KR20120038339A - Biomarker for diagnosis or confirming of progress stage of glioblastoma and the use thereof - Google Patents

Abstract

PURPOSE: A biomarker for diagnosing glioblastoma is provided to predict glioblastoma patient's prognosis and to develop anticancer drugs. CONSTITUTION: A biomarker for diagnosing or predicting glioblastoma includes survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2/3, or p16. A kit for diagnosing or predicting glioblastoma contains a material for measuring expression level of a gene encoding survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2/3 or p16 protein. The material is a primer with a sequence specific to the gene.

Description

Biomarker for the diagnosis or prognosis of glioblastoma and its use {Biomarker for Diagnosis or Confirming of Progress Stage of Glioblastoma and the Use Thereof}

The present invention relates to biomarkers for the diagnosis or prognosis of glioblastoma and its use, more particularly survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 Using a protein selected from the group consisting of biomarkers for diagnosis or prognosis of glioblastoma, measuring the expression level of the protein to provide information necessary for diagnosis or prognosis of glioblastoma It relates to the method of analysis.

Glioma (glioma) is a tumor that accounts for 60% of primary brain tumors (malignant tumors) still no special treatment other than radiation therapy. In particular, glioblastoma, which is classified as the most malignant, is highly resistant to radiation and chemotherapy compared to other cancers. Once diagnosed, the expected survival is only 1 year. In addition, there are cerebrovascular barriers that make drug delivery difficult and are relatively marginalized in the development of therapeutics due to a relatively lack of understanding of neurobiology.

Recent developments in systems biology and bioinformatics tools have reported significant genetic defects and gene expression patterns that are thought to be associated with glioblastoma development and progression. The discovery of representative molecular markers (molecular markers) is inconclusive, and there is a limit that the gene expression analysis remains at the transcript level, and thus cannot represent the degree of protein expression in actual cancer tissue.

In order to provide a biomarker for predicting glioblastoma prognosis, the present inventors introduced a tissue microarray (TMA) technique to analyze expression patterns of important proteins related to glioblastoma, and analyzed a new concept of image analysis and Analysis of these important proteins (survivin, cyclinE, DCC, TGF-β CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3, p16 ) by analyzing them through an optimized histochemical score (H-score) It has been confirmed that the present invention can be applied to confirm the progress of blastoma and has completed the present invention.

An object of the present invention is to provide a biomarker for the diagnosis or prognosis of glioblastoma selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 There is.

Another object of the present invention is to measure the expression level of a gene encoding a protein selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16. To provide a kit for diagnosing or prognostic glioblastoma, comprising a substance that can be.

Another object of the present invention, survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 to provide information necessary for the diagnosis or prognosis of glioblastoma It is to provide a method for analyzing protein comprising the step of measuring the amount of expression of the protein selected from the group.

In order to achieve the above object, the present invention is for the diagnosis or prognostic prediction of glioblastoma selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 Provide a biomarker.

The present invention can also measure the expression level of a gene encoding a protein selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16. Provided is a kit for diagnosing or predicting glioblastoma, comprising a substance.

The present invention is also selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 for providing information necessary for the diagnosis or prognosis of glioblastoma. It provides a protein analysis method comprising the step of measuring the expression level of the protein.

The proteins discovered through the analysis of the present invention can be applied to predict the prognosis of glioblastoma patients, thereby providing a kit for diagnosing glioblastoma prognosis using the same, and applied to the development of a new concept of targeted therapies and anticancer drugs targeting the discovered proteins. It can be useful. In addition, it is useful because it can bring a large ripple effect in the related industrial field.

FIG. 1 shows that the tissue microarray (TMA) image is analyzed by manual scoring method and optimized H-scoring method, and there is no difference according to the analysis method (A: survivin, B: APC).
Fig. 2 shows the clinical prognosis of 56 glioblastoma patients classified using 108 protein expression levels (A: protein expression level before and after normalization by boxplot, B: Hierarchical clustering result by heatmap and dendogram). Results: C: Results of Kaplan-Meier method for overall survival analyzed by Univariate survival analysis.
Figure 3 shows the results of the analysis of the expression of the predictive prognostic marker protein compared with TMA (82 cases) and public DNA microarray data (55 cases) using the tissue of another glioblastoma patient (A: Hierarchical clustering of 10 biomarkers, B: Kaplan-Meier survival curves of 2 separate patient groups, C: 9 target genes and euclidean distance between patients, D: Kaplan-Meier for each group survival curves compared to overall survival).
Figure 4 shows the expression of the marker protein for prognostic prognosis predicted in two isolated patient groups.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, in order to discover important proteins related to glioblastoma that can predict the prognosis of glioblastoma, the expression of glioblastoma-associated proteins is analyzed by tissue microarray (TMA) in patients with glioblastoma. The analysis was performed using conceptual image analysis and optimized histochemical score (H-score). As a result, glioblastoma patients were divided into patients with good glioblastoma prognosis and patients with poor glioblastoma prognosis. Survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and It was confirmed that the expression difference of p16 protein appears.

In other words, survivin, cyclinE, DCC, and TGF-β proteins were overexpressed in the patients with poor prognosis and CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 proteins were lower than those in the patients with poor prognosis. Confirmed.

The present invention relates to a biomarker for the diagnosis or prognosis of glioblastoma selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 in one aspect. will be.

In another aspect, the present invention provides a method for measuring the expression level of a gene encoding a protein selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16. It relates to a kit for diagnosing or prognostic glioblastoma, comprising a substance that can be.

In the present invention, the substance may be characterized as a primer having a complementary sequence specific for the gene encoding the protein.

In another aspect, the present invention consists of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 for providing information necessary for the diagnosis or prognosis of glioblastoma. It relates to a protein analysis method comprising measuring the expression level of a protein selected from the group.

In the present invention, the survivin, cyclinE, DCC and TGF-β Predicting the diagnosis or prognosis of glioblastoma by measuring protein overexpression and predicting the diagnosis or prognosis of glioblastoma by measuring the low expression of DC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 proteins Can be. In addition, the expression levels of survivin and cyclinE proteins were found to function as an important factor in dividing glioblastoma patients.

Glioblastoma is very important for predicting the prognosis of glioblastoma, because once expected, life expectancy is only 1 year. The expression of proteins detected by the factor can be distinguished into a patient group with a good prognosis and a patient group with a poor prognosis, which is an important criterion for the treatment of glioblastoma patients. Obtain the information necessary for diagnosing or predicting blastoma. In addition, since the diagnosis or prognosis of glioblastoma can be predicted by measuring the expression level of the proteins, it can be usefully used to develop a therapy for glioblastoma using this.

In the present invention, the tissue microarray (TMA) technique and the optimized histochemical score (H-score) were analyzed by immunoprofiling by integratively analyzing clinical results such as survival time in glioblastoma. Molecular markers were predicted to predict the prognosis of blastoma. A total of 56 glioblastoma patients were analyzed for the expression of 108 glioblastoma-associated proteins. As a result, the clusters with different protein expressions were divided into two groups (n = 19, 37). In addition, the survival time difference between the two groups was found to be statistically significant (p <0.05). Figure 2 shows the protein expression levels before and after normalization by boxplot analysis using the 'normalize.quantiles' function in the affy package of the R program, and also R program package (Comprehensive R archives network (CRAN). [Cited]; Available from : The results of Hierarchical clustering using http://cran.r-project.org/.) as a heatmap and dendogram, and the overall survival analyzed by Univariate survival analysis using Kaplan-Meier method. The results are shown.

In FIG. 3, hierarchical clustering of 10 biomarkers using the tissues of another glioblastoma patient was performed to express the expression of the prognostic marker protein, and the patient group was divided into two groups. Kaplan-Meier survival curves were used to verify the significance, and log-rank analysis showed statistically significant differences in overall survival. In addition, the euclidean distance between 9 genes and patients was verified, and Kaplan-Meier survival curves of each group were compared with overall survival.

In the present invention, the significant difference in expression of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3, p16 proteins in the patients with poor prognosis were also verified (q <0.05). In addition, we tested 82 independent glioblastoma samples and public DNA array data that were independently produced to verify whether the proteins identified through this study can be used as markers for predicting the prognosis of glioblastoma patients. Both protein and RNA expression levels were found to be able to distinguish between the two patient groups, which means that the protein can be used to predict the diagnosis or prognosis of glioblastoma by measuring its expression.

As a result of analyzing the expression levels of glioblastoma-related proteins, survivin, cyclinE, DCC and TGF-β proteins were overexpressed in patients with poor glioblastoma prognosis and CDC25B, Histone H1, p-EGFR , p-VEGFR2 / 3 and p16 proteins were found to be low expression (Table 1).

Code Protein Localization Adjusted p-Value Up / down regulated
at poor prognosis X50 cyclin E Nuclear 0.00744 Up X16 DCC Nuclear 0.0155 Up X41 survivin Nuclear 0.0295 Up X44 TGF β Cytoplasm 0.0104 Up X08 CDC25B Nuclear 0.00744 Down X24 Histone H1 (B419) Nuclear 0.0113 Down X78 p-EGFR Cytoplasm 0.0295 Down X75 p-VEGFR2 / 3 Cytoplasm 0.0122 Down X31 p16 Cytoplasm 0.00744 Down X31 p16 Nuclear 0.00744 Down

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

group Microarray  Collection of samples for (glioblastoma patients and tissue collection)

62 GBM samples were obtained from Samsung Medical Research Institute from January 2004 to December 2006. The ages of the patients ranged from 13 to 78 years, and their survival ranged from 22 to 913 days with a median value of 376.5 days. In addition, an independent 82 GBM sample (obtained from patients diagnosed with GBM between January 2004 and December 2007) for the verification of glioblastoma prognostic predictor proteins found in the present invention was obtained from Samsung Medical Research Institute. Got it.

Processing of brain tissue

Brain extraction was performed at an appropriate time after cell inoculation and treatment. To determine the biological status of the tumor before and after brain extraction and to examine histology, place the brain extracted in the brain matrix after brain harvest, slice the brain at 2 mm intervals, and slice one tissue around the cell line. The frozen block was made using the OCT compound. The remaining tissue sections were placed in a buffered 10% formalin solution and fixed at 4 ° C for 24 hours to form paraffin blocks. Frozen sections were made using a frozen slicer with a thickness of 8 um, and stored at -20 ° C. Paraffin sections were made with a 4 um thickness and stored at room temperature. In order to confirm the expression by immunohistochemical method, the tissue was prepared first through de-paraffin and re-hydration. The prepared tissues were colored with Gill's Hematoxylin and Eosin for 15-20 seconds and then stained with water. Then, after the dehydration process was mounted in permount. At this time, the control sample should react only with the secondary antibody and should not be stained. Tumorigenecity of each cell group was determined by measuring the size and number of stained tumors.

Histological examination

Paraffin sections (4-6 um) were attached to the coated slide glass and dried overnight. The tissues were subjected to de-paraffinization with xylene, followed by gradual processing of [100, 95, 80% ethanol, DW] samples according to alcohol percentage, and re-hydrated in PBS (pH 7.5). Samples were subjected to antigen retrival for 5 minutes in a microwave oven in water or treated with pepsin and left at 37 ° C. for 15 minutes, followed by washing with PBS. 3% H ₂ O ₂ was reacted with peroxidase in the tissue. The tissue was blocked with 5% normal horse serum / 1% normal goat serum, and the primary antibody cytosine deaminase antibody and 3 hours at room temperature and overnight at 4 ° C. After washing with water, the tissue was blocked with 5% normal horse serum / 1% normal goat serum, and HRP-coated secondary antibody was treated at room temperature for 1 hour. After 5-10 minutes of color development with DAB, the color development of DAB was confirmed on a microscope and the appropriate color development was determined. After 15-20 seconds of color development with Gill's Hematoxylin, background staining with water was performed, followed by dehydration and mounting with permount.

Tissue micro array ( TMA ) H- of the image scoring  Analysis by method

Sections were prepared from paraffin blocks prepared using tissues of each patient, and pathologically cancerous sites were selected by H & E staining. Tissue microarrays were prepared using Beecher Instruments (Sun Prairie, Wis.) MTA-1 instrument according to the company's standard protocol. The expression patterns of 78 important proteins known to be associated with glioblastoma were analyzed by immunostaining on the TMA slides prepared in this manner (expression and intracellular location information of each protein were established through existing research papers). After staining, the TAM slide is converted into an image file using Aperio's Scan Scope CS System, and the converted image is analyzed using Bioimagene's TissueMine (Bioimagene Co., USA) program. (negative), 1 (weak), 2 (moderate), and 3 (strong)]. After image analysis, H-score (0-300) was verified by setting the criteria of 0 negative% + 1 weak% + 2 moderate% + 3 strong% (McCarty KS et al ., Arch Pathol Lab Med. 1985; 109 (8): 716-21; Abd El-Rehim DM et al ., Int J Cancer. 2005; 116 (3): 340-50). Through this, as a result of analyzing the tissue microarray image using the manual scoring method and the optimized H-scoring method, it was confirmed that there is no difference according to the analysis method (FIG. 1).

Claims (6)

A biomarker for the diagnosis or prognosis of glioblastoma selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16.
It includes a substance capable of measuring the expression level of a gene encoding a protein selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16, Kit for diagnosis or prognosis of glioblastoma.
The kit for diagnosis or prognosis of glioblastoma according to claim 2, wherein the substance is a primer having a complementary sequence specific for the gene encoding the protein.
Expression of protein selected from the group consisting of survivin, cyclinE, DCC, TGF-β, CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 to provide information necessary for the diagnosis or prognosis of glioblastoma Analysis method of the protein comprising the step of measuring.
The method of claim 4, wherein the step of measuring the expression level of the protein is determined by overexpression of a protein selected from the group consisting of survivin, cyclinE, DCC, and TGF-β.
According to claim 4, The step of measuring the expression level of the protein CDC25B, Histone H1, p-EGFR, p-VEGFR2 / 3 and p16 characterized in that for determining the low expression of the protein selected from the group consisting of Analysis of Proteins.

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