JP2008292424A - Neoplasm detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for performing prognosis while more adequately determining a degree of malignancy of neoplasm than a conventional method. <P>SOLUTION: The method uses survivin and EGFR as markers for detecting neoplasm, and uses a degree of transfer or development of the survivin and the EGFR into a tissue as an index for detecting the existence of the neoplasm and determining a degree of malignancy of the neoplasm. Herein, the survivin and the EGFR in a neoplasm tissue specimen is subjected to immune tissue chemical dyeing or immune cell chemical dyeing, the ratio of the number of survivin positive cells and the number of EGFR positive cells to the number of total observed cells is measured, and a degree of malignancy of the neoplasm is determined from the value. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting a tumor such as glioma, and a diagnostic kit that can be used for detecting a tumor.

  Glioma (glioma), which is a type of tumor, is a tumor of glial cells that originally exist between neurons and support them in various ways. Among malignant tumors that develop in the brain, It accounts for about 1/3 of primary brain tumors. Of the gliomas, WHO Grade I, the least malignant, is a diffuse astrocytoma that occurs in the cerebellum of children, and since this tumor alone does not invade the surrounding brain so much, it can be cured by surgery alone. is there. WHO Grade II or higher glioma often recurs only by surgery, and chemotherapy and immunotherapy with radiation and anticancer drugs are performed after surgery. WHO Grade VI glioma is one of the most malignant brain tumors, and is called glioblastoma. Glioblastoma is still a disease that is still difficult to treat, and most patients relapse within a few months by surgery alone, so postoperative radiation therapy and chemotherapy are essential. The diagnosis of malignant glioma has been conventionally classified into four grades (grades I to VI) showing malignancy according to WHO criteria, and the prognosis is said to follow this grade. The determination of glioma malignancy is comprehensively determined from various angles by examining the patient's angiography, MRI, CT, pathological diagnosis, etc. This determination result is an index for determining the patient's treatment method It is considered as one of However, even when the grade of malignancy and the patient's prognosis did not match, or even in the same grade, the patient's prognosis varied.

  In order to compensate for the shortcomings of conventional tests, a method for prognosing glioma has been developed by using as an index the expression of a protein (apoptosis inhibitor) called survivin expressed in the brain tissue of glioma patients (see Patent Document 1). .

JP 2004-138522 A

  An object of the present invention is to provide a method and a reagent for determining a malignancy of a tumor such as glioma more appropriately and performing a prognosis as compared with a conventional method.

  Conventionally, glioma was diagnosed using only the expression of survivin expressed in the brain tissue of glioma patients as an index, but the malignancy of glioma was clearly determined and the prognosis could not be accurately diagnosed. In particular, there were some patients with prognostic malignancy even though the survivin expression level was low. By analyzing the relationship between the expression of glioma and survivin and EGF receptor (EGFR), the present inventors diagnosed in combination with not only survivin but also the expression of EGFR, and malignant tumors such as glioma By classifying the degrees in detail and finding that the prognosis can be accurately diagnosed, the present invention has been completed.

That is, the present invention is as follows.
[1] A method for detecting a tumor using survivin and EGFR as markers.
[2] A method for detecting the presence of a tumor by using the degree of transcription or expression of survivin and EGFR in a tissue as an index, measuring the transcription or expression of survivin and EGFR in the tissue, and the amount of transcription or expression of survivin and EGFR [1] The method for detecting a tumor according to [1], wherein it is determined that a tumor exists when the value is larger than that of a normal tissue.

[3] A method for determining the malignancy of a tumor using the degree of survivin and EGFR transcription or expression in tumor tissue as an index, measuring the transcription or expression of survivin and EGFR in the tumor tissue, and the amount of survivin and EGFR transcription Alternatively, a method for determining the malignancy of a tumor, wherein the malignancy of the tumor is determined to be high when the expression level is larger than that of a normal tissue.

[4] Immunohistochemical staining or immunocytochemical staining of survivin and EGFR in tumor tissue specimens, and then measuring the ratio of the number of survivin positive cells and EGFR positive cells to the total number of observed cells. [3] The method for determining the malignancy of a tumor according to [3], wherein the degree is determined.

[5] The method according to any one of [1] to [4], wherein the tumor is glioma and the tissue is brain tissue.
[6] A tumor diagnostic kit for detecting a tumor using survivin and EGFR as markers.
[7] The tumor diagnostic kit according to [6], which comprises at least an anti-survivin antibody and an anti-EGFR antibody, and detects a tumor using survivin and EGFR as markers.
[8] The tumor diagnostic kit according to [6] or [7], wherein the tumor is glioma.

  Diagnosis of tumors such as glioma using survivin index and EGFR index together makes it possible to make an appropriate prognosis and to establish an appropriate treatment method and treatment policy. Appropriate prognosis enables the selection of treatments suitable for each patient from radical treatment to terminal. In other words, it will be possible to select necessary and sufficient radiation therapy, chemotherapy, gene therapy, and appropriate relief measures for physical and mental distress, and maintain the quality of life (QOL) of tumor patients. Improvements can be made. Furthermore, if the expression level of EGFR can be suppressed for patients with high expression levels of EGFR and low survivin expression, the possibility of effective tumor treatment is suggested.

Hereinafter, the present invention will be described in detail.
In the method of the present invention, tumor is detected using survivin and EGF receptor (EGFR) as markers. Here, detection means determining whether a tumor is present, determining whether a patient is afflicted with a tumor, determining the malignancy of a tumor, and diagnosing the prognosis of a patient afflicted with a tumor Including things. In the present invention, tumor detection includes tumor malignancy determination and tumor prognosis, and tumor malignancy determination includes tumor prognosis.

  Survivin has been identified as one of the proteins containing an amino acid sequence homologous to the apoptosis-inhibiting domain, which is included in the apoptosis-inhibiting protein derived from baculovirus. Its action is to bind to active caspase-3 and inhibit its proteolytic activity. In addition, it is known that cells deficient in survivin are inhibited in proliferation, and are considered to be essential proteins for cell proliferation. This survivin is expressed in many human cancer cells and lymphomas in vivo, and is considered to be involved in the growth of cancer cells.

  EGFR (epidermal growth factor receptor) is a glycoprotein that transmits the physiological action of epidermal growth factor (EGF) into cells and is found in almost all tissue cells. It is known that EGFR expression increases in squamous cell carcinomas such as the esophagus and lung. However, opinions differ on whether EGFR measurement is effective for prognosis determination of glioma.

  In the present invention, a target tumor is not limited, and any tumor can be a target. Both benign and malignant tumors are targeted, but are particularly effective against malignant tumors. When malignant tumors are classified according to organs, brain, nerve tumor, bladder cancer, skin cancer, stomach cancer, lung cancer, liver cancer, lymphoma / leukemia, colon cancer, pancreatic cancer, anal / rectal cancer, esophageal cancer, uterine cancer, breast cancer, bone / Classified into osteosarcoma, leiomyoma, rhabdomyosarcoma, and other cancers. As described above, the target tumor is not particularly limited and includes any of the above tumors and cancers, but is particularly effective against glioma in brain tissue. In order to detect these tumors, determine the grade of malignancy, and diagnose the prognosis, it is only necessary to measure the transcription or expression of survivin and EGFR in the tissue where each tumor occurs.

  In the method of the present invention, tumors in the examined tissue can be detected by detecting either or both of survivin and EGFR transcription or expression in the tissue. Transcription of survivin and EGFR can be detected by measuring mRNA encoding survivin and mRNA encoding EGFR, and expression of survivin and EGFR can be detected by measuring survivin protein and EGFR protein.

  When detecting transcription of survivin and EGFR, tissue or cells may be collected as a biological sample, and mRNA encoding survivin and mRNA encoding EGFR contained in the sample may be measured. To measure mRNA, a part of the tissue is collected and used as a material. For example, when the subject is a glioma of brain tissue, a part of the brain tissue surgically removed by craniotomy or the like is collected and used as a material. mRNA can be measured by extracting mRNA from the collected tissue or cells. In addition, tissue section specimens can be prepared, or the collected cells can be fixed on a glass slide and stained by in situ hybridization. You may go. Alternatively, the extracted mRNA may be measured by a known RNA measurement method such as Northern blotting or RT-PCR. Extraction of mRNA should be performed according to known methods, for example, as described in Shinsei Kagaku Koza 2 Nucleic Acid I Separation and Purification Tokyo Chemical Doujin July 10, 1991 and Molecular Biology Experiment Protocol I Maruzen Co., Ltd. June 30, 1997 Can do. In situ hybridization can be performed, for example, as described in Molecular Biology Experiment Protocol III Maruzen Co., Ltd. August 30, 1997. In this case, in order to specifically measure mRNA encoding survivin and mRNA encoding EGFR, a probe or primer comprising a partial sequence complementary to the partial sequence of mRNA encoding survivin and mRNA encoding EGFR is used. . Survivin and EGFR nucleotide sequences are known (for example, survivin is registered with GenBank accession number U75285, EGFR is registered with NM_005228), and probes or primers are designed based on known nucleotide sequence information can do. The nucleotide sequence of survivin gene is represented by SEQ ID NO: 1, the amino acid sequence of survivin is represented by SEQ ID NO: 2, the nucleotide sequence of EGFR gene is represented by SEQ ID NO: 3, and the amino acid sequence of EGFR is represented by SEQ ID NO: 4. The primer or probe is a survivin gene or EGFR gene fragment, and the number of bases is 5 to 50, preferably 10 to 30, and more preferably 15 to 25.

  When detecting the expression of survivin and EGFR, tissue or cells may be collected as a biological sample, and survivin and EGFR contained in the sample may be measured. The tissue or cells may be collected as described above. At this time, protein can be extracted from the tissue or cells, survivin and EGFR in the extract can be measured, and further, immunostaining can be performed by immunohistochemistry or immunocytochemistry techniques. .

  For the measurement of the extracted protein, a known immunoassay method (immunoassay) such as ELISA, radioimmunoassay, or Western blotting may be used.

  The measurement by immunohistochemistry or immunocytochemistry is performed by preparing a sliced sample of the collected tissue or by fixing the cells of the collected tissue on a slide glass. The tissue should be fixed as necessary. For the fixation, a fixing solution such as a buffer solution containing neutral formalin, 10% formalin, acetone, methanol or the like can be used. The composition and fixing method of the fixing solution can be performed by known methods described in, for example, “Revised Enzyme Antibody Method”, edited by Keiichi Watanabe and Kazuho Nakane. The tissue after fixation is embedded in paraffin as necessary, and a paraffin section specimen having a thickness of about 1 to 5 μm is prepared with a thin cutting device such as a microtome. The paraffin section sample may be hydrophilized by removing paraffin by xylene or ethanol treatment at the time of measurement and immersing it in physiological saline or a buffer solution. Further, a frozen section can be used instead of the paraffin section.

  Staining may use an anti-cybabin antibody and an anti-EGFR antibody labeled with an enzyme, a fluorescent substance, a radioisotope, etc., or after binding the anti-cybabin antibody and the anti-EGFR antibody to cybabin and EGFR in a section sample, A secondary antibody that binds to an anti-cybabin antibody and an anti-EGFR antibody and labeled with an enzyme, a fluorescent substance, a radioisotope, or the like may be used.

  The anti-cybabin antibody and anti-EGFR antibody may be prepared as a monoclonal antibody or a polyclonal antibody by a known method. Commercially available anti-cybabin antibodies and anti-EGFR antibodies can also be used. The labeling of the antibody can be performed by a known method.

  Examples of the enzyme used for labeling the antibody include horseradish peroxidase and alkaline phosphatase. Examples of the fluorescent substance include fluorescein and rhodamine. Alternatively, staining may be performed using a known biotin-avidin complex.

  For immunocytochemistry, the collected cells may be fixed on a slide glass using formalin or the like, and Cyvabin and EGFR in the cells may be visualized by the same method as the immunohistochemistry method. In immunohistochemistry or immunocytochemistry, staining can be determined with a microscope or the naked eye, or an appropriate optical measuring device may be used. Staining by immunohistochemistry can be performed, for example, according to the description of Molecular Biology Experiment Protocol III Maruzen Co., Ltd. published on August 30, 1997. Immunohistochemical staining and immune cell staining methods include ABC (Avidin Biotin Complex) method, direct labeling enzyme antibody method, LSAB (Labelled Strept Avidin Biotin) method, SABC (Strept Avidin Biotin Complex) method, Envision (manufactured by DAKO) and Examples include a polymer method using MAX-PO (manufactured by Nichirei Co., Ltd.) and the like, but the present invention can be carried out by any method.

For example, the ABC method or SABC method is performed as follows.
Survivin and EGFR present on the tissue are captured by anti-survivin antibody and anti-EGFR antibody, respectively, to generate survivin / primary antibody complex and EGFR / primary antibody complex. Next, after washing the tissue, a secondary antibody labeled with biotin is added to an antibody against this primary antibody (generally, an antibody against the immunoglobulin of the animal from which the primary antibody was obtained), and a survivin / primary antibody complex and EGFR / primary antibody are added. Binding to the complex produces a triple complex consisting of survivin / primary antibody complex / biotin-labeled secondary antibody and EGFR / primary antibody complex / biotin-labeled secondary antibody. Next, horseradish peroxidase conjugated with avidin or streptavidin capable of covalently binding to biotin is reacted with the triple complex, and a color former is added to the complex to enzymatically develop color. The amount of survivin and EGFR is measured from this color development amount.

  As described above, when the transcription or expression of survivin and EGFR in a tissue or cell is detected, and the amount of transcription or expression of survivin and EGFR is increased, it can be determined that the patient is suffering from a tumor. For example, when measuring survivin and EGFR mRNA or protein in tissue or cell extracts, measure survivin and EGFR mRNA or protein per unit weight tissue or number of unit cells of a normal person not previously affected by a tumor. However, if the mRNA or protein level of survivin and EGFR is significantly higher than the normal value, it can be determined that the patient is suffering from a tumor.

  Detection of survivin and EGFR transcription or expression can be used to distinguish whether a patient suffering from a tumor is malignant or benign. It can also be used for prognosis diagnosis, particularly for predicting survival days. In this case, if the amount of survivin and EGFR transcription or expression is significantly increased compared to normal tissues, it can be determined that the tumor is a malignant tumor with a short survival rate, and the prognosis is poor. Can be determined. Furthermore, according to a technique of in situ hybridization or immunohistochemistry using a tissue, it is possible to distinguish which part of the tissue is normal and which part is a tumor.

Tumors can be detected and diagnosed from the survivin and EGFR levels measured as described above, but in order to more accurately identify the tumor malignancy or determine the prognosis of the tumor, The transcription or expression of survivin and EGFR per unit volume or area, or per unit cell number may be measured. For example, the survivin index (%) and EGFR index (%) are calculated by measuring the ratio of survivin positive cells and EGFR positive cells to the total number of observed cells, and from these values, the tumor malignancy or tumor prognosis is determined. It is preferable to do this. Specifically, when the number of cells is observed using a microscope, the survivin index (%) and the EGFR index (%) are calculated by the following formulas.
Survivin index (%) = 100 × (number of survivin positive cells in one visual field in microscopic observation) / (total number of cells in one visual field in microscopic observation)
EGFR index (%) = 100 × (number of EGFR positive cells in one visual field in microscopic observation) / (total number of cells in one visual field in microscopic observation)

  In the method of the present invention, the survivin index is low when the survivin index is less than 50%, and the survivin index is high when the survivin index is 50% or more. Also, when the EGFR index is less than 50%, the EGFR index is low, and when the EGFR index is 50% or more, the EGFR index is high. If the survivin index and EGFR index are high, it is determined that the tumor is a low-grade tumor with long survival and good prognosis, and if the survivin index and EGFR index are low, the survival rate It is determined that there is a short high-grade tumor. It is also possible to evaluate the malignancy of tumors such as WHO grade of glioma using survivin index and EGFR index as indices. The relationship between the survivin index and the EGFR index and grade is clarified by analyzing both indexes in each grade, and a cut-off value in grade determination can also be determined.

  As described above, using the survivin index and EGFR index as indicators, tumor prognosis can be diagnosed, and appropriate treatment suitable for each patient can be selected from radical treatment to terminal care. Leads to the maintenance and improvement of QOL (Quality of Life).

  Furthermore, tumor patients with a high EGFR index but a low survivin index have a high survival rate and a relatively good prognosis. Therefore, when the EGFR index is high and the survivin index is low, it can be determined that effective tumor therapy can be performed by suppressing EGFR or its downstream signal.

  The present invention further includes a detection reagent for detecting a tumor such as glioma using survivin and EGFR as markers, a detection reagent for determining the malignancy of the tumor, and a detection reagent and kit for diagnosing the prognosis of the tumor Is also included. The reagent and kit are a reagent and kit containing at least survivin and an EGFR gene fragment as a probe or primer, or a reagent and kit containing at least an anti-survivin antibody and an anti-EGFR antibody. The reagent and kit may further contain a labeled antibody that binds to the anti-survivin antibody and the anti-EGFR antibody.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Collect patient data
From 1996 to 2005, 99 patients with a clear detailed medical history were selected from the glioma patients at Nippon Medical School Hospital and Musashi Kosugi Hospital. Each case develops within 3 months before diagnosis and is selected as one that has not been previously diagnosed with glioma. All patients are trying surgically removed malignant tumors, chemotherapy and radiation therapy. The patient's operative age ranged from 8 to 82 years, with an average of 54 years. 55 men and 44 women died, and 78 of 99 died during the observation period. Survival days were defined as the number of days from the first craniotomy until death, and observations were made for up to 2000 days. The median is 383 days (55 weeks). The tissue excised during the operation was embedded in paraffin, then cut into 4 μm thick sections with a microtome, and stained with hematoxylin and eosin. Stained specimens were observed by at least two pathologists or brain surgeons, and pathological diagnosis and grade determination were made according to World Health Organization (WHO) guidelines. The results are shown in Table 1. The table shows the median number of cases, average age, sex, and survival days for each pathological diagnosis.

Immunostaining of glioma tissue Anti-survivin antiserum was prepared by the method described in the literature (Uematzu M et al., J Neurooncol 2005; 72: 231-238). An antibody against EGF receptor (EGFR) (clone 3C6) that has been reported to be associated with the glioma pathogenesis mechanism, which has been studied as a tumor marker due to its high expression in proliferating cells (clone K-2) Was purchased from Ventana (Arizona, USA). A tissue section of glioma having a thickness of 4 μm with a microtome was attached to a slide and stained with each antiserum and antibody. For the staining, a fully automatic staining apparatus NexES staining system (Ventana) was used. Staining conditions were deparaffinized, treated with denaturing buffer (CC1, Ventana) for 1 hour, reacted with each antiserum or antibody at 37 ° C for 32 minutes, and biotinylated secondary antibody at 37 ° C for 10 minutes with avidin binding After further reaction with peroxidase for 10 minutes, color was developed with 3 ′, 3-diaminobenzidine. For counterstaining, Mayer hematoxylin was used, and after dehydration, it was mounted with a soft mount (WAKO, Tokyo). A stained image with anti-survivin antiserum is shown in FIG. When stained with a survivin antibody, normal cells are not stained and glioma cells are stained brown. The cells stained brown were defined as survivin positive cells. The bar in the figure is 50 μm. Moreover, the dyeing | staining image by an anti- EGFR antibody is shown in FIG. When staining with an EGFR antibody, normal cells are not stained, and cells expressing a large amount of EGFR are stained brown. The cells stained brown were regarded as EGFR positive cells. The bar is 50 μm. Five fields were randomly selected for each field, and the number of positive cells stained with the antibody for 200 cells in each field was counted to obtain an index.

Evaluation of immunostained glioma tissue specimens The stained tissues were observed under a microscope. Five visual fields were randomly selected for one specimen, and the number of positive cells stained with the antibody was counted for 200 cells in each visual field. When the ratio of anti-survivin antiserum positive cells (survivin index) was 0 to less than 50%, the survivin index was low, and more than 50% was the survivin index high. When the anti-Ki-67 antiserum positive cell rate (Ki-67 index) was 0 to less than 10%, the Ki-67 index was low, and 10% or more was the Ki-67 index high. When the anti-EGFR antiserum positive cell rate (EGFR survivin index) is 0 to less than 50%, the EGFR index is low, and 50% or more is the EGFR index high. The results are shown in Table 2. Even within the same grade, the ratio of high for each index is different, indicating that it shows different pathological conditions.

Prognosis by Survivin Index Table 3 shows the results of analysis by Mantel-Cox Logrank test between each factor and the number of days of survival. Age, grade, survivin index, Ki-67 index, and EGFR index were all significant. In particular, the survivin and Ki-67 indices were extremely significant, P <0.0001. The Kaplan-Meier survival curve at this time is shown in FIG. 3A. As a result of analysis by Mantel-Cox Logrank test divided into high and low indices of survivin index, those with low expression clearly had high survival rate. Patients with a high survivin index had a median of 322 days and 1084 days for patients with a low survivin index (P <0.0001), with a significantly better prognosis for patients with a low survivin index. At this time, a survival curve was obtained by limiting only to grades II and III, which have a relatively good prognosis but a large variation in the prognosis (FIG. 3B). As a result of analysis by Mantel-Cox Logrank test divided into high and low indices of survivin index, those with low expression clearly had high survival rate. Again, the prognosis for patients with low survivin index was significantly better, with P = 0.0004. A survival curve was similarly obtained for only grade IV (FIG. 3C). Again, the prognosis for patients with significantly lower survivin index, P = 0.0207, was good. On the other hand, when the survival curves for the grades II and III were similarly obtained with the Ki-67 index, P = 0.0002, but when only the grade IV was analyzed, there was no significant difference with P = 0.2681. This result shows that the survivin index is more reliable than the Ki-67 index.

Association of survivin index with other variables
Table 4 shows the results of examining the correlation between the survivin index and other variables using Fisher's direct probability calculation method or χ ² test. There was no statistical association between survivin index and gender or EGFR index. On the other hand, there was a correlation between survivin index and age or grade (P = 0.0017 and 0.0002). This means that survivin expression is higher in older patients with higher malignancy. The correlation between Ki-67 and index was as high as P = 0.0002. This is thought to be due to the fact that both indexes show the same tendency of increasing cell proliferation.

Prognosis by survivin index and EGFR index combination Since the correlation between survivin index and EGFR index is low at P = 0.0573, it is highly possible that both indexes represent different pathologies of glioma. However, the EGFR index alone is high and low, with median survival days of 404 and 702 days, P = 0.0800 (Table 3), and is not a clear criterion for prognosis. Therefore, the high and low survivin index groups were classified according to the EGFR index, and the survival curve was examined (FIG. 4). Each of those diagnosed as having a low survivin index and those diagnosed as having a high survivin index were further classified according to the EGFR index to obtain a survival curve. Among those with a low survivin index, those with a low EGFR index had a higher survival rate and those with a higher expression had a lower survival rate. In other words, in the high survivin index group, the high or low EGFR index is not related to the survival days, but in the low survivin index group, the high EGFR index is low and the median survival days are 795 and 1509 days, P There was a big difference in prognosis with <0.0001. In particular, the low survivin and EGFR index groups included a total of 32 patients, of which 8 were classified as the most aggressive grade IV. The stained image is shown in FIG. The hematoxylin and eosin staining image shows that it is grade IV, but it was not stained with anti-survivin antiserum or anti-EGFR antibody. The bar is 50 μm. If a normal pathological examination (A: hematoxylin and eosin staining) diagnoses grade IV, it usually dies in less than one year, but both the survivin and EGFR indices are low (B: staining with anti-survivin antiserum) , C: Staining with anti-EGFR antibody), the prognosis was observed until 2000 days. One patient survived more than 2000 days, and the other survived 1933 days. Clearly, it differs greatly from the expected prognosis of normal glioma IV patients.

  The above results indicate that the prognosis can be diagnosed more accurately by combining the survivin index and the EGFR index.

  The method and reagent of the present invention enable detection of tumors such as malignant glioma, determination of malignancy, and prognosis.

It is a figure which shows the dyeing | staining image of the glioma tissue by anti-survivin antiserum. It is a figure which shows the dyeing | staining image of the glioma tissue by an anti-EGFR antibody. It is a figure which shows the survival curve of the glioma patient classified according to the survivin index, and is a figure which shows the result of having divided into two groups about the whole glioma tissue (grade II-IV) by using the level of the survivin index as an index. It is. It is a figure which shows the survival curve of the glioma patient classified according to the survivin index, and about the glioma tissue diagnosed as the glioma tissue (grade II to III), the analysis was divided into two groups with the level of the survivin index as an index. It is a figure which shows the result. It is a figure which shows the survival curve of the glioma patient classified by survivin index, and it is the result of having divided into two groups about the glioma tissue diagnosed as glioma tissue (grade IV) by using the level of survivin index as an index FIG. It is a figure which shows the survival curve of the glioma patient classified according to the combination of survivin index and EGFR index. It is a figure which shows the grade IV glioma tissue dyeing | staining image where both survivin index and EGFR * index are low. A is an image stained with hematoxylin and eosin, B is an image stained with anti-survivin antiserum, and C is an image stained with anti-EGFR antibody.

Claims (8)

  A method for detecting a tumor using survivin and EGFR as markers.   A method for detecting the presence of a tumor using the degree of transcription or expression of survivin and EGFR in an tissue as an index, measuring the transcription or expression of survivin and EGFR in a tissue, and the amount of transcription or expression of survivin and EGFR being normal tissue The method for detecting a tumor according to claim 1, wherein the tumor is determined to be present when the tumor is larger than the tumor.   A method for determining the malignancy of a tumor by using the degree of transcription or expression of survivin and EGFR in a tumor tissue as an index, measuring the transcription or expression of survivin and EGFR in a tumor tissue, and the amount of transcription or expression of survivin and EGFR A method for determining the degree of malignancy of a tumor, in which the degree of malignancy of a tumor is determined to be high when the value is larger than that of a normal tissue.   Immunohistochemical staining or immunocytochemical staining of survivin and EGFR in tumor tissue specimens, then measuring the ratio of the number of survivin positive cells and EGFR positive cells to the total number of observed cells, and determining the malignancy of the tumor from that value The method for determining the malignancy of a tumor according to claim 3.   The method according to any one of claims 1 to 4, wherein the tumor is glioma and the tissue is brain tissue.   A tumor diagnostic kit for detecting a tumor using survivin and EGFR as markers.   The tumor diagnostic kit according to claim 6, which comprises at least anti-survivin antibody and anti-EGFR antibody, and detects tumor using survivin and EGFR as markers.   The tumor diagnostic kit according to claim 6 or 7, wherein the tumor is glioma.

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