Method for the diagnosis of higher- and lower-grade astrocytoma using biomarkers and diagnostic kit thereof

Abstract

Disclosed is a method of diagnosing the presence of higher grade astrocytoma/glioblastoma (GBM) or lower-grade astrocytoma (DA or AA) in a human subject using secreted or plasma membrane associated biomarkers, which involves the detection of the expression levels of said genes, alone or in combination, in either tumor tissue samples or body fluids and a diagnostic kit thereof.

Claims

1. A method comprising: obtaining brain tissue from a subject having higher grade or lower grade astrocytoma; determining an expression level of a protein encoded by AP2A2 in the brain tissue obtained from the subject having higher grade or lower grade astrocytoma, wherein the expression level of protein is detected by ELISA; and administering a treatment to the subject having a higher grade or lower grade astrocytoma.

2. The method of claim 1, wherein protein is extracted from the brain tissue before determining the expression level of the protein encoded by AP2A2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

(1) FIG. 1. Heat map of SAM identified differentially regulated genes between glioblastoma and lower-grade astrocytoma.

(2) Normalized, log 2-transformed expression ratios of SAM identified differentially regulated genes were visualized using a dual color code with red and green indicating up- and down-regulation respectively, in the particular glioma sample compared to normal brain tissue. Grey square represents the missing data. Data was subjected to hierarchical clustering using TMEV software to obtain better visualization. Thirteen genes are upregulated in GBMs as against lower-grade astrocytomas and two genes vice versa.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention relates to a method of diagnosing the presence of glioblastoma or lower-grade astrocytoma in a biological sample. Diffuse infiltrating astrocytomas include the following entities: 1. Diffuse astrocytoma (DA; WHO Gr. II), 2. Anaplastic astrocytoma (AA; WHO Gr. III) and 3. Glioblastoma (GBM; WHO Gr. IV).

(4) The inventive method involves collecting or otherwise obtaining a test sample from suspected subject including of a bodily substance derived from the human subject, in which the sample contains human nucleic acid or protein originating from the subject, and quantitatively determining therein the level of expression of single or combination of genes selected from the group comprising of AP2A2, APOC3, BDNF, CALU, CXCL14, CXCL9, F11, GNRH1, LAD1, LAMP2, PRL, PSG9, SERPINC1, GPX3, SPARCL1. A characteristic higher expression of the any or a combination of the genes, AP2A2, APOC3, BDNF, CALU, CXCL14, CXCL9, F11, GNRH1, LAD1, LAMP2, PRL, PSG9 and SERPINC1, as compared to control sample from known healthy subject is diagnostic for the presence of glioblastoma, while the higher levels of GPX3 and/or SPARCL1 indicates the presence of lower-grade astrocytoma (see Table 1).

(5) This includes detection by means of measuring of proteins or specific nucleic acids, such as RNA or cDNA. The sample is preferably collected directly from the human subject's body. Preferred and convenient substances for sampling include blood, lymph or plasma, serum, cerebrospinal fluid, other biopsy sample of cellular material from brain tissue. Cellular material includes any sample containing human cells, including samples of tissue, expressed tissue fluids (e.g., lymph or plasma) or tissue wash and the like. Tissue samples that can be collected include, but are not limited to, cell-containing material from the brain. This includes normal brain tissue, tumor tissue, tumor-adjacent tissue, and/or blood plasma from a site within the brain.

(6) In accordance with the inventive methods, the tissue sample preferably contains cells that express a plurality of protein species and mRNA species, which proteins and/or mRNA species are detectably distinct from one another. obtaining and collecting the sample are used interchangeably herein and encompass sampling, resecting, removing from in situ, aspirating, receiving, gathering, and/or transporting the tissue sample or a concentrate, sediment, precipitate, supernatant, filtrate, aspirate, or other fraction of any of these. For example, conventional biopsy methods are useful for obtaining the tissue sample. These include percutaneous biopsy, laparascopic biopsy, surgical resection, tissue scrapes and swabs, sampling via stents, catheters, endoscopes, needles, surgical resection, and other known means. For example, to obtain a sample from inside the skull of the human subject; typically, Magnetic Resonance Imaging (MRI)-guided stereotactic techniques are employed, but other methods can be used.

(7) The blood sample can be collected from subjects and is allowed to clot at room temperature for no more than 72 hrs, and then centrifuged at 4 C. for 5 min at 1000 rpm. The serum (upper phase) is separated and stored at 20 C. until use. The sample is alternatively derived from cultured human cells, cell-free extracts, or other specimens indirectly derived from a subject's body, as well as from substances taken directly from a subject's body. Samples may be stored before detection methods are applied (for example nucleic acid amplification and/or analysis, or immunochemical detection) by well known storage means that will preserve nucleic acids or proteins in a detectable and/or analyzable condition, such as quick freezing, or a controlled freezing regime, in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), trehalose, glycerol, or propanediol-sucrose. Samples may also be pooled before or after storage for purposes of amplifying the nucleic acids specific for the said genes for analysis and detection, or for purposes of detecting the respective proteins.

(8) The sample is used immediately or optionally pre-treated by refrigerated or frozen storage overnight, by dilution, by phenol-chloroform extraction, or by other like means, to remove factors that may inhibit various amplification reactions. The level of expression in the sample for the said proteins or their messenger ribonucleic acid (mRNA) is then detected quantitatively or semi-quantitatively.

(9) Polynucleotides specific for the said genes, including mRNA species, are determined by base sequence similarity or homology to known nucleotide sequences. Base sequence homology is determined by conducting a base sequence similarity search of a genomics data base, such as the GenBank database of the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/BLAST/), using a computerized algorithm, such as PowerBLAST, QBLAST, PSI-BLAST, PHI-BLAST, gapped or ungapped BLAST, or the Align program through the Baylor College of Medicine server (www.hgsc.bcm.tmc.edu/seq_data). (Altschul S F, Madden T L, Schaffer A A, Zhang 3, Zhang Z, Miller W, Lipman D J. Nucleic Acids Res. (1997) 25(17), 3389-402; Zhang, J., Madden, T. L., (1997) Genome Res. 7(6), 649-56; Madden T L, Tatusov R L, Zhang J. (1996) Methods Enzymol. 266, 131-41).

(10) Preferably, polynucleotide sequences specific to the said genes, including an mRNA sequence, is at least 5 to 30 contiguous nucleotides long, more preferably at least 6 to 15 contiguous nucleotides long, and most preferably at least 7 to 10 contiguous nucleotides long. mRNA specific to any of the said genes can be, but is not necessarily, an mRNA species containing a nucleotide sequence that encodes a functional version of the said genes or fragments thereof. Also included among mRNAs specific to the said genes are splice variants.

(11) Quantitative detection of levels of mRNAs specific to the said genes or their proteins, or of other proteins or mRNA, species of interest in accordance with the present invention is done by any known method that provides a quantitative determination of expression. A quantitative method can be absolute or relative. An absolute quantitation provides an absolute value for the amount or level of expression in comparison to a standard, which amount or level is typically a mole, mass, or activity value normalized in terms of a specified mass of protein, mass of nucleic acid, number or mass of cells, body weight, or the like. Additionally, the quantitative or absolute value is optionally normalized in terms of a specified time period, i.e., expression level as a rate. A relative detection method provides a unitless relative value for the amount or level of expression, for example, in terms of a ratio of expression in a given sample relative to a control, such as normal tissue or the expression of a selected housekeeping gene. The skilled artisan is aware of other examples of quantitative and semi-quantitative detection methods.

(12) In accordance with the inventive methods, the expression level of the proteins encoded by the said genes is optionally detected by immunochemical means, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), immunoelectrophoresis, immunochromatographic assay or immunohistochemical staining, employing polyclonal or monoclonal antibodies or antibody fragments against the said gene products. Antibodies or antibody fragments that target the said proteins are available commercially or can be produced by conventional means.

(13) Similarly, the expression levels of other proteins of interest, in accordance with the inventive methods, can be detected by conventional immunochemical means as described above. Most preferably, quantitative or semi-quantitative detection of the expression level of mRNA species is accomplished by any of numerous methods of nucleic acid amplification (e.g., amplification of specific nucleic acid segments) in the form of RNA or cDNA, which RNA or cDNA amplification product is ultimately measured after amplification. The final amplification product of RNA or cDNA is measured by any conventional means, such as, but not limited to, densitometry, fluorescence detection, or any other suitable biochemical or physical assay system. Before amplification, it is, preferable to extract or separate mRNA from genomic DNA in the sample and to amplify nucleic acids remaining in that fraction of the sample separated from the DNA, to avoid false positives that are caused by amplification of contaminating genomic DNA in the original specimen.

(14) Histopathological means of classifying malignant tumors into grades are known for various kinds of malignant tumor, including astrocytomas. (Daumas-Duport C, Scheithauer B, O'Fallon J, Kelly P. (1988) Cancer 62, 2152-2165).

(15) The present inventive method can be used to diagnose the presence of glioblastoma or lower-grade astrocytoma.

(16) The foregoing descriptions of the methods of the present invention are only illustrative and by no means exhaustive. When these features of the present invention are employed, diagnostic and treatment decisions can be more appropriately optimized for the individual astrocytoma patient, and the prospects for his or her survival can be enhanced.

(17) Identification of Differentially Regulated Genes Between Glioblastoma, and Lower-Grade Astrocytoma (DA and AA)

(18) We obtained the expression profile of 18981 human genes using 19k cDNA microarrays (University Health Network, Canada) for twenty two samples of diffusely infiltrating astrocytoma comprising four diffuse astrocytoma (DA; Gr II), four AA (Gr. III) and fourteen GBM (Gr IV; six secondary and ten primary). Among the genes spotted on the microarray, 226 genes code for protein whose localization is either secreted or plasma membrane-associated. The expression data of these 226 genes was subjected to Significance Analysis of Microarrays using the two-class option to find out differentially regulated genes between lower-grade astrocytoma (DA/AA) and glioblastoma. While some of the found genes were already reported, some of them were novel. Of the found novel differentially regulated genes, two genes up regulated in LGA (DA/AA) as against GBM and thirteen genes are upregulated in GBM as against LGA (see FIG. 1).

EXAMPLES

(19) The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1: Tissue Collection

(20) Astrocytoma tissue samples were collected from patients, who underwent surgery at Sri Satya Sai Institute of Higher Medical Sciences and Manipal Hospital, Bangalore, India at the time of surgical resection. Controls comprised non-tumorous brain tissue samples (temporal lobe) collected from patients who underwent surgery for intractable epilepsy. A total of thirty-seven astrocytoma samples of different grades were used in this study. Tissues were bisected and one half was snap-frozen in liquid nitrogen and stored at 80 C. until RNA isolation. The other half was fixed in formalin and processed for paraffin sections and these were used to identify the histopathological grade and the type of astrocytoma.

(21) TABLE-US-00001 TABLE 1 Diagnostic markers for Glioblastoma and Lower-Grade Astrocytoma Fold LGA- GBM- difference Protein Mean Mean between SI locali- fold fold GBM & No Symbol Gene Name sation change* change* LGA Glioblastoma Specific Genes 1 AP2A2 adaptor- Plasma 1.66 1.18 1.96 NM_012305 related pro- mem- tein complex brane 2, alpha 2 subunit 2 APOC3 apolipo- secreted 1.46 1.08 1.58 NM_000040 protein C-III 3 BDNF brain- secreted 1.53 1.06 1.44 NM_170731 derived neurotrophic factor 4 CALU calumenin secreted 1.08 1.73 1.60 AL576538 5 CXCL14 chemokine secreted 1.03 1.59 1.55 KM_004887 (CXC motif) ligand 14 6 CXCL9 chemokine secreted 1.35 2.06 1.52 NM_002416 (CXC motif) ligand 9 7 F11 coagulation secreted 1.87 1.27 2.36 NM_J300128 factor XI 8 GNRH1 gonado- secreted 1.09 1.83 1.68 NM_000825 tropin- releasing hormone 1 9 LAD1 ladinin 1 secreted 1.30 1.05 1.37 NM_005558 10 LAMP2 lysosomal- Plasma 1.37 1.77 1.30 MM_002294 associated mem- membrane brane protein 2 11 PRL prolactin secreted 1.44 1.23 1.77 NM_000948 12 PSG9 pregnancy secreted 2.14 1.15 2.46 MM_002784 specific beta-1-gly- coprotein 9 13 SERPINC1 serine (or secreted 1.13 1.41 1.58 NM_00048S cysteine) proteinase inhibitor, clade C (anti- thrombin), member 1 Lower-Grade Astrocytoma (DA and AA) Specific Genes 14 GPX3 glutathione secreted 2.83 1.04 2.73 NM_002084 peroxidase 3 (plasma) 15 SPARCL1 SPARC- secreted 3.33 1.24 4.15 NM_001128 like 1 310 (mast9, hevin) *fold change is calculated to w.r.t. normal brain

Example 2: RNA Isolation

(22) Total RNA was extracted from the frozen tissue by a combination of the TRIzol method (Invitrogen, USA) and RNeasy Midi kit (Qiagen) according to the manufacturer's instructions. The RNA samples were quantified by measuring the absorbance using a spectrophotometer and visualized on a MOPS-Formaldehyde gel for quantity and quality assurance.

Advantages

(23) It provides a useful method for diagnosing the presence of glioblastoma and low grade astrocytoma. The method is useful both before and after clinical symptoms have appeared. The method can also be applied to monitor the effectiveness of anti-cancer treatments.

REFERENCE

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