KR20170037714A

KR20170037714A - Novel composition for prognosing lung cancer using fusion-gene

Info

Publication number: KR20170037714A
Application number: KR1020150136272A
Authority: KR
Inventors: 오충훈; 강근수; 반주연; 방만석; 이정주
Original assignee: 단국대학교 천안캠퍼스 산학협력단
Priority date: 2015-09-25
Filing date: 2015-09-25
Publication date: 2017-04-05
Also published as: KR101860181B1

Abstract

The present invention relates to a composition for predicting the prognosis of lung cancer, comprising a means for detecting a fusion gene specifically expressed in lung cancer tissue or a fusion protein expressed from said fusion gene, a kit for predicting the prognosis of lung cancer comprising said composition, To provide information for predicting the prognosis of lung cancer. The composition for predicting the prognosis of lung cancer of the present invention can be used for predicting the prognosis after the treatment of lung cancer by detecting a fusion gene that is specifically expressed only in the tissue of the cancerous region in the lung tissue of the patient having a certain level of lung cancer. It can be widely used to effectively treat lung cancer.

Description

[0001] The present invention relates to a composition for predicting the prognosis of lung cancer using a fusion gene,

The present invention relates to a composition for predicting the prognosis of lung cancer using a fusion gene. More particularly, the present invention relates to a composition for predicting the prognosis of lung cancer using a fusion gene, A composition for predicting prognosis, a kit for predicting lung cancer prognosis comprising the composition, and a method for providing information for predicting the prognosis of lung cancer using the kit or composition.

Lung cancer, a malignant tumor originating from the lung, is divided into small cell lung cancer and non-small cell lung cancer according to its tissue type. Although the small cell lung cancer is classified as a part of lung cancer by the location of the onset tissue, it is distinguished from the other lung cancer in terms of clinical course, treatment method and prognosis. In addition, non-small cell lung cancer is divided into adenocarcinoma, squamous cell carcinoma and large cell carcinoma according to the histological type.

In particular, small-cell lung cancer is a large mass, grayish white, and is known to proliferate along the bronchial wall. In many cases, surgical resection is difficult at the time of diagnosis, and malignancy is rapidly growing. It is known that chemotherapy or radiotherapy has a remarkable therapeutic effect while it is easily transferred to whole body through blood circulation. The brain, liver, bone, lung, adrenal gland, kidney, etc. have been reported as the main organs in which the small cell lung cancer is metastasized.

On the other hand, non-small cell lung cancer is classified into adenocarcinoma, squamous cell acinoma, and large-cell carcinoma as described above. First, adenocarcinomas occur mainly in the lungs, and they occur frequently in women or non-smokers. In many cases, the adenocarcinoma is metastasized even if the size is small, and the incidence of the adenocarcinoma is increasing recently. Next, squamous cell carcinoma is found mainly in the central lung, and it is known to grow up to the lumen of the bronchial tree, blocking the airway, is common to men, and is closely related to smoking. Finally, large cell carcinomas arise mainly in the vicinity of the lung surface (the distal end of the lung), about half in large bronchi, accounting for 4 to 10% of all lung cancers, and generally large cell size, It is known that prognosis is worse than other non - small cell lung cancer because it tends to proliferate and metastasize rapidly.

In particular, in the case of non-small cell lung cancer, the survival rate in the last 10 years is only about 10%. One of the main reasons for such a low survival rate is that the success rate of diagnosis of lung cancer is very low. As a method for improving the success rate of such lung cancer, a method for detecting a lung cancer-specific gene abnormality has been proposed, and various studies on lung cancer-specific gene mutation have been reported. However, There is a disadvantage that the criterion for judging whether or not an abnormality is present is ambiguous. In other words, the correlation between the level of mutation of a specific gene and the likelihood of lung cancer is unclear, and the clear criteria for the suspicion of lung cancer is ambiguous when a variety of lung cancer-associated genes are mutated. It is also common to all other cancer diagnoses.

Therefore, studies are being actively conducted to determine the criteria for determining whether a gene is abnormal, which can clearly determine whether or not a cancer has occurred. For example, Korean Patent No. 709633 discloses a technique for diagnosing the risk of lung cancer using XPG polymorphism markers for risk diagnosis of lung cancer. Korean Patent No. 884565 discloses a technique for diagnosing lung cancer-specific methylation marker genes (CDO1, CREM, FABP4, GOS2, HYAL1, LPXN, NFKBIA, RRAD, THBD, TNNC1 or TOM1).

However, the study on the diagnosis and treatment of lung cancer has been actively studied, but the method of predicting the prognosis of lung cancer has not been studied sufficiently. At present, postoperative clinical symptoms such as weight loss, loss of appetite, local symptoms, pathologic tumor stage, abdominal and inguinal examination, rectal examination, occult blood test, The prognosis is judged. However, this method has disadvantages such that the criterion is not objective, has a complicated procedure, and the result is not accurate.

Recently, as a diagnostic method for various cancers including lung cancer, a method of using a fusion gene specifically expressed in cancer cells has been developed. Such a fusion gene appears as a result of chromosomal rearrangement that occurs specifically in cancer tissue, for example, the BCR-ABL fusion gene found in patients with chronic myeloid leukemia (CML). Such a fusion gene is used for the early diagnosis of various cancers including lung cancer because it can clearly determine the onset of cancer according to its occurrence, but it is not used for prediction of cancer prognosis. As the cancer cells proliferate, a genetic variation is continuously generated, and chromosomal rearrangement is expected to occur continuously. Thus, it is expected that it is possible to use a specific fusion gene for cancer prognosis prediction, And a fusion gene which can be specifically used for predicting the prognosis of cancer selected from these fusion genes has not been reported yet.

Under these circumstances, the inventors of the present invention have found that five fusion genes can be used for predicting the prognosis of lung cancer, and as a result, .

One object of the present invention is to provide a fusion protein for the prediction of prognosis of lung cancer selected from the group consisting of CD74-ROS1, EML4-ALK, KSR1-LGALS9, KIF5B-RET and CLN6-CALML4 or a fusion protein expressed from the fusion gene A composition for predicting the prognosis of lung cancer.

Another object of the present invention is to provide a kit for predicting the prognosis of lung cancer comprising the above composition.

It is still another object of the present invention to provide a method for predicting the prognosis of lung cancer by treating a sample of a subject to be predicted of the prognosis of lung cancer and detecting the mRNA of the fusion gene for prediction of lung cancer or the fusion protein expressed from the fusion gene The present invention provides a method for providing information for predicting the prognosis of lung cancer.

The present inventors obtained a tissue sample of a normal region present in the lung tissue of a lung cancer patient and a tissue sample of a cancer-causing region, respectively, in order to find a fusion gene that can be specifically used for predicting the prognosis of lung cancer. Sequencing analysis showed that 251 fusion genes were expressed in normal tissues while 505 fusion genes were expressed in cancer tissues. Among the 505 fusion genes expressed in the cancer tissues, a fusion gene that is specifically expressed only in cancer tissues is firstly screened, and a fusion gene or cancer metastasis detected in two or more tissues among the firstly selected fusion genes And a fusion gene comprising a gene associated with tyrosine kinase that is a second-type gene. Among the 111 secondary fusion genes selected, five fusion genes (CD74-ROS1, EML4-ALK, and the like) specifically expressed in lung cancer tissues were selected based on the distance between the genes constituting the fusion gene and the similarity of the genes, KSR1-LGALS9, KIF5B-RET and CLN6-CALML4) were finally selected.

The present inventors investigated whether the final 5 selected fusion genes could be used in predicting the prognosis of lung cancer. In order to confirm whether or not the fusion genes of lung cancer tissues obtained from 100 patients Was used to confirm whether the respective fusion genes were detected. As a result, it was confirmed that the above five kinds of fusion genes were detected in the lung cancer tissue of the patient at a detection rate of 2 to 58%.

Since the fusion gene is specifically expressed in the lung cancer tissue of a patient who has not been treated even after treatment with lung cancer, it can be predicted that the patient's symptoms will be further aggravated by the fact that the fusion gene has been detected in the patient's sample These predictions were confirmed from the progression of the pathology of each patient.

That is, when the five kinds of fusion genes are detected in the patient's sample, the prognosis of lung cancer of the patient will be deteriorated, while if the five kinds of fusion genes are not detected in the patient's sample, The prognosis of a patient with lung cancer is being treated.

Therefore, it was found that the fusion gene can be used as a marker for predicting the prognosis of lung cancer. In addition, since the fusion gene is detected at different levels depending on the patient, these fusion genes can be used as markers for predicting the prognosis of lung cancer, either alone or in combination, It can be used as a marker for prediction. As described above, a technique of using five kinds of fusion genes as markers for predicting the prognosis of lung cancer has not been reported at all and has been developed for the first time by the present inventors.

In order to achieve the above object, the present invention provides a method for producing an anti-cancer agent, comprising the steps of: constructing a clone of CD74 (Cluster of Differentiation 74) -ROS1 (Eosinoderm microtubule-associated protein-like 4) kinase, KSR1, Galectin-9, KIF5B -RET, and ceroid-lipofuscinosis neuronal protein 6 (CALMODULIN- like 4). The composition for predicting the prognosis of lung cancer comprises the mRNA of the fusion gene for prediction of lung cancer prognosis or at least one agent for detecting the fusion protein expressed from the fusion gene.

The term "fusion gene for predicting lung cancer prognosis" of the present invention means a fusion gene in which two genes are specifically expressed in a lung cancer tissue derived from a lung cancer patient and not expressed in the normal tissue of the lung.

For the purpose of the present invention, the fusion gene includes a nucleotide sequence in which two genes on the same chromosome are bound in a head-to-tail form, or two genes on different chromosomes, tail < / RTI > At this time, a fusion gene in which two genes are combined in the form of head-to-tail may be composed of a form in which each gene of the complete nucleotide sequence is bound, or a nucleotide sequence in which some nucleotide sequences are mutated by deletion, Each gene may be composed of a combined form. In this case, the mutated base sequence region is not particularly limited, but may be, for example, 1 to 50% of the entire base sequence, as another example, 1 to 20%, and as another example, 1 to 10% Lt; / RTI >

For example, the fusion gene provided in the present invention is not particularly limited, but may be CD74-ROS1, which is composed of the nucleotide sequence of SEQ ID NO: 1, fused CD74 gene as a head gene and ROS1 gene as a tail gene; EML4-ALK consisting of the nucleotide sequence of SEQ ID NO: 2, the EML4 gene as the head gene and the ALK gene as the tail gene; KSR1-LGALS9 consisting of the nucleotide sequence of SEQ ID NO: 3 and fused with KSR1 gene as the head gene and LGALS9 gene as the tail gene; KIF5B-RET consisting of the nucleotide sequence of SEQ ID NO: 4 and fused with the KIF5B gene as the head gene and the RET gene as the tail gene; Or CLN6-CALML4 in which the CLN6 gene as the head gene and the CALML4 gene as the tail gene are fused.

Specific nucleotide sequences and protein information of each gene constituting the fusion gene are known in NCBI. For example, the nucleotide sequence and protein sequence of the CD74 (Cluster of Differentiation 74) gene are known as NM_001025158 and NP_001020329, respectively; The nucleotide sequence and protein sequence of the ROS1 gene (proto-oncogene tyrosine-protein kinase ROS) are known from NM_002944 and NP_002935, respectively; The nucleotide sequence and protein sequence of EML4 (Echinoderm microtubule-associated protein-like 4) gene are known in NM_001145076 and NP_001138548, respectively; Anaplastic lymphoma kinase (ALK) The nucleotide and protein sequences of the genes are known in NM_004304 and NP_004295, respectively; The nucleotide sequence and protein sequence of the KSR1 (kinase suppressor of ras 1) gene are known in XM_011525437.1 and XP_011523739.1, respectively; The nucleotide sequence and protein sequence of the LGALS9 (Galectin-9) gene are known in NM_002308 and NP_002299, respectively; The nucleotide sequence and protein sequence of the KIF5B (Kinesin-1 heavy chain 5B) gene are known in NM_004521 and NP_004512, respectively; The nucleotide sequence and protein sequence of the RET (receptor tyrosine kinase) gene are known from NM_000323 and NP_065681, respectively; The nucleotide sequence and protein sequence of the CLN6 (Ceroid-lipofuscinosis neuronal protein 6) gene are known in NM_017882 and NP_060352, respectively; The nucleotide sequence and protein sequence of the CALML4 (Calmodulin-like 4) gene are known in NR_104583.1 and NP_001273624.1, respectively.

The term "fusion protein" of the present invention means a protein that is expressed from the fusion gene and specifically detected in lung cancer tissue and can be used for prediction of prognosis of lung cancer.

For purposes of the present invention, the fusion protein may comprise a peptide sequence encoded from the nucleic acid sequence of the fusion gene, which may be detected by the antibody and used to predict the prognosis of lung cancer, A polypeptide expressed from the fusion gene CD74-ROS1 (SEQ ID NO: 1); A polypeptide expressed from the fusion gene EML4-ALK (SEQ ID NO: 2); A polypeptide expressed from the fusion gene KSR1-LGALS9 (SEQ ID NO: 3); A polypeptide expressed from the fusion gene KIF5B-RET (SEQ ID NO: 4); A polypeptide expressed from the fusion gene CLN6-CALML4 (SEQ ID NO: 5), and the like.

The fusion gene or the fusion protein can be used as a marker for predicting the prognosis of lung cancer because lung cancer is continuously expressed and specifically expressed in cancer tissues in which the disease is worsening. That is, when the fusion gene or the fusion protein is detected, it can be predicted that lung cancer has a poor prognosis, and when the fusion gene or the fusion protein is not detected, its prognosis is good.

The term " prognostic prediction "of the present invention is used in the same sense as" prognosis ", which means an act of predicting the progress and result of a disease in advance. More specifically, the prognosis prediction means that the progress of the disease after the treatment depends on the physiological or environmental condition of the patient, and it means all actions that predict the course of the disease after treatment considering the condition of the patient in a comprehensive manner .

For the purpose of the present invention, the prognosis prediction can be interpreted as predicting the disease-free survival rate or the survival rate of patients with lung cancer by anticipating the progress of the disease and the cure after the treatment of lung cancer. For example, predicting a "good prognosis" indicates a high level of disease-free survival or survival rate after treatment for lung cancer, suggesting that patients with lung cancer are more likely to be treated, and predictions of "poor prognosis" The disease-free survival rate or survival rate of patients after treatment is low, meaning that cancer is likely to recur from lung cancer patients or die from lung cancer.

The term " disease free survival "of the present invention means the possibility that a patient can survive without recurrence of cancer after treatment of lung cancer.

The term "survival rate" of the present invention means the possibility that, after treatment of lung cancer, the patient can survive regardless of recurrence of cancer.

On the other hand, lung cancer which can predict a prognosis by the fusion gene is not particularly limited, but examples thereof include small cell lung cancer; Or non-small cell lung cancer such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

The term "agent for measuring the mRNA level of a gene " of the present invention means an agent used in a method for measuring the level of mRNA transcribed from the fusion gene in order to confirm the expression of the fusion gene contained in the sample For example, RT-PCR, quantitative real-time PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase A primer base pair capable of specifically binding to a target gene used in a method such as RPA (RNase protection assay), Northern blotting, and DNA chip analysis, and a probe.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying Refers to a short nucleic acid sequence that serves to initiate DNA synthesis in the presence of a reagent for polymerization (i. E., DNA polymerase or reverse transcriptase) and a different four nucleoside triphosphate at a suitable buffer solution and temperature .

For the purpose of the present invention, a PCR amplification can be performed on the fusion gene using the composition containing the primer, and the prognosis of lung cancer can be predicted by detecting the fusion gene. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. For example, the present inventors detected the expression of a fusion gene consisting of the nucleotide sequences of SEQ ID NOS: 1 to 5 by a PCR method using a primer composed of polynucleotides of SEQ ID NOS: 11 to 20, The prognosis was predictable.

When a primer base pair composed of the polynucleotides of SEQ ID NOS: 11 to 20 is used as a component of a composition for predicting the prognosis of lung cancer, each primer base pair for detecting each fusion gene is used alone Or a combination of these primer base pairs. When a composition containing each primer base pair alone is used, the cost and time required for predicting the prognosis of lung cancer can be saved, and each primer base pair , The success rate of prediction of prognosis of lung cancer can be improved.

For example, primers capable of amplifying CD74-ROS1, one of the fusion genes for prediction of prognosis in lung cancer, can be primer base pairs consisting of SEQ ID NOS: 11 and 12; Primers capable of amplifying EML4-ALK may be primer base pairs consisting of SEQ ID NOS: 13 and 14; Primers capable of amplifying KSR1-LGALS9 can be primer base pairs consisting of SEQ ID NOS: 15 and 16; Primers capable of amplifying KIF5B-RET can be primer base pairs consisting of SEQ ID NOS: 17 and 18; The primers capable of amplifying CLN6-CALML4 can be primer base pairs consisting of SEQ ID NOS: 19 and 20.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few bases or a few hundred bases, which can be specifically bound to mRNA. The probe may be labeled with a labeling substance to detect a specific mRNA. Examples of the probe include an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. As shown in FIG.

For the purpose of the present invention, the probe can predict the prognosis of lung cancer by confirming whether or not the probe is complementarily hybridized with the mRNA of the fusion gene. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The term "labeling substance" of the present invention refers to a substance that assists in visually confirming the presence or absence of the probe. It generally includes enzymes, minerals, ligands, emitters, microparticles, redox molecules, Isotopes, and the like, but is not particularly limited thereto. For example, enzymes that can be used include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinesterase, Glucosoxidase, hexokinase, GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, beta -lactamase, and the like, and examples of the usable minerals include fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, And examples of ligands that can be used include biotin derivatives and the like, and available luminescent materials include acridinium ester, luciferin, luciferase, etc., and available microparticles Colloidal gold, colored latex, etc. The available redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like number, and a radioactive isotope available will include ^{3 H, 14 C, 32 P} , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re But is not limited thereto.

The primers or probes 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 many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The term "measurement of protein expression level" of the present invention means a process of confirming the presence and expression level of a protein expressed in the fusion gene in a biological sample obtained from the individual to predict the prognosis of lung cancer in a desired individual , But it can be usually performed by confirming the amount of the protein using an antibody or an aptamer that specifically binds to the protein of the gene. For this purpose, immunoprecipitation methods such as Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, FACS and protein chip assays can be used. However, the present invention is not limited thereto.

The term "antibody" of the present invention refers to a specific protein molecule that is indicated in the art as an antigenic site. For the purpose of the present invention, the antibody refers to an antibody that specifically binds to a protein expressed from the fusion gene of the present invention. The antibody may be obtained by cloning each gene into an expression vector according to a conventional method, A protein encoded by the gene can be obtained and can be produced from the obtained protein by a conventional method. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention and include all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used in the detection of proteins expressed from the fusion gene of the present invention 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 ') 2, F (ab') 2 and Fv.

The term "aptamer " of the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and is a single stranded nucleic acid (DNA, RNA, or modified nucleic acid) , And the presence of the target substance in the sample can be specifically confirmed through the binding. The preparation of the aptamer can be carried out by synthesizing an oligonucleotide having a selective and high binding ability to a target protein to be identified according to a general method of preparing an aptamer and then synthesizing an oligonucleotide having a 5'end or 3'end of the oligonucleotide -SH, -COOH, -OH or -NH2 so as to be able to bind to the functional group of the linker.

In the present invention, the aptamer may be an aptamer capable of specifically binding to a fusion protein expressed from a fusion gene for lung cancer prognosis prediction provided by the present invention, and may be an aptamer specifically binding to the fusion protein It can be a DNA aptamer that can do that.

In another aspect, the present invention provides a lung cancer prognostic prediction kit comprising the composition for predicting the prognosis of lung cancer.

The kit of the present invention can predict the prognosis of lung cancer by confirming the detection of the mRNA of the lung cancer-specific fusion gene or the fusion protein expressed therefrom. The kit for predicting the prognosis of lung cancer according to the present invention may contain not only an antibody capable of specifically detecting a primer, a probe or a fusion protein for detecting the expression of a lung cancer-specific fusion gene, but also one or more other components Compositions, solutions, or devices.

As a specific example, the kit of the present invention may be a kit containing necessary elements necessary for carrying out RT-PCR. In addition to the individual primer pairs specific for the fusion gene used as a marker, the RT-PCR kit also includes a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs) , Enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may be a prognosis prediction kit containing essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate on which a cDNA corresponding to the fusion gene or fragment thereof is attached as a probe, and a reagent, a preparation, an enzyme, and the like for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may comprise a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The coloring enzyme may be peroxidase, Alkaline Phosphatase may be used as the fluorescent substance, FITC, RITC or the like may be used as the fluorescent substance, and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

In another aspect, the present invention provides a method for predicting the prognosis of lung cancer, comprising the step of treating a sample of a subject to be predicted of the prognosis of lung cancer by detecting the mRNA of the fusion gene for prediction of lung cancer prognosis or a fusion protein expressed from the fusion gene And a method for providing information for predicting the prognosis of lung cancer.

The term "individual" of the present invention means an object to which the prognosis of lung cancer is to be predicted, and generally refers to an individual who has undergone lung cancer treatment after the onset of lung cancer. At this time, the above-mentioned individual may include, without limitation, humans and any animals that can cause lung cancer such as dogs, horses, cows, rats, goats, rabbits, chickens, ducks and geese.

The term "sample" of the present invention includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine which can be derived from the individual and capable of detecting a lung cancer- specific fusion gene or fusion protein But is not limited thereto.

Through the above method, it is possible to provide information for predicting whether the prognosis of the patient is good or bad by confirming whether or not the fusion gene or the fusion protein is detected in the patient's sample to predict the prognosis of lung cancer. That is, when the mRNA or the fusion protein of the fusion gene is detected in the sample, it can be determined that the prognosis of the individual is bad. On the other hand, when the mRNA or fusion protein of the fusion gene is not detected in the sample, It can be determined that the prognosis of the individual is good.

In order to detect the fusion gene at the mRNA level, RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection method, Northern blotting, and DNA chip technology may be used, but the present invention is not limited thereto.

In addition, in order to detect the fusion protein, a western blotting, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a radial immunodiffusion, an Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunohistochemical staining, immunohistochemical staining, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, A fluorescence activated cell sorter analysis, a protein chip technology, and the like can be used, but the present invention is not particularly limited thereto.

The composition for predicting the prognosis of lung cancer of the present invention can be used for predicting the prognosis after the treatment of lung cancer by detecting a fusion gene that is specifically expressed only in the tissue of the cancerous region in the lung tissue of the patient having a certain level of lung cancer. It can be widely used to effectively treat lung cancer.

FIG. 1 is a graph showing the results of comparing the levels of fusion genes expressed in normal tissues and cancer tissues of lung cancer patients, wherein N represents the number of fusion genes expressed in normal tissue samples, and T represents the number of fusion genes Indicates the number of fusion genes expressed in a tissue sample.

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

Example 1: Identification of fusion genes specific for lung cancer

Each lung tissue was obtained from 71 lung cancer patients obtained from NCBI Gene Expression Omnibus (GEO) and 10 lung cancer patients collected through clinical pathways, and lung tissues from the lung tissues obtained from the obtained lung tissues, Of lung tissue, respectively. Sequence data of each of these RNAs is obtained from each of the separated lung tissues, and the obtained RNA-sequence data is applied to a Fusion Map tool to determine whether or not each of the lung tissue of the normal region and the lung tissue of the cancer- The fusion gene was analyzed (Fig. 1).

FIG. 1 is a graph showing the results of comparing the levels of fusion genes expressed in normal tissues and cancer tissues of lung cancer patients, wherein N represents the number of fusion genes expressed in normal tissue samples, and T represents the number of fusion genes Indicates the number of fusion genes expressed in a tissue sample. As shown in FIG. 1, 251 fusion genes were expressed in normal tissues whereas 505 fusion genes were expressed in cancer tissues. Thus, various types of fusion genes were expressed in cancer tissues.

Of the 505 fusion genes expressed in the cancer tissues, the fusion genes that were not significantly expressed in the normal tissues and significantly expressed only in the cancer tissues were firstly screened. The primary selection of the fusion gene was screened for 63 different fusion genes from two or more lung cancer patients. Five fusion genes including the genes involved in the expression of the tyrosine kinase and the signal transduction pathways directly involved in the metastasis of cancer were finally selected from among the 63 selected fusion genes. At this time, it was confirmed that the final five selected fusion genes were CD74-ROS1, EML4-ALK, KSR1-LGALS9, KIF5B-RET and CLN6-CALML4.

Example 2: Verification of fusion genes specific for lung cancer

(CD74-ROS1, EML4-ALK, KSR1-LGALS9, KIF5B-RET and CLN6-CALML4) finally selected in Example 1 can be used to predict the prognosis of patients with lung cancer Respectively.

First, primers capable of amplifying the above five fusion genes were designed and synthesized.

CD74-ROS1 F: 5'-CCCGGAGAACCTGAGACACCTT-3 '(SEQ ID NO: 6)

CD74-ROS1 R: 5'-TGCCAGACAAAGGTCAGTGGGA-3 '(SEQ ID NO: 7)

EML4-ALK F: 5'-GGCAGTGTTTAGCATTCTTGGGG-3 '(SEQ ID NO: 8)

EML4-ALK R: 5'-GGTCACTGATGGAGGAGGTCTTG-3 '(SEQ ID NO: 9)

KSR1-LGALS9 F: 5'-GCAGGAGAGACCCAGCTTCAGC-3 '(SEQ ID NO: 10)

KSR1-LGALS9 R: 5'-GAGCTGAGAACGGTCCCATTG-3 '(SEQ ID NO: 11)

KIF5B-RET F: 5'-CGGCAACTTTAGCGAGTATAGATG-3 '(SEQ ID NO: 12)

KIF5B-RET R: 5'-TCCTTGACCACTTTTCCAAATTC-3 '(SEQ ID NO: 13)

CLN6-CALML4 F: 5'-CGGGGACATCGCCCACTACT-3 '(SEQ ID NO: 14)

CLN6-CALML4 R: 5'-CTCATTAAGGACGCCATCTGCA-3 '(SEQ ID NO: 15)

Next, total RNA was obtained from cancer tissues of 104 lung cancer patients who had undergone therapeutic treatment after lung cancer but were not yet treated, and each cDNA was synthesized from the obtained total RNA. At this time, clinical information on 104 patients who provided tissue samples is as shown in Table 1.

Clinical information of patients with lung cancer Check item division Measured value gender male
female
Unknown 74
19
11 Progression of lung cancer Ia
IB
IIa
IIb
IIIa
IIIb
IV
Unknown 16
27
18
7
8
One
2
25 Smoking Over smoking
Smoking
Non-smoking
Unknown 21
41
21
21 case history SQ (squamous carcinoma)
AD (adenocarcinoma)
Large
Unknown 45
54
3
2 age 65 years or older
Under 65
The 47
46
11

Using the synthesized cDNA as a template, qPCR using each of the synthesized primers was performed to detect each fusion gene, and the detection rate of each fusion gene was calculated (Table 2).

Detection of fusion gene in lung cancer tissue Fusion gene Detection rate (%) CD74-ROS1
EML4-ALK
KSR1-LGALS9
KIF5B-RET
CLN6-CALML4 2
9
35
10
58

As shown in Table 2, it was confirmed that the respective fusion genes were detected in lung cancer tissues at a detection rate of 2 to 58%.

Since the fusion gene is specifically detected in lung cancer tissue of a patient who has not been treated even after receiving treatment for lung cancer, the fusion gene is detected in the lung cancer tissue samples of the 104 patients, This prediction could be confirmed from the progress of the pathology of each of the above patients.

Therefore, it was found that the fusion gene can be used as a marker for predicting the prognosis of lung cancer. In addition, since the fusion gene is detected at different levels depending on the patient, these fusion genes can be used as markers for predicting the prognosis of lung cancer, either alone or in combination, It can be used as a marker for prediction.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> Novel composition for prognosing lung cancer using fusion-gene <130> KPA151042-KR <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 268 <212> DNA <213> Artificial Sequence <220> <223> CD74-ROS1 <400> 1 gcc caccttaaga acaccatgga gaccatagac tggaaggtct ttgagagctg gatgcaccat 120 tggctcctgt ttgaaatgag caggcactcc ttggagcaaa agcccactga cgctccaccg 180 aaagatgatt tttggatacc agaaacaagt ttcatactta ctattatagt tggaatattt 240 ctggttgtta caatcccact gacctttg 268 <210> 2 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> EML4-ALK <400> 2 ggatgttct 60 tactggagac tcaggtggag tcatgcttat atggagcaaa actactgtag agcccacacc 120 tgggaaagga cctaaagtgt accgccggaa gcaccaggag ctgcaagcca tgcagatgga 180 gctgcagagc cctgagtaca agctgagcaa gctccgcacc tcgaccatca tgaccgacta 240 caaccccaac tactgctttg ctggcaagac ctcctccatc agtgacctga aggaggtgcc 300 gcggaaaaac atcaccctca ttcg 324 <210> 3 <211> 226 <212> DNA <213> Artificial Sequence <220> <223> KSR1-LGALS9 <400> 3 gagatcctgt cggcctgctg ggctttcgac ctgcaggaga gacccagctt cagcctgctg 60 atggacatgc tggagaaact tcccaagctg gctgtcccct tttctgggac tattcaagga 120 ggtctccagg acggacttca gatcactgtc aatgggaccg ttctcagctc cagtggaacc 180 agaaccggcg gctctcccac cctggacact tctggaagtc agctga 226 <210> 4 <211> 292 <212> DNA <213> Artificial Sequence <220> <223> KIF5B-RET <400> 4 gcaactttag cgagtataga tgctgagctt cagaaactta aggaaatgac caaccaccag 60 aaaaaacgag cagctgagat gatggcatct ttactaaaag accttgcaga aataggaatt 120 gctgtgggaa ataatgatgt aaaggaggat ccaaagtggg aattccctcg gaagaacttg 180 gttcttggaa aaactctagg agaaggcgaa tttggaaaag tggtcaaggc aacggccttc 240 catctgaaag gcagagcagg gtacaccacg gtggccgtga agatgctgaa ag 292 <210> 5 <211> 304 <212> DNA <213> Artificial Sequence <220> <223> CLN6-CALML4 <400> 5 gcatggctct gtgagcgctg atgaggctgc ccgcacggct cccttccacc tcgacctctg 60 gtctctactc acactgcaga actgggttct ggactttggg cgtcccattg ccatgacgga 120 aatggagagc tggatttctc cacttttctg accattatgc acatgcaaat aaaacaagaa 180 gacccaaaga aagaaattct tctagccatg ttgatggtgg acaaggagaa gaaaggttac 240 gtcatggcgt ccgacctgcg gtcaaaactc acgagtctgg gggagaagct cacccacaag 300 gaag 304 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccggagaac ctgagacacc tt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgccagacaa aggtcagtgg ga 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggcagtgttt agcattcttg ggg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggtcactgat ggaggaggtc ttg 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcaggagaga cccagcttca gc 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gagctgagaa cggtcccatt g 21 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cggcaacttt agcgagtata gatg 24 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tccttgacca cttttccaaa ttc 23 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cggggacatc gcccactact 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctcattaagg acgccatctg ca 22

Claims

CD4 (Cluster of Differentiation 74) -ROSl (Proto-oncogene tyrosine-protein kinase ROS), EML4 (Echinoderm microtubule-associated protein-like 4) -ALK (Anaplastic lymphoma kinase), KSR1 (kinase suppressor of ras 1) -LGALS9 A fusion for the prediction of prognosis of lung cancer selected from the group consisting of Galectin-9), KIF5B (Kinesin-1 heavy chain 5B) -RET (receptor tyrosine kinase) and CLN6 (Ceroid-lipofuscinosis neuronal protein 6) -CALML4 (Calmodulin- A composition for predicting the prognosis of lung cancer, comprising at least one agent for detecting the mRNA of the gene or the fusion protein expressed from the fusion gene.

The method according to claim 1,
Wherein the fusion gene comprises a base sequence selected from the group consisting of SEQ ID NOS: 1 to 5.

The method according to claim 1,
Wherein the agent is a primer pair or probe that specifically binds to the fusion gene.

The method of claim 3,
Wherein the primer base pair consists of a polynucleotide selected from the group consisting of SEQ ID NOS: 6 to 15.

The method according to claim 1,
Wherein the agent is an antibody or an aptamer that specifically binds to the fusion protein.

6. A lung cancer prognostic prediction kit comprising the composition for predicting the prognosis of lung cancer according to any one of claims 1 to 5.

The method according to claim 6,
Wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit.

A composition for predicting the prognosis of lung cancer as set forth in any one of claims 1 to 5 is applied to a sample isolated from a subject to be predicted for prognosis of lung cancer to determine the mRNA of the fusion gene for prediction of lung cancer prognosis or the fusion protein expressed from the fusion gene The method comprising the steps of: detecting a prognosis of lung cancer.

9. The method of claim 8,
Wherein said subject is an individual who has undergone a lung cancer treatment procedure after the onset of lung cancer.

9. The method of claim 8,
Wherein said sample is a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine derived from an individual.

9. The method of claim 8,
The fusion gene can be detected by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay (RNase protection method, Northern blotting or DNA chip technology.

9. The method of claim 8,
The detection of the fusion protein can be carried out by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS assays, immunohistochemical staining, immunohistochemical staining, immunoprecipitation assays, (fluorescence activated cell sorter analysis) or protein chip technology.

9. The method of claim 8,
Wherein when the mRNA of the fusion gene for prediction of lung cancer prognosis or the fusion protein expressed from the fusion gene is detected from the sample, the prognosis of lung cancer is judged to be bad.