). Using fully sorted and trimmed bam files, the inventors have determined whether somatic cell mutations have been expressed.

Genomic DNA sequencing

After exon sequencing, candidate mutations were identified by Sanger sequencing. To detect the genomic mutation of the RhoA gene in samples of lymphoma patients deposited at Samsung Medical Center, a genome obtained from a formalin fixed paraffin embedded (FFPE) sample using an I-StarTaq-Maxime PCR PreMix kit (iNtRON) DNA was analyzed by PCR. The PCR product was electrophoresed on an agarose gel and sequenced using a nested primer.

RhoA activation assay

The amount of GTP-loaded RhoA was determined with a pull-down based RhoA activated assay kit (Cytoskeleton, Denver, CO) according to the manufacturer's instructions. Jurkat cells were transfected and maintained for 48 hours and then cultured in RPMI1640 medium containing 10% FBS. The cells were then washed with ice cold PBS and resuspended in lysis buffer. The cell lysate was transferred to a 1.5 ml refrigerated centrifuge tube and centrifuged at 10,000 xg for 1 minute at 4 [deg.] C. Protein concentration was determined using a Precision Red advance protein assay (Cytoskeleton, Denver, CO) and approximately 200-300 μg of protein was used for RhoA activation assay per prescription. GTP-bound RhoA protein in cell lysates was purified by reacting with rhoekin-RBD (Rho binding domain) glutathione affinity beads on a rotator at 4 ° C. The active GTP-bound RhoA protein in the cell lysate was bound to the beads while the inactive GDP-bound RhoA was removed through the wash step. Bead pellets were precipitated and resuspended in Laemmli buffer, followed by SDS-PAGE and immunoblotting using anti-RhoA and anti-HA antibodies.

Cell proliferation assays

After 48 hours of transfection, cells were plated in 96-well plates at a density of 5 x 10 ³ cells / well in triplicate in 100 μl RPMI 1640 medium containing 10% FBS and antibiotics. After plating, the cells were incubated at 37 [deg.] C in a humidity-maintained chamber. Cell proliferation was monitored with a cell counting kit-8 (Dojindo) for 4 consecutive days every 24 hours according to the manufacturer's instructions, and the absorbance value of each well was measured using a microplate reader (Spectra Max 180, Molecular Devices) at 450 nm.

In bito  Invasion assay

Cell infiltration assays were performed using Bio-Coat cell invasion chambers (Becton Dickinson; 8 μm pupil size) precoated with basement membrane matrigel after re-hydration according to the manufacturer's instructions. The Jurkat cells are transfected serum was loaded on the insert (upper chamber) at a density of 3 × 10 ⁵ cells / ml in the deficient medium. The lower compartments were filled with 750 μl medium containing 10% FBS. After incubation for 24 hours, the infiltrated cells were quantified by counting with a hemocytometer. Each experiment was repeated three times. Infiltration was determined by cell counting and was calculated using the following equation: Cell infiltration (%) = N / (lower N + upper N) × 100, where N represents the average number of cells in each compartment.

Experiment result

Whole exome sequencing and RNA-sequence analysis using an angioimmunoblastic T-cell lymphoma (AITL) sample

To identify the carcinogenic mechanism of lymphoma (AITL) and to develop an anticancer drug resistant therapeutic target, the present inventors conducted a total exome sequencing and RNA-sequence using a sample of AITL patients to investigate the mutant gene profile.

Blood samples obtained from lymphoma patients and controls (for example, normal persons) were used as templates to amplify the gene through exome + RNA-sequence (5 persons), exome sequencing (2 persons) and RNA- Mutations were analyzed (Figure 1). As can be seen from Fig. 1, mutations in which bases have been substituted in genes such as RhoA, TET2, and CD28 have been found. Among them, the mutation position of the RhoA gene is a mutation in which G is changed to G, which is the 50th nucleotide in the coding sequence (Sequence Listing GenBank Accession Number: AF498970.1), and the RhoA amino acid sequence of the second sequence (GGA) was substituted with valine (GTA) at the 17th glycine (GenBank Accession Number: AAM21117.1).

RhoA mutation detection in lymphoma patient samples

The RhoA mutations found through exome sequencing and RNA sequencing described above were confirmed using paraffin block patient samples collected at Samsung Medical Center. The genomic DNA was amplified by PCR method and the mutation position of RhoA was confirmed by Sanger sequencing.

RhoA G17V mutation detected in 27 AITL patients. Wild type 17/27 63.0% RhoA G17V
Mutation A
A / C
A / c
C / a 10/27 37.0% synthesis 27 100%

As shown in Table 1 above, PCR of 27 AITL patient samples with template showed that RhoA G17V mutation was detected in 10 patient samples (37.0%). The 10 patient samples in which the RhoA G17V mutation was detected were not only completely reversed in the nucleotide but also partially mutated and detected with the wild type (FIG. 2).

RhoA mutation detection in different types of lymphoma patients

To investigate whether the RhoA G17V mutation is specific to AITL, we have identified this mutation in other types of lymphoma.

Identification of RhoA G17V mutations in patients with different types of lymphoma. Lymphoma AITL PTCL NK / T Wild type 17/27 63.0% 12/13 92.3% 17/20 85.0% RhoA
G17V
Mutation A
A / C
A / c
C / a 10/27 37.0% 1/13 7.6% 3/20 15.0% synthesis 27 100% 13 100% 20 100%

As shown in Table 2, RhoA mutations at the same positions were detected in one of 13 (7.6%) of the PCTL samples and 3 of 20 (15.0%) of the NK / T samples, indicating that the RhoA G17V mutation &Lt; / RTI &gt; is found in T-cell-derived lymphoma.

Change of RhoA activity by RhoA G17V mutation

RhoA is a protein belonging to the Ras family and has GTPase activity and is associated with cell proliferation. Thus, the inventors investigated the GTPase activity of G17V mutated RhoA by the RhoA activation assay (Figure 3). The wild-type RhoA and the continuously activated form of the RhoA G14V mutation showed high GTPase activity, but the RhoA G17V mutation did not show any 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 effect of RhoA G17V mutation on cell proliferation, we used RhoA siRNA to eliminate endogenous RhoA and transfected wild-type RhoA or RhoA G17V mutant into Jurkat cells, respectively, Respectively. The G17V and T19N mutants lacking RhoA activity significantly increased the cell proliferation rate compared to the wild-type and G14V mutants (FIG. 4). Therefore, it was confirmed that the lower the RhoA activity, the greater the cell proliferation rate and the RhoA G17V mutation induces cell proliferation.

Effect of RhoA G17V Mutation on Cellular Infiltration

To investigate the effect of RhoA G17V mutation on cell invasion, we used RhoA siRNA to remove endogenous RhoA and transfected wild-type RhoA or RhoA G17V mutant into Jurkat cells, respectively, to induce invasiveness ) (Fig. 5). G17V and T19N mutants without RhoA activity were highly invasive compared to wild-type and G14V mutants, indicating that the lower the RhoA activity, the greater the cell invasion and the RhoA G17V mutation leads to an increase in cellular invasion .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

References

1. Koboldt, D. C., et al. (2012). "VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing." Genome research 22 (3): 568-576.

2. Li, H. and R. Durbin (2010). Fast and accurate long-read 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. Trapnell, C., et al. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25: 1105-1111.

6. Wang, K., et al. (2010). ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic acids research. 38: e164-e164.

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Claims (17)

Blood containing a primer or a probe that specifically binds to 10-100 consecutive nucleotide sequences comprising the substitution of G to T of the 50th nucleotide of the Rho homologue gene family member A gene (RhoA) A kit for diagnosing a tumor disease, wherein the hematologic tumor is a T-cell-derived lymphoma or leukemia.
The kit for diagnosing a hematologic malignancy according to claim 1, wherein the subject having the G to T substitution of the 50th nucleotide of the RhoA gene of the sequence listing 1 has a risk of hematologic tumors of 37% or more.
delete delete The method of claim 1, wherein the T-cell-derived lymphoma or leukemia is selected from the group consisting of angiimmunoblastic T-cell lymphoma (AITL), pancreatitis-like T-cell lymphoma (PTCL), NK- lymphocytic lymphomas, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, T-cell type anaplastic large cell lymphoma (T-cell type; ALCL) (T-cell leukemia / lymphoma), T-cell lymphocytic leukemia (T-cell lymphoma), and T-cell lymphoma prolymphocytic leukemia, T-cell granular lymphocytic leukemia, or aggressive NK-cell leukemia.
An antibody or aptamer that specifically binds to a mutant amino acid sequence in which the 17th amino acid of the amino acid sequence of the Rho homologue gene family member (RhoA) of SEQ ID No. 2 is substituted with Gly to Val amino acid sequence, A kit for the diagnosis of hematologic malignancy, wherein the hematologic tumor is a T-cell-derived lymphoma or leukemia.
7. The kit for diagnosing a hematologic malignancy according to claim 6, wherein the subject having the mutated amino acid sequence has a risk of hematologic tumors of 37% or more.
delete delete 7. The method of claim 6, wherein the T-cell derived lymphoma or leukemia is selected from the group consisting of angiimmunoblastic T-cell lymphoma (PIT), pancreatitis-like T-cell lymphoma (PTCL), NK- lymphocytic lymphomas, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, T-cell type anaplastic large cell lymphoma (T-cell type; ALCL) (T-cell leukemia / lymphoma), T-cell lymphocytic leukemia (T-cell lymphoma), and T-cell lymphoma prolymphocytic leukemia, T-cell granular lymphocytic leukemia, or aggressive NK-cell leukemia.
A method for providing information necessary for the diagnosis of T-cell derived lymphoma or leukemia 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 homologue gene family member (RhoA) gene of the first sequence of the sequence in the nucleic acid molecule of the step (a), wherein the 50th nucleotide is G- Wherein said subject is judged to have T-cell derived lymphoma or leukemia.
12. The method of claim 11, wherein the biological sample of interest is selected from the group consisting of blood, plasma, serum, tissue, cells, lymph, bone marrow, saliva, ocular fluid, semen, brain extract, spinal fluid, joint fluid, thymus fluid, A cell tissue fluid or a cell culture fluid.
delete 12. The method of claim 11, wherein the detection frequency of the single nucleotide polymorphic nucleotide in step (b) is at least 37%.
12. The method of claim 11, wherein step (b) is performed through gene amplification or microarray.
Rho homologous gene family (member A) gene, the first sequence of the sequence listing in which the 50 &lt; th &gt;
Rho homologous gene family (member A) protein, wherein the 17th amino acid is the second sequence of SEQ ID NO: 2 substituted by Gly to Val.

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