KR20120121941A - A composition for diagnosis of primary central nervous system lymphoma and a diagnosing kit comprising the same - Google Patents

Abstract

PURPOSE: A composition containing a formulation which measures gene expression level for diagnosing central nervous system lymphatic tumor is provided to develop a central nervous system lymphatic tumor-specific anticancer agent. CONSTITUTION: A composition for diagnosing central nervous system lymphatic tumor contains an agent which measures mRNA level of NPFFR2(neuropeptide FF receptor 2), C4orf7(chromosome 4 open reading frame 7), OSMR(oncostatin M receptor), EMCN(endomucin), TPO(thyroid peroxidase), FNDC1(fibronectin type III domain containing 1), COL12A1(collagen, type XII, alpha 1) or MSC(musculin) gene or protein expression level thereof. The agent for measuring mRNA level is a primer or probe which specifically binds to gene. The agent for measuring protein level contains an antibody which is specific to the protein.

Description

A composition for diagnosis of primary central nervous system lymphoma and a diagnosing kit comprising the same}

The present invention is NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC composition for diagnosing the central nervous system lymphoma comprising a preparation for measuring the expression level of at least one gene selected from the group consisting of, the central nervous system comprising the composition Lymphoma diagnostic kit, a method for diagnosing CNS lymphoma by measuring the expression level of the gene, a composition for predicting CNS lymphoma prognosis and a method for predicting prognosis.

Cancer is characterized by "uncontrolled cell growth," which results in the formation of cell masses called tumors that infiltrate surrounding tissues and, in severe cases, metastasize to other organs of the body. Academia is also called neoplasia.

Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, in many cases does not heal fundamentally, suffers the patient, and ultimately leads to death. More than 20 million people worldwide suffer from cancer, and more than 6 million people die of cancer each year, and 11 million people are expected to die by 2020, so cancer is an urgent need to find a cure. Disease.

Cancer varies from country to country, but in developed countries and Korea accounts for more than 20% of all deaths. However, despite many efforts, the exact cause or mechanism of cancer development is still unknown. Therefore, in addition to treating cancer, early detection and diagnosis of cancer is very important.

Lymphoma, on the other hand, is a malignant tumor that occurs in lymphoid tissue. Non-lymphoid lymphomas, which occur in non-lymphoid tissues, are known to occur mainly in the nose and throat and in the gastrointestinal tract and brain. The United States accounted for 2.1% of all cancer cases, the UK accounted for 2.8%, and our country accounted for 4%.

Among lymphomas, primary central nervous system lymphoma (PCNSL) is defined as lymphoma that is confined to the brain and brain stem, not systemic diseases. This is a term applied to the non-Hodgkin's lymphoma (NHL), which is limited to the central nervous system, and the optimal treatment of PCNSL is not yet known. Therefore, there is an urgent need for a method for early detection and diagnosis of central nervous system lymphoma.

Therefore, the present inventors have made efforts to identify markers for early diagnosis of central nervous system lymphoma. As a result, the present inventors have identified genes that are differentially low-expressed in central nervous system lymphoma compared to normal lymphoid tissues, and measuring the expression level of the central nervous system. It was found that lymphoma can be diagnosed early, and the present invention has been completed.

An object of the present invention is NPFFR2 (Neuropeptide FF receptor 2), C4orf7 (chromosome 4 open reading frame 7), OSMR (oncostatin M receptor), EMCN (endomucin), TPO (thyroid peroxidase), FNDC1 (fibronectin type III domain containing 1) COL12A1 (Collagen, type XII, alpha 1) and MSC (musculin) to provide a composition for diagnosing central nervous system lymphoma comprising an agent for measuring the expression level of one or more genes selected from the group consisting of MS or protein thereof.

Another object of the present invention to provide a central nervous system lymphoma diagnostic kit comprising the composition.

Another object of the present invention is NPFFR2 (Neuropeptide FF receptor 2), C4orf7 (chromosome 4 open reading frame 7), OSMR (oncostatin M receptor), EMCN (endomucin), TPO ( mRNA expression level of one or more genes selected from the group consisting of thyroid peroxidase (FNDC1), fibronectin type III domain containing 1 (FNDC1), COL12A1 (Collagen, type XII, alpha 1) and MSC (musculin) or the protein encoded by the gene Measuring the expression level; And comparing the expression level of the gene or the expression level of the protein encoded by the gene with the expression level of the gene of the normal control sample or the expression level of the protein. It is to provide a way.

Another object of the present invention is to provide a composition for predicting the prognosis of central nervous system lymphoma, comprising a preparation for measuring the expression level of mRNA or protein of the C4orf7 (chromosome 4 open reading frame 7) gene.

Another object of the present invention is to measure the mRNA expression level of the C4orf7 (chromosome 4 open reading frame 7) gene or the protein encoded by the gene from biological samples isolated from patients with CNS lymphoma; And comparing the expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level of the gene or the protein expression level of the gene in a normal control sample or a patient with a known central nervous system lymphoma patient. The aim is to provide a method for providing information necessary for predicting the prognosis of CNS lymphoma.

As one embodiment for achieving the above object, the present invention is an agent for measuring the expression level of mRNA or protein of one or more genes selected from the group consisting of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC It provides a composition for diagnosing central nervous system lymphoma comprising a.

In the present invention, "central nervous system lymphoma" is defined as lymphoma that is not systemic disease but is limited to the brain and brainstem. This is a term applied to non-Hodgkin's lymphomas (NHL) that are limited to those occurring in the central nervous system, and may preferably be primary central nervous system lymphoma (PCNSL).

In the present invention, "diagnosis" means confirming a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the onset of central nervous system lymphoma by confirming a decrease in expression of diagnostic markers of central nervous system lymphoma.

In the present invention, the term "diagnosis marker, diagnostic marker" is a substance that can diagnose CNS lymphoma cells from normal cells, and is a polypeptide which shows a decrease in cells with CNS lymphomas compared to normal cells. Or organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.). For the purposes of the present invention, the central nervous system lymphoma markers of the present invention may be genes and proteins encoded by them that exhibit specifically low levels of expression in central nervous system lymphoma cells, as compared to cells of lymphoid tissues such as normal lymph nodes.

Preferably, the central nervous system lymphoma marker may be one or more genes selected from the group consisting of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC genes and proteins encoded by the genes, and these genes and protein information may be It is registered in the National Center for Biotechnology Information (NCBI) (NM_004885, NM_152997, NM_003999.2, NM_016242, NM_000547, NM_032532, NM_004370, NM_005098).

In the present invention, "an agent for measuring the expression level of mRNA or protein of one or more genes selected from the group consisting of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC" is expressed in central nervous system lymphoma cells By identifying the decreasing expression level of the central nervous system lymphoma marker of the present invention, it is meant a molecule that can be used for detection of the marker, preferably an antibody, primer or probe specific for the marker.

The expression level of the central nervous system lymphoma markers can be known by confirming the expression levels of mRNAs or proteins encoded by genes of one or more genes selected from the group consisting of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC. have.

In the present invention, "mRNA expression level measurement" refers to a process of confirming the presence and expression level of mRNA of the central nervous system lymphoma marker gene in a biological sample to diagnose central nervous system lymphoma, by measuring the amount of mRNA. For this purpose, RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting (Northern) blotting) and DNA chips, but are not limited thereto.

Preferably, the agent for measuring mRNA level of the gene may be a primer pair or probe, the nucleic acid sequence of the NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSCNUSAP1 gene NM_004885, NM_152997, NM_003999.2, NM_016242 And NM_000547, NM_032532, NM_004370, NM_005098 (NCBI), one of ordinary skill in the art can design primers or probes that specifically amplify specific regions of these genes based on the sequences.

In the present invention, a "primer" is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is a starting point for template strand copying. Short nucleic acid sequence that functions. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

In the present invention, the central nervous system lymphoma can be diagnosed through PCR amplification using sense and antisense primers of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSCNUSAP1 polynucleotides. At this time, the length of PCR conditions, sense and antisense primers can be modified based on what is known in the art.

Preferably, as said primers, primers for COL12A1 are primer pairs of SEQ ID NOs: 1 and 2; Primers for C4orf7 include primer pairs of SEQ ID NOs: 3 and 4; Primers for FNDC1 include primer pairs of SEQ ID NOs: 5 and 6; Primers for NPFFR2 include primer pairs of SEQ ID NOs: 7 and 8; Primers for MSCs include primer pairs of SEQ ID NOs: 9 and 10; Primers for EMCN include primer pairs of SEQ ID NOs: 11 and 12; Primers for OSMR include primer pairs of SEQ ID NOs: 13 and 14; And primer pairs for SEQ ID NOs: 15 and 16 can be used for the TPO.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which can achieve specific binding with mRNA, and is labeled to confirm the presence or absence of a specific mRNA. Can be. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe.

In the present invention, hybridization may be performed using a probe complementary to NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1, and MSC polynucleotides, thereby diagnosing central nervous system lymphoma through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, 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 ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

According to a specific example of the present invention, the composition for detecting the central nervous system lymphoma diagnostic marker is each primer pair specific for at least one gene selected from the group consisting of NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC. It may include.

In the present invention, "measurement of expression level of protein" refers to a process of confirming the presence and degree of expression of a protein expressed in a central nervous system lymphoma marker gene in a biological sample in order to diagnose central nervous system lymphoma, specific for the protein of the gene. The amount of the protein is determined using an antibody that binds to the target.

As analytical methods for this, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Tissue immunity staining, immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), FACS and protein chips (protein chip) and the like, but are not limited thereto.

Preferably, the agent for measuring protein level may be an antibody.

In the present invention, "antibody" means a specific protein molecule directed to an antigenic site as it is known in the art. For the purposes of the present invention, the antibody specifically binds an antibody that specifically binds to a protein expressed by one or more genes selected from the group consisting of the markers of the present invention NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC. Means, such an antibody can be prepared by a conventional method from the obtained protein by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene. It may also include partial peptides that can be made from the protein.

The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

Antibodies used in the detection of CNS diagnostic markers of 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.

In another aspect, the present invention provides a kit for diagnosing CNS lymphoma, comprising the composition for diagnosing CNS lymphoma of the present invention.

Kit of the present invention is the central nervous system lymphoma diagnosis markers NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC expression level of one or more genes selected from the group consisting of identifying the marker by identifying the expression level of mRNA or protein Can be detected.

The kit for detecting markers of the present invention includes one or more other component compositions, solutions or devices suitable for analytical methods as well as primers, probes or optionally antibodies that detect the expression level of CNS diagnostic markers. May be included.

Preferably, the kit of the present invention is a primer, wherein the primer for COL12A1 is a primer pair of SEQ ID NOs: 1 and 2; Primers for C4orf7 include primer pairs of SEQ ID NOs: 3 and 4; Primers for FNDC1 include primer pairs of SEQ ID NOs: 5 and 6; Primers for NPFFR2 include primer pairs of SEQ ID NOs: 7 and 8; Primers for MSCs include primer pairs of SEQ ID NOs: 9 and 10; Primers for EMCN include primer pairs of SEQ ID NOs: 11 and 12; Primers for OSMR include primer pairs of SEQ ID NOs: 13 and 14; And primers for TPO can include primer pairs that are SEQ ID NOs: 15 and 16.

In the present invention, the kit for measuring the mRNA expression level of the central nervous system lymphoma markers may be a kit containing essential elements necessary for performing RT-PCR. In addition to the individual primer pairs specific for the marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases. Such enzymes, DNase, RNAse inhibitors, DEPC-water (DEPC-water), sterile water and the like can be included. In addition, it may include a primer pair specific to the gene used as a quantitative control, and preferably may include a primer pair of SEQ ID NO: 17 and SEQ ID NO: 18 specific to the GAPDH gene.

Preferably, the kit of the present invention may be a diagnostic kit including essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent labeled probe. In addition, the substrate may comprise cDNA corresponding to the quantitative control gene or fragment thereof.

As another specific example, the kit for measuring the expression level of the central nervous system lymphoma marker protein in the present invention is a secondary antibody, a chromogenic substrate, labeled with a substrate, a suitable buffer, a chromophore or a fluorophore for immunological detection of the antibody. And the like. The substrate may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase or an alkaline force. Fatase (Alkaline Phosphatase) can be used, the fluorescent material can be used FITC, RITC, etc., the color substrate substrate is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

As another aspect, the present invention provides a method for providing information necessary for diagnosing central nervous system lymphoma using the composition or diagnostic kit for diagnosing CNS lymphoma of the present invention.

The method is characterized in that NPFFR2 (Neuropeptide FF receptor 2), chromosome 4 open reading frame 7 (C4orf7), oncostatin M receptor (OSMR), endomucin (EMCP), thyroid peroxidase (TPO), Measuring mRNA expression level or expression level of at least one gene selected from the group consisting of FNDC1 (fibronectin type III domain containing 1), COL12A1 (Collagen, type XII, alpha 1) and MSC (musculin) Making; And comparing the expression level of the gene or the expression level of the protein encoded by the gene with the expression level of the gene or the expression level of the protein of the normal control sample.

Separation of mRNA from a biological sample can be carried out using a known process and mRNA levels can be measured by various methods.

In the present invention, the term "biological sample" includes tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and the like, in which the expression level of the central nervous system lymphoma markers differs due to the development of central nervous system lymphoma. This is not restrictive.

Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, DNA chip, and the like.

Through the detection methods, it is possible to compare the mRNA expression level in the normal control group and the mRNA expression level in patients with suspected central nervous system lymphoma, and to determine whether there is a significant decrease in the expression level in the central nervous system lymphoma marker gene by mRNA and suspected central nervous system lymphoma. The patient may be diagnosed as having actual central nervous system lymphoma.

Preferably, the mRNA expression level can be measured by reverse transcriptase polymerase reaction or DNA chip using a primer specific for the gene used as a central nervous system lymphoma marker.

The reverse transcriptase polymerase reaction is electrophoresis after the reaction to confirm the band pattern and the thickness of the band to determine whether the mRNA expression and the degree of genes used as diagnostic markers of central nervous system lymphoma, and compared with the control group, the generation of central nervous system lymphoma You can easily diagnose whether or not.

On the other hand, the DNA chip uses a DNA chip in which the nucleic acid corresponding to the central nervous system lymphoma marker gene or a fragment thereof is attached to a glass-like substrate with high density, and isolates the mRNA from the sample and labels the terminal or the inside with a fluorescent substance. CDNA probes can be prepared, hybridized to DNA chips, and read for the onset of CNS lymphoma.

In addition, the present invention comprises the steps of contacting the antibody specific for the protein encoded by the central nervous system lymphoma marker gene with a biological sample of a suspected central nervous system lymphoma to confirm the protein level by the antigen-antibody complex formation, and the reduction of the amount of protein formation It provides a method for providing information necessary for the diagnosis of CNS lymphoma, comprising the step of comparing with a protein level of a normal control sample.

The separation of proteins from biological samples can be carried out using known processes and protein levels can be measured in a variety of ways.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto.

Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in patients with suspected central nervous system lymphoma can be compared, and the significant expression level of the central nervous system lymphoma marker gene to protein is compared. By determining whether a decrease in the number of patients with central nervous system lymphoma suspected that the actual development of central nervous system lymphoma can be diagnosed.

In the present invention, "antigen-antibody complex" means a combination of central nervous system lymphoma marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed quantitatively through the magnitude of the signal of the detection label. It is measurable. Such a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto.

When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include, but are not limited to, 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, and the like. .

Preferably, the protein expression level may be measured by ELISA. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a labeled antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the complex of the antigen with the antibody attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is enzymatically developed by attaching the antibody to the solid support, reacting the sample, and then attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. Central nervous system lymphoma marker protein and antibody complex by confirming the degree of formation, it is possible to determine whether the development of central nervous system lymphoma.

Also, preferably, the protein expression level may be measured by Western blot using at least one antibody against the central nervous system lymphoma marker.

The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. By checking the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein produced by the expression of the gene can be confirmed to determine whether the central nervous system lymphoma develops. The detection method consists of a method for examining the expression level of the marker gene in the control group and the expression level of the marker gene in cells with central nervous system lymphoma. 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.

Also, preferably, the protein expression level may be measured by immunohistostaining with at least one antibody against the central nervous system lymphoma marker.

After collecting and fixing normal lymphoid tissue and suspected central nervous system lymphoma, paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several um and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

In addition, the protein expression level measurement may be performed using a protein chip in which at least one antibody against the central nervous system lymphoma marker is arranged at a predetermined position on the substrate and immobilized at a high density. In the method of analyzing a sample 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 and checked for the presence or expression level of the protein. It is possible to determine whether the nervous system lymphoma has developed.

As another aspect, the present invention provides a composition for predicting central nervous system lymphoma prognosis comprising an agent for measuring the expression level of mRNA of the C4orf7 (chromosome 4 open reading frame 7) gene or a protein thereof.

In the present invention, "prognosis" means an expectation for a medical ear (e.g., long-term viability, disease free survival rate, etc.), and includes a positive prognosis or negative prognosis, the negative prognosis being relapse, tumor growth, metastasis, weak Positive progression includes disease progression or mortality, such as resistance, and positive prognosis includes amelioration or stabilization of diseases such as disease-free conditions, tumor regression, and the like.

Preferably, the prognosis means a negative prognosis, and when it is confirmed that the expression level of the mRNA of the C4orf7 gene or a protein thereof is less than that of the control group, the prognosis may be predicted as a central nervous system lymphoma having a negative prognosis.

In the present invention, "prediction" means to estimate in advance with respect to the medical ear, and for the purposes of the present invention, the progress of the disease of the patient diagnosed with central nervous system lymphoma (progression, improvement, worsening, recurrence of central nervous system lymphoma) , Tumor growth, drug resistance, and death).

In one embodiment of the present invention, as a result of predicting the prognosis of the patients with central nervous system lymphoma based on the expression level of the C4orf7 gene, it was confirmed that the prognosis of the patient is poor when the C4orf7 gene is low expression (Fig. 5).

As another aspect, the present invention provides a kit for predicting the central nervous system lymphoma prognosis comprising the composition for predicting the central nervous system lymphoma prognosis.

Prognostic Prediction Kits of the present invention may include primers, probes, or antibodies that selectively recognize markers for measuring expression levels of C4orf7 markers, as well as one or more other component compositions, solutions or devices suitable for analytical methods. have.

Preferably, the kits of the present invention may comprise primer pairs having SEQ ID NOs: 3 and 4 as primers.

In yet another aspect, the present invention provides a method for measuring the mRNA expression level of a C4orf7 (chromosome 4 open reading frame 7) gene or a protein encoded by a biological sample isolated from a patient with central nervous system lymphoma; And comparing the expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level of the gene or the protein expression level of the gene in a normal control sample or a patient with a known central nervous system lymphoma patient. It provides a method for providing information necessary for predicting the prognosis of CNS lymphoma.

In the present invention, "biological sample isolated from a patient with central nervous system lymphoma" may be, but is not limited to, tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine from the patient, preferably formarin Fixed paraffin embedded tumor tissue.

In the present invention, "a central nervous system lymphoma patient with a known prognosis" refers to a patient diagnosed with a progression of the disease among patients diagnosed with central nervous system lymphoma, and a sample taken from these patients and a sample taken from a patient who wants to know a prognosis. By measuring and comparing gene expression levels in, we can accurately predict the prognosis of patients who want to know the prognosis. The method of measuring the mRNA expression level of the C4orf7 gene or the expression level of the protein encoded by the gene is as described above.

The present invention provides data useful for the treatment and prognosis of CNS lymphoma by providing markers for diagnosing the onset and prognosis of CNS lymphoma. In addition, the central nervous system lymphoma diagnostic markers may be usefully used for researches on developing central nervous system lymphoma-specific anticancer agents.

1 shows a Genome-wide frequency plot of copy number variation identified in 12 PCNSL and 13 normal peripheral blood samples.
2 shows the top 96 gene clusterings differentially expressed between tumor and normal groups.
3 shows metabolic pathways differentially expressed in PCNSL compared to normal lymph nodes.
Figure 4 shows the real-time RT-PCR results of genes that are differentially low expression in the PCNSL group compared to the normal lymph node group.
FIG. 5 shows Kaplan-Meier curves showing that low expression of the C4orf7 gene is associated with negative prognosis in PCNSL patients.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1: Preparation of Primary Central Nervous System Lymphoma Tissue

Fresh-frozen tissue samples from 13 patients with primary CNS Diffuse large B-cell lymphoma (PNS) using the Helsinki Declaration and a protocol approved by the Samsung Medical Center System Review Committee. Got it. Eight of the 13 samples were obtained by resection and five samples by stereotactic needle biopsy. All samples were confirmed by histological analysis. The quality of the frozen samples was confirmed by hematoxylin, eosin and CD20 immunohistochemical staining. All but one sample contained more than 80% malignant B cells, one of which was excluded from the study. Therefore, a total of 12 patients (6 males, 6 females) were enrolled in this study. The age range of patients diagnosed was 32 to 68 years (mean 54.5 years). A total of 12 samples were included in high-resolution array-based comparative genomic hybridization (aCGH). In view of the quality of tissue available after aCGH out of 12 samples, only 8 samples were used for further expression array analysis for RNA studies. Peripheral blood obtained from 13 healthy men was used as a control sample for aCGH, and fresh-frozen normal lymph nodes were used as control samples for expression arrays.

Example  2 : DNA  And gun RNA Extraction of

Standard proteinase K / RNAse and phenol-chloroform extraction from 12 freshly frozen tumor samples for high-resolution array-based comparative genomic hybridization (aCGH) DNA was extracted by (phenol-chloroform extraction). Peripheral blood was obtained from 13 healthy men to extract normal DNA. Total RNA was extracted from eight fresh tumor tissues and eight fresh-frozen normal lymph nodes through trizol-chloroform extraction for gene expression analysis.

Example  3: diagnosis of central nervous system lymphoma Marker  Sympathy

3-1: Experiment Method

<3-1-1> High Resolution Array-Based Comparative Dielectric Hybridization

High-resolution array-based comparative genomic hybridization (aCGH) was performed using an Agilent Human Genome CGH Microarray Kit 4 × 44K containing about 44000 probes consisting of 60-mer oligonucleotides. Labeling and hybridization were done using the protocol provided by the manufacturer. Briefly, 1.5 μg of test DNA and reference DNA (reference DNA) were digested using AluI and Rsa I (Promega) and purified using a QIAprep Spin Miniprep kit (QIAGEN). Test sample DNA and reference DNA were labeled by random priming using either Cy3-dUTP or Cy5-dUTP using Agilent Genomic DNA Labeling Kit PLUS. After the labeling reaction, the labeled and reference samples were mixed respectively and concentrated using Microcon YM-30 filters (Millipore). Probe denaturation and preannealing with Cot-1 DNA followed by hybridization at 65 ° C. for 40 hours. Thereafter, washing was performed using Agilent Oligo CGH in four steps. Specifically, washing with washing buffer 1 for 5 minutes at room temperature, washing with washing buffer 2 for 1 minute at 37 ° C., washing with acetonitrile for 1 minute at room temperature, and Agilent's stabilizer and drying solution for 30 seconds at room temperature. . All slides were scanned with an Agilent DNA microarray scanner. Data were acquired using Agilent Feature Extraction Software Version 9, and common aberration detection method with statistical algorithms z-score, 2.5 and 6.0 sensitivity thresholds, respectively. 2) and a moving average window of 0.2 Mb were analyzed by Agilent CGH Analytics Version 4.0 software.

<3-1-2> gene expression Microarray

Gene expression profiling analysis was performed using an Agilent Oligo Microarray Kit 4 X 44K according to the Agilent One-Color Microarray-based Gene Expression Analysis Protocol. Briefly, 0.5 μg of total RNA was amplified and labeled using the Agilent Quick Amp Labeling Kit. After labeling, cRNA was purified using RNeasy Mini-spin column (QIAGEN), cyanine 3 dye concentration, RNA absorbance ratio (260 nm / 280 nm) and cRNA using NanoDrop ND-1000 UV-VIS spectrophotometer. Quantification was made for concentration. Linearly amplified cRNA labeled with cyanine 3 was labeled with 10X blocking agent, nuclease-free water and 25X fragmentation buffer provided in the Agilent Gene Expression Hybridization Kit (Agilent Technologies). buffer) and incubated at 60 ° C. for 30 minutes for RNA fragmentation. After addition of 2X GEx hybridization buffer Hi-RPM, 17 hours at 10 rpm, 65 ° C. in a SureHyb DNA Microarray Hybridization Chamber in a DNA Microarray Hybridization Oven (Agilent Technologies) The fragmented RNA amplified by the reaction was hybridized to a 4 × 44K Whole Human Genome Oligo Microarray Chip (Agilent Technologies) containing 44000 cDNA clones. After hybridization, slides were washed for 1 minute each using Gene Expression wash buffers 1 and 2 and scanned with an Agilent DNA microarray scanner-G2565BA (Agilent Technologies). Feature Extraction Software Version 9 (Agilent Technologies) was used to extract and analyze the signal. To verify the amplification process, amplified and unamplified RNAs were labeled and hybridized and compared.

<3-1-3> Gene Expression Analysis

In order to identify genes that are differentially expressed between tumors and normal lymph nodes, the family-wise error rate (FWER) for the control of multiple-testing errors is the variance proposed by Huber et al. After normalization using a variance-stabilizing transformation method (VSN), it was calculated using software R (Version 2.9.1; http://www.r-project.org). Genes with significant differences in expression levels identified by gene expression microarray analysis (FWER, P <0.05) were used to obtain the biological features and meanings associated with the large gene list. visualization and integrated discovery) in bioinformatics resources. To identify gene variations in differentially expressed genes, the identified genes were mixed with aCGH data obtained from Agilent CGH Analytics Version 4.0 software to correlate common abnormal regions from aCGH. In addition, metabolic pathway analysis of PCNSL gene expression data sets was performed using KEGG / Biocarta pathway gene sets and gene set enrichment analysis to identify metabolic pathways that specifically affect PCNSL.

<3-1-4> Quantitative Real Time RT - PCR

To verify gene expression, cDNA was synthesized from 2 μg of total RNA from 6 fresh tumor samples and 5 normal reactive lymph node samples using the Superscript III RNA Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. It was. Real-time RT-PCR was performed using ABI 7900 HT Fast Real-Time PCR System (Applied Biosystems). NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC genes and reference genes (GAPDH) were amplified and detected using dsDNA-specific CYBR Green dye (Applied Biosystems). PCR reactions were prepared in 10 μl of final volume containing 5 μl of 2 × Power SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μl of cDNA and 0.2 μl of forward and reverse primers, respectively, at a concentration of 10 pmol / μl. DNA polymerase was activated at 95 ° C. for 10 minutes, denatured 45 times at 95 ° C. for 15 seconds and then annealed and extended at 60 ° C. for 1 minute. Each measurement was performed in duplicate. The quality of the PCR product was monitored using post-PCR melt-curve analysis. The threshold cycle (Ct), i.e. the number of fractional cycles of the amplified target that reached a fixed threshold, was determined. The concentration ratio of each of the eight genes to GAPDH was 2 ^ΔCt (ΔCt = ΔC _{tested gene} △ C _{reference gene} ).

Primers used for the PCR reaction were as follows: COL12A1: forward (SEQ ID NO: 5'-AGTGCATCAACGGGATACAG), reverse (SEQ ID NO: 2'5'-ACATGCAATTCCCTCTCTGC); C4orf7: forward (SEQ ID NO: 5'-CATATCCATTTCGCCCACTT), reverse (SEQ ID NO: 5'-TTTCGCTAGGAAGGGGAGTT); FNDC1: forward (SEQ ID NO: 5'-TCCCCAATGACCTGAAGAAG), reverse (SEQ ID NO: 6'5'-GCTTTGTCCCAGTCCACAAT); NPFFR2: forward (SEQ ID NO: 5'-GCCCCTGTGG ACTCTAATGA), reverse (SEQ ID NO: 8: 5'-TGGGATTGACACTGCTGTTG); MSC: forward (SEQ ID NO: 5'-CGCTTAAATCGGACTGGAAC), reverse (SEQ ID NO: 10: 5'-GGGAAGGGTCAAGGAGAATC); EMCN: forward (SEQ ID NO: 11: 5'-TCACACCAACAACTGGAACAA), reverse (SEQ ID NO: 12: 5'-TCAGTGGTTGTGGCTTTCAA); OSMR: forward (SEQ ID NO: 13'5'-TCTGAGCTCCCTTTGGAATG), reverse (SEQ ID NO: 14'5'-GGAACTCCAGTTGCTCCAGA); TPO: forward (SEQ ID NO: 15: 5'-GAGGGATTACATCCCCAGGA), reverse (SEQ ID NO: 16: 5'-GAACACGTTGGACACAGTGG); And GAPDH: forward (SEQ ID NO: 17: 5'-GCACCGTCAAGGCTGAGAA), reverse (SEQ ID NO: 18: 5'-AGCATCGCCCCACTTGATT).

<3-1-5> Statistical Analysis

Differences in mRNA expression levels between tumor and normal samples were calculated using the Mann-Whitney U test. Overall survival was determined using the Kaplan-Meier method, and survival curves were compared using a log-rank test. Survival was measured from the day of surgery. All tests were 2-sided and statistical significance was set to P threshold values below 0.05. Statistical analysis was performed using SPSS version 18 (SPSS Inc.).

3-2: Experimental Results

<3-2-1> of Primary Central Nervous System Lymphoma Copy number  transition

The data were entered into Gene Expression Omnibus (accession number GSE25298). As shown in FIG. 1, genome mutations were frequent in all 12 primary central nervous system lymphomas (PCNSL), but genome mutations were rare in all normal samples obtained from healthy men. None (FIG. 1). Specifically, the recurrent gain region (≥50%) is 1p36.33, 3q21.3, 3q22.1, 3q29, 7p22.3, 7q11.23, 7q22.1, 7q36.1, 8q24.3 , 9q34.11, 9q34.3, 10q26.3, 11p15.5, 11q13.1, 11q23.3, 14q32.33, 15q24.1, 16p13.3, 16p11.2, 16q24.3, 17p13.3, 17q25 .3, 19p13.3, 19q13.2, 19q13.31-33, 19q13.41-43, 20q13.33, 21q22.3 and 22q13.33. In 9 patients (75%) the most frequent gain area was 17p13.3 with TMEM93 and P2RX5 and 22q13.33 with IL17REL and MLC1. Recurrent loss areas (≥50%) are 6p21.32, 6q14.1-3, 6q15, 6q16.1-3, 6q21, 6q22.1-2, 6q22.31-33, 6q23.1- 3, 6q24.1-2, 6q26, 8p22, 8p21.3, 9p21.3, 14q32.33, p11.31 and 22q11.23. Deletion was most frequently observed at 9p21.3 with CDKN2A (8 patients; 66.7%).

<3-2-2> Identification of genes differentially expressed in primary CNS lymphoma

Data was entered into Gene Expression Omnibus (accession number GSE25297). Raw data from 8 tumors and 8 normal lymph nodes excluded one tumor and one normal sample showing inadequate quality. After normalization using the VSN method, 96 genes (FWER, P <0.05) that were differentially expressed in the tumor group compared to the normal group were detected.

As a result, as shown in FIG. 2, the clustering of these top 96 genes showed distinct separation between the two groups (FIG. 2). The genes included 27 up-regulated genes (Table 1) and 69 down-regulated genes (Table 2). 111 enhanced biologic terms were generated by functional annotation analysis using DAVID bioinformatics resources. Most of these terms were related to signaling, the properties of the extracellular matrix (ECM), and cell adhesion. Functional annotation groups, including OSMR, CCND3, TPO and IL3RA, were associated with Janus kinase (JAK) -signal transducers and activators of transcription (STAT) signaling. Biological terms that satisfy a P significant threshold of less than 0.01 are shown in Table 3.

<3-2-3> Primary CNS Lymphoma Specific Metabolic pathways  Confirm

Metabolic pathway activity (false discovery rate [FDR] Q value <0.05) of primary CNS lymphomas clearly distinguished from normal lymph nodes was observed in cytokine-receptor interactions; Cell adhesion and migration; Cell death; And mediate functions such as signaling through Bad, Notch, JAK-STAT and NF-KB metabolic pathways (Table 4).

Expression signature analysis confirmed that the constitutive genes of the JAK-STAT metabolic pathway, including OSMR, CCND3 and TPO genes, were down-regulated in primary CNS lymphoma. In primary CNS lymphoma, other genes associated with JAK-STAT metabolic pathways, such as the STAT3, JAK1, AKT3, IL22RA1, and IL12A genes, are up-regulated, while SOCS1 and SOCS3, which inhibit JAK-STAT signaling, are down-regulated (FIG. 3A). Changes in gene expression affecting cell adhesion were clearly distinguished between normal and tumor groups (FIG. 3B).

<3-2-3> Copy number  Identify changes in gene expression associated with mutations

The region containing significant common aberrations in copy number was associated with a data set of the top 96 differentially expressed genes (FWER, P <0.05) obtained from the expression array. This correlational analysis identified eight genes (NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1 and MSC). All eight genes were downregulated in gene expression microarrays and deleted in high resolution array based comparative genome hybridization of two or more patients (> 15%). The same eight genes were found to be neither deleted nor amplified in all 13 normal samples from healthy men. The FNDC1 and COL12A1 genes were identified as the most frequently deleted genes in five patients (41.7%). Data for each patient is shown in Table 5.

<3-2-4> NPFFR2 , C4orf7 , OSMR , EMCN , TPO , FNDC1 , COL12A1  And MSC Quantitative Real Time for RT - PCR  result

RNA expression levels for 11 fresh-frozen tissue samples (6 tumors and 5 normal lymph nodes) were measured and C _t was calculated for all 8 genes. MRNA expression levels of eight genes were normalized by reference gene (GAPDH). The concentration ratio of each of the eight genes to GAPDH was calculated.

As a result, as shown in FIG. 4, all eight genes except TPO had significantly lower mRNA expression levels in the primary CNS group than in the normal lymph node group (Mann-Whitney U test, P <0.05). MRNA expression levels of TPO were not significantly lower in the tumor group than in the normal group (mean concentration ratio = 0.001 [tumor], 0.093 [normal], P = 0.1), and C4orf7 showed the largest difference (mean concentration ratio = 0.007 [ Tumor], 12.4333 [normal], P = 0.006; FIG. 4).

Example  4 : NPFFR2 , C4orf7 , OSMR , EMCN , TPO , FNDC1 , COL12A1  And MSC of Yes Significance Confirmation

Independent sets of 23 FFPE tissue samples from primary CNS DLBCL patients were examined to confirm the prognostic significance of the eight differentially expressed genes. All patients received chemotherapy regimens based on high-dose methotrexate after diagnosis. Of these, 22 patients received additional whole-brain radiation therapy. Total RNA was extracted from the 23 FFPE samples and cDNA was synthesized for it. Quantitative real-time RT-PCR was performed as described in Example 3 above, and concentration ratios were calculated for each gene. Various expression levels were tested using the log-rank test and Kaplan-Meier method to identify cut-off points for the most significant mRNA concentration ratios in relation to prognosis.

As a result, as shown in FIG. 5, when the cut-off point was set to less than 0.5 or 0.5 among 8 genes, low expression of C4orf7 mRNA was associated with low survival rate (P = 0.0425). The median and mean values of the C4orf7 concentration ratio in the 23 FFPE tumor samples were 0.58 and 0.71, respectively (range, 0.11 to 2.49; FIG. 5).

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> A composition for diagnosis of primary central nervous system          lymphoma and a diagnosing kit comprising the same <130> PA110096KR <160> 18 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for COL12A1 <400> 1 agtgcatcaa cgggatacag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for COL12A1 <400> 2 acatgcaatt ccctctctgc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for C4orf7 <400> 3 catatccatt tcgcccactt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for C4orf7 <400> 4 tttcgctagg aaggggagtt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for FNDC1 <400> 5 tccccaatga cctgaagaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for FNDC1 <400> 6 gctttgtccc agtccacaat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NPFFR2 <400> 7 gcccctgtgg actctaatga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NPFFR2 <400> 8 tgggattgac actgctgttg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MSC <400> 9 cgcttaaatc ggactggaac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MSC <400> 10 gggaagggtc aaggagaatc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for EMCN <400> 11 tcacaccaac aactggaaca a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for EMCN <400> 12 tcagtggttg tggctttcaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for OSMR <400> 13 tctgagctcc ctttggaatg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for OSMR <400> 14 ggaactccag ttgctccaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TPO <400> 15 gagggattac atccccagga 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TPO <400> 16 gaacacgttg gacacagtgg 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 17 gcaccgtcaa ggctgagaa 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 18 agcatcgccc cacttgatt 19

Claims (12)

Neuropeptide FF receptor 2 (NPFFR2), chromosome 4 open reading frame 7 (C4orf7), oncostatin M receptor (OSMR), endomucin (EMCM), thyroid peroxidase (TPO), fibronectin type III domain containing 1 (FNDC1), COL12A1 (Collagen, Type XII, alpha 1) and MSC (musculin) composition for diagnosing central nervous system lymphoma comprising an agent for measuring the expression level of one or more genes selected from the group consisting of MS or protein thereof.
The composition of claim 1, wherein the agent for measuring the level of mRNA of the gene comprises a primer or probe that specifically binds to the gene.
The primer of claim 2, wherein the primers for COL12A1 in the primers are SEQ ID NOS: 1 and 2; Primers for C4orf7 include primer pairs of SEQ ID NOs: 3 and 4; Primers for FNDC1 include primer pairs of SEQ ID NOs: 5 and 6; Primers for NPFFR2 include primer pairs of SEQ ID NOs: 7 and 8; Primers for MSCs include primer pairs of SEQ ID NOs: 9 and 10; Primers for EMCN include primer pairs of SEQ ID NOs: 11 and 12; Primers for OSMR include primer pairs of SEQ ID NOs: 13 and 14; And the primers for TPO are primer pairs of SEQ ID NOs: 15 and 16.
The composition of claim 1, wherein the agent for measuring the expression level of the protein comprises an antibody specific for the protein.
Central nervous system lymphoma diagnostic kit comprising the composition of any one of claims 1 to 4.
The kit of claim 5, wherein the kit is selected from the group consisting of an RT-PCR kit, a DNA chip kit, and a protein chip kit.
From biological samples isolated from patients with suspected central nervous system lymphoma, Neuropeptide FF receptor 2 (NPFFR2), chromosome 4 open reading frame 7 (C4orf7), oncostatin M receptor (OSMR), endomucin (EMNP), thyroid peroxidase (TPO), and fibronectin (FNDC1) measuring mRNA expression level of at least one gene selected from the group consisting of type III domain containing 1), COL12A1 (Collagen, type XII, alpha 1) and MSC (musculin) or the expression level of the protein encoded by the gene; And
Providing information necessary for diagnosing central nervous system lymphoma, including comparing the expression level of the gene or the expression level of the protein encoded by the gene with the expression level of the gene or protein expression level of the normal control sample. How to.
The method of claim 7, wherein the method of measuring mRNA expression level is selected from the group consisting of reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting and DNA chip. How.
According to claim 7, Method for measuring the expression level of the protein, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, Complement fixation assay, FACS and protein chip.
C4orf7 (chromosome 4 open reading frame 7) gene composition for predicting the central nervous system lymphoma prognosis comprising an agent for measuring the expression level of mRNA or protein thereof.
Central nervous system lymphoma prognosis kit comprising the composition of claim 10.
Measuring the mRNA expression level of a C4orf7 (chromosome 4 open reading frame 7) gene or a protein encoded by the gene from a biological sample isolated from a patient with central nervous system lymphoma; And
Comparing the expression level of the gene or the expression level of the protein encoded by the gene with the mRNA expression level of the gene or the protein expression level of the gene in a normal control sample or a central nervous system lymphoma patient sample with known prognosis, A method of providing information necessary for predicting the prognosis of CNS lymphoma.

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