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    • 专利摘要:
    The present invention provides a method for diagnosing prognosis of astrocytic tumors which comprises the step of predicting a survival rate after surgery of a patient by detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors and a method for diagnosing malignancy of astrocytic tumors which comprises the step of detecting amplification of human chromosome 7 short arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors.
    公开号:CA2288531A1
    申请号:C2288531
    申请日:1999-11-04
    公开日:2000-12-04
    发明作者:Katsumi Harada；Haruhide Ito；Kohsuke Sasaki；Takafumi Nishizaki；Hisashi Kubota；Satoshi Ozaki
    申请人:Sumitomo Electric Industries Ltd；
    IPC主号:C12Q1-68
    专利说明:
    SPECIFICATION A method for diagnosing astrocytic tumors by detecting chromosomal aberration The present invention relates to a method for diagnosing astrocytic tumors, more particularly a method for diagnosing malignancy or prognosis of astrocytic tumors. More specifically, the present invention relates to a method for diagnosing astrocytic tumors by detecting amplification of a specific region of chromosome derived from a patient. Cancer is a disease where some cells abnormally grow and invade into surrounding normal tissues to destroy their normal functions. Since 1981, cancer is the primary factor of death in Japan, and it is socially necessary to rapidly establish a method for effectively diagnosing and treating the disease.Up to date, a variety of research on cancer have been made in gene level. As a result, it has been revealed that deletion or amplification of a part of chromosome correlates to carcinogenesis . For example, it has been found that Rb gene of human chromosome 13 is always deficient in retinoblastoma, and it has been found that p53 gene of human chromosome 17 short arm is deficient in a variety of cancers. These genes are tumor suppressor gene and it is thought that deletion of these genes causes carcinogenesis. Further, it is found that amplification of a specific region of the chromosome occurs in certain cancers . Recently, a theory is proposed that cancer arises from multi-step mutations of deletion and/or amplification of not one but several genes in genome. As mentioned above, relationship between carcinogenesis and chromosome aberration has been actively studied, but the relationship has not been fully revealed and mechanism of carcinogenesis has not yet been revealed. As one approach to examine relationship between carcinogenesis and chromosome aberration, a method for analyzing DNA of cancer cells using CGH (comparative genomic hybridization) method is known. For example, some unique structural changes of chromosomes have been reported in meningioma, astrocytic tumors, biliary region tumors, clear cell adenocarcinoma of the ovary, non-clear cell adenocarcinoma of the ovary, cancer of the uterine body, hepatocellular carcinoma, pituitary tumor, gastric cancer, esophagus squamous cell carcinoma and mouth squamous cell carcinoma, and among them, as to malignant astrocytic tumors, amplification of chromosome 7 short arm, chromosome 7 long arm, chromosome 20 long arm and chromosome 8 long arm and deletion of chromosome 10 short arm, chromosome 9 short arm, chromosome 10 long arm, chromosome 13 long arm and chromosome 22 long arm have been frequently observed. However, useful structural change of the above-mentioned chromosomes in diagnosing malignancy or prognosis of astrocytic tumors has not been determined. Analysing the above-mentioned chromosomal information in detail must be useful to appropriate diagnosing astrocytic tumors. Sum_m__ary of the Invent son One object to be achieved by the invention is to reveal relationship between specific structural change of the chromosome in astrocytic tumors, more specifically the presence or absence of amplification of chromosome 7 short arm or amplification of chromosome 8 long arm, and malignancy or prognosis of astrocytic tumors. In addition, another object to be achieved by the present invention is to provide a method for diagnosing malignancy or prognosis of astrocytic tumors by revealing relationship between a specific structural change of the chromosome and malignancy or prognosis of astrocytic tumors . Furthermore, another object to be achieved by the present invention is to provide a method for diagnosis which can be conveniently performed in a single step. In order to achieve the above-mentioned objects, the present inventors analyzed DNA of cancer cells of astrocytic tumors at various stages using CGH(comparative genomic hybridization) method. As a result, it has been found that detection of the presence or absence of amplification of chromosome 7 short arm or amplification of chromosome 8 long arm is useful for diagnosis of malignancy and prognosis of astrocytic tumors, and the present invention has been completed. Thus, according to the present invention, a method for diagnosing astrocytic tumors is provided, which comprises the step of detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors. In one embodiment of the present invention, the method for diagnosing prognosis of astrocytic tumors is provided, which comprises the step of predicting a survival rate after surgery of a patient by detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient at grade IIIor grade IV of astrocytic tumors. In another embodiment of the present invention, the method for diagnosing malignancy of astrocytic tumors is provided, which comprises the step of detecting amplification of human chromosome 7 short arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors. In further another embodiment of the present invention, the method for diagnosing astrocytic tumors is provided, which comprises the step of detecting amplification of human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient at grade II of astrocytic tumors. As one embodiment of carrying out the method for diagnosis of the present invention, the method for diagnosis is provided, which comprises the steps of:competitively hybridizing DNA of cancer cells derived from a patient which is labeled with a first label and DNA of normal cells which is labeled with a second label to chromosomes of normal cells; and observing a chromQSOmal region to which DNA of cancer cells is hybridized by the first label, observing a chromosomal region to which DNA of normal cells is hybridized by the second label, and observing a chromosomal region to which both DNAs are hybridized by a mixture of the first and second labels, thereby distinguishing the labels in each region so as to detect the amplification. As another embodiment of carrying out the method for diagnosis of the present invention, the method for diagnosis is provided, which comprises the steps of:competitively hybridizing DNA of cancer cells derived from a patient which is labeled to be detected as a first color and DNA of normal cells which is labeled to be detected as a second color to chromosomes of normal cells; and observing a chromosomal region to which DNA of cancer cells is hybridized by the first color, observing a chromosomal region to which DNA of normal cells is hybridized by the second color, and observing a chromosomal region to which both DNAs are hybridized by the third color formed by mixture of the first and second colors, thereby distinguishing the colors in each region so as to detect the amplification. Fig. 1 is a graph which shows survival rate after surgery of patients having amplification of chromosome 7 short arm or amplification of chromosome 8 long arm. Fig.2 is an illustration which shows a result of classification by age of patients having amplification of chromosome 7 short arm or amplification of chromosome 8 long arm. Fig.3 is an illustration which shows a proliferative activity (MIB-1 index) of cancer cells in patients having amplification of chromosome 7 short arm or amplification of chromosome 8 long arm.prPfPrrP~ Embodiments for Carrying out the Invention The method for carrying out the invention is described in detail below. The method for diagnosing astrocytic tumors according to the present invention is characterized in that aberration of chromosome region specific for astrocytic tumors is detected. Therefore, the diagnosis of the present invention may be carried out by any means which is capable of detecting change of chromosomal region, aberration of which is intended to be detected. Thus, in the method for diagnosis of astrocytic tumors according to the present invention, any method may be used which can detect amplification of human chromosome 7 short arm or human chromosome 8 long arm. As such a method for detecting chromosomal aberration, in situ hybridization may be used. The in situ hybridization is a method where a labeled nucleic acid probe is hybridized with DNA of chromosomes and then, the label is detected, thereby the chromosomal region to which the nucleic acid probe was hybridized is revealed. By using this method, the probe which was prepared by labeling genome DNA obtained from cells of tumor tissues of a patient can be hybridized to chromosomes of normal cells . The chromosomal region of normal cells where the probe could not be detected represents a region which is deleted in the chromosomes of cancer cells . The probe which was prepared by labeling genome DNA obtained from normal cells can be hybridized to chromosomes prepared from tumor tissues of patients. The chromosomal region of cancer cells to which the probe did not hybridize represents a region which is amplified in the chromosomes of cancer cells. However, when an excess amount of probe is used, the probe may be hybridized also to an amplified region and therefore the amount of the probe to be used must be suitably adjusted in such a way that amplified region can be detected. As mentioned above, in the case where either one of deletion or amplification of chromosomes of cancer cells is detected, in situ hybridization method can be effectively applied. When it is necessary to simultaneously detect deletion and amplification of chromosomes of cancer cells in a single procedure, CGH method is preferably used. In the method for diagnosis of astrocytic tumors of the present invention, CGHmethod can be used. In CGH method, DNA labeled with a first label and another DNA labeled with a second label are simultaneously hybridized to chromosomes, and the chromosomal region to which each DNAwas hybridized is revealed by detecting the 2 labels respectively (Du Manoir, S. et al., Hum. Genet., 90:590-610, 1993 ) . In the method for diagnosis of the present invention, DNA probe prepared from DNA of cancer cells of a patient and labeled with a first label and DNA probe prepared from DNA of normal cells and labeled with a second label are simultaneously hybridized to chromosomes of normal cells. In the regions amplified in cancer cells among the chromosomes, the first label is observed since DNA of cancer cells are dominantly hybridized. In the regions deleted in cancer cells, the second label is observed since only DNA of normal cells are hybridized. In the regions unchanged in cancer cells and normal cells, the first and second labels are observed as a mixed color since both DNAs are competitively hybridized. Thus, deletion and amplification of chromosomes in cancer can be detected in a single procedure. In the following, the method for diagnosis of the present invention using CGH method which is a preferred embodiment of the present invention is specifically described. DNA of cancer cells used in the method for diagnosis of the present invention is prepared from the cells collected from tumor tissues of a patient with suspected astrocytic tumors. The terms "cancer cells" and "tumor tissues" of a patient used herein include cells and tissues which are already in a cancerous state as well as those which are in a precancerous state. The term "astrocytic tumors" used herein has a broad sense which includes malignant astrocytic tumors, primary malignant astrocytic tumors and metastatic malignant astrocytic tumors. DNA of normal cells used in the method for diagnosis of the present invention is DNA of cells derived from a healthy donor having no cancer. The type of tissues and cells to be collected is not particularly limited. The method for diagnosis of the present invention can be carried out without problem, so far that DNA of cancer cells and DNA of normal cells to be prepared contain the segments corresponding to the chromosomal region, amplification of which is to be detected ( namely, chromosome 7 short arm or chromosome 8 long arm) . However, genome DNA is usually used in order to avoid complexity of selecting DNA of a specific chromosomal region. As genome DNA, a full length genome may be used, but it is preferred to use a smaller DNA ( for example, within 10-1000kb in length) which was obtained from the collected cells by using a suitable restriction enzyme according to a method known to a skilled person in the art. The length of each DNA used in hybridization as a probe is preferably 200-1000 bp. However, DNA having a length out of this range may be used. DNA of cancer cells and DNA of normal cells are respectively labeled with different labels (a first label and a second label ) before hybridization. The type of the labels is not particularly limited so long as each label can be detected distinguishably with each other after hybridization. Therefore, spectrally, biochemically, chemically or immunologically detectable labels can be broadly used. For example, it is possible to use fluorescent dyes, electron density reagents, enzymes, biotin, digoxygenin and the like. A preferred label is those which are colored and can therefore be visually recognized. Among them, it is particularly preferred that a combination of labels are selected in such a way that the color of a first label and the color of a second label are mixed to form a third color which is distinguishable from these two colors. For example, by selecting a label of which color is green as a first label and a label of which color is red as a second label, yellow can be observed when two colors are mixed. By choosing labels in such a way that 3 colors which are distinguishable from each other are formed, and hybridizing DNA, a region which is deleted in cancer, one which is amplified in cancer and one which is unchanged in cancer can be visually and easily distinguished. Examples of fluorescent dye of green which can be used in the present invention include FITC (fluorescein isothiocyanate). Examples of fluorescent dye of red include TRITC (tetramethylrhodamine isoth,iocyanate), rhodamine and Texas Red. The condition under which DNA of cancer cells and DNAof normal cells are hybridized to chromosomes is not particularly limited. As chromosomes to which DNA of cancer cells and DNA of normal cells are hybridized, chromosome samples prepared from cells such as lymphocytes can be used. Also, hybridization may be performed using unfixed chromosomes and then, the chromosomes may be dropped onto a slide glass or etc. , and fixed. The method of analysis after hybridization is appropriately determined depending on the type of the labels . When fluorescent label is used as a label, analysis can be performed by visually observing the sample using high resolution fluorescence microscopy and the like. Also, the image observed by fluorescence microscopy is read by a color image scanner and the colors at each point are resolved into 3 colors, and quantification can be performed as to which of the colors of green and red is major at each point ( namely, ratio of each labeling material at each point can be determined). As mentioned above, malignancy or prognosis of astrocytic tumors can be diagnosed by detecting amplification of specific chromosome (namely, chromosome 7 short arm or chromosome 8 long arm) in the chromosomes of tissues of astrocytic tumors derived from a patient according to the present invention. Namely, as is understood from the following Examples ( see Fig. 1 ) , it has now been found that the survival rate after surgery of a patient with the amplification of chromosome 7 short arm is generally lower than that of a patient with the amplification of chromosome 8 long arm. Therefore, a survival rate after surgery can be predictable by examining the presence or absence of amplification of chromosome 7 short arm or amplification of chromosome 8 long arm before surgery, and this method can be a useful tool for judgment of necessity of surgery. Also, as is understood from the following Examples ( see Fig.3 ) , it has now been found that cells with amplification of chromosome 7 short arm shows a significantly high proliferative activity (MIB-1 index) than cells with amplification of chromosome 8 long arm. Therefore, when amplification of human chromosome 7 short arm is detected in the chromosomes of cancer cells derived from a patient with astrocytic tumors, it can be diagnosed that the malignancy of this astrocytic tumor is generally high. MIB-1 index indicates a distribution of Ki-67 nuclear antigen, which distribution and amount of expression change with cell cycle, as a frequency of cells at mitonic growth phase, by an immuno-staining method using anti-Ki-67 antibody. The determination of MIB-1 index can be carried out by preparing a suitable cell slice and then immuno-staining the slice with anti-Ki-67 ( MIB-1 ) antibody ( for example, DAB method or ABC method). MIB antigen is present in nucleolus, and although it is not intended to be bound by any theory, it is thought that the determination of MIB-1 index reflects a determination of ratio of cells in cell growth state in either staining intensity or staining positivity (pattern). Also, p53 can be stained using anti-p53 antibody, and co-staining using both anti-Ki-67 (MIB-1 ) antibody and anti p53 antibody is thought to be useful for evaluation of malignancy. Furthermore, as is understood from the following Examples, in the chromosomes of the patients in the cases of Grade II of astrocytic tumors which turned to be malignant, it has been found that amplification of human chromosome 8 short arm was observed in a high frequency ( 75$ ) , while amplification of chromosome 7 short arm was not observed. Therefore, it is considered that the cases of astrocytic tumors at Grade II which show amplification of chromosome 8 long arm may probably become malignant and therefore need an active therapy. By the method for diagnosis of the present invention based on the above findings, early diagnosis of astrocytic tumors becomes possible, and it is expected that an effective cancer therapy can be achieved. As a specific region of chromosome to be used in the diagnosis, a more limited region (locus) is preferred. For example, effect of background can be decreased, and as a result, more reliable diagnosis can be achieved by using amplification of chromosome 8q24.1-24.2 in the diagnosis than using amplification of chromosome 8 long arm in the diagnosis. Moreover, by limiting the locus on the chromosome, DNA of this locus can be efficiently amplified by using YAC vector etc. Thus, the limitation of locus on the chromosome may be qualitatively different from the designation of either of short or long arm of the chromosome. Therefore, it is preferred in the present invention that detection of amplification of chromosome 7p12-13 or chromosome 8q24.1-24.2 is used to diagnose astocytic tumors. Moreover, according to the present invention, the prognosis of a patient can be objectively predicted, and a therapy which is thought to be the most suitable to each patient can be selected. Namely, an active therapy such as the use of anti-tumor agent or radiation may be applied to a patient who was judged as poor prognosis, and such a therapy need not be applied to a patient who was judged as good prognosis. This improves QOL of cancer patients and also gives a great benef it in view of medical economy. Thefollowing Examples illustrate the present invention more in detail, but the scope of the present invention is not limited by the Examples. Example 1: Analysis of chromosomal aberration in astrocytic tumors (1) Preparation of chromosome sample Using cells having normal chromosomes (blood cells of normal person), a chromosome sample was prepared by the following procedure.lOml of blood was collected from each person, and was diluted approximately 1 .5 times with culture medium (RPMI 1640, 10$FCS, PHA(20,u 1/ml)). The diluted blood was gently poured into a tube (Ficol Paque: Pharmacia), and centrifuged at 2, OOOrpm for 20 minutes . The layer of leukocytes was taken out, and dilution with the same medium and centrifugation at 1,200 rpm for 5 minutes were repeated twice . The same medium was added to the sample and was fully stirred, and then 15 ml of sample was placed in each of 2 dishes of lOcm, and cultivation was started. At 48 hours after cultivation was started, thymidine (Sigma) was added to a final concentration of 300 ,u.g/ml. After cultivation for 18 hours, the washing with the same medium by centrifugation at 1,200 rpm for 5 minutes was repeated three times. After cultivation for 6 hours, colcemid (Gibco) was added to a final concentration of 0.05 ,ug/ml. After 1 hour, the same medium was added to the sample, and the sample was washed by centrifugation at 1,200 rpm for 5 minutes, and was subjected to hypotonic treatment with 3-5m1 of 0.075 M KC1 for 30 minutes at room temperature. Carnoy's fixative (methanol : acetic acid - 3:1) was then added several times until the color of erythrocytes disappeared whereby the cells were fixed. The fixed cells were dropped onto slides and stored at -80°C until use.(2) Preparation of genome DNA Using tumor tissues from patients with astrocytic tumors (49 patients; 4 patients of grade I, 10 patients of grade II, 15 patients of grade I II and 20 patients of grade IV ) , genome DNA was prepared by the following procedure. The solid tumor tissue of 5mm square or more was moistened with PBS and cut into small pieces with an ophthalmologic scissors . The cell suspension was charged into a syringe ( lml ) and discharged from it and this procedure was repeated several times, and then the cell suspension was filtered through nylon mesh. The cell suspension was poured into a centrifugation tube and 0.2% NaCl was added thereto and the tube was placed for a few minutes. The suspension was centrifuged at 2,000 rpm for minutes, and then the supernatant was discarded and the precipitate was resuspended in a small amount of PBS . The cell suspension was placed in a microtube and centrifuged at 2, OOOrpm for 5 minutes, and the supernatant was discarded. Then, DNAwas prepared using the nucleic acid extracting reagent SEPAGENE ( trade name, Sanko Juuyaku Inc . ) in accordance with its instructions. Also, genome DNA for use as DNA of normal cells was prepared from lymphocytes of blood from a healthy donor in accordance with the following procedure. 20 ml of blood with anti-coagulant, heparin, was collected from a healthy donor, and was diluted twice with PBS . The diluted blood was gently layered on 30 ml Ficoll solution in a centrifugation tube, and placed for 10 minutes . Then, the tube was centrifuged at 2, 000 rpm for 20 minutes to form a milk white layer of lymphocytes under a pale yellow layer. The milk white layer was taken into another centrifugation tube containing 30 ml Ficoll solution and PBS was added thereto. The tube was centrifuged at 2, 000 rpm for 5 minutes and the supernatant was removed by aspirator.3 ml of 0.2% NaCl was added to cause hemolysis, and the sample was centrifuged at 2, 000 rpm for 5 minutes, and the supernatant was removed by aspirator. Then, DNA was prepared using the nucleic acid extracting reagent SEPAGENE(trade name, Sanko Juuyaku Inc.) in accordance with its instructions.(3) Preparation of labeled DNA DNA of astrocytic tumor cells was labeled with FITC which emits green fluorescence, and DNA of normal cells was labeled with TRITC which emits red fluorescence. DNA was labeled by mixing dTTP, dNTP mixture, labeled d-UTP, template DNA, nick translation buffer, nick translation enzyme and water and incubating the mixture for an appropriate period in accordance with the instructions of Nick Translation Kit (Vysis).(4) Hybridization (FISH method) (A) Preparation of single strand DNA probe Human COT-1 DNA (synthetic DNA for suppressing non-specific hybridization; 20,u1; 400ng of DNA) , TRITC-labeled DNAof normal cells (10,u1; 200ng of DNA), FITC-labeled DNA of astrocytic tumor cells (10~C,cl; 200ng of DNA), sodium acetate ( 4,u1 ) and 100 ethanol ( lml ) were mixed and centrifuged 14, 000 rpm for 30 minutes at 4°C. The supernatant was discarded by decantation and was fully dried in the dark. Then, 10 ,u 1 of Master Mix #1 was added and fully pipetted. Then, the sample was treated at 75 °C for 5 minutes to prepare a single strand sample.(B) Preparation of single-stranded chromosome sample DNA of chromosome sample was treated with 70~formamide/2xSSC (pH7.0) at 75°C for 2.5 minutes to be single stranded. The sample was then successively treated with 70~ethanol, 85~ ethanol and 100 ethanol each for 2 minutes to be dehydrated, and was air-dried. By analyzing the location and number of bands on the chromosome, it was confirmed that the chromosome was normal one.(C) Hybridization Onto a slide glass of the chromosome sample was mounted 9.5 ,u 1 of the single-stranded probe-mix respectively. The glass was covered with a cover glass of 18x18 mm, and shielded with a paper bond. The glass was placed on a hot plate for about minutes, and hybridization was performed in COZincubator at 37°C for 2-3 days. After hybridization, washing the sample with 50$formamide/2xSSC (pH7.0) at 45°C for 10 minutes was repeated three times. Then, washing the sample with 2xSSC at 45°C for 10 minutes and with PN buffer at room temperature for 10 minutes was repeated twice, and washing the sample with a distilled water at room temperature for 5 minutes was repeated twice. Then, 8 ,ul of 0.1-0.2mg/ml DAPI-antifade was mounted on the sample, and the sample was covered with a cover glass of 18x18 mm.
    (5) Fluorescence microscopic analysis The slide glass was analyzed under fluorescence microscopy, and fluorescence microscopic photography was done(color photograph). In each chromosome, the regions with green corresponds to regions which indicates DNA amplification specific for astrocytic tumors. As a result, the sample could be classified into the group with amplification of chromosome 7 short arm ( 17 cases in total;cases of Grade I I I and 12 cases of Grade IV ) , the group with amplification of chromosome 8 long arm (11 cases in total; 4 cases of Grade II, 2 cases of Grade III and 5 cases of Grade IV), and the other group. Further, the patients with amplification of chromosome 7 short arm and those with amplification of chromosome 8 long arm were classified by age, and the results as shown in Fig.2 were obtained. As is understood from the results in Fig.2, the group with amplification of chromosome 8 long arm was observed frequently in younger generation (p=0.018). The common region of amplification was chromosome 7p12-13 and chromosome 8q24.1-24.2. In the cases of Grade II which turned to be malignant, amplification of chromosome 8 long arm was observed in a high frequency ( 75$ ) , and it was considered that the cases of Grade II which show amplification of chromosome 8 long arm tend to be malignant and therefore need an active treatment. Further, it was found that 40% of the cases of Grade IIIand Grade IV with amplification of chromosome 8 long arm can survive for more than 80 months. Example 2: Analysis of the presence or absence of p53 accumulation in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 of long arm As mentioned hereinbefore, the co-staining of MIB-1 and p53 is considered to be useful for the evaluation of malignancy, and the increase or decrease of p53 (the increase of mutated p53 or the decrease of normal p53 ) is considered to be indicative for carcinogenesis. Therefore, the presence or absence of p53 ( tumor suppression gene product ) accumulation in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 long arm were analyzed by immunostaining. Specifically, the following procedure was performed using anti-p53 antibody. Tissue samples prepared from each subject were subjected to deparaffin treatment (twice with xylene each for 5 minutes, with 100% ethanol for 3 minutes, with flowing water for 5 minutes, and with PBS for 5 minutes ) , and then were subjected to microwave treatment if necessary. Then, the samples were treated with methanol containing 0.3% hydride peroxide (hydride peroxide 0 . 5m1; pure methanol 50m1 ) at room temperature for 20 minutes for the treatment of endogenous peroxidase inhibition, and washed with flowing water for 5 minutes, and washed with cold PBS three times each for 5 minutes. 100,u,1 of 10% normal goat serum solution in PBS was prepared per each sample and used to treat the samples in a moisture box at room temperature for 30 minutes and the serum was removed.100,u1 of the first antibody (anti-p53 antibody, Dako) diluted with PBS (approximately 50-100 times for monoclonal (mouse IgG) antibody, or approximately 200-1000 times for polyclonal ( rabbit IgG ) antibody ) , was prepared per the sample, and was added to the samples and reacted in a moisture box either at room temperature for 60 minutes or 4°C overnight. After reaction with the first antibody, the samples were washed with cold PBS three times each for 20 minutes.100,U.1 of the secondary antibody (biotinated anti-mouse IgG or biotinated anti-rabbit IgG antibody) diluted 100 times with PBS was prepared per the sample, and was added to the samples and reacted in a moisture box at room temperature for 30 minutes . After reaction with the secondary antibody, the samples were washed with cold PBS three times each for 10 minutes.100,u1 of a solution of avidin-peroxidase contained in the avidin/biotin complex kit was prepared per the sample 30 minutes before use. This solution was added to the samples and reacted in a moisture box for 60 minutes . After reaction, the samples were washed with cold PBS three times each for 20 minutes. DAB tablet ( 1 tablet ) was dissolved in a distilled water (50m1) and 3-4 drops of hydrogen peroxide were added thereto immediately before use. The samples were placed in the DABsolution, and reacted for 5 minutes . After color of the samples becomes brown, the samples were washed with flowing water for minutes to stop the reaction. The samples were washed with flowing water for 5 minutes, and treated with Meyer's hematoxylin for 1 minute, and washed with flowing water for 5 minutes. The samples were treated with 70$ absolute ethanol and then with carbol xylene each for 5 minutes, and the treatment with xylene for 5 minutes was repeated twice, and the sampled were sealed. The obtained results are shown below. Table 1: Relationship of amplification of chromosome 7 short arm or amplification of chromosome 8 long arm with p53 accumulation p53 number of the cases number of the cases accumulation with amplification of with amplification of chromosome 7 short arm chromosome 8 lonc~~ arm absence(-) 15 3 _resence ~( + ) 2 6 As is understood from the results in Table 1, the decrease of p53 was observed in a significant high frequency (p=0.008) in the cases with amplification of chromosome 7 short arm. Therefore, it is suggested that the cases with amplification of chromosome 7 short arm having tendency of decreased p53 tend to be malignant. In other words, p53 is one of the tumor suppression genes, and it is considered that the greater the expression (accumulation) becomes, the more the cell is benign. Example 3: Analysis of the survival rate after surgery in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 long arm The survival rate after surgery in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 long arm was analyzed. The results obtained are shown in Fig.l. As is understood from the results in Fig.l, in the cases of Grade IIIor Grade IV, the survival rate of the cases with amplification of chromosome 7 short arm was s ignif icantly lower than that with amplification of chromosome 8 long arm ( p=0 . 035 ) . In the cases with amplification of chromosome 8 long arm, the cases of long survive and the cases showing growth inhibition by radio-chemotherapy were found. Example 4: Analysis of proliferative activity in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 long arm MIB-1 index (as defined below) was analyzed in the cases with amplification of chromosome 7 short arm or amplification of chromosome 8 long arm. MIB-1 index = number of cells stained with MIB-1 antibody/total number of cells (in certain visual field) The procedure was the same as in Example 2 except in that anti-MIB-1 antibody (Immunotech Inc.) was used in place of anti-p53 antibody. The results obtained are shown in Fig.3. As is understood from the results in Fig.3, the cases with amplification of chromosome 7 short arm had a significantly higher MIB-index than that with amplification of chromosome 8 long arm, and had a greater proliferative activity. Moreover, the cases with a high value of MIB-1 index had a tendency of showing a rapid growth of tumors. According to the present invention, it has been revealed that it is possible to diagnose malignancy or prognosis of astrocytic tumors by detecting amplification of specific regions of chromosome from the tissue of astrocytic tumors, i.e. , chromosome 7 short arm or chromosome 8 long arm. Namely, according to the present invention, it has been revealed that it is possible to predict a survival rate after surgery of a patient by detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors. More particularly, it has been found that, in Grade III or IV, the survival rate after surgery of the patients with amplification of human chromosome 8 long arm is higher than those with amplification of human chromosome 7 short arm, and this information is useful for diagnosing prognosis of astrocytic tumors. Moreover, according to the present invention, it has been revealed that the proliferative activity is significantly higher in the case where amplification of human chromosome 7 short arm was detected in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors, than in the case where amplification of human chromosome 8 long arm was detected, and therefore, it has become possible to diagnose malignancy and prognosis of astrocytic tumors by detecting amplification of human chromosome 7 short arm. Furthermore, according to the present invention, it has been revealed that the cases with amplification of human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors of Grade II has a high tendency of being malignant, and therefore it has become possible to diagnose astrocytic tumors, in particular diagnose whether further therapy is necessary or not, by detecting amplification of human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors of Grade II.
    权利要求:
    Claims (6)
    [1] 1. A method for diagnosing astrocytic tumors which comprises the step of detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors.
    [2] 2. The method of claim 1 which is a method for diagnosing prognosis of astrocytic tumors which comprises the step of predicting a survival rate after surgery of a patient by detecting amplification of human chromosome 7 short arm or human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient at grade III or grade IV of astrocytic tumors.
    [3] 3. The method of claim 1 which is a method for diagnosing malignancy of astrocytic tumors which comprises the step of detecting amplification of human chromosome 7 short arm in the chromosomes of cancer cells derived from a patient suffering from astrocytic tumors.
    [4] 4. The method of claim 1 which is a method for diagnosing astrocytic tumors which comprises the step of detecting amplification of human chromosome 8 long arm in the chromosomes of cancer cells derived from a patient at grade II of astrocytic tumors.
    [5] 5. The method for diagnosis of any one of claims 1-4 which comprises the steps of:competitively hybridizing DNA of cancer cells derived from a patient which is labeled with a first label and DNA of normal cells which is labeled with a second label to chromosomes of normal cells; and observing a chromosomal region to which DNA of cancer cells is hybridized by the first label, observing a chromosomal region to which DNA of normal cells is hybridized by the second label, and observing a chromosomal region to which both DNAs are hybridized by a mixture of the first and second labels, thereby distinguishing the labels in each region of the chromosome so as to detect amplification of human chromosome 7 short arm or human chromosome 8 long arm.
    [6] 6. The method for diagnosis of any one of claims 1-4 which comprises the steps of:competitively hybridizing DNA of cancer cells derived from a patient which is labeled to be detected as a first color and DNA of normal cells which is labeled to be detected as a second color to chromosomes of normal cells; and observing a chromosomal region to which DNA of cancer cells is hybridized by the first color, observing a chromosomal region to which DNA of normal cells is hybridized by the second color, and observing a chromosomal region to which both DNAs are hybridized by the third color formed by mixture of the first and second colors, thereby distinguishing the colors in each region of the chromosome so as to detect an amplification of human chromosome 7 short arm or human chromosome 8 long arm.
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    引用文献:
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    法律状态:
    2003-11-04| FZDE| Dead|
    优先权:
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    US32562999A| true| 1999-06-04|1999-06-04||
    US09/325,629||1999-06-04||
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