KR101346955B1 - Composition for predicting the recurrence possibility and survival prognosis of brain tumor and kit comprising the same - Google Patents

Abstract

The present invention relates to a method of using chromosome 17p related genes, MYC / MYCN genes and WNT related genes as marker genes for predicting the risk of recurrence and survival prognosis of brain tumors. Specifically, the present invention provides a method for predicting the risk of recurrence and survival prognosis of brain tumor by complex detection of loss of chromosome 17p-related genes, increased expression of MYC / MYCN genes, and increased expression of WNT-related genes. A composition and kit for predicting recurrence risk and survival prognosis of brain tumors. Therefore, the present invention not only predicts and prevents recurrence of brain tumors including medulloblastoma in advance, but also usefully increases the survival rate of patients with recurring brain tumors by predicting the survival prognosis of patients treated for brain tumors. Can be used.

Description

Composition for predicting the recurrence possibility and survival prognosis of brain tumor and kit comprising the same}

The present invention is a brain tumor specific marker gene, a composition for predicting the recurrence and survival prognosis of brain tumors, including the chromosome 17p-related genes, MYC / MYCN gene, and the agent to measure the expression level of the genes related to WNT, the composition Kit for predicting the recurrence and survival prognosis of a brain tumor, including the method of measuring the expression level of the marker gene to provide information necessary for predicting the recurrence and survival prognosis of the brain tumor, and testing the protein encoded by the marker gene The present invention relates to a method of screening a test substance that treats a substance to promote or inhibit the activity of a protein as a recurrence inhibitor of brain tumor.

Brain tumors are the fourth most common tumors in the whole body, and are the second most common tumors in children. The incidence of brain tumors is about 10 per 100,000 population, and the most common glioma among primary brain tumors accounts for about 35% of the total and is malignant. Mutations that have been known so far as one of the causes of brain tumors include radiation, chemicals, viruses, and the like, and the incidence of brain tumors is known to increase even when immune function is reduced, such as AIDS. Recently, due to environmental pollution, there is a growing interest in the association between chemicals and cancer, but there is no clear causal relationship between the occurrence of brain tumors.

The mechanisms by which a brain tumor causes clinical symptoms include symptoms caused by an increase in brain pressure, symptoms caused by the brain tumor compressing the surrounding brain, symptoms caused by the brain tumor electrically stimulating the surrounding brain nerve (epilepsy seizures), and symptoms caused by hormonal abnormalities. Included. Without active treatment, both malignant and benign brain tumors have fatal consequences.

The incidence of medulloblastoma, known as one of these brain tumors, is about 5 to 9 per 1 million people per year. Medulloblastoma accounts for about 4% of all intracranial tumors and 20% of pediatric brain tumors. The onset age ranges from 3 to 8 years and 80% of patients are under 16 years old. The gender ratio is 2: 1, which is more common in men. In the case of medulloblastoma, the fourth ventricle is closed and hydrocephalus is often accompanied by an excess of cerebrospinal fluid in the ventricles. Accordingly, symptomatic intracranial hypertension with increased cerebral pressure is the most common initial symptom. Characteristic headache, vomiting is common, papillary edema, which causes the swelling of the optic nerve to the retina due to the increase in the pressure of the brain, can be accompanied, and abduction nerve paralysis that can cause eye disorders.

Clinical treatments for brain tumors including medulloblastoma include surgical surgery and chemotherapy. Surgical surgery is difficult to extract because the boundary between tumor and normal brain tissue is unclear. Radiation therapy is performed on the nerve axis after surgery, and chemotherapy may be applied after the radiation treatment. In particular, when the tumor recurs, radiation therapy, intrathecal methotrexate injection, or chemotherapy is applied. At present, due to the remarkable development of combination therapy including surgery and radiation therapy, chemotherapy increased the 5-year survival rate by 70%, but has a better prognosis when histologically showing desmoplastic features. have. On the other hand, in serious risk groups and infants under 3 years of age, they still have low survival rates, and side effects and sequelae from combination therapy are still a challenge.

In order to determine the prognosis of existing patients with brain tumors, it is common to synthesize the course of clinical treatment, radiotherapy and chemotherapy, and to determine the survival prognosis including the possibility of recurrence by considering the malignancy, location, and age of brain tumors. This method, however, has various differences according to the types of patients, and the result of clinical treatment is also different, and thus it is not an accurate prognostic method.

Under these circumstances, the present inventors have made diligent efforts to develop a more accurate method of determining 5-year survival prognosis of a medulloblastoma patient, a type of brain tumor. As a result, the expression and expression of marker genes with increased or decreased expression in medulloblastomas specifically. In the case of confirming the amount, it was confirmed that not only a more accurate prediction of the possibility of recurrence and 5-year survival prognosis of the medulloblastoma patients, but also the prediction of the actual treatment result was completed.

It is an object of the present invention to provide a composition for predicting recurrence and survival prognosis of brain tumors, including an agent for measuring expression levels of chromosome 17p-related genes, MYC / MYCN gene, and WNT-related genes as brain tumor specific marker genes. .

Another object of the present invention to provide a kit for predicting the recurrence and survival prognosis of brain tumors comprising the composition.

Still another object of the present invention is to provide a method of measuring the expression level of the marker gene and providing information necessary for predicting recurrence and survival prognosis of brain tumor.

Another object of the present invention is to treat a test substance to a protein encoded by each gene selected from the group consisting of chromosome 17p related genes, MYC / MYCN gene, and WNT related genes to promote or inhibit the activity of the protein. It relates to a method for screening a test substance as a relapse inhibitor of brain tumor.

As one embodiment for achieving the above object, the present invention is MYC having five or more genes selected from the group consisting of chromosome 17p related genes having the nucleotide sequence of SEQ ID NO: 1 to 92, the base sequence of SEQ ID NO: 93 and 94 And mRNA expression levels of at least two genes selected from the group consisting of one or more genes of MYCN genes and WNT related genes having the nucleotide sequences of SEQ ID NOs: 95-106, or expression levels of proteins encoded by the genes. It relates to a composition for predicting the recurrence and survival prognosis of brain tumors, including the agent to be measured by.

As marker genes for predicting recurrence and survival prognosis of brain tumors in the composition according to the present invention, chromosome 17p-related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92 are ZNF594 (NM_032530), ELAC2 (NM_018127), PRPF8 (NM_006445), INPP5K (NM_130766), MED31 (NM_016060), C17orf81 (NM_203413), RNF167 (NM_015528), MIS12 (NM_024039), SMG6 (NM_017575), C17orf85 (NM_018553), PSMB6 (NM_002798), NF232D, NT232 MAP2K4 (NM_003010), TMEM107 (NM_032354), SENP3 (NM_015670), RNASEK (BC040148), SRR (NM_021947), DERL2 (NM_016041), EFNB3 (NM_001406), KIAA0753 (NM_014804), PELP1 (N_1_02) 143,143 TSR1 (NM_018128), COX10 (NM_001303), PITPNA (NM_006224), CRK (NM_016823), MYH10 (NM_005964), C1QBP (NM_001212), TMEM93 (NM_001014764), MPDU1 (NM_004870), CYB5446171 (NM_00444) C17orf61 (BC030270), NDEL1 (NM_001025579), ZNF18 (NM_144680), TXNDC17 (NM_032731), DVL2 (NM_004422), SCO1 (NM_004589), ANKFY1 (NM_016376), SLC25A11 (NMG3003NM_003562M) PK (NM_013276), PAFAH1B1 (NM_000430), DPH1 (NM_001383), CNTROB (NM_001037144), NUP88 (NM_002532), ZZEF1 (NM_015113), UBE2G1 (NM_003342), TIMM22 (NM_0133N) ZM153 FXR2 (NM_004860), VAMP2 (NM_014232), PHF23 (NM_024297), GPS2 (NM_004489), RABEP1 (NM_004703), TRAPPC1 (NM_021210), GLOD4 (NM_016080), ACADVL (NM_000018), TTC19 (N161) MINK1 (NM_153827), LOC100128288 (XM_001724933), KRBA2 (NM_213597), POLR2A (NM_000937), FAM57A (NM_024792), PFAS (NM_012393), RPAIN (NM_001033002), SNORA67 (NR_002912A, NB_002912A) MED11 (NM_001001683), NCOR1 (NM_006311), ZBTB4 (NM_020899), ARRB2 (NM_004313), SPAG7 (NM_004890), CAMKK1 (NM_032294), RPA1 (NM_002945), C17orf68 (NM_0250NM_018), UBB KCNAB3 (NM_004732), CTNS (NM_004937), SNORD49B (NR_003043), RNMTL1 (NM_018146), OR1D5 (NM_014566), TMEM88 (NM_203411), GEMIN4 (NM_015721), GABARAP (NM_007278M), ABRN_02162, 02BN162 . All of the chromosome 17p related genes according to the present invention are located on chromosome 17p11-13 and are differentially expressed genes (DEGs) expressed in a brain tumor patient group. Loss of expression of these genes acts as a poor prognostic marker in predicting the recurrence and survival of brain tumors.

Further, as marker genes for predicting recurrence and survival prognosis of brain tumors in the composition according to the present invention, MYC and MYCN genes having the nucleotide sequences of SEQ ID NOs: 93 and 94 have GenBank Accession Numbers NM_002467 and NM_005378, respectively. These genes are differentially expressed genes (DEGs) in a group of brain tumor patients, and their increased expression acts as a poor prognostic marker in predicting recurrence and survival of brain tumors.

In addition, as a marker gene for predicting recurrence and survival prognosis of brain tumors in the composition according to the present invention, WNT related genes having the nucleotide sequences of SEQ ID NOs: 95 to 106 are AXIN2 (NM_004655), DKK1 (NM_012242), and DKK2 (NM_014421). , DKK4 (NM_014420), FZD10 (NM_007197), FZD6 (NM_003506), LEF1 (NM_016269), PLCB1 (NM_182734), SMAD3 (NM_005902), TP53 (NM_000546), WIF1 (NM_007191) and W57168 (N). The genes are differentially expressed genes (DEGs) in the brain tumor patient group, and the increased expression group has a good prognosis and the normal expression group serves as a poor prognostic marker in predicting the recurrence and survival of the brain tumor.

Accordingly, the present invention provides chromosome 17p-related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92, MYC and MYCN genes having the nucleotide sequences of SEQ ID NOs: 93 and 94, and WNT related genes having the nucleotide sequences of SEQ ID NOs: 95 to 106 These genes were identified as marker genes for predicting recurrence and survival prognosis of brain tumors, and their expression levels were measured at the mRNA or protein level to confirm that the recurrence and survival prognosis of brain tumors can be predicted more accurately.

In a preferred embodiment, the present invention measures the expression level of two or more genes selected from WNT related genes having a nucleotide sequence of SEQ ID NOS: 95-106 in a subject to predict the recurrence and survival prognosis of brain tumors. After selecting the subjects with reduced expression and measuring the expression level of five or more genes selected from chromosome 17p related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92 in the selected subjects, the chromosome 17p was confirmed. Expression levels of one or more of MYC and MYCN genes having the nucleotide sequences of SEQ ID NOs: 93 and 94 in the subjects whose loss of related genes were confirmed can be determined to have a poor prognosis for brain tumors. have. The present invention more accurately predicts the recurrence and survival prognosis of brain tumors by combining three prognostic predictive parameters: increased expression of WNT related genes, loss of chromosome 17p related genes and increased expression of MYC / MYCN genes. There are features of the invention.

Thus, the composition of the present invention may include an agent for measuring the expression level of the mRNA of the genes having a nucleotide sequence of SEQ ID NO: 1 to 92 or proteins encoded by these genes in order to detect the loss of chromosome 17p-related genes. In addition, the composition of the present invention may include an agent for measuring the expression level of mRNA of the genes having the nucleotide sequences of SEQ ID NOs: 93 and 94 or the protein encoded by these genes in order to detect increased expression of MYC / MYCN genes. have. The composition of the present invention may also include an agent for measuring the expression level of genes having the nucleotide sequences of SEQ ID NOS: 95 to 106 to detect a decrease in the expression of mRNA of WNT related genes or proteins encoded by these genes. .

As used herein, the term “brain tumor” refers to primary brain tumors that arise from brain tissue or meninges that envelop the brain, and secondary brain tumors that occur in the skull or surrounding structures or that have metastasized to the brain from other cancers of the body. Primary brain tumors are largely divided into malignant and benign brain tumors. Since the brain is surrounded by a skull, even benign brain tumors, if not treated, will press on the brain and cause an increase in brain pressure, leading to the same fatal consequences as malignant brain tumors. Brain tumors that can be applied to the present invention and predict their recurrence and survival prognosis include medulloblastoma, meningioma, schwannoma, pituitary adenoma, glioma, and the like, preferably medulloblastoma.

In the present invention, the term "medulloblastoma" is a malignant, invasive embryonic brain tumor, which occurs in the cerebellum, mainly in children, tends to differentiate into neural tissues, and refers to a kind of brain tumor that metastasizes well along the cerebrospinal fluid. . Primary brain tumors most commonly seen in children under 18 years of age, usually occur in the cerebellum (velum) to grow into the fourth ventricle. Occurrence in the cerebellum hemisphere is more common in adults. There is a high tendency to metastasize, the most common metastasis is the subarachnoid space of the brain and spine, and the most common metastasis to the bone marrow outside the nervous system.

As used herein, the term "prognosis" refers to the progression and cure of a disease, such as the possibility of recurrence, metastatic spread of neoplastic diseases such as brain tumors, and the possibility of brain tumor-related death or progression, including drug resistance. For the purposes of the present invention, prognosis means survival prognosis of a brain tumor, preferably a prognosis of a patient who has surgically resected a brain tumor, in particular, a medulloblastoma. Although the 5-year survival rate for determining postoperative cure depends on the degree of cancer progression, the present invention is a marker gene for predicting the recurrence and survival prognosis of brain tumors, the increased expression of WNT-related genes, the loss of chromosome 17p-related genes, By detecting the increased expression of MYC and MYCN genes in combination, it is possible to accurately and easily predict the recurrence and survival prognosis of brain tumors, preferably medulloblastomas.

As used herein, the term "prediction" means that the patient responds favorably or unfavorably to a therapy, such as chemotherapy or radiation therapy, such that the patient is treated, for example, by removal of certain therapeutic agents, and / or primary tumors, And / or survival and / or viability after treatment with chemotherapy for a certain period of time without cancer recurrence. Prediction according to the present invention confirms whether a brain tumor, preferably a medulloblastoma patient, responds favorably to a given treatment regimen, including, for example, administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, or the like, It means predicting the long-term survival of the patient after the treatment regimen.

As used herein, the term "prognostic marker gene of brain tumor" refers to a marker gene capable of confirming survival prognosis by predicting whether a brain tumor will relapse within 5 years after surgery or chemotherapy to a patient diagnosed with brain tumor. Marker genes for predicting prognosis of brain tumors according to the present invention include chromosome 17p-related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92, MYC and MYCN genes having the nucleotide sequences of SEQ ID NOs: 93 and 94, and SEQ ID NOs: 95 to 106 WNT related genes having the base sequence of are included. As a marker gene for predicting the prognosis of brain tumors, if the loss of chromosome 17p-related genes, increased expression levels of MYC and MYCN genes, and non-increased expression levels of WNT-related genes are detected from the individual, there is a high probability of recurrence of the brain tumor and survival prognosis. It can be predicted as bad.

As a marker gene for predicting the prognosis of brain tumors according to the present invention, chromosome 17p-related genes having nucleotide sequences of SEQ ID NOs: 1 to 92, MYC and MYCN genes having nucleotide sequences of SEQ ID NOs: 93 and 94, and SEQ ID NOs: 95 to 106 The characteristics of the WNT-related genes having the nucleotide sequence of are summarized in Tables 1 to 22 below.

The present invention reveals a specific relationship between the predictability of recurrence and survival prognosis of brain tumors, especially medulloblastoma, and the change in the expression level of the genes, and the genes may be predictive markers for the survival prognosis of medulloblastoma, a type of brain tumor. It was first identified. Specifically, the present inventors synthesized cDNA by extracting total mRNA from tissue samples of pediatric brain tumor patients diagnosed with medulloblastoma, and labeled the Affymetrix gene chip. The labeled cDNA was hybridized with the Affymatrix Human Gene 1.0 ST Array chip, and a gene whose expression rate was specifically changed in the medulloblastoma was identified. As a result, there were 134 genes with low specific expression in patients with poor prognosis of medulloblastoma, of which 92 were expressed in human autosomal 17.

In order to further confirm the association between the loss of chromosome 17 and the poor prognosis, array CGH was applied to 15 representative samples to compare the chromosome 17 and the genome modification. As a result of the analysis, the low expression rate gene was found to be the gene expressed at 17p13 to 17p12 of the locus of chromosome 17.

In addition, a complex analysis of independent medullary blastoma populations was performed to investigate patients with no chromosome 17 but with poor prognosis. The high expression rates of MYC and MYCN were associated with poor prognosis of medullary blastoma. Therefore, it was confirmed that the state of the 17p and MYC / MYCN is important in determining the prognosis of the medulloblastoma.

As used herein, the term "agent for measuring the expression level of mRNA and protein" refers to chromosome 17p-related genes of SEQ ID NOs: 1 to 92, SEQ ID NOs: 93 and 1, which are marker genes for predicting the prognosis of brain tumors, preferably medulloblastomas. It refers to a molecule that can be used to measure the expression level of MYC and MYCN genes of 94 and WNT related genes of SEQ ID NOS: 95-106 or proteins encoded by these genes, preferably the marker gene It may be an antisense oligonucleotide, primer or probe capable of binding, or an antibody capable of specifically identifying the expression level of the protein encoded by the marker gene.

The agent for measuring the mRNA level of the prognostic marker gene of the brain tumor according to the present invention is preferably an antisense oligonucleotide, primer pair or probe, and specifically amplifies specific regions of these genes based on the nucleotide sequence of the marker gene. Primers or probes can be designed. Since the base sequence of the possibility of recurrence and survival prognostic marker gene of the brain tumor according to the present invention is registered in the GenBank and known in the art, those skilled in the art specifically amplify specific regions of these genes based on the base sequence. Primers or probes can be designed.

The term "antisense" in the present invention refers to a nucleotide sequence and an inter-subunit backbone in which an antisense oligomer is capable of hybridizing with a target sequence in RNA by Watson-Crick base pairing to form a heteroduplex with the mRNA typically in the target sequence ≪ / RTI > Oligomers may have exact sequence complementarity or similarity to the target sequence. This antisense oligomer can alter the processing of mRNA that blocks or inhibits translation of mRNA and produces splice variants of mRNA.

As used herein, the term “primer” refers to template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperatures. Refers to a single-stranded oligonucleotide that can act as a starting point. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. In the present invention, after performing PCR amplification using the forward and reverse primers for the prognostic marker gene of the brain tumor according to the present invention, it is possible to predict the possibility of recurrence of the brain tumor and predict the 5-year survival prognosis through the amplification of the PCR product. PCR conditions, length of the forward and reverse primers can be modified based on what is known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a short number of bases to hundreds of bases, which is capable of specific binding with mRNA. You can check the presence. Probes can be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, after hybridization is carried out using a probe complementary to the nucleotide sequence of the prognostic marker gene of the brain tumor according to the present invention, it is possible to predict the possibility of recurrence of the brain tumor and predict the 5-year survival prognosis through hybridization.

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 methylation, capping, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methylphosphonate, phosphoester, phosphoro Amidate, carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

The expression level of the prognostic marker gene of the brain tumor according to the present invention can be known by confirming the expression level of the mRNA or the protein encoded by the gene. In the present invention, "mRNA expression level measurement" is a process of confirming the presence and expression level of mRNA of the marker gene according to the present invention in a biological sample in order to predict the recurrence and survival prognosis of brain tumor. Can be. Analytical methods for this include RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting ), But not limited to DNA microarray chip.

In the present invention, "protein expression level measurement" is a process of confirming the presence and degree of expression of a protein expressed from a marker gene according to the present invention in a biological sample in order to predict the possibility of recurrence and 5-year survival prognosis of a brain tumor. Check the amount of protein using an antibody that specifically binds to the protein. Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunity Electrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.

Agents for measuring protein levels are preferably antibodies. As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed from a prognostic marker gene of a brain tumor according to the present invention, which antibody is cloned into an expression vector according to a conventional method. The protein encoded therefrom can be obtained and prepared from conventional proteins by conventional methods.

The form of the antibody suitable for the present invention is not particularly limited, and a part thereof is included in the antibody of the present invention as long as it is a polyclonal antibody, a monoclonal antibody or an antigen-binding agent, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used to predict the prognosis of brain tumors according to the 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 antigen binding function and includes Fab, F (ab '), F (ab') ₂ and Fv.

The terms “differentially expressed gene”, “differential gene expression” and their synonyms used interchangeably, as compared to their expression in normal or control subjects, indicate that their expression is disease, specifically cancer, eg For example, genes that are activated at higher or lower levels in a subject suffering from a brain tumor. The term also includes genes whose expression is activated at higher or lower levels in different stages of the same disease. It will also be appreciated that differentially expressed genes can be activated or inhibited at the nucleic acid level or the protein level, or can produce different polypeptide products through alternating splicing. Such differences can be demonstrated, for example, by changes in mRNA levels, surface expression, secretion or other distribution of the polypeptide. Differential gene expression can be used to compare expression between two or more genes or gene products thereof, or to compare ratios of expression between two or more genes or gene products thereof, or even two differently processed genes of the same gene. Comparison of the products may include the differences between normal subjects and diseases, in particular those suffering from cancer, or between various stages of the same disease. Differential expression can be used to identify both quantitative and qualitative differences in the pattern of transient or cell expression in a gene or its expression product, for example, between normal and diseased cells, or between cells undergoing different disease events or disease stages. Include. For the purposes of the present invention, “differential gene expression” is at least about 2 times, preferably at least about 4 times between the expression of a given gene in normal and diseased subjects or at various stages of disease development in a diseased subject. More preferably at least about 6 times and most preferably at least about 10 times.

In another aspect, the present invention relates to a kit for predicting recurrence and survival prognosis of a brain tumor comprising the composition.

Specifically, the kit of the present invention comprises at least five genes selected from the group consisting of chromosome 17p related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92, among MYC and MYCN genes having the nucleotide sequences of SEQ ID NOs: 93 and 94; And at least one gene and an agent for measuring the mRNA expression level of at least two genes selected from the group consisting of WNT related genes having the nucleotide sequences of SEQ ID NOs: 95-106, or the expression level of the protein encoded by the gene. Can be.

The kit of the present invention checks the mRNA expression level or protein expression level of the marker gene expressed in brain tumors, preferably in medulloblastoma, whether or not there is a loss of chromosome 17p-related genes in the test subject, and the expression level of MYC / MYCN gene is increased. It is possible to predict the treatment outcome and survival prognosis of brain tumors by complex detection of increased or not increased expression level of WNT related genes. The kit of the present invention includes one or more other components suitable for analytical methods as well as primers, probes for measuring the expression level of prognostic marker genes of brain tumors, especially medulloblastomas, or antibodies that selectively recognize proteins encoded from the marker genes. Compositions, solutions or devices can be included.

Specifically, the kit for measuring the mRNA expression level of the marker genes in the present invention may be a kit containing the necessary elements for performing RT-PCR. RT-PCR kits include test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Tag-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC, as well as individual primer pairs specific for the marker gene. -May include DEPC-water, sterile water, and the like.

In addition, the kit for measuring the expression level of the protein expressed by the coding of the prognostic marker genes in the present invention is a secondary antibody labeled with a substrate, suitable buffer, chromophore or fluorescent substance for immunological detection of the antibody and Color substrates and the like. In the above description, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like can be used, and chromogenic enzymes include peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used, and the chromogenic substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazolin- Sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

In addition, the kit of the present invention may be in the form of a microarray for predicting the recurrence and survival prognosis of a brain tumor comprising one or more of the prognostic marker genes according to the present invention. The microarray may comprise DNA or RNA polynucleotide probes. The microarray is a conventional microarray except that the probe includes a probe specific for the nucleotide sequence of chromosome 17p-related genes, MYC / MYCN genes, and WNT-related genes as a prognostic marker gene according to the present invention. The microarray of the present invention is useful for predicting recurrence and survival prognosis of brain tumors, especially medulloblastomas, by detecting loss of chromosome 17p-related genes, increased expression levels of MYC / MYCN genes, and non-increased levels of WNT-related genes. Information can be provided.

Methods of preparing microarrays by immobilizing probes on prognostic marker genes of brain tumors according to the invention on a substrate are well known in the art. For example, the DNA microarray may include, but is not limited to, micropipetting using a piezo electric method or a method using a spotter in the form of a pin. Probes for marker genes can be immobilized on a substrate. The substrate of the microarray of the present invention is preferably coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, It is not. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but the present invention is not limited by these examples.

In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection involves labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance, such as a substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating a signal from the labeling substance. The hybridization result can be read by detecting.

In another aspect, the present invention provides a method of providing information necessary to predict the likelihood of recurrence and survival prognosis of a brain tumor.

Specifically, the information providing method according to the present invention,

1) measuring mRNA expression levels of two or more genes selected from the group consisting of WNT related genes having a nucleotide sequence of SEQ ID NOs: 95 to 106 from a sample of an individual, or an expression level of a protein encoded by the gene;

2) 5 or more selected from the group consisting of chromosome 17p-related genes having the nucleotide sequences of SEQ ID NOs: 1 to 92 in the individual sample in which the expression level of the WNT related genes was not increased in comparison with the normal control in step 1). Measuring the mRNA expression level of a gene or the expression level of a protein encoded by the gene;

3) mRNA expression level of at least one of MYC and MYCN genes of SEQ ID NOs: 93 and 94 or the expression level of the protein encoded by the gene in the individual sample in which the loss of chromosome 17p related genes was confirmed in step 2) compared to the normal control group Measuring; And

4) In step 3), the individual sample having increased expression levels of MYC and MYCN genes compared to the normal control sample may include determining that the brain tumor has a high probability of recurrence and a poor survival prognosis.

As used herein, the term "sample of an individual" refers to a sample used to predict clinical outcome after treatment from a subject undergoing surgical or chemical treatment for a brain tumor, i.e., recurrence and survival prognosis, and includes tissue, cells, Samples such as whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like. In a specific embodiment of the present invention, a sample of a brain tumor, in particular, a medulloblastoma patient.

According to the method of the present invention, the expression level of the prognostic marker gene of the brain tumor according to the present invention or the protein encoded therefrom is measured from a sample of an individual who has undergone surgical or chemical treatment for a brain tumor, particularly a medulloblastoma, and this is normal subject. Compare with the expression level measured from the sample. As a result, the expression level of two or more genes among the WNT related genes having the nucleotide sequences of SEQ ID NOs: 95 to 106 among the marker genes of the present invention was not increased, and 5 of the chromosome 17p related genes of SEQ ID NOs: 1 to 92 were increased. The loss of more than one gene is confirmed, and if the expression level of one or more of the MYC / MYCN genes of SEQ ID NOs: 93 and 94 is activated, there is a high possibility of recurrence of the brain tumor of the individual and a poor 5-year survival prognosis. It can be determined.

Analytical methods for measuring mRNA levels include reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real time reverse transcriptase polymerase, RNase protection assay, Northern blot, DNA microarray chip, and the like. The method of analysis is not limited.

Through the analysis method, it is possible to compare the mRNA expression level of the normal control sample and the mRNA expression level of the brain tumor, preferably the medulloblastoma patient sample, and to determine whether the marker gene is lost or significantly increased or decreased in the mRNA expression level. Can predict the probability of relapse and 5 year survival prognosis.

The mRNA expression level can be measured using a reverse transcriptase polymerization method or DNA microarray chip, preferably using primers specific for the marker gene.

In a preferred embodiment of the present invention, by performing a reverse transcriptase polymerization reaction specific to the marker gene and electrophoresis to confirm the band pattern and the thickness of the band to determine the mRNA expression and extent of the prognostic marker gene of the brain tumor and the control and By comparison, the probability of recurrence and 5-year survival prognosis for brain tumors, preferably for medulloblastoma patients, can be predicted.

On the other hand, DNA DNA microarray chip is a DNA chip in which the nucleic acid corresponding to the marker gene or a fragment thereof is attached to a glass-like substrate with high density, and isolates the mRNA from the sample, the end or the inside of which is labeled with a fluorescent material cDNA probes are prepared, hybridized to DNA chips, and then predictable for recurrence and 5-year survival prognosis after treatment of patients with brain tumors, preferably medulloblastoma. Specifically, the analysis method using the DNA microarray chip may be performed including the following steps.

1) separating the mRNA of the marker gene according to the invention from a sample of the individual;

2) synthesizing mRNA isolated from a sample of the subject and mRNA of a normal control group with cDNA and labeling each of them with a different fluorescent substance;

3) hybridizing each cDNA labeled with another fluorescent material with a DNA microarray chip to which a probe for a marker gene is immobilized; And

4) analyzing the hybridized DNA microarray chip and comparing the mRNA expression level of the marker gene with the mRNA expression level of the normal control group.

The fluorescent material is preferably selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), and rhodamine, but is not limited thereto. All fluorescent materials known to the art can be used. In addition, the DNA microarray chip may be used such as 36 k Human V4.0 OpArray oligo microarray (Operon, Germany) or Whole human genome oligo microarray (Agilent, USA), but is not limited thereto, brain tumor according to the present invention As long as the probe for the survival prognostic marker gene can be loaded, it can be used without limitation.

Assays for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS And protein chips, but are not limited thereto. Through the above analysis method, it is possible to compare the amount of antigen-antibody complex formation in the normal control group with the amount of antigen-antibody complex formation in brain tumors, preferably in medulloblastoma patients, and to determine whether the protein expression level is significantly increased or decreased. , The possibility of recurrence after treatment and preferably the 5-year survival prognosis for patients with brain tumors, preferably medulloblastoma.

As used herein, the term “antigen-antibody complex” refers to a combination of a marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be measured quantitatively through the magnitude of a signal of a detection label. .

Protein expression levels can be measured, for example, using an ELISA. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. The degree of complexation of the marker protein with the antibody can be confirmed to predict the likelihood of recurrence and 5-year survival prognosis after treatment of a brain tumor, preferably a medulloblastoma patient.

In addition, Western blot using one or more antibodies to the marker can be used. Isolate the whole protein from the sample, electrophoresis it to separate proteins according to size, transfer to nitrocellulose membrane and react with the antibody, and the amount of the antigen-antibody complex produced therefrom is labeled using a labeled antibody. How to check. Accordingly, the amount of protein produced by the expression of the gene can be confirmed to predict the possibility of recurrence and 5-year survival prognosis after treatment of brain tumors, preferably medulloblastoma patients. The detection method consists of examining the expression level of the marker protein in the control group and the expression level of the marker protein in the brain tumor patients. mRNA or protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above.

Immunohistochemical staining using one or more antibodies to the marker can also be performed. After collecting and immobilizing tissue from a brain tumor, preferably a medulloblastoma patient, paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several μm and attached to a glass slide to prepare a tissue slice slide, whereby antibodies specific for the marker protein according to the present invention are reacted by known methods. Then, the unreacted antibody is washed and removed, and the expression level of the marker protein can be observed under a microscope by reacting with a coloring reagent for observing the immune response.

In addition, a protein chip in which one or more antibodies against the marker are arranged at predetermined positions on a substrate and immobilized at high density can be used. In the method using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the presence or expression level of the protein, and thus to a brain tumor, preferably a medulloblastoma. The likelihood of recurrence and 5-year survival prognosis can be predicted after treatment of the patient.

Furthermore, the present invention is a test substance that promotes or inhibits the activity of a protein by treating a protein encoded by each gene selected from the group consisting of chromosome 17p-related genes, MYC / MYCN gene, and WNT-related genes. The present invention relates to a method for screening as a recurrence inhibitor of brain tumor.

Specifically, in the screening method of the present invention,

1) one or more of the chromosome 17p related genes of SEQ ID NOS: 1 to 92 are lost, the expression level of one or more of the MYC and MYCN genes of SEQ ID NOs: 93 and 94 is activated, and the WNT related genes of SEQ ID NOs: 95 to 106 Contacting the test substance with cells in which the expression level of at least one of the cells is reduced; And

2) measuring the mRNA expression level of the gene or the expression level of the protein encoded from the gene; And

3) screening a test substance that controls the expression level of the test substance-treated cells with the expression level of untreated cells, the loss of chromosome 17p related genes, the increased expression of MYC and MYCN genes, and the expression of WNT related genes. .

As used herein, the term "test agent" includes any substance, molecule, element, compound, entity, or combination thereof. But are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or chemical compound, or a combination of two or more substances. Unless otherwise indicated, the agents, materials and compounds may be used interchangeably.

Test substances that can be screened or identified by the methods of the invention include polypeptides, beta-turnmimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, Oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogs or combinations thereof. The test materials can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Preferably, the test substance may be a peptide, for example, a peptide having about 5 to 30, preferably about 5 to 20, more preferably about 7 to 15 amino acids. The peptide may be a naturally occurring protein, a random peptide or a cleavage of a "biased" random peptide.

The test substance may also be "nucleic acid ". The nucleic acid test material may be a naturally occurring nucleic acid, a random nucleic acid, or a "biased" random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used similar to those described above.

In addition, the test substance may be a small molecule (eg, a molecule having a molecular weight of about 1,000 or less). Preferably, a high throughput assay can be applied to the method for screening small molecules.

Wherein the expression level of the gene of interest is a method as described above at the mRNA and / or protein level, such as mRNA expression level RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blot, DNA microarray chip, protein expression level, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS , Protein chips, and the like.

The predictive technology of recurrence risk and survival prognosis of brain tumor according to the present invention predicts the risk of recurrence of brain tumor in a patient who has been treated by developing the brain tumor and relapses through selection of appropriate treatment and special management for patients with high risk of recurrence of brain tumor. This can improve the survival rate of patients with brain tumors by delaying time and preventing recurrence.

1 shows the division into five subpopulations by unsupervised hierarchical clustering using 915 probe sets according to the greatest variability of expression (IQR> 1). The symbol represents the 5-year survival of the patient. □ (good prognosis); ■ (bad prognosis).
2 shows autonomic estimated hierarchical clustering with statistical backing using expression patterns of 915 probe sets. Red is the value of the approximately unbiased (AU) test, and green is the value of the BP (bootstraping) analysis.
3 shows a heatmap for each particular genetic marker previously reported and the association between clinical characteristics and five clusters defined as autonomically estimated clustering.
4A-4F show complex analyzes of medulloblastoma populations.
FIG. 4A shows autonomic estimated hierarchical expression using expression patterns of 156 transcripts of chromosomes 17p13 and 17p12 obtained from data of Kool and Fattet except for probe sets with low expression rate (less than 5%) or variability (IQR <0.25) Indicate clustering.
FIG. 4B shows autonomic estimated hierarchical clustering using the expression patterns of 138 transcripts of chromosomes 17p13 and 17p12 obtained from Thompson's data, excluding probe sets with low expression rate or variability.
4C to 4E are biplot showing the relative expression rates of MYC and MYCN including normal brain data (4c: Seoul National University Children's Hospital data, 4d: Kool and Fattet data, 4e: Thompson's data).
4F shows biplots between mean expression levels of MYC / MYCN and PC1 of expression patterns of transcripts on chromosomes 17p13 and 17p12.
FIG. 5 shows autonomic estimated hierarchical clustering by statistical backing using expression patterns of 207 transcripts derived from chromosomes 17p13 and 17p12. Red is the value of the approximately unbiased (AU) test, and green is the value of the BP (bootstraping) analysis.
6a to 6f show the analysis of medulloblastoma through Seoul National University Children's Hospital data.
FIG. 6A shows autonomic estimated hierarchical clustering using expression patterns of 207 transcripts of 17p13 and 17p12 except for a probe set with low expression rate (less than 5%) or variability (IQR <0.25).
6B shows the association of defined clusters with clinical characteristics.
Figure 6c is a heat map showing the expression pattern of the chromosome 17 chromosome depending on the position of the chromosome.
6D shows chromosomal modification of 17p.
6E shows PC plots calculated from the expression patterns of 207 17p transcripts.
FIG. 6F is a Kaplan-Meier curve showing the overall survival of a subpopulation as determined by autonomic estimated hierarchical clustering analysis.
7A to 7C are diagrams comparing chromosome modifications and expression patterns on chromosome 17.
8A-8B show chromosomal segregation of informative genes used to predict 5-year survival.
8A shows the partial region under the ROC curve (pAUC, p = 0.2) corresponding to each probe set chromosome region of chromosome 17p, the P value obtained by -log ₁₀ in the form of Student's t-test, and the signal to noise ratio ( signal-to-noise-ratio).
8B shows log ₂ −values of the two patients with the least loss of chromosome 17p.
Figure 9 shows the bi-plot of the relative expression level of chromosome 17p and the average of MYC / MYCN expression by Seoul National University Children's Hospital data.
Figure 10 shows a bi-plot of the relative expression level of chromosome 17p and the average of MYC / MYCN expression by the data by Kool and Fattet data.
FIG. 11 shows a biplot of the relative expression level of chromosome 17p and the mean of MYC / MYCN expression by Thomson's data.
12A-12F show Kaplan-Meier curves and log-rank test for overall survival of subgroups defined by the status of chromosome 17p or the expression level of MYC / MYCN. 12a, 12c and 12e are shown for all ages, and 12b, 12d and 12f are shown for ages 3 and older.
13A-13D show Kaplan-Meier curves and logarithms for the overall survival of seven (13a, 13b) or five (13c, 13d) subpopulations, depending on the expression level of MYC / MYCN and the status of chromosome 17p. Indicates a rank check. 13a and 13c are shown for all ages, and 13b and 13d are shown for ages 3 and older.
14A-14G show Kaplan-Meier curves and log-rank tests for overall survival when divided into seven subgroups for all ages.
15A-15G show Kaplan-Meier curves and log-rank tests for overall survival when divided into seven subgroups for ages 3 years and older.
16A-16D show Kaplan-Meier curves and log-rank tests for overall survival when divided into five subgroups for all ages.
17A-17D show Kaplan-Meier curves and log-rank tests for overall survival when divided into five subgroups for ages 3 years and older.
18A-18C show Kaplan-Meier curves for the effect of age when divided into seven subgroups. FIG. 18A shows the curves of the high MYC / MYCN and chromosome 17p non-deleting population, FIG. 18B the middle or high MYC / MYCN and the chromosome 17p missing population, and FIG. 18C shows the curves of the middle MYC / MYCN and chromosome non-dissipating population.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention in detail, but the scope of the present invention is not limited by these examples.

Example  1: Gene Expression Analysis and Hierarchical Clustering

1-1: Patient Characterization

Between 1998 and 2008, 30 cases of medulloblastoma were collected from patients diagnosed with medulloblastoma and surgically treated at Seoul National University Children's Hospital (SNUCH). The clinical data were obtained and analyzed with the approval of the Institutional Review Board of Seoul National University Children's Hospital. The relationship between the characteristics of the patients used in the experiment and the two or five year survival rate is shown in Table 23 below. The total median follow-up time was 51 months (range of 11-101 months). The two-year survival rate for 30 children was 83.3%, and the five-year survival rate for 54 children was 54.2%, except for six who did not have 5-year survival. There was no significant difference in age and gender between the extended and poor survival groups.

1-2: RNA  Ready and Microarray  process

Total RNA was extracted using a Qiagen RNeasy Mini kit (Hilden, Germany), and then amplified and labeled according to the Affymetrix Genechip Whole Transcript Sense Target Labeling protocol. The labeled cDNA obtained was hybridized to Affymetrix Human Gene 1.0 ST array (Santa Clara, Calif.) And scanned. The background of the scanned raw expression values was corrected and normalized and then summed using Robust Multiarray Averaging (RMA). The Log ₂ conversion data obtained for subsequent analysis was used.

1-3: Analysis of Gene Expression Data

Assays for the detection of differentially expressed genes (DEGs) were performed on 24 patients with 5-year survival. We applied modified t-statistics based on an empirical Bayes approach. Low diversity noninformative probe sets were removed by performing nonspecific filtering because the probe sets lack sensitivity in estimating different expressions. The BH FDR p-value (Benjamini-Hochberg false discovery rate-adjusted p-value) was used to correct multiple test errors. Expression values were normalized between samples with the median of each probe set and the inter-quartile range (IQR), and autonomous hierarchical clustering analysis was performed using Manhattan distance measurement and Word linkage algorithms. Statistical support for clustering was obtained by BP (bootstrapping) analysis and approximately unbiased (AU) test. Fisher's exact test was used to assess the significance of the association.

1-4: Array comparison dielectric matching method array comparative genomic hybridization , Arrangement CGH )

Array CGH was performed using an Agilent 180K array (Santa Clara, Calif.) On 15 medulloblastoma tissue samples. The patient's DNA was labeled with cyanine 3'-dUTP, and the set of samples (Promera, Madison, WI) as a reference sample was labeled with cyanine 5'-dUTP. After hybridization and microarray scanning of labeled DNA, the background was modified and the dye-localized standardized untreated log ₂ -ratios were extracted using Agilent Feature Extraction Software. By applying a recently developed algorithm utilizing 38 calibration profiles obtained from undisclosed clinical genetic studies, performed using peripheral blood samples collected from normal pediatric or nonmalignant patients ( .... van de Wiel MA , Brosens R, Eilers PH, Kumps C, Meijer GA, Menten B, et al Smoothing waves in array CGH tumor profiles Bioinformatics 2009; 25: 1099-104), log 2 - noise from the ratio Further removal. Then, we applied a circular binary segmentation (CBS) algorithm and merged the segmented log ₂ -ratios.

1-5: According to gene expression pattern Medulloblastoma Molecular  Layering

Based on previous reports that gene expression patterns can be successfully used to classify heterogeneous medulloblastoma into several genetically homologous subpopulations, the present invention utilizes a set of 915 probes with the largest expression variability (IQR> 1.0). Unsupervised hierarchical clustering was performed. Thirty medulloblastoma patients were divided into five subpopulations by statistical support exceeding the 95% bootstrap value (FIGS. 1 and 2). Genetic markers for each previously reported subtype identified four subgroups of six (c1-c6): MYC (c1), SHH (c3), photoreceptor (c5) and WNT (c6). However, the neurogenic group (c2) was not distinguished from the mixed group (c4) so that patients were placed in the c2 or c4 population based on the expression patterns of CRX and GRM1 / GRM8, which are known as specific markers of the neurogenic and mixed groups, respectively. (FIG. 3). Despite very clear subclusters, the five clusters identified through autonomously estimated clustering did not show a significant association with 5-year survival or 2-year survival prognosis (FIG. 3).

1-6: Prognosis of 5-year survival outcome Marker

In order to detect reliable characteristics of 5-year survival in medulloblastoma, the present invention was confirmed using expression patterns obtained from 24 patients whose 5-year survival status was confirmed. To correct for multiple test errors, the BH FDR method was applied at 0.2 significance level, resulting in poor expression of 66 probe sets (corresponding 63 genes, 2 non-coding RNAs, and 1 transcript). Significant changes were made in patients with prognosis (Tables 24-28).

All 63 differentially expressed genes were all down regulated in patients with poor survival prognosis and located on chromosome 17p. The results suggest that low expression of the gene located at 17p in medulloblastoma may be a determinant of poor prognosis. Adopting a less stringent significance level (p <0.01), 92 of the 134 down-regulated probe sets resulted in chromosome 17p.

Since most of the differentially expressed genes correspond to transcripts from 17p, the present invention examined the association between the expression changes of these transcripts and the 5-year survival results. Hierarchical clustering of 17p transcripts from 30 medulloblastoma patients showed a clear cluster with 17p loss (FIG. 4A). Clustering of the 17p missing group was supported by 100% statistical AU / BP values (FIG. 5). A high level of significant association was observed between the 5 year prognosis and the two populations (p = 0.00022) (FIG. 6B). Other clinical factors such as histology, two-year prognosis, and overall survival were also significantly associated with the populations identified by the expression pattern of chromosome 17p gene (FIG. 6B).

Focusing on a group of 17p missing patients, array CGH was applied to 15 representative samples selected from three populations to compare gene expression patterns on chromosome 17 and genome modifications. It was found that the expression pattern was very consistent with the abnormalities in the copy number or diploid (FIGS. 7A-7C), which enabled inferences for the abnormalities of chromosome 17 in other samples that did not perform array CGH (FIG. 7A). 6c, d). Six of eight patients in the chromosome 17p loss group showed isochromosome 17q, while the other two patients showed only 17p loss without a 17q increase (MB_09 and MB_24). This suggests that loss of chromosome arm 17p is a major factor for poor prognosis in medulloblastoma.

To investigate whether there is a reliable differential expression of differentially expressed or informative genes for 5-year survival outcomes, we calculated by introducing three values related to the informativeness of each probe set for chromosome 17p. The values were plotted at positions on the chromosomes of. These three numerical parameters are the partial region under the ROC curve (pAUC, p = 0.2), the P value obtained by -log ₁₀ in the form of the Student's t-test, and the signal-to-noise-ratio to be. The results indicated that the informative gene was locally isolated at the 17p terminal region corresponding to the 17p13-17p12 locus (FIGS. 8A and 8B), which was observed in two patients with poor prognosis and minimal loss in the 17p region. Coincident with the missing area. In addition, the PC plot of FIG. 6E shows identifiable segregation of 17p missing populations showing poor survival, and consistently, the Kaplan-Meier curve of FIG. 6F will stratify patients based on hierarchical clustering of 17p transcripts. When shown, there was a significant survival difference.

Example  2: independent Medulloblastoma Cohort  Complex analysis

2-1. Data collection for analysis

As indicated in Cho et al., J Clin Oncol, 2010, three independent cohorts (Kool et al., PLoS One. 2008; 3: e3088; Fattet et al., J Pathol. 2009; 218: 86-94; and Thompson et al., J Clin Oncol. 2006; 24: 1924-31). Microarray data of normal brains were obtained from the NCBI GEO ftp site (registered numbers: GSE17757, GSE11882, GSE2361, and GSE13471). After preprocessing the data using the RMA approach, the datasets from the Cool Cohort and Partet Cohort were pretreated together because they used the same microarray platform. To rule out the possibility of death due to surgical complications, data from two patients who died within one month of surgery (A317, MB117) were excluded. A total of 176 medulloblastoma samples including 30 patients who visited Seoul National University Children's Hospital were used for analysis (Tables 29 to 46).

2-2: 17p, MYC , And MYCN Stratification and Survival Analysis of Patients According to Their Status

In each patient, the relative expression levels of specific genes or genes (mean of MYC, MYCN, MYC and MYCN, major components of 17p) were calculated as follows: For a given dataset, expression values were expressed as mean and standard deviation. The relative positions were determined by standardizing and rescaling the normalized values by calculating minimum and maximum expressions of 0 and 1, respectively. The patients were then divided into seven or five subgroups, depending on the combination of 17p status and the expression level of MYC / MYCN. In the subgrouping of the two classes, the status of 17p was divided into two categories, namely missing or non-deleted, determined by unsupervised clustering of the expression pattern of chromosome 17p. When divided into seven subgroups, the status of MYC / MYCN is high (relative level of mean of MYC, MYCN, or MYC / MYCN> 0.7), low (relative level of mean of MYC, MYCN, and MYC / MYCN ≤ 0.15), and 3 levels of medium (other than high and low patients). When divided into five subgroups, the status of MYC / MYCN is high (relative level of mean of MYC, MYCN, or MYC / MYCN> 0.5), low (relative level of mean of MYC, MYCN, and MYC / MYCN ≤ 0.5) into two levels. On the other hand, the WNT group (group with increased expression of WNT-related genes) was always pre-determined as an independent subgroup regardless of 17p and MYC / MYCN status. Kaplan-Meier analysis and log-rank test were performed to assess the difference in survival among defined subgroups. Depending on age, histology and the presence of seeding, a multivariate Cox proportional risk model was applied to 17p status and relative expression values of MYC and MYCN.

2-3: Independent Medulloblastoma Cohort  And complex analysis thereof

Other medulloblastoma data were used to investigate the association between 5-year survival and 17p expression. However, a significant proportion (approximately 30%) of the 17p missing group showed a good five year survival prognosis, while about 40% of patients in the 17p non missing group had a poor five year survival prognosis (FIGS. 4A and 4B). This indicates that, as with the results using SNUCH data, only a loss of 17p may not be a major factor in determining 5-year survival for poor prognosis.

Therefore, the present inventors focused on three children (MB_01, MB_10, and MB_14) belonging to the group showing no 5-year survival prognosis but no 17p loss in the SNUCH data (FIG. 6A). One child (MB_01) had very high MYC expression (relative expression: 0.94, second highest MYC expression level), and the other child had high MYCN expression (relative expression: 0.72, fourth highest MYCN expression level). (FIG. 3 and Tables 29 to 46). Therefore, the present inventors assume that the expression level of MYC or MYCN is another inducer of poor prognosis in medulloblastoma, and plots the relative expression levels of MYC and MYCN together with the expression levels of normal brain for 5-year survival and The correlation with the expression level of MYC / MYCN was examined (FIGS. 4A-4F).

Most patients with MYC and MYCN expression levels that were identical to those of normal brains had very good 5-year survival results. The biplot of the mean expression of MYC / MYCN and the relative level of expression of 17p Principal Component 1 confirmed that patients with low levels of MYC / MYCN had no prolonged survival and exhibited prolonged survival. And MYC / MYCN status can be seen as an important factor in determining the survival prognosis of medulloblastoma patients (Fig. 4f, 9, 10, 11). In addition, Kaplan-Meier curves and log-rank tests confirmed a significant association between 5-year prognosis and 17p and MYC / MYCN status (FIGS. 12A-12F).

In addition, we defined WNT populations as independent subgroups regardless of 17p or MYC / MYCN status, in both full age and children over 3 years of age when patients were divided into seven or five groups by these two factors. Significant differences in survival were found between the subgroups (FIGS. 13A-17D). When subdivided into five subgroups, the predicted five-year survival probability ranged from 19 to 81%, corresponding to the high MYC (N) / 17p loss group and the low MYC (N) / 17p non-loss group, respectively. Table 47. Survival was the highest in patients with normal range of MYC (N) expression levels and 17p non-deletion (FIGS. 13A-13F, Table 47). Children under 3 years were not found in the WNT group and in the group showing normal range of MYC (N) expression levels (relative expression ≤ 0.15). This may partially explain the previous report showing a worse prognosis.

Multivariate Cox-Regression Survival Analysis also showed that reduced survival was associated with age under 3 years, suggesting that age is one factor that influences survival outcomes even after adjustment of MYC / MYCN and 17p status. (Table 48). When excluding data from children under 3 years of age, the expected survival was particularly increased in the high MYC (N) / 17p non-loss group among the 7 subgroups (Table 47). These results suggest that the effect of age may be altered by the condition of MYC / MYCN and chromosome 17p. Indeed, the undesirable effects of low age were noticeable in groups with poor prognostic markers, such as high MYC / MYCN or 17p missing groups (FIGS. 18A-18C).

Attach an electronic file to a sequence list

Claims (22)

MRNA expression levels of chromosome 17p-related genes consisting of the nucleotide sequences of SEQ ID NOs: 1 to 92, MYC and MYCN genes consisting of the nucleotide sequences of SEQ ID NOs: 93 and 94, and WNT related genes consisting of the nucleotide sequences of SEQ ID NOs: 95 to 106, or Composition for predicting the risk of recurrence and survival prognosis of brain tumors comprising an agent for complexly measuring the expression level of the protein encoded by the gene.
The composition of claim 1, wherein the brain tumor is a disease selected from the group consisting of medulloblastoma, meningioma, schwannoma, pituitary adenoma, and glioma.
The composition of claim 1, wherein the agent for measuring the mRNA expression level of the gene is an antisense oligonucleotide, primer pair or probe that specifically binds to the gene.
The composition of claim 1, wherein the agent for measuring the expression level of the protein is an antibody specific for the protein encoded by the gene.
A kit for predicting recurrence risk and survival prognosis of a brain tumor comprising the composition of claim 1.
The kit of claim 5, wherein the kit is an RT-PCR kit, a DNA microarray chip kit, or a protein chip kit.
According to claim 6, wherein the RT-PCR kit is a chromosome 17p related gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 92, MYC and MYCN gene consisting of the nucleotide sequence of SEQ ID NO: 93 and 94, and bases of SEQ ID NO: 95 to 106 Kit comprising a primer pair specific for the WNT related gene consisting of the sequence.
According to claim 6, wherein the DNA microarray chip kit is a chromosome 17p related gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 92, MYC and MYCN gene consisting of the nucleotide sequence of SEQ ID NO: 93 and 94, and SEQ ID NO: 95 to 106 of Kit comprising a probe specific for the WNT-related gene consisting of a nucleotide sequence.
According to claim 6, wherein the protein chip kit is a chromosome 17p-related gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 92, MYC and MYCN gene consisting of the nucleotide sequence of SEQ ID NO: 93 and 94, and the base sequence of SEQ ID NO: 95 to 106 Kit comprising an antibody specific for a protein encoded by the WNT-related gene consisting of.
The kit of claim 5, wherein the brain tumor is a disease selected from the group consisting of medulloblastoma, meningioma, schwannoma, pituitary adenoma, and glioma.
1) measuring the mRNA expression level of the WNT related gene consisting of the nucleotide sequence of SEQ ID NOs: 95 to 106 or the expression level of the protein encoded by the gene from a sample of the isolated individual;
2) In the individual sample confirmed that the expression level of the WNT-related genes was not increased compared to the normal control in step 1), by the mRNA expression level of the chromosome 17p-related gene consisting of the nucleotide sequence of SEQ ID NO: 1 to 92 or by the gene Measuring the expression level of the encoded protein;
3) mRNA expression levels of the MYC and MYCN genes consisting of the nucleotide sequences of SEQ ID NOs: 93 and 94 or mRNA encoded levels of the proteins encoded in the individual samples in which the loss of chromosome 17p-related genes compared to the normal control in step 2) Measuring the expression level; And
4) The risk of recurrence and survival prognosis of the brain tumors is determined in step 3), wherein the individual samples having increased expression levels of the MYC and MYCN genes compared to the normal control samples are determined to have a high probability of recurrence of the brain tumor and a poor survival prognosis. How to provide the information needed to make predictions.
The method of claim 11, wherein said mRNA expression level is measured using an antisense oligonucleotide, primer pair, or probe that specifically binds said gene.
The method of claim 11, wherein the mRNA expression level is any one selected from the group consisting of reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real time reverse transcriptase polymerase, RNase protection assay, Northern blot, and DNA microarray chip. It is measured using.
The method of claim 11, wherein said protein expression level is measured using an antibody specific for a protein encoded by said gene.
The method of claim 11, wherein the protein expression level of Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS And it is measured using any one selected from the group consisting of protein chip method.
The method of claim 11, wherein the brain tumor is a disease selected from the group consisting of medulloblastoma, meningioma, schwannoma, pituitary adenoma, and glioma.
1) The chromosome 17p-related gene consisting of the nucleotide sequences of SEQ ID NOs: 1 to 92 is lost, and the expression levels of the MYC and MYCN genes consisting of the nucleotide sequences of SEQ ID NOs: 93 and 94 are activated, and the nucleotide sequences of SEQ ID NOs: 95 to 106 are activated. Contacting the test substance with cells in which the expression level of the WNT-related genes is not increased; And
2) measuring the mRNA expression level of the gene or the expression level of the protein encoded by the gene; And
3) screening a test substance that controls the expression level of the test substance treated cells compared to the expression level of untreated cells, the loss of chromosome 17p related genes, the increased expression of MYC and MYCN genes, and the expression of WNT related genes. , Screening inhibitors of brain tumor recurrence.
18. The method of claim 17, wherein the brain tumor is a disease selected from the group consisting of medulloblastoma, meningioma, schwannoma, pituitary adenoma, and glioma.
18. The method of claim 17, wherein said mRNA expression level is measured using oligonucleotides, primer pairs or probes that specifically bind said gene.
18. The method according to claim 17, wherein the mRNA expression level is selected from the group consisting of reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real time reverse transcriptase polymerase, RNase protection assay, Northern blot and DNA microarray chip. It is measured by.
18. The method of claim 17, wherein the protein expression level is measured using an antibody specific for a protein encoded by the gene.
18. The method of claim 17, wherein the protein expression level is Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS And it is measured using any one selected from the group consisting of protein chip method.

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