KR102267407B1 - Novel NTRK1 fusion gene as colon cancer marker and the uses thereof - Google Patents

Abstract

It relates to a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) and NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the same. In addition, the present invention relates to a composition for diagnosing colon cancer comprising the fusion protein, the fusion gene, or an agent for measuring a transcript of the fusion gene, and a kit for diagnosing colon cancer comprising the same. In addition, the present invention relates to a method of providing information necessary for diagnosing colorectal cancer, including measuring the level of the NTRK1 fusion gene, a transcript thereof, or a protein thereof, and a method of providing information necessary for predicting a prognosis of colorectal cancer treatment. In addition, it relates to a screening method for a colorectal cancer therapeutic agent comprising measuring the expression level of the NTRK1 fusion gene, and to a pharmaceutical composition for preventing or treating colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient. By providing a diagnostic marker specific for colorectal cancer according to the present invention, accurate colorectal cancer diagnosis is possible, predicting the clinical prognosis through stratification of colorectal cancer patients, predicting the effectiveness of treatment for colorectal cancer, In particular, it becomes possible to predict the effectiveness of colorectal cancer treatment by NTRK1 protein inhibitors. Accordingly, it is possible to limit the administration of the drug to colorectal cancer patients who are considered to be ineffective to administer the drug, and thus it becomes possible to realize efficient and safe customized colorectal cancer treatment.

Description

Novel NTRK1 fusion gene as colon cancer marker and the uses thereof

It relates to a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) and NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the same. In addition, the present invention relates to a composition for diagnosing colon cancer comprising the fusion protein, the fusion gene, or an agent for measuring a transcript of the fusion gene, and a kit for diagnosing colon cancer comprising the same. In addition, the present invention relates to a method of providing information necessary for diagnosing colorectal cancer, including measuring the level of the NTRK1 fusion gene, a transcript thereof, or a protein thereof, and a method of providing information necessary for predicting a prognosis of colorectal cancer treatment. In addition, it relates to a screening method for a colorectal cancer therapeutic agent comprising measuring the expression level of the NTRK1 fusion gene, and to a pharmaceutical composition for preventing or treating colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient.

Colorectal cancer is the second and third most common cancer in cancer deaths, accounting for about 13% of all cancers. In the United States, it is reported that about 50,310 people die from colorectal cancer every year and 136,830 new cases of colorectal cancer occur. In Korea, colorectal cancer ranks fourth in mortality rate after lung cancer, liver cancer, and stomach cancer. In 1980, 5.8% of all cancers accounted for 6.1% in 1985, 8.2% in 1995, 11.2% in 2002, and 12.9% in 2012. In 2011, the death rate from colorectal cancer increased by 5.6%, while the death rate from gastric cancer, liver cancer, and cervical cancer decreased.

With the development of early diagnosis, curative surgery, and chemotherapy using colonoscopy, the 5-year survival rate of all colorectal cancer patients is reported to be about 64.7%. However, the 5-year survival rate of stage 1 colorectal cancer patients improved to 89.8%, and the 5-year survival rate for stage 2–3 colorectal cancer patients improved to 70.5%, while the 5-year survival rate for late stage colorectal cancer patients was 12.9%, stagnating for the past 30 years. However, it is difficult to improve the survival rate through existing treatments. The extended period of disease-free survival of end-stage colorectal cancer patients by targeted therapies such as Erbitux (Merck) and Avastin (Roche), which were recently approved for insurance benefits, were 0.9 months (p = 0.048) and 1.4 months (p), respectively, compared to conventional chemotherapy. = 0.0023), indicating insufficient clinical efficacy. Compared with breast cancer and lung cancer, genomic research is insufficient for colorectal cancer, and accordingly, patient stratification at the molecular level is insufficient. Therefore, in order to increase the survival rate of colorectal cancer patients, it is urgent to stratify patients at the molecular level and discover new customized colorectal cancer targets.

It is known that various genetic changes such as various types of cancer genes and tumor suppressor genes are involved in the development of colorectal cancer. In fact, colorectal cancer is the cancer for which genetic changes that occur during the carcinogenesis process have been identified the most. In colorectal cancer, a change in one oncogene or tumor suppressor gene cannot cause cancer alone. In order for normal colonic mucosal cells to progress to colorectal cancer through the stage of adenoma, several cancers are required over many years. Changes in related genes must be accumulated, which is called a multi-step change of genes in the development of colorectal cancer. What is important here is not the sequence of genetic changes at each stage, but the sum of the ultimately cumulative changes in genes. Genetic changes involved in the development of colorectal cancer include mutations in cancer genes and tumor suppressor genes, DNA methylation abnormalities, and mutations in DNA repair genes.

Since the formation of cancer proceeds in a complex relationship between various genes and their interactions, a genomic approach is being made to discover new cancer genes and therapeutic targets by examining and comparing the expression and mutation of all genes. . Genes whose expression is specifically increased or decreased in cancer cells are reported to be involved in the survival and proliferation of cancer cells, such as cell division, cell signaling, cytoskeleton, cell movement, cell defense, expression of genes and proteins, and intracellular metabolism. is becoming Advances in cancer genomics have contributed to stratification of patients, such as breast cancer and lung cancer, and to discover targets for customized treatment of patients by class. However, the lack of evaluation of the clinical effectiveness of the discovered target is evaluated as a major cause of clinical research failure of customized anticancer therapeutics researched and developed through cancer genomics.

The NTRK1 fusion gene has been reported across a variety of carcinomas. A TPM3-NTRK1 fusion gene has been reported in colorectal cancer and papillary thyroid cancer (Martin-Zanca et al., Nature, 1986, vol. 319, pp. 743-748; Wilton et al., Cytogenetics and cell genetics, 1995, vol. 68, 122- 124), myosin phosphatase Rho interacting protein (MPRIP) or CD74-NTRK1 fusion gene has been reported in lung cancer (Vaishnave et al., Nature medicine, 2013, vol. 19, pp. 1469-1472; Patent No. WO 2014071358 A2), a TP53 or lamin A/C (LMNA)-NTRK1 fusion gene has been reported in Spitzoid melanoma (Wiesner et al., Nature communications, 2014 vol. 5, 3116; Patent No. WO 2013059740 A1), a RAB GTPase activating protein 1-like: RABGAP1L)-NTRK1 fusion gene has been reported in gallbladder cancer (Ross et al., The oncologist, 2014, Vol. 19, 235~ 242), a neurofascin (NFASC) or brevican (BCAN)-NTRK1 fusion gene was reported in glioblastoma multiforme (Kim et al., PloS one, 2014, Vol. 9, e91940). However, while the role of the NTRK1 fusion gene as an oncogene in some carcinomas has been identified, the role, frequency, and clinical significance of the NTRK1 fusion gene as an oncogene in colorectal cancer has not been comprehensively identified.

The present inventors have made intensive research efforts to stratify colorectal cancer patients, predict prognosis, and discover genes that can be a target of treatment through genomic research of colorectal cancer. As a result, LMNA-NTRK1 and TPM3- in Korean and Chinese colorectal cancer tissues The NTRK1 fusion gene and its expression are significantly increased in a specific patient group, and the NTRK1 fusion gene and the fusion transcript and fusion protein encoded by it are factors that determine the clinical prognosis of colorectal cancer. By finding out, the present invention was completed.

One object of the present invention is to provide a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) and NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the same.

Another object of the present invention is to provide a composition for diagnosing colon cancer comprising the fusion protein, the fusion gene, or an agent for measuring a transcript of the fusion gene.

Another object of the present invention is to provide a kit for diagnosing colon cancer comprising the composition for diagnosing colon cancer.

Another object of the present invention is to provide a method for providing information necessary for diagnosing colorectal cancer, comprising measuring the level of the NTRK1 fusion gene, its transcript, or its protein.

Another object of the present invention is to provide a method for providing information necessary for predicting the prognosis of colorectal cancer treatment, which includes measuring the level of the NTRK1 fusion gene, its transcript, or its protein.

Another object of the present invention is to provide a screening method for a therapeutic agent for colorectal cancer, comprising measuring the expression level of the NTRK1 fusion gene.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient.

As a result of repeated intensive studies to achieve the above object, the present inventors conducted TPM3-NTRK1 fusion by next-generation transcriptome sequencing of transcripts extracted from 150 cases of colorectal cancer clinical tissue and 50 normal colorectal clinical tissues. Transcripts and LMNA-NTRK1 fusion transcripts were detected. The TPM3-NTRK1 fusion gene was generated by an inversion between the q21.3 and q23.1 regions of chromosome 1, and the LMNA-NTRK1 fusion gene was generated by a deletion between the q22 and q23.1 regions of the chromosome 1.

The novel fusion gene and its encoded fusion transcript and fusion protein were retrieved from 3 cases (2%) of 147 Korean colon cancer clinical tissues. From the above facts, it was found that the expression of fusion transcripts and fusion proteins in colorectal cancer was caused by gene fusion.

The three cases of colorectal cancer clinical tissues in which NTRK1 fusion transcripts were confirmed did not have somatic mutations in known colorectal cancer genes such as KRAS, NRAS, and PIK3CA. In addition, when the NTRK1 fusion gene was forcibly expressed in a non-neoplastic NIH3T3 cell line, in vitro and in vitro tumorigenicity were obtained. From the above facts, it was revealed that NTRK1 gene fusion is a driver oncogene in colorectal cancer.

The fusion protein of the present invention was confirmed in 29.2% of Korean colorectal cancer patients and 25.6% of Chinese colorectal cancer patients, and the fusion gene is observed with high frequency in colorectal cancer of Northeast Asians such as Koreans and Chinese. It was found that the causative gene was In addition, the survival rate of colorectal cancer patients having the fusion gene of the present invention or the fusion protein encoded therein is lower than the survival rate of colorectal cancer patients not having the fusion gene of the present invention or the fusion protein encoded therein, so that the fusion gene of the present invention has colorectal cancer It was found that it is possible as a diagnostic marker to predict the clinical prognosis of a patient.

It is thought that the above gene fusion results in improved activation of NTRK1 protein, and in colorectal cancer patients in which such activation has occurred, an inhibitor for NTRK1 protein is considered to be therapeutically effective. For this reason, the present inventors are able to predict the efficacy of treatment with a drug by targeting the fusion gene in colorectal cancer, and in this prediction, it is possible to predict the efficacy of treatment with a drug for patients determined to be effective. When a drug is administered, it has been found that effective treatment is possible, and the present invention has been completed.

Accordingly, the present invention provides a method for diagnosing the clinical prognosis of a colorectal cancer patient using the fusion gene between the TPM3 gene or the LMNA gene and the NTRK1 gene, the fusion transcript and the fusion protein encoded therein, and colorectal cancer by the NTRK1 protein inhibitor. It relates to a method for judging the effectiveness of treatment, and a method for treating colorectal cancer using the judging of the efficacy, and more particularly, to provide the following inventions.

As one aspect for achieving the above object, the present invention provides a fusion protein of LMNA (Lamin A) or TPM3 (Tropomyosin 3) and NTRK1 (Neurotrophic Tyrosine Kinase, Receptor, Type 1) and a fusion gene polynucleotide encoding the same do.

Specifically, the present invention provides a fusion protein consisting of the following structural formula.

N-[fragment containing the N terminus of LMNA (Lamin A)]-[fragment containing the C terminus of Neurotrophic Tyrosine Kinase (NTRK1, Receptor, Type 1)]-C, or

N-[fragment containing the N terminus of TPM3 (Tropomyosin 3)]-[fragment containing the C terminus of NTRK1]-C.

In addition, the present invention provides a fusion gene polynucleotide consisting of the following structural formula.

5'-[fragment comprising the 5' end of the LMNA gene]-[fragment comprising the 3' end of the NTRK1 gene]-3', or

5'-[fragment comprising the 5' end of TPM3]-[fragment comprising the 3' end of NTRK1 gene]-3'.

That is, the present invention relates to a fusion protein in which a fragment including the N-terminus of LMNA or a fragment including the N-terminus of TPM3 and a fragment including the C-terminus of NTRK1 are linked, and a fragment including the 5' end of the LMNA gene or TPM3 gene The present invention relates to a fusion protein in which a fragment including a 5' end and a fragment including a 3' end of the NTRK1 gene are linked, wherein the LMNA, TPM3 or NTRK1 may be of human origin. The fusion protein of the present invention may be mixed with the NTRK1 fusion protein, or, in some cases, the LMNA-NTRK1 fusion protein or the TPM3-NTRK1 fusion protein.

As shown in the Examples of the present invention, the present inventors first discovered a fusion example of LMNA protein and NTRK1 protein in a human colorectal cancer patient. In addition, the frequency and clinical prognosis of colorectal cancer patients having TPM3 gene or LMNA gene fusion with NTRK1 gene and the fusion transcript and fusion protein encoded therein were confirmed for the first time through large-scale colorectal cancer patient cohort analysis (Table 1).

In the present invention, the TPM3 gene encoding the TPM3 protein may be derived from a human and is located on human chromosome 1 (q21.3), and the TPM3 protein encoded therefrom is a polypeptide having a total amino acid length of 285aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein. For example, the TPM3 gene is GenBank accession no. It may have a nucleotide sequence provided by NM_152263, and the TPM3 protein may be a protein having an amino acid sequence encoded by NM_152263. In particular, the TPM3 protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and the TPM3 gene may be composed of the nucleic acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, the fragment including the N-terminus of TPM3 has an amino acid sequence encoded by the nucleotide sequence up to the 8th exon of NM_152263 (from 154134289 to 154142876 base positions based on the (-) strand on chromosome 1). It may be a single nucleotide (g) that does not form a codon at the 3' end of exon 8 (Table 2 and Table 3). In particular, the fragment including the N-terminus of TPM3 may be composed of the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

The LMNA gene encoding the LMNA protein may be of human origin and is located on human chromosome 1 (q22), and the LMNA protein encoded therefrom is a polypeptide having a total amino acid length of 664aa. The LMNA protein or LMNA protein fragment is the N-terminal fusion partner of the LMNA-NTRK1 fusion protein. The LMNA gene is GenBank accession no. It may have a nucleotide sequence provided by NM_170707, and the LMNA protein may be a protein having an amino acid sequence encoded by NM_170707. In particular, the LMNA protein may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27, and the LMNA gene may be composed of the nucleic acid sequence of SEQ ID NO: 28, but is not limited thereto.

In the present invention, the fragment including the N-terminus of LMNA may have an amino acid sequence encoded by the nucleotide sequence up to the 6th exon of NM_170707 (base positions 156084504 to 156105740 bases relative to the (+) strand on chromosome 1). , one nucleotide (c) that does not form a codon at the 3' end of exon 6 may be present (Tables 6 and 7). In particular, the fragment including the N-terminus of the LMNA may consist of the amino acid sequence of SEQ ID NO: 31, but is not limited thereto.

The NTRK1 gene encoding the NTRK1 protein may be of human origin and is located on human chromosome 1 (q23.1), and the NTRK1 protein encoded therefrom is a protein with a total amino acid length of 796aa. NTRK1 protein or NTRK1 protein fragment is the C-terminal fusion partner of the TPM3-NTRK1 fusion protein. For example, the NTRK1 gene is GenBank accession no. It may have a nucleotide sequence provided by NM_001012331, and the NTRK1 protein may be a protein having an amino acid sequence encoded by NM_001012331. In particular, the NTRK1 protein may be a polypeptide composed of the amino acid sequence of SEQ ID NO: 7, and the NTRK1 gene may be composed of the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 13, but is not limited thereto.

In the present invention, the fragment including the C terminus of NTRK1 is exon 9 (156830671 to 156844363 base positions based on the (+) strand on chromosome 1) or exon 11 (156830671 based on the (-) strand on chromosome 1) of NM_001012331 It may have an amino acid sequence encoded by the nucleotide sequence from ~156845312 base position) or exon 12 (156830671 to 156845872 base positions based on the (-) strand on chromosome 1) to the last exon. At this time, the first two nucleotides (ac) of the 3' end of exon 9 and the first two nucleotides (gc) of the 3' end of exon 11 and the first two nucleotides of the 3' end of exon 12 ( gt) may be in a form that does not form a codon, and is linked to one nucleotide (g) additionally included in the TPM3 protein fragment as described above when fused with a TPM3 protein fragment to a codon (gac or ggc or ggt) to encode one amino acid (D or G). In particular, the fragment including the C terminus of NTRK1 may be composed of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, but is not limited thereto.

In the present specification, the break point (or fusion site) of the fragments of the fusion partner protein is described based on the exon of the gene encoding them, and the N-terminus of the fragment (in the case of the C-terminal fusion partner) Alternatively, the amino acid sequence of the encoded protein fragment is not affected by cleavage at any point in the intron region between the exon included at the end of the C-terminus (in the case of an N-terminal fusion partner) and the exon to be cleaved and removed. The cut point may be any point among the intron positions.

Meanwhile, in the present invention, the LMNA-NTRK1 fusion gene may have the nucleotide sequence of SEQ ID NO: 33, and the LMNA-NTRK1 fusion protein may have the nucleotide sequence of SEQ ID NO: 34, but is not limited thereto. In addition, in the present invention, the TPM3-NTRK1 fusion gene may have the nucleotide sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and the TPM3-NTRK1 fusion protein has SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26 may be, but is not limited thereto.

Unless otherwise stated in the present invention, all nucleotide sequences determined by sequencing the gene, transcript or transcript-derived cDNA or DNA molecule described herein may be determined using an automated next-generation sequencing sequencer or Sanger method sequence sequencer. and all amino acid sequences encoded by the determined nucleotide sequence can be determined using an automated peptide sequencer. The nucleotide sequence determined by this automated approach may contain some errors compared to the actual sequence. For example, the automatically determined nucleotide sequences are typically about 90% or more, specifically about 95% or more, more specifically about 99% or more, more specifically about 99.9% or more sequences from the actual nucleotide sequence of the sequenced cDNA or DNA molecule. may have homology. A single insertion or deletion in the nucleotide determined by comparison with the actual sequence may be included, and such an insertion or deletion causes a frameshift in translation of the nucleotide sequence so that the encoded amino acid sequence is completely different from the actual amino acid sequence. can be quite different.

In another aspect, the present invention provides a composition for diagnosing colon cancer comprising the fusion protein, the fusion gene, or an agent for measuring a transcript of the fusion gene.

In the present invention, the fusion protein and the fusion gene are as described above.

As used herein, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to determine whether colon cancer has occurred.

As used herein, the term “colon cancer” refers to cancer occurring in the mucous membrane, which is the innermost surface of the large intestine, and refers to rectal cancer, colon cancer, and anal cancer collectively.

Meanwhile, in the present invention, the fusion protein and fusion gene of LMNA or TPM3 and NTRK1 can be used as a diagnostic marker for diagnosing colorectal cancer, and in the present invention, the term "diagnosis marker, diagnostic marker or diagnostic marker" A substance that can distinguish colon cancer cells from normal cells and diagnose them. Polypeptides or nucleic acids (eg, mRNA), lipids, glycolipids, glycoproteins, and sugars that show an increase in colorectal cancer cells compared to normal cells. organic biomolecules such as (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purpose of the present invention, the colorectal cancer diagnostic marker is LMNA-NTRK1 (Lamin A-Neurotrophic Tyrosine Kinase, Receptor, Type 1) or TPM3-NTRK1 (Tropomyosin 3-Neurotrophic Tyrosine Kinase, Receptor, Type 1) fusion gene. These are genes whose expression is increased in a tissue or cell.

Selection and application of significant diagnostic markers determine the reliability of diagnostic results. A significant diagnostic marker means a marker with high reliability so that a result obtained by diagnosis is accurate, thus having high validity and showing consistent results even during repeated measurements. The colorectal cancer diagnostic marker of the present invention shows the same results even in repeated experiments with genes whose expression always increases due to direct or indirect factors with the onset of colorectal cancer, and the difference in expression level is very large compared to the control group, resulting in erroneous results They are highly reliable markers with little probability of dropping . Therefore, a diagnosis result based on a result obtained by measuring the expression level of a significant diagnostic marker of the present invention is reasonably reliable. At this time, except for genes expressed in almost the same amount in normal colonic epithelial cells and colon cancer cells, for example, genes whose expression is increased more than twice in colorectal cancer tissue compared to genes expressed in normal tissue used as a control. can select them.

In a specific embodiment of the present invention, the fusion gene of the TPM3 gene or the LMNA gene and the NTRK1 gene is a causative gene in colorectal cancer. It was confirmed that the probability of diagnosis of poor prognosis due to malignancy of colorectal cancer and treatment with NTRK1 protein inhibitor was high.

Therefore, the NTRK1 fusion gene or the fusion transcript and fusion protein encoded therein of the present invention have been confirmed to be specifically found or expressed in patients with solid cancer, specifically colorectal cancer, and thus the fusion gene or the fusion transcript and fusion protein encoded therein The protein can be usefully used as a prognostic diagnostic marker for solid cancer, specifically colorectal cancer.

The expression level of the fusion gene of the present invention in a biological sample can be confirmed by checking the amount of the fusion protein such as a transcript of the fusion gene, particularly mRNA. Preferably, the agent for measuring the level of the fusion gene mRNA may be a composition for diagnosing colon cancer comprising a primer that specifically binds to the gene.

In the present invention, "mRNA expression level measurement" refers to a process of determining the presence and expression level of mRNA of colorectal cancer marker genes in a biological sample in order to diagnose colon cancer, and the amount of mRNA is measured. Analytical methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip, but is not limited thereto.

In the present invention, the primer specifically binding to the mRNA of the fusion gene is a primer that specifically binds to the mRNA of the fusion gene, the fusion point is selected such that the region amplified by the primer includes the fusion point of the two genes to be fused. It may be a primer designed to be placed in between, and when the two genes to be fused constituting the fusion gene are the TPM3 gene and the NTRK1 gene, a primer specific for the nucleotide sequence encoding the N terminus of the TPM3 protein and the C terminus of NTRK1 It is a primer pair including a primer specific to a nucleotide sequence, and in particular, may be a primer pair consisting of the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 36, but is not limited thereto.

In addition, when the two genes to be fused constituting the fusion gene are the LMNA gene and the NTRK1 gene, a primer specific for the nucleotide sequence encoding the N-terminus of the LMNA protein and a primer specific for the nucleotide sequence encoding the C-terminus of NTRK1 are used. A primer pair including, in particular, may be a primer pair consisting of the nucleotide sequences of SEQ ID NO: 37 and SEQ ID NO: 38, but is not limited thereto.

On the other hand, the "primer" of the present invention is a nucleic acid sequence having a short free three-terminal hydroxyl group, which can form a base pair with a complementary template and refers to a short nucleic acid sequence that functions as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having a sequence of 7 to 50 nucleotides as primers specific for each marker gene. Primers may incorporate additional features that do not change the basic properties of the primer to serve as the starting point for DNA synthesis.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, “encapsulation”, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phospho poroamidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked residues, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.) ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention may also be modified using labels capable of providing a detectable signal, either directly or indirectly. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

In the present invention, the "agent for measuring a fusion gene" is a process of confirming the existence of the fusion gene of the present invention, which is a colorectal cancer marker gene, in a biological sample for diagnosing colon cancer, for example, specifically to the fusion gene. It may include a binding probe or primer. In particular, as the probe specifically binding to the fusion gene, a probe having the characteristics of Table 8 may be used, but the present invention is not limited thereto. In a specific example of the present invention, measurements were made using a probe of the NTRK1 Split FISH Probe (Cat# FS0024, Taiwan) sold by Abnova.

In the present invention, "measurement of protein expression level" refers to a process of confirming the presence and expression level of a protein expressed from the fusion gene of the present invention, which is a colorectal cancer marker gene, in a biological sample for diagnosing colon cancer. The amount of the protein can be confirmed by using an antibody that specifically binds to the protein of the fusion gene.

In particular, in the present invention, the antibody that specifically binds to the protein of the fusion gene may be one that binds to LMNA or near the fusion site of TPM3 and NTRK1 or to the C terminus of NTRK1, but is not limited thereto. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, and rocket. Immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited thereto. .

Preferably, the agent for measuring the level of the protein may be a composition for diagnosing colon cancer comprising an antibody specific for the protein.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

As described above, since a novel colorectal cancer marker fusion protein has been identified in the present invention, it can be easily prepared using a technique well known in the art to generate an antibody using it.

The polyclonal antibody can be produced by a method well known in the art for obtaining a serum containing the antibody by injecting the above-described colon cancer marker protein antigen into an animal and collecting blood from the animal. Such polyclonal antibodies can be prepared from hosts of any animal species, such as goats, rabbits, sheep, monkeys, horses, pigs, bovine dogs and the like.

Monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6:511-519), or a phage antibody library (Clackson et al, Nature, 352:624). -628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

In another aspect of the present invention, there is provided a kit for diagnosing colon cancer comprising the composition for diagnosing colon cancer according to the present invention.

Specifically, the colorectal cancer diagnostic kit may be a reverse transcription polymerase-PCR (RT-PCR) kit, a DNA chip kit, or a protein chip kit. In particular, the colorectal cancer diagnostic kit may further include one or more other component compositions, solutions or devices suitable for the analysis method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include primers specific for the nucleic acid sequence of the control gene. Other Reverse Transcription Polymerase Reaction Kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another aspect, it may preferably be a diagnostic kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, and the like for preparing a fluorescent probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

Also preferably, it may be a diagnostic kit comprising essential elements necessary for performing a protein chip or performing ELISA. A protein chip kit or an ELISA kit includes an antibody specific for a marker protein. Antibodies are antibodies with high specificity and affinity for each marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibody, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and substrates thereof or capable of binding the antibody. other materials and the like.

In another aspect, the present invention provides a method of providing information necessary for diagnosing colorectal cancer, comprising measuring the level of the NTRK1 fusion gene, its transcript, or its protein, in order to provide information necessary for diagnosing colorectal cancer. to provide. Specifically, measuring the level of the NTRK1 fusion gene, its transcript or its protein from an isolated sample of an individual suspected of colon cancer; and comparing the measured expression level of the fusion gene, its transcript or its protein with the expression level of the fusion gene, its transcript, or its protein in a normal control sample. A method of providing information necessary for diagnosing colon cancer to provide.

In the present invention, the NTRK1 fusion gene includes a polynucleotide constituting the 5' end of the LMNA gene or the TPM3 gene; and a polynucleotide constituting the 3' end of the NTRK1 gene.

In the present invention, "individual" refers to an animal that can develop colon cancer, and is not limited as long as colon cancer is suspected. In addition, as a disease for which information necessary for diagnosis can be obtained through the above method, the expression of a fusion gene of the TPM3 gene or the LMNA gene and the NTRK1 gene, or the expression of the NTRK1 gene depending on the fusion of the NTRK1 gene is limited. It may not, in particular, be colon cancer.

In the present invention, as used herein, the subject's sample is a tissue in which the level of mRNA of the LMNA-NTRK1 or TPM3-NTRK1 fusion gene, which is a colorectal cancer marker, or its protein is different, for example, cells, tissues, organs, body fluids (blood, lymph fluid, etc.) ), digestive fluid, sputum, alveolar-bronchial lavage fluid, urine, feces, as well as nucleic acid extracts (genomic extracts, transcriptome extracts, cDNA preparations or aRNA preparations prepared from transcriptomic extracts) or proteins obtained from these biological samples Extracts include, but are not limited to. do. In addition, the sample may have been subjected to formalin fixation treatment, alcohol fixation treatment, freezing treatment or paraffin embedding treatment. In addition, the gene, transcript, cDNA, or protein can be prepared by selecting a known method suitable for it, taking into consideration the type and state of the sample, and the like.

Through the above detection methods, by comparing the expression level of the NTRK1 fusion gene, its transcript, or its protein in the normal control with the expression level in the suspected colorectal cancer individual, it is possible to diagnose whether the colorectal cancer-suspected individual actually has colorectal cancer. That is, after measuring the expression level of the marker of the present invention in cells suspected of colorectal cancer, measuring the expression level of the marker of the present invention in normal cells and comparing both, the expression level of the marker of the present invention is normal If the expression is more from the cells estimated to be colorectal cancer than that of the cells, the cells estimated to be colorectal cancer can be judged as colorectal cancer. That is, when the expression level of the fusion gene, its transcript, or protein thereof measured from the isolated sample of an individual suspected of colon cancer in the above method is higher than the expression level of the fusion gene, its transcript, or its protein in the normal control sample, the large intestine It may be characterized in that it is diagnosed as cancer.

As an analysis method for measuring the level of mRNA or protein thereof of the LMNA-NTRK1 or TPM3-NTRK1 fusion gene, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc., but are not limited thereto. Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the antigen-antibody complex formation amount in the colorectal cancer suspect can be compared, and the LMNA-NTRK1 or TPM3-NTRK1 fusion protein in the colon cancer marker gene By judging whether or not there is a significant increase in the expression level, it is possible to diagnose whether a patient suspected of colon cancer actually develops colorectal cancer.

In the present invention, the term antigen-antibody complex refers to a combination of a colorectal cancer marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formation can be quantitatively measured through the size of a signal of a detection label. .

The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto. When an enzyme is used as the detection label, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Therase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphophenolpyruvate deca Voxylase, β-latamase, and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrine, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4 W(CN)8 , [Os(bpy) 3 ] 2+ , [RU(bpy) 3 ] 2+, [MO(CN) 8 ] 4-, and the like. Radioisotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like.

The protein expression level is preferably measured using an ELISA method. ELISA is a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support, and an indirect ELISA using a labeled antibody recognizing an antigen attached to a solid support. Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and antigen, reacts with another antibody that recognizes an antigen in a complex of an antibody attached to a solid support and antigen, and then a labeled antibody that recognizes the antibody It includes various ELISA methods, such as an indirect sandwich ELISA using a secondary antibody. More preferably, after attaching an antibody to a solid support and reacting the sample, a labeled antibody recognizing the antigen of the antigen-antibody complex is attached to enzymatically develop color or for an antibody recognizing the antigen of the antigen-antibody complex It is detected by a sandwich ELISA method in which a labeled secondary antibody is attached to enzymatically develop color. By confirming the degree of formation of a complex between the colorectal cancer marker protein and the antibody, it is possible to determine whether colorectal cancer occurs.

Also, preferably, Western blot using one or more antibodies against the colorectal cancer marker is used. The total protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to a nitrocellulose membrane to react with the antibody. In a method of confirming the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein generated by gene expression can be checked to determine whether colon cancer occurs. The detection method consists of a method of examining the expression level of the marker gene in the control group and the expression level of the marker gene in the colorectal cancer-infected cells. The level of the peptide fragment can be expressed as an absolute (eg, μg/ml) or relative (eg, relative intensity of signal) difference of the above-described marker protein.

Also, preferably, one or more antibodies against the colorectal cancer marker are arranged at a predetermined position on a substrate and immobilized at a high density using a protein chip. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, the separated protein is hybridized with the protein chip to form an antigen-antibody complex, and the presence or expression level of the protein is checked by reading it, You can check if you have colon cancer.

Specifically, in order to detect the fusion protein, the fusion gene, or the transcript in the clinical sample of a specific colorectal cancer suspect of the present invention, (a) the fusion protein, the fusion gene encoding it, and the fusion gene correspond to the clinical sample treating (adding) and reacting a substance that interacts with one or more selected from the group consisting of a transcription product; and (b) detecting the reactant obtained through the above process. It may further include the step of preparing a clinical sample prior to step (a), and the step of preparing the clinical sample may include obtaining (separating) a clinical sample from a subject suspected of colon cancer. In step (a), the interacting substance is a compound, antibody, aptamer, and the fusion gene or transcript (all or part; such as a fusion site) that specifically binds to the fusion protein (all or a part; for example, a fusion site). site) binding to nucleic acid molecules (eg, primers, probes, aptamers, etc.), and may be at least one selected from the group consisting of compounds. One or more immunochemical labeling materials selected from the group consisting of free radicals, radioactive isotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophages, coenzymes, etc. are attached to these interacting substances, or can be used together with the labeling substances. . In step (b), the reactant is a complex produced by interaction (binding) between one or more selected from the group consisting of the fusion protein, fusion gene, and transcript obtained in step (a) and a substance interacting therewith (complex). When the reactant is detected in the step of detecting the reactant, it may be determined that the fusion protein, fusion gene or transcript is present.

The substance that interacts with the fusion gene encoding the NTRK1 fusion protein or a corresponding transcript may be a nucleic acid molecule capable of hybridizing with the fusion gene or transcript. For example, the nucleic acid molecule is an antisense oligonucleotide (eg, siRNA, microRNA, etc.) capable of specifically hybridizing with the fusion gene in the clinical sample, the fusion site of the fusion gene, or a transcript corresponding to the fusion gene or the fusion site. , a probe (eg, 5 to 100 bp, 5 to 50 bp, 5 to 30 bp, or 5 to 25 bp), and may be at least one selected from the group consisting of an aptamer. For example, a nucleic acid molecule capable of hybridizing with a fusion gene encoding the fusion protein or a transcript molecule corresponding to the fusion gene is 50 to 250 consecutive, specifically 100 to 200 bases including the fusion site in the fusion gene. 20 to 100, specifically 25 to 50 nucleotide sequences adjacent to both ends of the polynucleotide fragment, or a sequence complementary thereto, and 20 to 100 bp, or 25 to 50 bp in length to amplify the polynucleotide fragment consisting of may be a pair of primers. The 'hybridization is possible' means that the detection target nucleotide sequence is completely complementary, or has a nucleotide sequence that is 80% or more (eg 80-100%), specifically 90% or more (eg 90-100%) complementary nucleotide sequence. It means that it can specifically bind to a target gene or transcript.

In another example, the substance that specifically interacts with the NTRK1 fusion gene or its transcript is at least one selected from the group consisting of free radicals, radioactive-isotopes, fluorescent dyes, chromogenic substrates, enzymes, bacteriophages, coenzymes, etc. A chemical labeling substance may be attached or used together with the labeling substance. For example, a probe for fluorescence in situ hybridization capable of hybridizing to each fusion gene and a primer pair for polymerase chain reaction can be used (Tables 8 and 9).

In another example, the detection of the NTRK1 fusion protein is the fusion protein or a substance that interacts with the NTRK1 protein fusion-dependently expressed in colorectal cancer, such as a substance that specifically binds (eg, an antibody, an aptamer or compound) using a conventional assay method for detecting the interaction between the fusion protein and the substance (eg, antibody or aptamer), that is, formation of a complex, eg, immunochromatography, immunohistochemistry ), enzyme linked immunosorbent assay, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, western blotting blotting) and flow cytometry (cell sorting).

In another aspect, the present invention provides a method for providing information necessary for predicting a colorectal cancer treatment prognosis, comprising measuring the level of the NTRK1 fusion gene, its transcript, or its protein from an isolated sample of a colorectal cancer patient. to provide.

Specifically, the present invention comprises the steps of measuring the level of an NTRK1 fusion gene, a transcript thereof or a protein thereof from an isolated sample of a colorectal cancer patient; and comparing the measured expression level of the fusion gene, its transcript or its protein with the fusion gene, its transcript, or its protein expression level in a normal control sample, providing information necessary for predicting the prognosis of colorectal cancer treatment How to

The step of measuring the level of the NTRK1 fusion gene, its transcript, or its protein is almost the same as described in the method for providing information necessary for diagnosing colorectal cancer.

However, there is a difference in the aspect that the sample to be measured is an isolated sample of a colorectal cancer patient rather than an isolated sample of a suspected colon cancer individual, and a normal control group in the present invention is a normal group and a colorectal cancer patient group in which the NTRK1 fusion gene is not expressed. there is

In the present invention, the method for providing information necessary for predicting colorectal cancer treatment prognosis is the NTRK1 fusion gene, its transcript, or its protein expression level of a normal control sample from a sample isolated from a colorectal cancer patient. When it is higher than the protein expression level, it may be characterized in that the prognosis of colorectal cancer treatment is predicted to be worse than when it is the same or lower than the protein expression level. In addition, comparing the expression level of the NTRK1 fusion gene, its transcript, or its protein includes determining whether the NTRK1 fusion gene, its transcript, or its protein is expressed. That is, it includes predicting that a colorectal cancer patient expressing the NTRK1 fusion gene, a transcript thereof, or a protein thereof will have a poorer colorectal cancer treatment prognosis than a colorectal cancer patient not expressing the expression.

In addition, in the present invention, the method for providing information necessary for predicting the treatment prognosis for colorectal cancer is a fusion gene, its transcript, or its protein expression level of a normal control sample from a sample isolated from a colorectal cancer patient. When it is higher than its protein expression level, it may be characterized in that it is predicted that the colorectal cancer treatment efficacy by the NTRK1 protein inhibitor is high.

On the other hand, in the present invention, the method of providing information necessary for predicting the prognosis of colorectal cancer treatment is to treat isolated cells of colorectal cancer patients with an NTRK1 fusion protein inhibitor, and the NTRK1 fusion gene of the NTRK1 fusion gene, its transcript, or its protein. It may include the step of measuring the level.

In the present invention, the 'NTRK1 protein inhibitor' to be evaluated for the efficacy of colorectal cancer treatment is not particularly limited as long as it is a substance capable of directly or indirectly inhibiting the function of NTRK1 protein. Substances capable of inhibiting the function of NTRK1 protein, specifically, tyrosine kinase, specifically, other tyrosine kinases may be used. As known NTRK1 protein inhibitors applicable to the present invention, for example, (5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15 -tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanoibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacene-13 (6H )-one (generic name: lestaurtinib: compounds targeting FLT3, JAK2, TRKA, TRKB and TRKC), N-[5-((2R)-2-methoxy-2-phenyleta noyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]pipeazol-3-yl]-4-(4-methylpiperazin-1-yl)benzamide (generic name: Danu) Sertib (danusertib): compounds targeting ARK1, RET, ABL, FGFR1, TRKA), N,1,4,4-tetramail-8-((4-(4-methylpiperazin-1-yl) Phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (generic name: milciclib: CDK2, TRKA, TRKB, TRKC compounds targeting), RXDX-101 (codenames: compounds targeting ROS1, ALK, TRKA), TSR-011 (codenames: compounds targeting TRKA, TRKB, TRKC, ALK), PLX-7486 ( Codenames: compounds targeting CSF1R, TRKA, TRKB, TRKC), NMS-E628 (codenames: compounds targeting ROS1, JAK2, ALK, TRKA), compounds with codenames AZD-7451 (TRKA, TRKB, A compound targeting TRKC), a compound with the code name NMS-P626 (a compound targeting TRKA), a compound with the code name RXDX-102 (a compound targeting TRKA), a compound with the code name ARRY-47 (TRKA, TRKB, compounds targeting TRKC).

In a specific embodiment of the present invention, the 10-year survival period of a cohort of 216 Korean colorectal cancer patients was analyzed according to the expression of the protein encoded by the NTRK1 fusion gene, and the NTRK1 fusion protein was expressed in 63 colorectal cancer patients expressing the NTRK1 fusion protein. It was confirmed that the survival period was significantly shortened compared to 153 colorectal cancer patients who did not express this (FIG. 12). This confirms that the prognosis of colorectal cancer treatment is poor when the NTRK1 fusion gene and the fusion protein expressed therefrom are measured in colorectal cancer patients.

In another aspect, the present invention provides a screening method for a colorectal cancer therapeutic agent comprising the step of measuring the expression level of the NTRK1 fusion gene, specifically, treating a candidate material for colorectal cancer treatment to cells expressing the NTRK1 fusion gene to do; and measuring the expression level of the NTRK1 fusion gene.

In the present invention, the NTRK1 fusion gene is as described above.

In the present invention, measuring the expression level of the NTRK1 fusion gene includes a method of measuring the level of a transcript of the fusion gene and a protein thereof, and a method of measuring the level of the transcript of the fusion gene or the fusion protein. The method for measuring the level is the same as described above.

In the screening method for a colorectal cancer therapeutic agent of the present invention, in particular, when the expression level of the NTRK1 fusion gene is lowered by treatment with a candidate substance for colorectal cancer treatment, it may be determined as a colorectal cancer therapeutic agent.

Specifically, a method for comparing the increase or decrease in the expression of the NTRK1 fusion gene in the presence and absence of a candidate for colorectal cancer treatment can be usefully used for screening colorectal cancer therapeutic agents. A substance that indirectly or directly reduces the concentration of the NTRK1 fusion gene (LMNA-NTRK1 or TPM3-NTRK1) or a protein expressed therefrom can be selected as a therapeutic agent for colorectal cancer. That is, the expression level of the marker NTRK1 fusion gene of the present invention is measured in colorectal cancer cells in the absence of a candidate for colorectal cancer treatment, and the expression level of the marker NTRK1 fusion gene of the present invention is measured in the presence of a candidate for treatment of colorectal cancer. After comparing the two, a substance that reduces the expression level of the marker of the present invention in the presence of a candidate for treatment of colorectal cancer compared to the level of expression of the marker in the absence of a candidate for treatment of colorectal cancer can be predicted as a therapeutic agent for colorectal cancer.

In another aspect, the present invention provides a material screened as a colorectal cancer treatment agent through the screening method of the colorectal cancer treatment agent.

In a specific embodiment of the present invention, the screening method for a colorectal cancer therapeutic agent by inhibiting the function of the NTRK1 fusion protein was confirmed using the colorectal cancer cell line KM12. Restautinib and crizotinib, which are multiple tyrosine kinase inhibitors, were purchased from Torqueless Bioscience (UK), and ARRY-470, an NTRK1 protein-specific inhibitor, was synthesized by LG Life Sciences, and the compound provided by the Korea Advanced Institute of Science and Technology was used. As a result of screening through the screening method of the present invention, compound 8 (5f in the present invention) and compound 27 (6s in the present invention) disclosed in Korean Patent Laid-Open No. 10-2013-0106186 were screened. The screened compound 8(5f) is a compound represented by Formula 1 below, and Compound 27(6s) is a compound represented by Formula 2 below.

[Formula 1]

[Formula 2]

Each of the inhibitors was treated at a concentration of 10 μM at 0.64 nM for 4 days, and the degree of inhibition of cell proliferation was confirmed using the ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium, USA).

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer comprising an NTRK1 fusion protein inhibitor as an active ingredient.

The NTRK1 fusion protein inhibitor may act by inhibiting the activity of the fusion protein or the expression of the fusion gene. As described above, the NTRK1 fusion protein inhibitor is not particularly limited as long as it is a substance capable of directly or indirectly inhibiting the function of the NTRK1 protein, specifically, the function of the NTRK1 tyrosine kinase. Substances that inhibit other tyrosine kinases may be used as long as they can inhibit NTRK1 tyrosine kinase. As a known RET tyrosine kinase inhibitor applicable to the present invention, as described above, for example, (5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7, 8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanoibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-inda Sen-13(6H)-one (generic name: lestaurtinib: compounds targeting FLT3, JAK2, TRKA, TRKB and TRKC), N-[5-((2R)-2-methoxy- 2-phenylethanoyl)-1,4,5,6-tetrahydropyrrolo[3,4-c]piperazin-3-yl]-4-(4-methylpiperazin-1-yl)benzamide (generic name: danusertib: compound targeting ARK1, RET, ABL, FGFR1, TRKA), N,1,4,4-tetramail-8-((4-(4-methylpiperazine) -1-yl) phenyl) amino) -4,5-dihydro-1H-pyrazolo [4,3-h] quinazoline-3-carboxamide (generic name: milciclib: CDK2, TRKA , TRKB, compounds targeting TRKC), RXDX-101 (codenames: compounds targeting ROS1, ALK, TRKA), TSR-011 (codenames: compounds targeting TRKA, TRKB, TRKC, ALK) , PLX-7486 (codenames: compounds targeting CSF1R, TRKA, TRKB, TRKC), NMS-E628 (codenames: compounds targeting ROS1, JAK2, ALK, TRKA), codenames AZD-7451 (compound targeting TRKA, TRKB, TRKC), code name NMS-P626 (compound targeting TRKA), code name RXDX-102 (compound targeting TRKA), code name ARRY-47 may be a compound of (a compound targeting TRKA, TRKB, or TRKC), in particular, ARRY-470, a compound represented by the following Chemical Formula 1 or a compound represented by Chemical Formula 2.

[Formula 1]

[Formula 2]

In addition, the NTRK1 fusion protein inhibitor may be an NTRK1 fusion gene expression inhibitor, and may be selected from the group consisting of NTRK1 fusion gene antisense oligonucleotides, siRNA, shRNA and microRNA. In addition, the NTRK1 fusion protein inhibitor may be an antibody that specifically binds to the NTRK1 fusion protein.

As long as such an antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding property, a part thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, and in addition to the novel antibody, antibodies already known in the art may be included. The antibody is another aspect for achieving the above object, and the present invention provides an LMNA-NTRK1 or TPM3-NTRK1 fusion gene expression inhibitor or a protein inhibitor expressed from the fusion gene as an active ingredient for the prevention or treatment of colorectal cancer. Provided is a pharmaceutical composition for use. It includes functional fragments of antibody molecules as well as complete forms having full lengths of two heavy chains and two light chains, as long as they have the properties of binding to specifically recognize them. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') ₂ and Fv.

The pharmaceutical composition for the treatment of colorectal cancer of the present invention is used in a method of removing the NTRK1 fusion protein having an activity of increasing cancer migration in the serum of a colorectal cancer patient using an antibody specific for the NTRK1 fusion protein, It can cure colon cancer. The present inventors confirmed that the compound exhibiting the NTRK1 fusion protein inhibitory effect through screening has the effect of inhibiting colorectal cancer cell proliferation, and identified that the NTRK1 fusion gene or a fusion protein expressed therefrom can be a target for colorectal cancer treatment did.

Such a composition for treating colorectal cancer includes, without limitation, substances capable of promoting anticancer activity by specifically binding to the NTRK1 fusion gene or a protein expressed therefrom, for example, proteins, aptamers, peptides, carbohydrates, compounds can be

The composition of the present invention may be administered together with a pharmaceutically acceptable carrier, and for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. may be used. In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses.

In another aspect, the present invention provides a method for preventing or treating colon cancer, comprising administering to an individual a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising the NTRK1 fusion protein inhibitor as an active ingredient.

The NTRK1 fusion protein inhibitor, subject and composition of the present invention are as described above.

The composition of the present invention can be used alone, but in order to increase the therapeutic efficiency, radiation therapy or chemotherapy (cell growth arrest or cytotoxic substance, antibiotic-type substance, alkylating agent, antimetabolic substance, hormone agent, immune agent, interferon-type substance) Substance, cyclooxygenase inhibitor, metallomatrix protease inhibitor, telomerase inhibitor, tyrosine kinase inhibitor, anti-growth factor receptor substance, anti-HER substance, anti-EGFR substance, anti-angiogenic substance, farnesyl transferase inhibitor , ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin conjugates, topoisomerase I inhibitors, topoisomerase II inhibitors, etc.) can be used in combination with other anticancer therapies.

The term, administration, as used in the present invention, means introducing the composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is selected according to the type of the inhibitor or the type of colorectal cancer, but the target tissue It can be administered through any general route as long as it can be reached. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, intrathecal administration may be made, but is not limited thereto does not

The effective dosage range of the composition of the present invention includes sex, body surface area, disease type and severity, age, drug sensitivity, administration route and excretion rate, administration time, treatment period, target cells, expression level, etc. in other medical fields It may vary depending on various factors well known in the art, and can be easily determined by experts in the art.

By providing a diagnostic marker specific for colorectal cancer according to the present invention, accurate colorectal cancer diagnosis is possible, predicting the clinical prognosis through stratification of colorectal cancer patients, predicting the effectiveness of treatment for colorectal cancer, In particular, it becomes possible to predict the effectiveness of colorectal cancer treatment by NTRK1 protein inhibitors. Accordingly, it is possible to limit the administration of the drug to colorectal cancer patients who are considered to be ineffective to administer the drug, and thus it becomes possible to realize efficient and safe customized colorectal cancer treatment.

1 is a diagram of the NTRK1 gene by NTRK1 gene fusion in three colorectal cancer clinical tissues among 150 colorectal cancer clinical tissues and 50 normal colorectal clinical tissues derived from the same patient through next-generation transcriptome sequencing using Illumina's HiSeq 2000. A figure showing the increased expression of transcripts and a figure showing the data analysis result.
Figure 2 is a figure showing that NTRK1 is fused with LMNA or TPM3 by analyzing the nucleotide sequence of the fusion transcript encoded from the NTRK1 fusion gene by Sanger sequencing, and a figure showing the data analysis results.
3 is a diagram showing the expression of NTRK1 protein specific to colorectal cancer having an NTRK1 fusion gene by analyzing the expression of NTRK1 protein in a pair of normal colon cancer and normal colon clinical tissues in which the NTRK1 fusion gene is confirmed at the transcript level.
FIG. 4 shows (a) colon cancer and NTRK1 fusion gene at the transcript and protein level through next-generation transcriptome sequencing; (b) A figure showing the fusion of the NTRK1 gene specific to colon cancer by analyzing the NTRK1 gene fusion in a normal colon clinical tissue pair at the DNA level through fluorescence in situ hybridization. Yellow arrows indicate NTRK1 gene isolated by gene fusion. The ruler represents 5 micrometers.
5 is a diagram showing a single fusion site of the LMNA gene and the NTRK1 gene and three types of fusion sites of the TPM3 gene and the NTRK1 gene.
6 is a diagram showing the structure of a fusion protein encoded by a single fusion gene of an LMNA gene and an NTRK1 gene and three types of a fusion gene of a TPM3 gene and an NTRK1 gene.
7 is a colorectal cancer clinical tissue with NTRK1 fusion gene by analyzing somatic mutations of colorectal cancer-specific oncogenes and suppressor genes in 147 colorectal cancer clinical tissues through next-generation transcriptome sequencing analysis. The figure confirms that there is no somatic mutation.
Figure 8 shows that LMNA-NTRK1 and TPM3-NTRK1 fusion genes are forcibly expressed in a non-neoplastic NIH3T3 cell line to evaluate the presence or absence of cell mass formation (colony formation assay). Expression of the NTRK1 fusion gene induces in vitro tumorigenicity of the NIH3T3 cell line. is a picture that shows As a negative control, an empty vector without the NTRK1 fusion gene was forcibly expressed, and as a positive control, a KM12 colorectal cancer cell line containing the TPM3-NTRK1 fusion gene was used.
9 shows the expression of the NTRK1 fusion gene by forcibly expressing the LMNA-NTRK1 and TPM3-NTRK1 fusion genes in the non-neoplastic NIH3T3 cell line and inoculating them under the dorsal subcutaneous tissue of immune-deficient nude mice to evaluate the presence or absence of tumor formation. Figure showing the induction of in vitro tumorigenicity of the NIH3T3 cell line. As a positive control, the KM12 colorectal cancer cell line containing the TPM3-NTRK1 fusion gene was used.
10 is a cohort of Korean colorectal cancer patients by analyzing the expression of the protein encoded by the NTRK1 gene in a tissue microarray derived from a cohort of 216 Korean colorectal cancer patients and a tissue microarray derived from a cohort of 472 Chinese colorectal cancer patients. This is a figure confirming the expression of NTRK1 protein in 29.1% of patients and 25.6% of the Chinese colorectal cancer cohort.
11 is a diagram showing by fluorescence direct conjugation that the higher the NTRK1 protein expression level, the higher the NTRK1 gene fusion frequency among clinical tissues derived from a cohort of Korean colorectal cancer patients in which the expression of the protein encoded by the NTRK1 gene was confirmed. Yellow arrows indicate NTRK1 gene isolated by gene fusion. The ruler represents 10 micrometers.
12 shows the 10-year survival period of a cohort of 216 Korean colorectal cancer patients according to the expression of the protein encoded by the NTRK1 gene. 63 colorectal cancer patients expressing NTRK1 protein and 153 colorectal cancer patients not expressing NTRK1 protein The figure shows that the survival period is significantly shortened compared to the patient.
13 is a KM12 colorectal cancer cell line with a TPM3-NTRK1 fusion gene, among tyrosine kinase inhibitors, restaurtinib (lestaurtinib), which has inhibitory ability on NTRK1 protein, and crizotinib (crizotinib) and NTRK1 without inhibitory ability on NTRK1 protein; By applying compound 8(5f) and compound 27(5s) and ARRY-470 disclosed in Korean Patent Application Laid-Open No. 10-2013-0106186, which are protein-specific inhibitors, by concentration, KM12 cell proliferation was further reduced in the NTRK1-specific inhibitor is the picture shown.
14 is a diagram showing that 118.5 million aligned reads were produced with a median value through next-generation transcriptome sequencing, and the level at which an error could occur in one base out of 100 was confirmed to be 94.3%.
15 is a diagram confirming three outlier clinical tissues by analyzing the entire clinical tissues by PCA (Principle Component Analysis) based on the expression levels of 18,725 genes whose expression is recognized in 80% or more of clinical tissues.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example One : LMNA or TPM3 - NTRK1 Fusion gene and encoded by it all-in-one water and fusion proteins

In the present invention, a fusion example of LMNA protein and NTRK1 protein in colorectal cancer was first disclosed in the present invention through the following examples. In addition, the frequency and clinical prognosis of colorectal cancer patients having TPM3 gene or LMNA gene and NTRK1 gene fusion, and the fusion transcript and fusion protein encoded therein were confirmed for the first time in the present invention through large-scale colorectal cancer patient cohort analysis.

1-1. NTRK1 Wow fusion protein genes that make up

First, the NTRK1 fusion gene specifically present in colorectal cancer tissues identified in the present invention is summarized in Table 1 below.

Colorectal cancer-specific NTRK1 fusion gene donor gene domain acceptor gene domain Distance (Mb) TPM3 1q21.3 NTRK1 1q23.1 2.6 LMNA 1q22 NTRK1 1q23.1 0.6

The NTRK1 fusion gene of the present invention is cleaved and fused at the break point (or fusion site) of the protein fragments listed in Table 1 above. The fragment is encoded at any point in the intron region between the exon included at the end of the N-terminal (in the case of a C-terminal fusion partner) or C-terminus (in the case of an N-terminal fusion partner) of the fragment and the exon to be cleaved and removed. Since the amino acid sequence of the protein fragment is not affected, the actual cleavage point may be any of the intron positions.

1-2. TPM3 - NTRK1 fusion gene and TPM3 - NTRK1 fusion protein

In the present invention, the TPM3 gene encoding the TPM3 protein is a human-derived TPM3 gene and is located on human chromosome 1 (q21.3), and the TPM3 protein encoded therefrom is a protein having a total amino acid length of 285aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, the TPM3 protein is human-derived TPM3, and the NTRK1 gene encoding it is located on human chromosome 1 (q21.3). The TPM3 protein encoded therefrom is a protein with a total amino acid length of 285 aa. The TPM3 protein or TPM3 protein fragment is the N-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, the GenBank accession no. The TPM3 gene having the nucleotide sequence of NM_152263 was used.

The fragment of the TPM3 protein was used to have an amino acid sequence encoded by the nucleotide sequence up to the 8th exon of NM_152263 (from 154134289 to 154142876 base positions based on the (-) strand on chromosome 1). One nucleotide (g) that did not form a codon was present at the 3' end of exon 8 encoding the TPM3 protein used in the present invention. The gene encoding the fragment of the TPM3 protein and the TPM3 protein encoded therefrom are summarized in Tables 2 and 3 below.

Gene encoding a fragment of the TPM3 protein TPM3 gene
Accession No. TPM3 protein coding region
(CDS) TPM3 protein fragment coding region:
exon basis TPM3 protein fragment coding region:
cDNA standard chromosomal
cutting position 5' end cut position
base sequence NM_152263 118~975 (858bp)
(SEQ ID NO: 1) exon 1 to exon 8 118-891 + 1nt (g) (775bp) (SEQ ID NO: 2) chr1: 154142876 5'-ATT GAT GAC CTG GAA g-3'
(SEQ ID NO: 3)

Fragment of TPM3 protein Total length of TPM3 protein
(Accession No.) TPM3 protein fragment site N-terminal cut position
amino acid sequence 285aa (NP_689476)
(SEQ ID NO: 4) 1-258aa + 1nt (g) (amino acid sequence: SEQ ID NO: 5) N-IDDLE + 1 nt (g) (amino acid sequence: SEQ ID NO: 6)

In the present invention, NTRK1 protein is human-derived NTRK1, and the NTRK1 gene encoding it is located on human chromosome 1 (q23.1). The NTRK1 protein encoded therefrom is a protein with a total amino acid length of 796aa. NTRK1 protein or NTRK1 protein fragment is the C-terminal fusion partner of the TPM3-NTRK1 fusion protein.

In the present invention, GenBank accession no. The NTRK1 gene having the nucleotide sequence of NM_001012331 was used.

The fragment of the NTRK1 protein is exon 9 (156830671 to 156844363 base positions relative to the (+) strand on chromosome 1) or exon 11 (156830671 to 156845312 base positions relative to the (-) strand on chromosome 1) of NM_001012331 or Those having an amino acid sequence encoded by the nucleotide sequence from exon 12 (base positions 156830671 to 156845872 based on the (-) strand on chromosome 1) to the last exon were used. At this time, the first two nucleotides (ac) of the 3' end of exon 9 and the first two nucleotides (gc) of the 3' end of exon 11 and the first two nucleotides of the 3' end of exon 12 ( gt) had a sequence that did not form a codon. The sequences that do not form the codon are linked to one nucleotide (g) additionally included in the TPM3 protein fragment as described above when fused with the TPM3 protein fragment to form a codon (gac or ggc or ggt) to form one amino acid (D or G) was encoded. The genes encoding the fragments of the NTRK1 protein and the NTRK1 protein expressed therefrom are summarized in Tables 4 and 5 below.

Gene encoding fragment of NTRK1 protein NTRK1 gene
(Accession No.) Coding region of NTRK1 protein (CDS) NTRK1 protein fragment coding region:
exon basis NTRK1 protein fragment coding region:
cDNA standard cleavage site on chromosome 3' end direction cleavage site base sequence NM_002529 52~2447 (2396bp)
(SEQ ID NO: 7) exon 9 to exon 17 2nt (ac) +1236-2447 (1214bp) (SEQ ID NO: 8) chr1:156844363 5'-ac ACT AAC AGC ACA TCT -3' (SEQ ID NO: 9) NM_002529 52~2447 (2396bp)
(SEQ ID NO: 7) exon 11 to exon 17 2nt (gc) + 1393-2447 (1055bp) (SEQ ID NO: 10) chr1:156845312 5'- gc CCG GCT GTG CTG GCT -3' (SEQ ID NO: 11) NM_002529 52~2447 (2396bp)
(SEQ ID NO: 7) exon 12 to exon 17 2nt (gt) + 1540-2447 (908bp) (SEQ ID NO: 12) chr1:156845872 5'-gt GTT CAC CAC ATC AAG -3' (SEQ ID NO: 13)

Fragment of NTRK1 protein Total length of NTRK1 protein (Accession No.) NTRK1 protein fragment site C-terminal cleavage site amino acid sequence 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (ac) + 400-796 (amino acid sequence: SEQ ID NO: 15) 2nt (ac) + TNSTS-C (amino acid sequence: SEQ ID NO: 16) 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (gc) + 453-796 (amino acid sequence: SEQ ID NO: 17) 2nt (gc) + PAVLA-C (amino acid sequence: SEQ ID NO: 18) 796aa (NP_002520)
(SEQ ID NO: 14) 2nt (gt) + 502-796 (amino acid sequence: SEQ ID NO: 19) 2nt (gt) + VHHIK-C (amino acid sequence: SEQ ID NO: 20)

The fusion gene (TPM3-NTRK1 fusion gene) encoding the TPM3-NTRK1 fusion protein in which the TPM3 protein or a fragment thereof and the NTRK1 protein or a fragment thereof is fused is a nucleotide encoding the TPM3 protein or a fragment thereof as described above at the 5' end. The molecule and the 3' end were prepared to contain a nucleotide molecule encoding the NTRK1 protein or a fragment thereof as described above.

As can be seen in the table above, in the present invention, the nucleotide sequence from exon 1 to exon 8 of NM_152263 at the 5' end of the TPM3-NTRK1 fusion gene in the present invention and exon 9 or exon 11 or exon 12 to exon 17 of NM_002529 at the 3' end A fusion gene in which nucleotide sequences were linked was prepared. Specifically, in the present invention, the nucleotide sequence from the 118th to the 892th of NM_152263 (SEQ ID NO: 2) at the 5' end in the present invention and the nucleotide sequence from the 1234th or 1391th or 1538th to the 2447th of NM_002529 at the 3' end (SEQ ID NO: 2) No. 8; SEQ ID NO: 10; SEQ ID NO: 12) linked TPM3-NTRK1 fusion gene (SEQ ID NO: 21: a nucleotide sequence comprising SEQ ID NO: 3 and SEQ ID NO: 9; SEQ ID NO: 22: comprising SEQ ID NO: 3 and SEQ ID NO: 11 nucleotide sequence; SEQ ID NO: 23: a nucleotide sequence including SEQ ID NO: 3 and SEQ ID NO: 13) was prepared.

In addition, the TPM3-NTRK1 fusion protein encoded from the TPM3-NTRK1 fusion gene prepared above in the present invention has the amino acid sequence from the 1st to the 258th of NP_689476 on the N-terminal side (SEQ ID NO: 5) and the 400th or NP_002520 on the C terminal A fusion protein (SEQ ID NO: 24: an amino acid sequence comprising SEQ ID NO: 6 and SEQ ID NO: 16; SEQ ID NO: 25) to which the 453th or 502th to 796th amino acid sequences (SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20) are linked : amino acid sequence comprising SEQ ID NO: 6 and SEQ ID NO: 18; SEQ ID NO: 26: amino acid sequence comprising SEQ ID NO: 6 and SEQ ID NO: 20).

1-3. NMNA - NTRK1 fusion gene and NMNA - NTRK1 fusion protein

The LMNA gene encoding the LMNA protein typically performed in the present invention is a human-derived LMNA gene and is located on human chromosome 1 (q22), and the LMNA protein encoded therefrom is a protein having a total amino acid length of 664aa. The LMNA protein or LMNA protein fragment is the N-terminal fusion partner of the LMNA-NTRK1 fusion protein.

In the present invention, GenBank accession no. The TPM3 gene having the nucleotide sequence of NM_170707 was used.

As the fragment of the LMNA protein, one having an amino acid sequence encoded by the nucleotide sequence up to the 6th exon of NM_170707 (base positions 156084504 to 156105740 bases on the (+) strand on chromosome 1) was used. One nucleotide (c) that does not form a codon was present at the 3' end of exon 6 encoding the LMNA protein used in the present invention. Genes encoding fragments of the LMNA protein and LMNA proteins encoded therefrom are summarized in Tables 6 and 7 below.

Gene encoding fragment of LMNA protein LMNA gene
(Accession No.) LMNA protein coding region (CDS) LMNA protein fragment coding region: exon reference LMNA protein fragment coding region: cDNA reference cleavage site on chromosome 5' end direction cleavage site nucleotide sequence NM_170707 250-2244 (1995 bp) (SEQ ID NO: 27) exon 1 to exon 6 250-1233 + 1nt (c) (985bp) (SEQ ID NO: 28) chr1:156105740 5'-GAG GAC TCA CTG GCC c-3' (SEQ ID NO: 29)

Fragments of LMNA Proteins Overall length of LMNA protein (Accession No.) LMNA protein fragment site N-terminal cleavage site amino acid sequence 664aa (NP_733821) (SEQ ID NO: 30) 1-328aa + 1nt (c) (amino acid sequence: SEQ ID NO: 31) N-EDSLA + 1 nt (g) (amino acid sequence: SEQ ID NO: 32)

In the present invention, the fusion gene (LMNA-NTRK1 fusion gene) encoding the LMNA-NTRK1 fusion protein in which the LMNA protein or a fragment thereof is fused with the NTRK1 protein or a fragment thereof (LMNA-NTRK1 fusion gene) includes the LMNA protein or a fragment thereof as described above at the 5' end. It was prepared to include the encoding nucleotide molecule and the nucleotide molecule encoding the NTRK1 protein or a fragment thereof described in Example 1-2 at the 3' end.

Specifically, in the present invention, an LMNA-NTRK1 fusion gene was prepared in which the nucleotide sequence from exon 1 to exon 6 of NM_170707 on the 5' end and the nucleotide sequence from exon 11 to exon 17 of NM_002529 on the 3' end were linked. Specifically, in the present invention, the nucleotide sequence from the 250th to the 1234th (SEQ ID NO: 28) of NM_170707 on the 5' end and the nucleotide sequence from the 1391th to the 2447th of NM_002529 on the 3' end are linked (SEQ ID NO: 10) to the LMNA -NTRK1 fusion gene (SEQ ID NO: 33: a nucleotide sequence comprising SEQ ID NO: 29 and SEQ ID NO: 11) was prepared.

In addition, the LMNA-NTRK1 fusion protein encoded from the LMNA-NTRK1 fusion gene prepared in the present invention has the amino acid sequence from the 1st to the 328th amino acid sequence of the LMNA protein on the N-terminal side (SEQ ID NO: 31) and the NTRK1 protein on the C-terminal side It was a fusion protein (SEQ ID NO: 34: an amino acid sequence comprising SEQ ID NO: 32 and SEQ ID NO: 18) to which the amino acid sequence from the 453th to the 796th of the (SEQ ID NO: 18) was linked.

Example 2: NTRK1 Fusion gene or its transcript measure

In the present invention, the frequency and clinical prognosis of colorectal cancer patients having the NTRK1 fusion gene (TPM3 gene or fusion gene of LMNA gene and NTRK1 gene) and the fusion transcript and fusion protein encoded therein in colorectal cancer patients. To measure the NTRK1 fusion gene or its transcript. Various methods are available for this purpose, but in the present invention, a probe for fluorescence in situ hybridization (FISH) that can be hybridized to the NTRK1 fusion gene (NTRK1 Split FISH Probe, Cat# FS0024, Abnova, Taiwan) was used, and NTRK1 fusion transcription was performed. A primer for chain polymerization that is hybridizable to the product was prepared.

Probe for fluorescence direct conjugation that can be hybridized to NTRK1 fusion gene junction site Length label material 5' end of SEQ ID NO:7
(chr1: 156390000~156814000) about 420 kb TexRed 3' end of SEQ ID NO:7
(chr1: 156851000~157630000) about 780 kb FITC

Primer for chain polymerization that can be hybridized to NTRK1 fusion transcript fusion gene fusion site Forward Primer Sequence Reverse Primer Sequence TPM3-NTRK1 SEQ ID NO: 21;
SEQ ID NO: 22;
SEQ ID NO:23 5'- AAG AAG ATA AAT ATG AGG -3'
(SEQ ID NO: 35) 5'-CCG TGC CGC ATA TAC TCA AA -3'
(SEQ ID NO: 36) LMNA-NTRK1 SEQ ID NO: 33 5'-CCA GGT GGA GCA GTA TAA GAA G -3'
(SEQ ID NO: 37) 5'- TGT GGG TTC TCG ATG ATG TG -3'
(SEQ ID NO: 38)

In addition, the detection of the NTRK1 fusion protein is a substance that interacts with the fusion protein or NTRK1 protein that is expressed dependently on the fusion of the NTRK1 gene in colorectal cancer, such as a substance that specifically binds (eg, an antibody, an aptamer, or a compound) A conventional assay method for detecting the interaction between the fusion protein and the substance (eg, antibody or aptamer), that is, the formation of a complex, using, for example, immunochromatography, immunohistochemistry, enzyme enzyme linked immunosorbent assay, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, western blotting and It can be detected by flow cytometry cell sorting or the like. For example, antibodies for immunohistologic examination for detecting NTRK1 protein are exemplified in Table 10 below. In the present invention, an antibody for immunohistochemistry that detects NTRK1 protein (Anti-NTRK1 Antibody EP1058Y (Cat# TA301109, OriGene, USA)) was used.

Antibody for immunohistochemistry that interacts with NTRK1 fusion protein antigen site of action voice contrast positive control Other applications Tyrosine at position 791 of the NTRK1 protein amino acid sequence and its surrounding peptides Kinase domain C-terminus of NTRK1 protein lymphocytes ganglion cells Western blotting, fluorescence immunoassay, flow cytometry

Example 3: Experiments using clinical tissue

Fresh frozen clinical tissues used in the present invention were collected from colorectal cancer patients who underwent surgical resection at Pusan National University Hospital and Hwasun Chonnam National University Hospital from 2008 to 2012, Hwasun Chonnam National University Hospital was stored and sold at the base bank. Colorectal cancer tissues were selected according to the guidelines of the International Cancer Genome Consortium (ICGC). For example, only clinical tissues composed of more than 60% cancer cells and less than 20% necrotic cells or normal cells after pathological diagnosis were used as clinical tissues. Colorectal cancer clinical tissues collected from colorectal cancer patients with a family history of colorectal cancer were excluded. Finally, 150 cases of colorectal cancer tissues and 50 cases of normal colon tissues were used.

Example 4: microsatellite instability analysis ( microsatellite instability analysis : MSI )

After extracting total DNA from the formalin-fixed paraffin-embedded tissue after formalin-fixed paraffin-embedded among the colorectal cancer clinical tissues of Example 3, using Bethesda markers (BAT26, D5S346, BAT25, D17S250, D2S123) Sieve instability was investigated.

Specifically, total DNA was extracted using the QIAmp DNA FFPE Tissue kit (Kiagen, Germany). DNA purity and concentration were measured with an ND-100 spectrophotometer (Nanodrop Technology, USA). Microsatellite instability was determined according to the length and signal intensity of the amplification product obtained after multiplex chain polymerization. When the chain polymerization reaction failed due to incompleteness of the extracted DNA, etc., microsatellite instability was determined by considering the total number of somatic mutations in major genes including DNA polymerase epsilon (POLE).

Example 5: Next Generation transcript sequencing ( RNA - seq )

Total RNA was extracted with RNAiso Plus (Takada Bio, Japan), pretreated with DNase I (Kiagen), and then purified with RNeasy Mini kit (Kiagen). RNA integrity was analyzed by Bioanalyer (Agilent, USA), and RNA from clinical tissue of colorectal cancer having an RNA integrity index (RNA Integrity Number: RIN) of 6 or higher was applied to RNA-seq. A library for RNA-seq was prepared with TruSeq RNA sample preparation kit (Illumina, USA). For example, mRNA was recovered as magnetic beads to which poly-T oligo was attached, and then fragmented by acoustic shearing. Primary cDNA was prepared from fragmented mRNA using reverse transcriptase and random hexamer, and secondary cDNA was prepared using DNA polymerase I and RNase H. Secondary cDNA was connected to the adapter and then the cDNA library was completed through chain polymerization. The cDNA library was sequenced using HiSeq 2000 (Illumina), and about 100 million paired-end reads with a length of 101 bp were produced.

Example 6: sequencing

In Example 5, the paired-end reads produced through next-generation transcriptome sequencing were performed using GSNAP and TopHat under conditions that allow 5% mismatch, National Center for Biotechnology Information (NCBI, USA) It was aligned with the human reference genome (hg 19) provided by Paired-end reads were also extracted from public databases (36,742 mRNAs from the RefSeq database, 73,671 mRNAs from the USCS database and 161,214 mRNAs from the Ensembl database) extracted from public databases by aligning them to a human reference transcript for mRNA splicing. Misalignment caused by the was minimized.

Example 7: Fusion gene analysis

In-frame fusion genes were searched through the GFP algorithm and cross-verified with the deFuse and FusionMap algorithms. For example, a fusion gene through GFP was searched under the following conditions.

(1) Discordant read pairs aligned with different donor genes and acceptor genes constituting the fusion transcript and reads aligned with exonic fusion breakpoints (exon- If spanning reads exist, accept them.

(2) Accept if the strand orientation of the donor and recipient gene DNA strands accounts for gene fusion through chromosomal translocations or inversions or deletions.

(3) Excludes cases with high nucleotide sequence homology (E value < 0.01).

(4) Excludes illogical read pairs with a distance of less than 10bp.

The fusion gene was confirmed through the following conditions.

(1) Accept the fusion gene retrieved from two algorithms of GFP or deFuse or FusionMap.

(2) If there are more than 10 lead pairs aligned with the donor gene and the recipient gene, it is accepted.

(3) Accept when there are more than 10 reads aligned to the fusion site.

(4) If the donor gene and the recipient gene exist in the same chromosome and the distance between genes is more than 100Kb, it is accepted.

(5) Out-frame fusion genes are excluded.

(6) Acceptance is accepted if the exons of the recipient gene present after the fusion site are overexpressed compared to the exons of the unfused recipient gene.

Example 8: aberrant gene ( differentially expressed gene : DEG ) analysis

Aberrant genes were analyzed using GenePattern software of Broad Institute, USA. Reads produced through next-generation transcriptome sequencing and aligned with TopHat were assembled into transcripts using Cufflinks. The number of aligned reads for each gene was standardized by the FPKM (Fragments Per Kilobase of exon per Million) method, and then expressed as the expression level for each gene. Based on the expression level of 18,725 genes with FPKM of 0 or more in 80% or more of the clinical tissues, the entire clinical tissues were analyzed by PCA (Principle Component Analysis), and 3 outlier clinical tissues (6 pairs) were removed. Through PCA analysis, 18,725 genes obtained from a total of 147 colorectal cancer clinical tissues and 47 normal clinical tissues were subjected to aberrant gene analysis. Aberrant genes specific to colorectal cancer clinical tissues were searched for by pairwise 2-sided t-test (p < 0.05) and Benjamini-Hochart multiple comparison correction (FDR < 0.05).

Example 9: somatic mutation ( non - synonymous somatic mutation ) analysis

Single nucleotide variances (SNVs) were searched by aligning reads produced through next-generation transcriptome sequencing to human reference transcripts through GSNAP. Single nucleotide mutations were searched through the following conditions.

(1) If the number of position-specifically aligned leads is two or more, it is accepted.

(2) If the average base quality of the above position is 20 or more, it is accepted.

(3) Accept when the allele frequency of the position is 3% or more.

(4) Accept when the number of leads aligned in the above position is 10 or more.

For gene annotation, refer to RefSeq, and germline variants were removed through the following conditions.

(1) Compared with dbSNP137, cases where the frequency of minor allele among all clinical tissues is 1% or more are excluded.

(2) Compared with the 59 Korean reference genomes, germline mutations observed in normal individuals are excluded.

(3) Germ cell mutations observed in the normal colon tissues of 47 cases used in the present invention are excluded.

Considering that a higher-than-expected frequency of somatic mutation was observed in colorectal cancer clinical tissues without a normal colonic tissue pair, somatic mutations were searched based on negative selection.

Example 10: reverse transcription chain polymerization reaction ( reverse transcriptase - polymerase chain reaction: RT - PCR )and Sanger sequencing ( Sanger sequencing )

The NTRK1 fusion transcript was confirmed by reverse transcription chain polymerization and Sanger sequencing. The conditions of the chain polymerization reaction are summarized in Table 11 below.

Chain polymerization conditions for confirming NTRK1 fusion transcripts donor gene host gene primer Chain Polymerization Conditions chain polymerization reaction product TPM3 (exon 8) NTRK1 (exon 9 or exon 11 or exon 12) SEQ ID NO: 35 and SEQ ID NO: 36 Denaturation: 94 degrees 30 seconds
Annealing: 55.7 degrees 30 seconds
extension
72 degrees 30 seconds
30 total 406bp
553bp
712bp LMNA (exon 6) NTRK1 (exon 11) SEQ ID NO: 37 and SEQ ID NO: 38 Metamorphosis: 94 degrees 30 seconds
Bonding: 54 degrees 30 seconds
Height: 72 degrees 30 seconds
30 total 354bp

The product of the chain polymerization reaction was inserted into TOPO TA vector (Life Technologies, USA) and then sequenced by Sanger method.

Example 11: Direct fluorescence bonding ( fluorescence in situ hybridization : FISH )

The NTRK1 fusion gene was identified using a split FISH probe that binds to the 5' and 3' ends of the NTRK1 gene, respectively (Abnova, Taiwan). For example, after formalin fixation, the paraffin-embedded sections of colon cancer and normal clinical tissues were paraffin-removed, treated with protease, and then denatured at 75°C. Sections of denatured clinical tissues were incubated for 17 hours in ThermoBrite (Abot, USA) together with the probes listed in Table 8. Cross-sections of cultured clinical tissues were counterstained using DAPI (Abot) after washing, and the distance between TexRed and FITC signals was observed.

Example 12: Immunohistological analysis ( immunohistochemistry ) and clinicopathological ( clinicopathological ) analysis

Focusing on the NTRK1 fusion protein's expression dependent on NTRK1 gene fusion, the expression of the NTRK1 fusion protein was confirmed immunohistologic using the NTRK1 protein-specific antibody listed in Table 10 (Origin, USA). Brain ganglion cells and lymphocytes were used as positive and negative controls for immunohistologic analysis of NTRK1 fusion protein expression, respectively.

The expression frequency of NTRK1 fusion protein was analyzed immunohistochemically using tissue microarrays constructed from a cohort of 216 Korean colorectal cancer patients and 472 Chinese colorectal cancer patients (Biomax, USA). Through immunohistological analysis of NTRK1 fusion protein expression, Korean colorectal cancer patients were stratified into NTRK1 fusion protein positive and negative patients, and the stratified patient group was colorectal cancer location, colon cancer invasion depth, and perineural invasion. ), lymphovascular emboli, lymphnode metastasis, microsatellite instability, and 10-year survival rates were compared.

Example 13: NIH3T3 in cell lines NTRK1 Expression of fusion gene

The cDNAs of TPM3, LMNA and NTRK1 genes were purchased from Origin. LMNA (exon 6)-NTRK1 (exon 11) and TPM3 (exon 8)-NTRK1 (exon 9 or exon 11 or exon 12) fusion transcripts were prepared through overlap extension chain polymerization. The prepared fusion transcript was cloned into pcDNA3.1 (Life Technologies) equipped with IRES-GFP using a Cold Fusion Cloning kit (System Bioscience, USA). After the cloned fusion gene was confirmed through raw sequencing, TPM3-NTRK1 and LMNA-NTRK1 fusion transcript expression vectors were completed. The NIH3T3 cell line was purchased from the Korea Cell Line Bank. The fusion transcript expression vector was introduced into the NIH3T3 cell line using FUGENE 6 (Roche, Germany). The transformed NIH3T3 cell line was selected using G418 (Like Technologies), and was completed through flow cytometry analysis using GFP protein expression.

Example 14: NTRK1 fusion gene neoplastic analysis

The tumorigenicity of the TPM3-NTRK1 or LMNA-NTRK1 fusion gene was confirmed by evaluating the in vitro cell aggregation ability and in vivo tumorigenicity of the transformed NIH3T3 cell line. For the in vitro cell agglomeration capacity, a total of 1,200 transformed NIH3T3 cells were introduced into 0.3% upper agarose and then cultured on 0.5% lower agarose for 3 weeks to observe the number and size of the formed cell agglomerates. The NIH3T3 cell line introduced with the empty vector without the fusion gene and the KM12 colorectal cancer cell line with the TPM3-NTRK1 fusion gene were used as negative and positive controls, respectively. The formed cell agglomerates were stained with 0.05% crystal violet and then counted under an optical microscope. In vivo neoplasticity was observed after inoculation of a total of one million transformed NIH3T3 cells or KM12 cells into the dorsal subcutaneous tissue of immune-deficient nude mice. Five nude mice were used in each experimental group. When a measurable tumor was formed after cell inoculation, the size of the tumor was measured for a total of 32 days after cell inoculation at 3-day intervals.

Example 15: Drug screening

The screening method for colorectal cancer treatment drug through inhibition of NTRK1 fusion protein was confirmed using the KM12 cell line. Restautinib and crizotinib, which are multiple tyrosine kinase inhibitors, were purchased from Torqueless Bioscience (UK), and ARRY-470, an NTRK1 protein-specific inhibitor, was synthesized by LG Life Sciences, and the compound provided by the Korea Advanced Institute of Science and Technology was used. As a result of screening through the screening method of the present invention, compound 8 (5f in the present invention) and compound 27 (6s in the present invention) disclosed in Patent Publication No. 10-2013-0106186 were screened. The screened compound 8(5f) is a compound represented by Formula 1 below, and Compound 27(6s) is a compound represented by Formula 2 below.

[Formula 1]

[Formula 2]

Each of the inhibitors was treated at a concentration of 10 μM at 0.64 nM for 4 days, and the degree of inhibition of cell proliferation was confirmed using the ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium, USA).

As a result of screening for drugs capable of treating colorectal cancer by inhibiting the activity of the NTRK1 fusion gene or the NTRK1 fusion protein encoded therein, restaurtinib, which has activity on the NTRK1 protein among multiple tyrosine kinase inhibitors, is KM12 at a concentration of about 10.7 nM. The growth of cells was inhibited by 50%, and ARRY-470, a selective inhibitor of NTRK1 protein, inhibited the growth of KM12 cells by 50% at about 3.2 nM. On the other hand, crizotinib, which has low activity on NTRK1 protein, inhibited the growth of KM12 cells by 50% at a concentration of about 184.8 nM. Compound 8 (5f in the present invention) and compound 27 (6s in the present invention) disclosed in Korean Patent Application Publication No. 10-2013-0106186 with high selectivity for NTRK1 protein showed relatively low cell permeability in KM12 cells at 544.2 nM and 929.1 nM was confirmed to inhibit the growth of 50% (see FIG. 13).

Example 16: Observed in Korean colorectal cancer clinical tissue in-frame fusion gene

Through the next-generation transcriptome sequencing analysis, a median value of 118.5 million aligned reads was produced, and the level at which an error could occur in one base out of 100 was confirmed to be 94.3% (see FIG. 14 ). As a result of PCA analysis, 3 clinical tissues were identified as outliers, and 3 pairs of colon cancer and normal colonic clinical tissues including the abnormal clinical tissues were excluded from the present invention (see FIG. 15 ). Therefore, 147 cases of colorectal cancer clinical tissue and 47 cases of normal colon clinical tissue were used in the present invention. As a result of fusion gene analysis using GFP or deFuse or FusionMap algorithm, nine in-frame fusion genes were identified (see Table 12).

In-frame fusion gene observed in Korean colorectal cancer clinical tissue Sample ID Stage Discordant paired-end reads spanning reads Distance
(bp) donor gene
(Donor gene) FPKM Location conferred gene
(Acceptor) FPKM Location LGP088T Ⅲ 27 153 2620933 TPM3 59 chr1:154142876 NTRK1 31 chr1:156844363 LGC012T Ⅲ 25 132 2620933 TPM3 94 chr1:154142876 NTRK1 20 chr1:156845312 TPM3 94 chr1:154142876 NTRK1 20 chr1:156845872 LGC026T II 11 15 675664 LMNA 145 chr1:156105740 NTRK1 32 chr1:156845312 LGC007T Ⅲ 21 197 771740 PTPRK 17 chr6:128841404 RSPO3 31 chr6:127469793 LGC054T Ⅲ 36 140 771740 PTPRK 15 chr6:128505577 RSPO3 37 chr6:127469793 LGC030T Ⅲ 56 374 2667510 NAGLU 255 chr17:40693224 IKZF3 55 chr17:37922746 LGP047T II 45 206 1020112 GTF3A 641 chr13:27999075 CDK8 93 chr13:26923209 LGP024T II 24 98 55463 RAD51AP1 7 chr12:4662255 AKAP3 18 chr12:4737971 LGP031T II 17 53 28829615 RASA1 11 chr5:86564807 LOC644100 19 chr5:115394422

The confirmed fusion gene was a fusion gene between the tyrosine dephosphorylation enzyme, receptor type, K (tyrosine phosphatase, receptor type, K: PTPRK) gene and R-spondin 3 (R-spondin 3: RSPO3) gene in 2 cases of colorectal cancer. It was confirmed in clinical tissues (1.4%), and a fusion gene between the N-acetylglucosaminidase (NAGLU) gene and the IKAROS family zinc finger 3 (IKAROS family zinc finger 3: IKZF3) gene was found. It was confirmed in 1 case of colorectal cancer clinical tissue (0.7%), and a fusion gene between the general transcription factor IIIA (GTF3A) gene and the cyclin-dependent kinase 8 (CDK8) gene was found in 1 case. It was confirmed in colorectal cancer clinical tissue (0.7%), and a fusion gene between the RAD51 associated protein 1 (RAD51AP1) gene and the A kinase anchor protein 3: AKAP3 gene was found in one case of colorectal cancer. Confirmed in clinical tissues (0.7%), RAS p21 protein activator 1: RASA1 gene and ADP-ribosylation factor-like 14 effector protein-like: LOC644100 A fusion gene between genes was identified in one case of colorectal cancer clinical tissue (0.7%), and lamin A/C (lamin A/C: LMNA) or tropomysosin 3 (tropomysosin 3: TPM3) gene and neurotrophic tyrosine kinase, receptor , a type 1 (neurotrophic tyrosine kinase, receptor, type 1: NTRK1) gene fusion gene was identified in 3 cases of colorectal cancer clinical tissue (2.0%). Among the above fusion genes, the NTRK1 gene is a gene encoding a TrkA protein fixed to the cell membrane, and the TPM3-NTRK1 fusion gene has been reported in colorectal cancer and papillary thyroid cancer, and myosin phosphatase Rho interacting protein (myosin phosphatase Rho) in lung adenocarcinoma. A fusion gene between the interacting protein: MPRIP gene or CD74 gene and NTRK1 gene has been reported, a fusion gene between the TP53 gene or LMNA gene and NTRK1 gene has been reported in Spitzoid melanoma, and RAB gtipase-like activity in gallbladder cancer. A fusion gene between the RAB GTPase activating protein 1-like (RABGAP1L) gene and the NTRK1 gene has been reported. In polymorphic orphan species, a fusion between a neurofascin (NFASC) gene or a brevica (BCAN) gene and an NTRK1 gene has been reported. genes have been reported. However, the frequency and clinical significance of the NTRK1 fusion gene in colorectal cancer have not been identified.

Example 17: NTRK1 gene, NTRK1 Analysis of fusion gene and its protein

Expression of the NTRK1 gene exon was observed only in colorectal cancer clinical tissues in which the NTRK1 fusion gene was identified (see FIG. 1 ). As a result of analyzing the NTRK1 fusion transcript using reverse transcription chain polymerization and Sanger sequencing, the TPM3-NTRK1 fusion gene was cut at the chr1: 154142876 position in the TMP3 gene to preserve SEQ ID NO: 2, and specifically, SEQ ID NO: 3 to 5' conserved terminally and truncated at positions chr1:156844363 or chr1:156845312 or chr1:156845872 in the NTRK1 gene to preserve SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12, specifically SEQ ID NO: 9 or SEQ ID NO: 11 or sequence It was confirmed that No. 13 was fused in a form conserved in the 3' end direction. The LMNA-NTRK1 gene was cut at position chr1:156105740 of the LMNA gene to preserve Serial code 28, specifically, SEQ ID NO: 29 was conserved in the 5' end direction, and was cut at position chr1: 156845312 in the NTRK1 gene to obtain SEQ ID NO: 10 It was confirmed that the fusion was conserved, specifically, SEQ ID NO: 11 was conserved in the 3' end direction (refer to FIG. 2).

As a result of immunohistologic analysis using an antibody against NTRK1 protein, specifically NTRK1 protein encoded by NTRK1, specifically an antibody that binds to the C-terminal region of TrkA protein, TRKA expression was observed in colorectal cancer clinical tissues in which NTRK1 fusion transcript was confirmed. was confirmed (see FIG. 3 ). The NTRK1 fusion gene was reconfirmed by fluorescence direct fusion analysis for the NTRK1 gene. The 5' end of the NTRK1 gene was spliced with a TexRed-labeled probe, and the 3' end of the NTRK1 gene was spliced with a FITC-labeled probe. As a result, NTRK1 fusion transcripts and fusion proteins were isolated from clinical tissues of colorectal cancer. Red and green probe signals were confirmed (refer to FIG. 4), and combined red and green probe signals were confirmed in normal colon clinical tissue (refer to FIG. 5). As a result of estimating the NTRK1 fusion gene and the fusion protein, it was confirmed that the NTRK1 fusion protein has a well-conserved tyrosine kinase domain in the NTRK1 protein (see FIG. 6 ).

Example 18: NTRK1 fusion gene neoplastic Confirm

The oncogenicity of the NTRK1 fusion gene has been reported in papillary thyroid cancer and lung adenocarcinoma. However, the oncogenicity of the NTRK1 fusion gene in colorectal cancer has not been sufficiently verified. In silico and in vitro and in vivo evaluations were performed to confirm the tumorigenicity of the TPM3-NTRK1 fusion gene and the LMNA-NTRK1 fusion gene. As a result of in silico analysis of the neoplasticity of the NTRK1 fusion gene based on the results of the somatic mutation analysis, somatic mutations of the representative colorectal cancer gene were not observed in colorectal cancer clinical tissues in which the NTRK1 fusion gene was confirmed (refer to FIG. 7). Through this, it was confirmed that the NTRK1 fusion gene acts as a driver oncogene of colorectal cancer. NIH3T3 cells transformed through forced expression of the NTRK1 fusion gene formed a cell mass similar to that of the KM12 colorectal cancer cell line in vitro. In contrast, the NIH3T3 cell line introduced with the empty vector without the NTRK1 fusion gene did not form a cell mass (see FIG. 8 ). The transformed NIH3T3 cell line was inoculated into immune-deficient nude mice, and the NTRK1 fusion gene was introduced in the NIH3T3 cell line, and measurable tumors were formed from the 18th day after the cell inoculation. On the 32nd day after the cell inoculation, the KM12 colorectal cancer cell line and the KM12 colorectal cancer cell line It developed into a tumor at a similar level (see FIG. 9).

Example 19: NTRK1 protein expression and NTRK1 Correlation of fusion genes

Considering that NTRK1 protein is expressed in an NTRK1 gene fusion-dependent manner, tissue microarrays and NTRK1 protein, specifically TrkA encoded by NTRK1 gene, were constructed from a cohort of 216 Korean colorectal cancer patients and 472 Chinese colorectal cancer patients. As a result of observing the frequency of the NTRK1 fusion gene using an antibody that binds to the C-terminal part of the protein, it was confirmed that the NTRK1 protein was expressed in 29.2% of colon cancer clinical tissues derived from Korean colorectal cancer patients, and 25.6% of the Chinese colon. It was confirmed that NTRK1 protein was expressed in colorectal cancer clinical tissues derived from cancer patients (see FIG. 10 ). In order to confirm the correlation between NTRK1 protein expression and the NTRK1 fusion gene, direct fluorescence fusion for the NTRK1 gene was performed in clinical tissues of Korean colorectal cancer patients whose NTRK1 protein expression was confirmed from a cohort of Korean colorectal cancer patients. The frequency of the isolated NTRK1 fluorescence direct junction signal was observed in clinical tissues in which NTRK1 protein expression was confirmed (p < 0.0192) (see FIG. 11 and Table 13). Therefore, it was confirmed that the NTRK1 fusion gene is a part of NTRK1 protein expression, specifically, the gene responsible for 42.9% of the expression.

Correlation between NTRK1 protein expression and NTRK1 fusion gene Sample ID Cytoplasmic TrkA expression NTRK1 fusion P value IHC FISH Colon_50_FISH01 Strong frequently detected 0.0192 Colon_50_FISH02 Strong Not detected Colon_50_FISH03 Strong Not available Colon_50_FISH04 Strong frequently detected Colon_50_FISH05 Strong Not detected Colon_50_FISH06 Moderate frequently detected Colon_50_FISH07 Moderate Not detected Colon_50_FISH08 Mild Not detected Colon_50_FISH09 Mild Not detected Colon_50_FISH10 Mild Not detected Colon_50_FISH11 negative Not detected Colon_50_FISH12 negative Not detected Colon_50_FISH13 negative Not detected Colon_50_FISH14 negative Not detected Colon_50_FISH15 negative Not detected

Example 20: NTRK1 fusion protein and Survival rate of colorectal cancer patient group

A cohort analysis was performed on 216 Korean colorectal cancer patients who could be tracked among colorectal cancer patients who received the clinical sample. Among the 10-year survival rates of 216 Korean colon cancer patients, 42 patients (24.1%) died during the follow-up period.

First, according to the results of analyzing clinical samples, a total of 216 Korean colorectal cancer patients were stratified into NTRK1 fusion protein-positive patients and NTRK1 fusion protein-negative patients according to the expression of NTRK1 protein. As a result of clinical pathological analysis between the stratified colorectal cancer patient groups, the colorectal cancer patient group in which the NTRK1 fusion gene or the NTRK1 fusion protein encoded therein was confirmed had colorectal cancer located in the left colon (p = 0.0504), and the colorectal cancer invasiveness was high (p = 0.0437), positive perineuronal infiltration or lymphatic vascular embolism was observed (p = 0.0429), lymph node metastasis was high (p = 0.015), and microsatellite was stable (p = 0.0024) (see Table 14). .

Clinicopathological characteristics of colorectal cancer patients (Korean) cohort N Cytoplasmic TrkA P value (-) (+) age (years) 216 70.35 70.22 0.4675 Size (cm) 216 5.199 5.548 0.2959 gender 0.3351 Male 118 85 33 Female 98 68 30 location 0.0504 right colon 87 67 20 left colon 129 86 43 Histological type 0.0761 well 16 13 3 Moderate 178 120 58 Poor 10 10 0 Mucinous 12 10 2 Invasion depth
(T1-2 vs. T3) 0.0437 T1 12 10 2 T2 23 19 4 T3 181 124 57 Perineural invation
(PNI) 0.1315 negative 149 109 40 Positive 67 44 23 Lymphovascular emboli (LVE) 0.2229 negative 142 103 39 Positive 74 50 24 PNI/LVE 0.0429 negative 119 90 29 Positive 97 63 34 Lymphnode metastasis
(N0 vs N1-2) 0.015 N0 124 95 29 N1a 40 23 17 N1b 32 22 10 N2a 13 9 4 N2b 7 4 3 Microsatellite status 0.0024 MSS 180 119 61 MSI-L 6 5 One MSI-H 30 29 One

As a result of analyzing the 10-year survival rate of the colorectal cancer hierarchical group, the colorectal cancer patient group in which the NTRK1 fusion protein was confirmed had a significantly lower survival rate than the colorectal cancer patient group in which the NTRK1 fusion protein was not identified (p = 0.0411, risk = 0.5346, 95% confidence interval = 0.2548 to 0.9722) (see Figure 12).

Example 21: Conclusion

As described above, according to the present invention, the prognosis of colorectal cancer patients can be improved by the detection of the TPM3-NTRK1 fusion gene or its encoded fusion transcript and the fusion protein or the NTRK1 gene transcript and protein dependent on the fusion of the NTRK1 gene. It becomes possible to diagnose and predict the effectiveness of colorectal cancer treatment with NTRK1 protein inhibitors.

Inhibitors having an inhibitory effect on NTRK1 protein have already been introduced into clinical studies for the treatment of various carcinomas. For example, restautinib is being studied for its therapeutic effect on prostate cancer, neuroblastoma, and myeloid leukemia, danucertib is being studied for its therapeutic effect on prostate cancer, non-small cell lung cancer, and chronic myelogenous leukemia, and myrciclib is being studied for its therapeutic effect on thyroid cancer and other solid cancers, RXDA-101 for other solid cancers, TSR-011 for non-small cell lung cancer, NMS-P626 for colorectal cancer, and RXDX-102 for other carcinomas The effect is being studied.

As such, fusion of the NTRK1 gene or expression of the NTRK1 gene transcript and protein dependent thereon occurs in a plurality of carcinomas. The NTRK1 fusion gene is separated from mutations in colon cancer genes such as KRAS, NRAS, and PIK3CA. Therefore, the NTRK1 fusion gene can be a target of an existing NTRK1 protein inhibitor, specifically, a tyrosine tyrosine kinase inhibitor having inhibitory activity against NTRK1 tyrosine kinase. In addition, the existence of the NTRK1 fusion gene is recognized in Chinese colon cancer patients including Koreans. Therefore, the method of the present invention is very useful while increasing the efficiency of colorectal cancer treatment in Korean and Chinese colorectal cancer patients.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> LG Life Sciences Ltd. <120> Novel NTRK1 fusion gene as colon cancer marker and the uses it <130> KPA140037-EN-P1 <150> KR 10-2014-0004491 <151> 2014-01-14 <160> 38 <170> KopatentIn 2.0 <210> 1 <211> 858 <212> DNA <213> TPM3 cds <400> 1 atgatggagg ccatcaagaa aaagatgcag atgctgaagt tagacaagga gaatgctctg 60 gatcgggcag agcaagctga agctgagcag aagcaggcag aagaaagaag taaacagctg 120 gaggatgagc tggcagccat gcagaagaag ctgaaaggga cagaggatga gctggacaag 180 tattctgaag ctttgaagga tgcccaggag aagctggaac tggcagagaa gaaggctgct 240 gatgctgagg ctgaggtggc ctccttgaac cgtaggatcc agctggttga agaagagctg 300 gaccgtgctc aggagcgcct ggccactgcc ctgcaaaagc tggaagaagc tgaaaaagct 360 gctgatgaga gtgagagagg tatgaaggtt attgaaaacc gggccttaaa agatgaagaa 420 aagatggaac tccaggaaat ccaactcaaa gaagctaagc acattgcaga agaggcagat 480 aggaagtatg aagaggtggc tcgtaagttg gtgatcattg aaggagactt ggaacgcaca 540 gaggaacgag ctgagctggc agagtctaag tgttctgagc tggaggagga gctgaagaat 600 gtcaccaaca acctcaagtc tcttgaggct caggcggaga agtactctca aaaagaagat 660 aaatatgagg aagaaatcaa gattcttact gataaactca aggaggcaga gacccgtgct 720 gagtttgctg agagatcggt agccaagctg gaaaagacaa ttgatgacct ggaagatgag 780 ctctatgccc agaaactgaa gtacaaggcc attagcgagg agctggacca cgccctcaat 840 gacatgacct ctatataa 858 <210> 2 <211> 775 <212> DNA <213> TPM3 fragment cDNA <400> 2 atgatggagg ccatcaagaa aaagatgcag atgctgaagt tagacaagga gaatgctctg 60 gatcgggcag agcaagctga agctgagcag aagcaggcag aagaaagaag taaacagctg 120 gaggatgagc tggcagccat gcagaagaag ctgaaaggga cagaggatga gctggacaag 180 tattctgaag ctttgaagga tgcccaggag aagctggaac tggcagagaa gaaggctgct 240 gatgctgagg ctgaggtggc ctccttgaac cgtaggatcc agctggttga agaagagctg 300 gaccgtgctc aggagcgcct ggccactgcc ctgcaaaagc tggaagaagc tgaaaaagct 360 gctgatgaga gtgagagagg tatgaaggtt attgaaaacc gggccttaaa agatgaagaa 420 aagatggaac tccaggaaat ccaactcaaa gaagctaagc acattgcaga agaggcagat 480 aggaagtatg aagaggtggc tcgtaagttg gtgatcattg aaggagactt ggaacgcaca 540 gaggaacgag ctgagctggc agagtctaag tgttctgagc tggaggagga gctgaagaat 600 gtcaccaaca acctcaagtc tcttgaggct caggcggaga agtactctca aaaagaagat 660 aaatatgagg aagaaatcaa gattcttact gataaactca aggaggcaga gacccgtgct 720 gagtttgctg agagatcggt agccaagctg gaaaagacaa ttgatgacct ggaag 775 <210> 3 <211> 16 <212> DNA <213> TPM3 5'terminal cleavage site seq <400> 3 attgatgacc tggaag 16 <210> 4 <211> 285 <212> PRT <213> TPM3 full length <400> 4 Met Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys 1 5 10 15 Glu Asn Ala Leu Asp Arg Ala Glu Gln Ala Glu Ala Glu Gln Lys Gln 20 25 30 Ala Glu Glu Arg Ser Lys Gln Leu Glu Asp Glu Leu Ala Ala Met Gln 35 40 45 Lys Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Ala 50 55 60 Leu Lys Asp Ala Gln Glu Lys Leu Glu Leu Ala Glu Lys Lys Ala Ala 65 70 75 80 Asp Ala Glu Ala Glu Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val 85 90 95 Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln 100 105 110 Lys Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met 115 120 125 Lys Val Ile Glu Asn Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu 130 135 140 Gln Glu Ile Gln Leu Lys Glu Ala Lys His Ile Ala Glu Glu Ala Asp 145 150 155 160 Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp 165 170 175 Leu Glu Arg Thr Glu Glu Arg Ala Glu Leu Ala Glu Ser Lys Cys Ser 180 185 190 Glu Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu 195 200 205 Glu Ala Gln Ala Glu Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu 210 215 220 Glu Ile Lys Ile Leu Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala 225 230 235 240 Glu Phe Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp 245 250 255 Leu Glu Asp Glu Leu Tyr Ala Gln Lys Leu Lys Tyr Lys Ala Ile Ser 260 265 270 Glu Glu Leu Asp His Ala Leu Asn Asp Met Thr Ser Ile 275 280 285 <210> 5 <211> 258 <212> PRT <213> TPM3 fragment <400> 5 Met Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys 1 5 10 15 Glu Asn Ala Leu Asp Arg Ala Glu Gln Ala Glu Ala Glu Gln Lys Gln 20 25 30 Ala Glu Glu Arg Ser Lys Gln Leu Glu Asp Glu Leu Ala Ala Met Gln 35 40 45 Lys Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Ala 50 55 60 Leu Lys Asp Ala Gln Glu Lys Leu Glu Leu Ala Glu Lys Lys Ala Ala 65 70 75 80 Asp Ala Glu Ala Glu Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val 85 90 95 Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln 100 105 110 Lys Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met 115 120 125 Lys Val Ile Glu Asn Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu 130 135 140 Gln Glu Ile Gln Leu Lys Glu Ala Lys His Ile Ala Glu Glu Ala Asp 145 150 155 160 Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp 165 170 175 Leu Glu Arg Thr Glu Glu Arg Ala Glu Leu Ala Glu Ser Lys Cys Ser 180 185 190 Glu Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu 195 200 205 Glu Ala Gln Ala Glu Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu 210 215 220 Glu Ile Lys Ile Leu Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala 225 230 235 240 Glu Phe Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp 245 250 255 Leu Glu <210> 6 <211> 5 <212> PRT <213> TPM3 N terminus cleavage site seq <400> 6 Ile Asp Asp Leu Glu 1 5 <210> 7 <211> 2391 <212> DNA <213> NTRK1 CDS <400> 7 atgctgcgag gcggacggcg cgggcagctt ggctggcaca gctgggctgc ggggccgggc 60 agcctgctgg cttggctgat actggcatct gcgggcgccg caccctgccc cgatgcctgc 120 tgcccccacg gctcctcggg actgcgatgc acccgggatg gggccctgga tagcctccac 180 cacctgcccg gcgcagagaa cctgactgag ctctacatcg agaaccagca gcatctgcag 240 catctggagc tccgtgatct gaggggcctg ggggagctga gaaacctcac catcgtgaag 300 agtggtctcc gtttcgtggc gccagatgcc ttccatttca ctcctcggct cagtcgcctg 360 aatctctcct tcaacgctct ggagtctctc tcctggaaaa ctgtgcaggg cctctcctta 420 caggaactgg tcctgtcggg gaaccctctg cactgttctt gtgccctgcg ctggctacag 480 cgctgggagg aggagggact gggcggagtg cctgaacaga agctgcagtg tcatgggcaa 540 gggcccctgg cccacatgcc caatgccagc tgtggtgtgc ccacgctgaa ggtccaggtg 600 cccaatgcct cggtggatgt gggggacgac gtgctgctgc ggtgccaggt ggaggggcgg 660 ggcctggagc aggccggctg gatcctcaca gagctggagc agtcagccac ggtgatgaaa 720 tctgggggtc tgccatccct ggggctgacc ctggccaatg tcaccagtga cctcaacagg 780 aagaacgtga cgtgctgggc agagaacgat gtgggccggg cagaggtctc tgttcaggtc 840 aacgtctcct tcccggccag tgtgcagctg cacacggcgg tggagatgca ccactggtgc 900 atccccttct ctgtggatgg gcagccggca ccgtctctgc gctggctctt caatggctcc 960 gtgctcaatg agaccagctt catcttcact gagttcctgg agccggcagc caatgagacc 1020 gtgcggcacg ggtgtctgcg cctcaaccag cccacccacg tcaacaacgg caactacacg 1080 ctgctggctg ccaacccctt cggccaggcc tccgcctcca tcatggctgc cttcatggac 1140 aaccctttcg agttcaaccc cgaggacccc atccctgtct ccttctcgcc ggtggacact 1200 aacagcacat ctggagaccc ggtggagaag aaggacgaaa caccttttgg ggtctcggtg 1260 gctgtgggcc tggccgtctt tgcctgcctc ttcctttcta cgctgctcct tgtgctcaac 1320 aaatgtggac ggagaaacaa gtttgggatc aaccgcccgg ctgtgctggc tccagaggat 1380 gggctggcca tgtccctgca tttcatgaca ttgggtggca gctccctgtc ccccaccgag 1440 ggcaaaggct ctgggctcca aggccacatc atcgagaacc cacaatactt cagtgatgcc 1500 tgtgttcacc acatcaagcg ccgggacatc gtgctcaagt gggagctggg ggagggcgcc 1560 tttgggaagg tcttccttgc tgagtgccac aacctcctgc ctgagcagga caagatgctg 1620 gtggctgtca aggcactgaa ggaggcgtcc gagagtgctc ggcaggactt ccagcgtgag 1680 gctgagctgc tcaccatgct gcagcaccag cacatcgtgc gcttcttcgg cgtctgcacc 1740 gagggccgcc ccctgctcat ggtctttgag tatatgcggc acggggacct caaccgcttc 1800 ctccgatccc atggacctga tgccaagctg ctggctggtg gggaggatgt ggctccaggc 1860 cccctgggtc tggggcagct gctggccgtg gctagccagg tcgctgcggg gatggtgtac 1920 ctggcgggtc tgcattttgt gcaccgggac ctggccacac gcaactgtct agtgggccag 1980 ggactggtgg tcaagattgg tgattttggc atgagcaggg atatctacag caccgactat 2040 taccgtgtgg gaggccgcac catgctgccc attcgctgga tgccgcccga gagcatcctg 2100 taccgtaagt tcaccaccga gagcgacgtg tggagcttcg gcgtggtgct ctgggagatc 2160 ttcacctacg gcaagcagcc ctggtaccag ctctccaaca cggaggcaat cgactgcatc 2220 acgcagggac gtgagttgga gcggccacgt gcctgcccac cagaggtcta cgccatcatg 2280 cggggctgct ggcagcggga gccccagcaa cgccacagca tcaaggatgt gcacgcccgg 2340 ctgcaagccc tggcccaggc acctcctgtc tacctggatg tcctgggcta g 2391 <210> 8 <211> 1196 <212> DNA <213> NTRK1 fragment cDNA <400> 8 acactaacag cacatctgga gacccggtgg agaagaagga cgaaacacct tttggggtct 60 cggtggctgt gggcctggcc gtctttgcct gcctcttcct ttctacgctg ctccttgtgc 120 tcaacaaatg tggacggaga aacaagtttg ggatcaaccg cccggctgtg ctggctccag 180 aggatgggct ggccatgtcc ctgcatttca tgacattggg tggcagctcc ctgtccccca 240 ccgagggcaa aggctctggg ctccaaggcc acatcatcga gaacccacaa tacttcagtg 300 atgcctgtgt tcaccacatc aagcgccggg acatcgtgct caagtgggag ctgggggagg 360 gcgcctttgg gaaggtcttc cttgctgagt gccacaacct cctgcctgag caggacaaga 420 tgctggtggc tgtcaaggca ctgaaggagg cgtccgagag tgctcggcag gacttccagc 480 gtgaggctga gctgctcacc atgctgcagc accagcacat cgtgcgcttc ttcggcgtct 540 gcaccgaggg ccgccccctg ctcatggtct ttgagtatat gcggcagggg gacctcaacc 600 gcttcctccg atcccatgga cctgatgcca agctgctggc tggtggggag gatgtggctc 660 caggccccct gggtctgggg cagctgctgg ccgtggctag ccaggtcgct gcggggatgg 720 tgtacctggc gggtctgcat tttgtgcacc gggacctggc cacacgcaac tgtctagtgg 780 gccagggact ggtggtcaag attggtgatt ttggcatgag cagggatatc tacagcaccg 840 actattaccg tgtgggaggc cgcaccatgc tgcccattcg ctggatgccg cccgagagca 900 tcctgtaccg taagttcacc accgagagcg acgtgtggag cttcggcgtg gtgctctggg 960 agatcttcac ctacggcaag cagccctggt accagctctc caacacggag gcaatcgact 1020 gcatcacgca gggacgtgag ttggagcggc cacgtgcctg cccaccagag gtctacgcca 1080 tcatgcgggg ctgctggcag cgggagcccc agcaacgcca cagcatcaag gatgtgcacg 1140 cccggctgca agccctggcc caggcacctc ctgtctacct ggatgtcctg ggctag 1196 <210> 9 <211> 17 <212> DNA <213> NTRK1 3' terminal cleavage site seq <400> 9 acactaacag cacatct 17 <210> 10 <211> 1037 <212> DNA <213> NTRK1 fragment cDNA <400> 10 gcccggctgt gctggctcca gaggatgggc tggccatgtc cctgcatttc atgacattgg 60 gtggcagctc cctgtccccc accgagggca aaggctctgg gctccaaggc cacatcatcg 120 agaacccaca atacttcagt gatgcctgtg ttcaccacat caagcgccgg gacatcgtgc 180 tcaagtggga gctgggggag ggcgcctttg ggaaggtctt ccttgctgag tgccacaacc 240 tcctgcctga gcaggacaag atgctggtgg ctgtcaaggc actgaaggag gcgtccgaga 300 gtgctcggca ggacttccag cgtgaggctg agctgctcac catgctgcag caccagcaca 360 tcgtgcgctt cttcggcgtc tgcaccgagg gccgccccct gctcatggtc tttgagtata 420 tgcggcacgg ggacctcaac cgcttcctcc gatcccatgg acctgatgcc aagctgctgg 480 ctggtgggga ggatgtggct ccaggccccc tgggtctggg gcagctgctg gccgtggcta 540 gccaggtcgc tgcggggatg gtgtacctgg cgggtctgca ttttgtgcac cgggacctgg 600 ccacacgcaa ctgtctagtg ggccagggac tggtggtcaa gattggtgat tttggcatga 660 gcagggatat ctacagcacc gactattacc gtgtgggagg ccgcaccatg ctgcccattc 720 gctggatgcc gcccgagagc atcctgtacc gtaagttcac caccgagagc gacgtgtgga 780 gcttcggcgt ggtgctctgg gagatcttca cctacggcaa gcagccctgg taccagctct 840 ccaacacgga ggcaatcgac tgcatcacgc agggacgtga gttggagcgg ccacgtgcct 900 gcccaccaga ggtctacgcc atcatgcggg gctgctggca gcgggagccc cagcaacgcc 960 acagcatcaa ggatgtgcac gcccggctgc aagccctggc ccaggcacct cctgtctacc 1020 tggatgtcct gggctag 1037 <210> 11 <211> 17 <212> DNA <213> NTRK1 3' terminal cleavage site seq <400> 11 gcccggctgt gctggct 17 <210> 12 <211> 890 <212> DNA <213> NTRK1 fragment cDNA <400> 12 gtgttcacca catcaagcgc cgggacatcg tgctcaagtg ggagctgggg gagggcgcct 60 ttgggaaggt cttccttgct gagtgccaca acctcctgcc tgagcaggac aagatgctgg 120 tggctgtcaa ggcactgaag gaggcgtccg agagtgctcg gcaggacttc cagcgtgagg 180 ctgagctgct caccatgctg cagcaccagc acatcgtgcg cttcttcggc gtctgcaccg 240 agggccgccc cctgctcatg gtctttgagt atatgcggca cggggacctc aaccgcttcc 300 tccgatccca tggacctgat gccaagctgc tggctggtgg ggaggatgtg gctccaggcc 360 ccctgggtct ggggcagctg ctggccgtgg ctagccaggt cgctgcgggg atggtgtacc 420 tggcgggtct gcattttgtg caccgggacc tggccacacg caactgtcta gtgggccagg 480 gactggtggt caagattggt gattttggca tgagcaggga tatctacagc accgactatt 540 accgtgtggg aggccgcacc atgctgccca ttcgctggat gccgcccgag agcatcctgt 600 accgtaagtt caccaccgag agcgacgtgt ggagcttcgg cgtggtgctc tgggagatct 660 tcacctacgg caagcagccc tggtaccagc tctccaacac ggaggcaatc gactgcatca 720 cgcagggacg tgagttggag cggccacgtg cctgcccacc agaggtctac gccatcatgc 780 ggggctgctg gcagcgggag ccccagcaac gccacagcat caaggatgtg cacgcccggc 840 tgcaagccct ggcccaggca cctcctgtct acctggatgt cctgggctag 890 <210> 13 <211> 17 <212> DNA <213> NTRK1 3' terminal cleavage site seq <400> 13 gtgttcacca catcaag 17 <210> 14 <211> 796 <212> PRT <213> NTRK1 full length <400> 14 Met Leu Arg Gly Gly Arg Arg Gly Gln Leu Gly Trp His Ser Trp Ala 1 5 10 15 Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu Ile Leu Ala Ser Ala Gly 20 25 30 Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu 35 40 45 Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly 50 55 60 Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln Gln His Leu Gln 65 70 75 80 His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Glu Leu Val 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp Leu Gln 145 150 155 160 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Pro Glu Gln Lys Leu Gln 165 170 175 Cys His Gly Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly 180 185 190 Val Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp Val Gly 195 200 205 Asp Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln 210 215 220 Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val Met Lys 225 230 235 240 Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 245 250 255 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly 260 265 270 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 275 280 285 Gln Leu His Thr Ala Val Glu Met His His Trp Cys Ile Pro Phe Ser 290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Ala 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr 340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly 355 360 365 Gln Ala Ser Ala Ser Ile Met Ala Ala Phe Met Asp Asn Pro Phe Glu 370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 385 390 395 400 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe 405 410 415 Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu 420 425 430 Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe 435 440 445 Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met 450 455 460 Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu 465 470 475 480 Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr 485 490 495 Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu 500 505 510 Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu 515 520 525 Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys 530 535 540 Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu 545 550 555 560 Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe 565 570 575 Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met 580 585 590 Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala 595 600 605 Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu 610 615 620 Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr 625 630 635 640 Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys 645 650 655 Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser 660 665 670 Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met 675 680 685 Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe 690 695 700 Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile 705 710 715 720 Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala 725 730 735 Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys 740 745 750 Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro 755 760 765 Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu 770 775 780 Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 785 790 795 <210> 15 <211> 397 <212> PRT <213> NTRK1 fragment <400> 15 Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro 1 5 10 15 Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe 20 25 30 Leu Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys 35 40 45 Phe Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala 50 55 60 Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr 65 70 75 80 Glu Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln 85 90 95 Tyr Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val 100 105 110 Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala 115 120 125 Glu Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val 130 135 140 Lys Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg 145 150 155 160 Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe 165 170 175 Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr 180 185 190 Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp 195 200 205 Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly 210 215 220 Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val 225 230 235 240 Tyr Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn 245 250 255 Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met 260 265 270 Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr 275 280 285 Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys 290 295 300 Phe Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu 305 310 315 320 Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu 325 330 335 Ala Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala 340 345 350 Cys Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu 355 360 365 Pro Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala 370 375 380 Leu Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 385 390 395 <210> 16 <211> 344 <212> PRT <213> NTRK1 fragment <400> 16 Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser Leu His Phe 1 5 10 15 Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser 20 25 30 Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala 35 40 45 Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu 50 55 60 Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu 65 70 75 80 Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu 85 90 95 Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu 100 105 110 Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr 115 120 125 Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp 130 135 140 Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala 145 150 155 160 Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu 165 170 175 Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu 180 185 190 His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln 195 200 205 Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Ile Tyr 210 215 220 Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg 225 230 235 240 Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser 245 250 255 Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly 260 265 270 Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile 275 280 285 Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val 290 295 300 Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His 305 310 315 320 Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro 325 330 335 Pro Val Tyr Leu Asp Val Leu Gly 340 <210> 17 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site aa seq <400> 17 Pro Ala Val Leu Ala 1 5 <210> 18 <211> 295 <212> PRT <213> NTRK1 fragment site <400> 18 Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu Gly 1 5 10 15 Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu Leu 20 25 30 Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala 35 40 45 Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr 50 55 60 Met Leu Gln His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr Glu 65 70 75 80 Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp Leu 85 90 95 Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly 100 105 110 Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu Ala 115 120 125 Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu His 130 135 140 Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln Gly 145 150 155 160 Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Ile Tyr Ser 165 170 175 Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg Trp 180 185 190 Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp 195 200 205 Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys 210 215 220 Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile Thr 225 230 235 240 Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val Tyr 245 250 255 Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His Ser 260 265 270 Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro Pro 275 280 285 Val Tyr Leu Asp Val Leu Gly 290 295 <210> 19 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site seq <400> 19 Val His His Ile Lys 1 5 <210> 20 <211> 5 <212> PRT <213> NTRK1 C terminus cleavage site seq <400> 20 Val His His Ile Lys 1 5 <210> 21 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 21 attgatgacc tggaagacac taacagcaca tct 33 <210> 22 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 22 attgatgacc tggaaggccc ggctgtgctg gct 33 <210> 23 <211> 33 <212> DNA <213> TPM3-NTRK1 fusion site seq <400> 23 attgatgacc tggaaggtgt tcaccacatc aag 33 <210> 24 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 24 Ile Asp Asp Leu Glu Asp Thr Asn Ser Thr Ser 1 5 10 <210> 25 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 25 Ile Asp Asp Leu Glu Gly Pro Ala Val Leu Ala 1 5 10 <210> 26 <211> 11 <212> PRT <213> TPM3-NTRK1 fusion site aa seq <400> 26 Ile Asp Asp Leu Glu Gly Val His His Ile Lys 1 5 10 <210> 27 <211> 1995 <212> DNA <213> LMNA CDS <400> 27 atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60 ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat 120 cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180 cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240 tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc 300 cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360 accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg 420 ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480 gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540 aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg 600 aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660 cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720 ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780 aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840 aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900 agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960 cgagacctgg aggactcact ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020 aaggagcggg agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag 1080 gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg caagctcttg 1140 gagggcgagg aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc 1200 cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa aaagcgcaaa 1260 ctggagtcca ctgagagccg cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320 gccgtggagg aggtggatga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag 1380 gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440 taccggttcc caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca 1500 ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca gaacacctgg 1560 ggctgcggga acagcctgcg tacggctctc atcaactcca ctggggaaga agtggccatg 1620 cgcaagctgg tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac 1680 ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740 ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc 1800 agcggctcag gagcccaggt gggcggaccc atctcctctg gctcttctgc ctccagtgtc 1860 acggtcactc gcagctaccg cagtgtgggg ggcagtgggg gtggcagctt cggggacaat 1920 ctggtcaccc gctcctacct cctgggcaac tccagccccc gaacccagag cccccagaac 1980 tgcagcatca tgtaa 1995 <210> 28 <211> 985 <212> DNA <213> LMNA fragment cDNA <400> 28 atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60 ctgtcgccca cccgcatcac ccggctgcag gagaaggagg acctgcagga gctcaatgat 120 cgcttggcgg tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180 cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240 tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc 300 cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa agcgcgcaat 360 accaagaagg agggtgacct gatagctgct caggctcggc tgaaggacct ggaggctctg 420 ctgaactcca aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc 480 gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540 aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg 600 aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga gaccaagcgc 660 cgtcatgaga cccgactggt ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720 ctggcggatg cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag 780 aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840 aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac 900 agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga ggcgaagctt 960 cgagacctgg aggactcact ggccc 985 <210> 29 <211> 16 <212> DNA <213> LMNA 5' terminal cleavage site seq <400> 29 gaggactcac tggccc 16 <210> 30 <211> 664 <212> PRT <213> LMNA full length <400> 30 Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met 340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425 430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu 435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Lys Phe Thr Leu Lys Ala Gly Gln Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly 580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Val Gly 595 600 605 Gly Pro Ile Ser Ser Gly Ser Ser Ala Ser Ser Val Thr Val Thr Arg 610 615 620 Ser Tyr Arg Ser Val Gly Gly Ser Gly Gly Gly Ser Phe Gly Asp Asn 625 630 635 640 Leu Val Thr Arg Ser Tyr Leu Leu Gly Asn Ser Ser Pro Arg Thr Gln 645 650 655 Ser Pro Gln Asn Cys Ser Ile Met 660 <210> 31 <211> 328 <212> PRT <213> LMNA fragment <400> 31 Met Glu Thr Pro Ser Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala 325 <210> 32 <211> 5 <212> PRT <213> LMNA N terminus cleavage site seq <400> 32 Glu Asp Ser Leu Ala 1 5 <210> 33 <211> 33 <212> DNA <213> LMNA-NTRK1 fusion site seq <400> 33 gaggactcac tggcccgccc ggctgtgctg gct 33 <210> 34 <211> 11 <212> PRT <213> LMNA-NTRK1 fusion site aa seq <400> 34 Glu Asp Ser Leu Ala Gly Pro Ala Val Leu Ala 1 5 10 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TPM3-NTRK1 Forward Primer Sequence <400> 35 aagaagataa atatgagg 18 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TPM3-NTRK1 Reverse Primer Sequence <400> 36 ccgtgccgca tatactcaaa 20 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LMNA-NTRK1 Forward Primer Sequence <400> 37 ccaggtggag cagtataaga ag 22 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMNA-NTRK1 Reverse Primer Sequence <400> 38 tgtgggttct cgatgatgtg 20

Claims (42)

N-[fragment containing the N terminus of LMNA (Lamin A)]-[fragment containing the C terminus of Neurotrophic Tyrosine Kinase (NTRK1, Receptor, Type 1)]-C, or
a fusion protein consisting of N-[fragment containing the N terminus of TPM3 (Tropomyosin 3)]-[fragment containing the C terminus of NTRK1]-C; a fusion gene encoding the fusion protein; Or a composition for diagnosing colorectal cancer comprising an agent for measuring the transcript of the fusion gene, wherein the fragment including the N-terminus of the LMNA comprises amino acids 1 to 328 from the N-terminus in a human-derived LMNA protein, and wherein The fragment including the N-terminus of TPM3 will include the amino acids 1 to 258 from the N-terminus in the human-derived TPM3 protein, a composition for diagnosing colon cancer.
The composition for diagnosing colon cancer according to claim 1, wherein the LMNA, TPM3 or NTRK1 is human-derived.
The composition for diagnosing colon cancer according to claim 2, wherein the human-derived LMNA consists of the amino acid sequence of SEQ ID NO: 27.
delete The composition for diagnosing colon cancer according to claim 1, wherein the fragment including the N-terminus of the LMNA consists of the amino acid sequence of SEQ ID NO: 31.
According to claim 2, wherein the human-derived TPM3 consists of the amino acid sequence of SEQ ID NO: 4, colon cancer diagnostic composition.
delete The composition for diagnosing colon cancer according to claim 1, wherein the fragment including the N-terminus of TPM3 is composed of the amino acid sequence of SEQ ID NO: 5.
The composition for diagnosing colon cancer according to claim 2, wherein the human-derived NTRK1 consists of the amino acid sequence of SEQ ID NO: 7.
The method of claim 1, wherein the fragment including the C-terminus of NTRK1 comprises amino acids 400 to 796, 453 to 796, or 502 to 796, from the N-terminus in human NTRK1 protein. Phosphorus, a composition for diagnosing colon cancer.
The composition for diagnosing colon cancer according to claim 1, wherein the fragment including the C-terminus of NTRK1 is composed of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.
delete delete delete delete delete delete The composition for diagnosing colon cancer according to claim 1, wherein the transcript of the fusion gene is mRNA.
The composition for diagnosing colon cancer according to claim 18, wherein the agent for measuring the mRNA of the fusion gene comprises a primer that specifically binds to the mRNA of the fusion gene.
The colon according to claim 19, wherein the primer specifically binding to the mRNA of the fusion gene is a primer designed to sandwich the fusion point so that the region amplified by the primer includes the fusion point of two genes to be fused. A composition for diagnosing cancer.
The method according to claim 20, wherein when the two genes to be fused are the TPM3 gene and the NTRK1 gene, a primer specific to the nucleotide sequence encoding the N-terminus of the TPM3 protein and a primer specific to the nucleotide sequence encoding the C-terminus of NTRK1 are included. Which is a primer pair, colon cancer diagnostic composition.
The composition for diagnosing colon cancer according to claim 21, wherein the primer specifically binding to the mRNA of the fusion gene is a primer pair consisting of the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 36.
20. The method of claim 19, wherein when the fusion gene is an LMNA gene and an NTRK1 gene, a primer comprising a primer specific for the nucleotide sequence encoding the N-terminus of the LMNA protein and a primer specific for the nucleotide sequence encoding the C-terminus of NTRK1. A pair, a composition for diagnosing colon cancer.
The composition for diagnosing colon cancer according to claim 23, wherein the primer specifically binding to the mRNA of the fusion gene is a primer pair consisting of the nucleotide sequences of SEQ ID NO: 37 and SEQ ID NO: 38.
The composition for diagnosing colon cancer according to claim 1, wherein the agent for measuring the fusion gene comprises a probe or primer that specifically binds to the fusion gene.
The composition for diagnosing colon cancer according to claim 1, wherein the agent for measuring the NTRK1 fusion protein comprises an antibody specific for the protein.
The composition for diagnosis of colorectal cancer according to claim 26, wherein the antibody specific for the NTRK1 fusion protein binds to the C-terminus of the NTRK1 protein.
A kit for diagnosing colon cancer comprising the composition of claim 1.
The kit for diagnosing colon cancer according to claim 28, which is an RT-PCR kit, a DNA chip kit, or a protein chip kit.
Measuring the level of the NTRK1 fusion gene, its transcript, or its protein from an isolated sample of a subject suspected of colon cancer; And Comprising the step of comparing the measured expression level of the fusion gene, its transcript or its protein expression level with the fusion gene, its transcript, or its protein expression level of a normal control sample, a method of providing information necessary for diagnosing colon cancer , the gene is 5'-[fragment comprising the 5' end of the LMNA gene]-[fragment comprising the 3' end of the NTRK1 gene]-3', or
5'-[fragment comprising the 5' end of TPM3]-[fragment comprising the 3' end of NTRK1 gene]-3'.
31. The method of claim 30, wherein the NTRK1 fusion gene comprises: a polynucleotide constituting the 5' end of the LMNA gene or the TPM3 gene; and a polynucleotide constituting the 3' end of the NTRK1 gene, a method for providing information necessary for diagnosing colorectal cancer.
The method of claim 30, wherein the expression level of the fusion gene, its transcript, or protein thereof measured from the isolated sample of a subject suspected of colon cancer is higher than the expression level of the fusion gene, its transcript, or its protein in the normal control sample, A method of providing information necessary for diagnosing colon cancer, characterized in that it is diagnosed as colon cancer.
Measuring the level of the NTRK1 fusion gene, its transcript, or its protein from the isolated sample of a colorectal cancer patient; and comparing the measured expression level of the fusion gene, its transcript or its protein with the fusion gene, its transcript, or its protein expression level in a normal control sample, providing information necessary for predicting the prognosis of colorectal cancer treatment As a method, the gene is 5'-[fragment comprising the 5' end of the LMNA gene]-[fragment comprising the 3' end of the NTRK1 gene]-3', or 5'-[the 5' end of TPM3 Fragment comprising]-[fragment comprising the 3' end of NTRK1 gene]-3'.
34. The method of claim 33, wherein when the expression level of the fusion gene, its transcript, or its protein from the isolated sample of a colorectal cancer patient is higher than the expression level of the fusion gene, its transcript, or its protein of the normal control sample, the colorectal cancer treatment prognosis is A method of providing information necessary for predicting a colorectal cancer treatment prognosis, characterized in that the prediction is worse than the same or lower case.
34. The method of claim 33, wherein when the expression level of the fusion gene, its transcript, or its protein from the sample isolated from a colorectal cancer patient is higher than that of the normal control sample, the NTRK1 protein inhibitor is used. A method of providing information necessary for predicting a colorectal cancer treatment prognosis, characterized in that predicting that the colorectal cancer treatment efficacy is high.
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