WO2001077385A1 - Method of detecting cancer - Google Patents

Abstract

A technique for conveniently and quickly evaluating the malignancy of cancer (in particular, gastric cancer) or detecting cancer (in particular, gastric cancer) at a high reliability. A method of evaluating the malignancy of cancer (in particular, gastric cancer) or a method of detecting cancer (in particular, gastric cancer) based on the expression doses of at least 8 genes, each showing a change in expression dose depending on the malignancy of gastric cancer, or difference in expression between a test sample and a control sample; a kit therefor; and an array wherein the above genes are immobilized on a support.

Description

 Specification

 Method of detecting cancer

 The present invention provides a method for easily and quickly evaluating cancer malignancy with high reliability, a method for evaluating malignancy of cancer, particularly gastric cancer, an array and a kit used for the evaluation method, simple and high reliability The present invention relates to a method for detecting cancer, particularly gastric cancer, and a kit therefor. Background art

 Cancer has been the leading cause of death in Japan since 1981, with gastric cancer being the most frequent cancer. In recent years, it has been clarified that there is a multi-stage carcinogenesis mechanism until normal cells become cancerous [Phiron ER (Fearon. ER) et al., Cell, Vol. 61, No. 759- Pp. 767 (1992), Sugimura T., Science, 258, 603-607 (1992)]. Specifically, canceration of normal cells requires accumulation of multiple genetic abnormalities, including DNA repair genes, tumor suppressor genes, and oncogenes. In general, gene instability and inactivation of tumor suppressor genes are thought to be involved in the development of cancer. Also, activation of cancer genes and overexpression of Z or growth factors are thought to be involved in cancer progression and malignancy. Furthermore, genes encoding genes that degrade extracellular matrix molecules and genes encoding proteins that regulate cell motility or adhesion are thought to be involved in translocation and invasion.

Gene instability includes gene instability associated with abnormalities in the DNA mismatch repair system and chromosomal instability. As an example of the former, the chain length of the simple repetitive sequence existing in the genome is different between the cancerous part and the non-cancerous part of the same individual (microsatellite instability) [Thibodeau. SN, et al., Science, Vol. 260, pp. 8 16—8 19 (1993)], and as an example of the latter, And rearrangement between chromosomes. The rearrangement between the chromosomes causes the expression of a protein that is not found in normal cells. On the other hand, even if the protein is expressed in normal cells, it may affect the expression level. In fact, in human chronic myeloid leukemia, rearrangement between chromosomes caused fusion of the bcr and c-abl genes, confirming the expression of hybrid mRNA transcribed from the bcr-abl fusion gene, which is not present in normal cells. Have been. Furthermore, it has been confirmed that leukemia develops when the bcr-abl fusion gene is introduced into animals [Watson, JD, et al., Molecular Biology of Recombinant DNA, Second Edition; 9 pages (1992)].

 Examples of inactivation of the tumor suppressor gene include inactivation of the P53 gene. The cause of inactivation is thought to be a deletion in the gene or a point mutation that occurs in a specific part of the coding region (Nigro, JM et al., Nature, Vol. 342. 705-708 (19989), Malkin D. et al., Science, 250, 1233-123 (1990)]. Also, deletions and point mutations in the p53 gene have been observed in many types of cancer. For example, in gastric cancer, the deletion and point mutation of the p53 gene are found in 60% or more cases of early stage cancer (Yokozaki H., et al., Journal of Cancer Research and Clinical). Oncology (Journal of Cancer Research and Clinical Oncology), Vol. 119, pp. 67-70 (1992)) o

On the other hand, the P16 / MTS1 gene is known as a gene that is inactivated by homo-deletion. Specifically, high frequency homozygous deletions have been observed in gliomas, spleen cancer, bladder cancer, etc. [Cairns, P., et al., Nature genetics, 1st ed. Volume 1, pages 210-212 (1995)]. pl6 protein regulates the cell cycle. It has been suggested that the abnormal expression of P16 is involved in canceration of cells (Okamoto A. et al., Proceedings of the National Academy of Sciences of the USA (Pr. oceedings of the National Academy of Sciences of the United States of America). Vol. 91, No. 11045-11049 (1994)].

 Activation of an oncogene includes, for example, insertion mutation of a virus near an oncogene or rearrangement between chromosomes. For example, viral insertion mutations have been identified in chicken lymphomas caused by the avian leukosis virus (ALV). In this case, ALV DNA is inserted in the vicinity of C-myc, which is a kind of gene, and normal C-myc is overexpressed by strong virus enhancer and oral motor, It was confirmed that different new sequences were expressed. In some types of human B-cell tumors, c-myc, an oncogene, is placed under the strong transcription signal of immunoglobulin due to interchromosomal rearrangement, and its expression level increases. It has been confirmed that In this case, the c-myc expression product in cancer cells did not differ from that expressed in normal cells, and it is considered that canceration was caused by an increase in the expression level of c-myc [Watson, JD, et al., Molecular Biology of Recombinant DNA, 2nd Edition; Maruzen Co., Ltd., pp. 305-308 (1992)].

 Examples of overexpression of a growth factor include overexpression of C-Met encoding a hepatocyte growth factor receptor. This abnormal expression of C-Met is not observed in normal mucosa from the early stage of gastric carcinogenesis.The expression of mRNA having a length of 6.0 kilobases was observed. (Kuniyasu.H. , Et al., International Journal of Cancer ^ Vol. 55, pp. 72-75 (1993)], Gene amplification is frequently observed in metastatic cancer, and gene amplification and cancer A correlation has been confirmed with the degree of malignancy of tumors [Kunias H. et al., Biochemical and Biophysical Research Communications ^ Vol. 189, pp. 227-232 (1992) ) 0

Examples of correlations between genetic abnormalities and cancer malignancy include c-Met above Amplification and / or overexpression of the oncogene C-erbB2 gene in breast cancer, ovarian cancer, gastric cancer, uterine cancer, etc. [Wright C. (Wright) et al., Cancer Research, Vol. 49, No. 2087- 209 p. (19989), Safari B. (Saffari.B.) Et al., Cancer Research, Vol. 55, 56.93-- 56.98 p. (1995)], 1 tissue of gastric cancer Of the oncogene K-sam gene and overexpression of Z or its overexpression in low-differentiated adenocarcinoma of the type [Tahara, E. et al., Gastric Cancer, Tokyo Springer (Springer-Ver lag), published in 1993, pp. 209-217], respectively. In order for cancer cells to invade tissues and metastasize, extracellular matrix must be degraded, and various enzymes belonging to the matrix meta-oral proteinase (MMP) family, representative of degrading enzymes, are in excess compared to normal cells. [Goldberg GI. Et al., Cancer Treatment Research, Vol. 53, pp. 701-708 (1991)]. In gastric cancer, it has been reported that the mRNA of matrilysin (MMP-7), which is expressed relatively specifically by cancer cells, was overexpressed in more than 60% of the cases [Senoyu A. et al. Clinical & Experimental Metastasis, Vol. 16, pp. 31-321 (1998)].

Malignant tumors cannot grow unless tumor blood vessels are regenerated, and they can grow at an accelerated rate by inducing angiogenesis. Tumor angiogenesis is controlled by the expression balance of various regulatory factors, angiogenesis promoting factors and suppressors, but the most important factor is vascular endothelial cell growth factor VEGF. A positive correlation has been found between VEGF expression and enhanced angiogenesis in various solid tumors [Shiva M et al., Advanced Cancer Research, Vol. 67, pp. 281 (19) 9 5)] In addition, it has been reported that the prognosis of VEGF-high expressing tumors is poor in gastric cancer, esophageal cancer, colorectal cancer, etc. [Toy M et al., Molecular Medicine, Volume 35, Vol. 1206—1215 pages ( 1 9 9 8) D o

 Recently, the first report has been made that the classification of cancer, which was previously only possible by pathological diagnosis, was made possible by simultaneously measuring the expression of a large number of genes using a DNA microarray. In other words, Golab TR et al. Screened 6817 genes of a person immobilized on a DNA microarray and were able to classify acute leukemia (ALL or AML) mainly by changing the expression level of 50 genes. (Science, Vol. 28, Vol. 5, pp. 53-53 (p. 199 9)). As described above, at the level of each gene, information on genes involved in the development and progression of cancer, and information on the gene abnormality are increasing. However, since the mechanism of carcinogenesis is multi-stage and requires the accumulation of multiple mutations, the determination of a single gene or a random combination of a small number of genes is still sufficient for practical use. At present, no definitive diagnosis or prognosis has been made.

 In fact, even today, the diagnosis of cancer and the determination of its progress and differentiation are all performed by pathological diagnosis. However, pathologically, there are cases with good prognosis even for cancers showing almost the same degree of progression and differentiation, while there are cases with high malignancy that relapse or metastasize early. In some cases, there are many cases showing sensitivity to anticancer drugs and radiation irradiation, but showing resistance in other cases. Therefore, at present it is impossible to predict these before treatment or early after surgery. Disclosure of the invention

Predicting the diagnosis, progression, differentiation, etc. of cancer can prevent unnecessary treatment, greatly minimizing the burden on patients and establishing a treatment policy suitable for individual patients. Can contribute. It is also thought that this will lead to a reduction in medical expenses. Therefore, a first object of the present invention is to provide a gene that can be used as an index for determining the degree of malignancy of cancer, particularly gastric cancer, which has a large number of patients, and in particular, a large number of genes whose expression status changes as the cells become cancerous or develop. And easily and quickly assess cancer malignancy with high reliability It is an object of the present invention to provide a method for evaluating the degree of malignancy of cancer based on analysis of the expression pattern of the gene or a method for detecting cancer. Further, a second object of the present invention is to provide an array or a kit used for evaluating the above-mentioned cancer aggressiveness or detecting cancer cells.

 In order to achieve the above object, the present inventors not only individually compare the intracellular expression levels of a large number of genes in cancer tissues, particularly in cancer tissues and gastrointestinal cancer patients, but also have gene expression patterns. Analysis has revealed a large number of genes involved in malignant transformation of cancer, especially gastric cancer. The present inventors have devised an appropriate technique for conducting a pattern analysis of gene expression by a combination of these genes, and have found that it is possible to evaluate the malignancy of cancer, thereby completing the present invention.

 That is, the gist of the present invention is:

 [1] The following steps:

 (1) measuring the expression level of at least eight genes whose expression level changes in accordance with the malignancy of the cancer in the test sample; and

 (2) Step of evaluating the malignancy of cancer based on the expression level of each gene obtained in (1)

A method for evaluating the degree of malignancy of cancer, characterized by performing

 (2) a nucleic acid or a fragment thereof corresponding to at least eight genes whose expression level changes according to the malignancy of the cancer is immobilized in a predetermined region on the support, Array to evaluate,

 (3) a primer or Z or probe for detecting mRNA or a fragment thereof expressed from at least eight genes whose expression level changes depending on the grade of cancer malignancy, Evaluation or cancer detection kit,

[4] for evaluation of cancer malignancy, comprising an antibody or a fragment thereof to a polypeptide or a fragment thereof encoded by at least eight genes whose expression level changes depending on the malignancy of the cancer Or a kit for detecting cancer, and [5] Expression of at least eight genes or polypeptides encoded by the genes whose expression levels change depending on the malignancy of the cancer is examined between the test sample and the control sample. A method for detecting cancer, comprising detecting cancer cells as an indicator that the test sample contains cancer cells when there is a difference in the expression of the gene or polypeptide from the sample.

About. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a tree diagram of a classification of gastric cancer patients obtained by Hierarchical clustering using [natural logarithmic conversion value of relative expression ratio] of 105 genes.

 FIG. 2 is a tree diagram of expression patterns of each gene in each gastric cancer patient obtained by Hierarchical clustering using [natural logarithmic conversion value of relative expression ratio] of 105 genes.

 FIG. 3 is a tree diagram of classification of gastric cancer patients obtained by Hierarchical clustering using the expression intensity of 105 genes.

 FIG. 4 is a tree diagram of the expression pattern of each gene in each gastric cancer patient obtained by Hierarchical clustering using the expression intensity of 105 genes.

 FIG. 5 is a tree diagram of classification of gastric cancer patients obtained by Hierarchical clustering using [natural logarithmic conversion value of relative expression ratio] of 20 genes.

 FIG. 6 is a tree diagram of the expression pattern of each gene in each gastric cancer patient obtained by Hierarchical clustering using [natural logarithmic conversion value of relative expression ratio] of 20 genes. BEST MODE FOR CARRYING OUT THE INVENTION

As used herein, “cancer malignancy” is determined by the degree of invasion and the presence or absence of metastasis as primary factors. As a result, factors that affect the 5-year survival rate after surgery and other procedures are considered. Point.

 Specifically, cancer that remains in the mucosa and submucosa and is not metastasized is judged to be of low malignancy, and the 5-year survival rate is expected to be high (over 90%). On the other hand, if the cancer spreads deeply from the submucosa to the muscular layer, the subserosa, and the serosal surface, and the cancer has spread to lymphatic, hematogenous, invasive, or disseminated, the malignancy is judged to be high, and 5 years The survival rate is low. Among them, disseminated metastasis to the peritoneum, liver metastasis, distant lymph node metastasis, or when the cancer spreads to other organs, the prognosis is very poor, and the 5-year survival rate is low (20% Less than) . In addition, the presence or absence of sensitivity to an anticancer agent or radiation is raised as a secondary factor. If the patient has sensitivity to the anticancer agent or radiation, the degree of malignancy is low, and if the resistance is high, the degree of malignancy is determined to be high.

1. The method for evaluating the malignancy of cancer of the present invention, particularly gastric cancer

 The method for evaluating the malignancy of cancer, particularly gastric cancer, according to the present invention comprises the following steps:

 (1) measuring the expression level of at least eight genes in the test sample whose expression level changes depending on the malignancy of cancer, particularly gastric cancer; and

 (2) Step of evaluating the malignancy of cancer, particularly gastric cancer, based on the expression level of each gene obtained in (1)

One of the features is to perform According to the evaluation method of the present invention, since the expression levels of at least eight genes whose expression levels change according to the malignancy of cancer, particularly gastric cancer described below, are measured, the method is simple, and quickly and with high reliability. It has an excellent effect of being able to evaluate the malignancy of cancer, especially gastric cancer.

Examples of the test sample include a sample derived from a living body such as blood; urine; feces; and a tissue removed by a surgical technique. In the method for evaluating the malignancy of cancer, particularly gastric cancer, of the present invention, a lesion tissue collected with a biopsy forceps is desirable from the viewpoint of preoperative diagnosis. In this specification, an individual serving as a source of the test sample may be referred to as a “sample”. The gene J whose expression level changes according to the malignancy of the cancer used in the evaluation method of the present invention is a gene that can serve as an index for the evaluation of malignancy of cancer, particularly of gastric cancer, and It refers to a gene whose expression level changes along with it, in other words, a gene whose expression is significantly induced or suppressed.

 Such genes can be analyzed, for example, by analyzing the copy number of genes in the genome and the pattern of chromosomal rearrangement, comparing the expression levels of gene products in normal cells and cancerous cells, and determining the differences between the two cells. It can be detected by identifying one. Examples of the gene product include mRNA transcribed from a gene and a protein that is a translation product. In the detection of the gene used in the present invention, it is efficient to use mRNA as an index, for which various techniques have been developed for analysis with the advance of gene manipulation technology.

 Methods for confirming changes in gene expression using mRNA as an index include the Northern Hybridization method, RT-PCR method, subtraction method, differential display method, and the like. These methods are appropriately selected. Thus, the gene used in the present invention can be found. Furthermore, methods for simultaneously detecting changes in the expression of a large number of genes, such as hundreds or thousands, include methods using DNA arrays (such as DNA chip hybridization analysis and DNA macroarray hybridization). Analysis etc.) are known.

The “gene whose expression level changes according to the malignancy of the cancer” can be obtained by using a DNA array on which a nucleic acid corresponding to a human-derived gene or a fragment thereof is immobilized. For example, a DNA microarray on which a fragment of a gene suggested to have some association with cancer is immobilized (IntelliGene Human Cancer CHIP, manufactured by Takara Shuzo Co., Ltd.) and used in the present invention. Can be found. The aforementioned “gene whose expression level changes depending on the malignancy of the cancer” can be obtained, for example, by the following gene selection method. Gene selection method:

 For example, mRNA is prepared from a cancerous lesion tissue collected from a cancer patient, and a reverse transcription reaction is performed using the mRNA as a type II. At this time, for example, a labeled cDNA can be obtained by using an appropriately labeled primer or labeled nucleotide. The labeling method is not particularly limited, and examples thereof include a compound containing a radioisotope, a fluorescent substance, and a label using a ligand such as biotin.

 Next, hybridization is performed between the labeled cDNA and a DNA array in which a nucleic acid corresponding to an appropriate gene or a fragment thereof is immobilized. Hybridization may be performed by a known method, and the conditions may be appropriately selected from those suitable for the DNA array-labeled cDNA to be used. For example, it can be carried out under the conditions described in Molecular-cloning, A laboratory manual, 2nd edition, pages 9.52 to 9.55 (1 989).

Further, under the same hybridization conditions as described above, hybridization of nucleic acid derived from a control sample (a sample derived from a healthy person, a sample derived from a non-lesion site, etc.) and the DNA array is performed. In these human samples, it takes time from collection to measurement of gene expression level, and mRNA may be degraded by the action of RNase. In order to measure the difference in gene expression between the test sample and the control sample, it is necessary to correct the mRNA levels of both using a standard gene with relatively small fluctuations in expression. Furthermore, when competitive hybridization is performed on a single DNA array using the two types of fluorescence described below, it is necessary to correct the intensity difference between the two types of fluorescent substances. Examples of the nucleic acid used for the purpose of such correction include a nucleic acid derived from a non-lesion site; a housekeeping gene [eg, glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene, cyclophilin gene, actin gene, and Tubulin gene, phospholipase A2 gene, etc.), and to confirm that the nucleic acid is not a non-specific hybridization. Examples of the negative control include nucleic acids derived from a heterologous organism such as plasmid pUC18.

 Next, by comparing the results of hybridization with the sample derived from the lesion and the results of hybridization with the control sample, genes with different expression levels can be detected in both cases. Specifically, for an array hybridized with the nucleic acid sample labeled by the above method, an appropriate signal is detected according to the labeling method used, and the cancer lesion of each gene on the array is detected. The expression level in the origin sample and the control sample can be compared. Preferably, if a multi-wavelength detection fluorescence analyzer capable of detecting a plurality of labels, for example, two types of fluorescence, is used, the difference between the gene expression level in a sample derived from a cancer lesion and the gene expression level in a control sample is determined. Can be compared on the same DNA array by competitive hybridization. For example, a sample derived from a cancerous lesion is fluorescently labeled with Cy5-dUPP, and a control nucleic acid sample is fluorescently labeled with Cy3-dUPP. By mixing the two in equal amounts and performing hybridization with the DNA array, the difference in the gene expression level between the two can be detected as the difference in signal color and fluorescence intensity. Genes having a significant difference in the signal intensity obtained in this way are genes whose expression level changes as the cells become cancerous, and are genes that can be used as indicators of canceration, cancer, particularly malignancy of gastric cancer. is there. The “gene whose expression level changes in accordance with the degree of malignancy of cancer” used in the present invention is more preferably a gene having a larger expression level difference as an index.

 The criteria for selecting a target gene in the above-described gene selection method are as follows:

 (I) the expression intensity in the test sample or control sample is at least twice the expression intensity of the negative control gene,

 (II) High correlation with cancer malignancy

Is mentioned. The specific sorting method is described in Example 1. As for the gene obtained as described above, its expression changes as the cells become cancerous. Any gene may be used, for example, (1) a group of genes involved in oncogenes and tumor suppressors, (2) a group of genes involved in nuclear receptors or transcription coupling of nuclear receptors, (3) ) Genes involved in kinase-type transmission, (4) genes involved in receptor-type kinase, (5) genes involved in intermediate filament markers, (6) cell cycle, Genes involved in growth cycle (cell cycle & growth regulators), (7) genes involved in apoptosis (apoptosis), (8) DNA damage response, repair & recombination (9) genes involved in receptors (receptors), (10) genes involved in cell death and development regulators,

1 1) Genes involved in cell adhesion, motility & invasion (1 2) Genes involved in angiogenesis regulators, (1 3) Cell invasion ( invasion regulators),

(14) genes that contribute to cell-cell interactions; (15) Rho family GTPa se and its regulators (Rho family small GTPases

& their regulator), (1 6) Growth factors and cytokines

(growth factors & cytokines), and (17) genes involved in energy metabolism.

More specifically, the expression levels of 105 genes listed in Tables 1 and 2 below and 51 genes further listed in Tables 6 and 7 described below were measured to evaluate the malignancy of cancer. Can be performed.

Table 2

本発明の癌、 特に胃癌の悪性度の評価方法においては、 正確な評価を得る観点 から、 上記遺伝子のうち、 本発明において発現量を測定される遺伝子は、 少なく とも 8種であり、 好ましくは少なくとも 2 0種であり、 より好ましくは 3 0種以 上であり、 さらに好ましくは 4 0種以上であり、 特に好ましくは 5 0種以上であ ることが望ましく、 評価方法における操作の容易性、 特に後述の Hierarchical c lustering により遺伝子発現のパターン解析を行なう場合の操作の容易性の観点 から、 1 0 0種以下であることが望ましい。

In the method for evaluating the malignancy of cancer, particularly gastric cancer, of the present invention, from the viewpoint of obtaining an accurate evaluation, among the above genes, at least eight genes whose expression levels are measured in the present invention are preferably used. It is at least 20 types, more preferably 30 types or more, further preferably 40 types or more, particularly preferably 50 types or more. In particular, from the viewpoint of easiness of the operation when performing pattern analysis of gene expression by Hierarchical clustering described later, it is desirable that the number be 100 or less.

 As a specific example of the “gene whose expression level changes depending on the malignancy of the cancer”, preferably, an apoptosis inhibitor Sano ikubin (apoptosis inhibi tor survivin (Genebank registration number: U75285)) gene,

 Type I collagen Hiichi 2 Ccol lagen type I a -2 (Genebank registration number: J03464)] gene,

 Ι type Π collagen pro-α-1 chain (collagen type I I I pro-alpha-1 (Genebank accession number: X14420))

Fibronectin precursor (FN) (Genebank registration number: X02761)] gene,

 MM P-1; gastrointestinal collagenase C matrix metal loproteinase 1; intestinal col lagenase (Genebank registration number: M13509)] gene,

MM P-7; uterine matrilysin Cniatrix metal loproteinase 7; matri lysin, ut erine (Genebank accession number: Z11887)] gene,

 U-plasminogen activator Curokinase-type plasminogen activator pre cursor (EC 3.4.21.73); U-plasminogen act ivator (UP A); plasminogen activa tor. Urokinase (Genebank registration number: X02419)] Gene,

Perkinase-type plasminogen activator receptor Curokinase-type plasminogen activator receptor; GPI-anchored from precursor (U-PAR); monocyte act ivat ion antigen M03; CD87 antigen (Genebank registration number: U09937)] Child,

Alpha-2-macroglobulin (alpha-2-Μ) (Genbank accession number: M11313)

 Platelet-derived growth factor receptor precursor Cbeta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank registration number: J03278)] gene,

 BIGH3 (Genebank accession number: M77349) gene,

 XVI I type 1 collagen chain (collagen type XVI I alpha-1 (Genebank registration number: AF018081))

Growth hormone-dependent insulin-like growth factor-binding protein (growth hormone-dep endent insul in-like growth factor-binding protein (Geneno'nk registration number: M35878)) gene,

Hepatoma-derived growth factor (HDGF) (Genebank registration number: D16431) gene,

Cinsul in-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: X16302)] gene,

Type IV col lagenase (gene bank accession number: M55593) gene,

osteonectin) isparc precursor secreted protein acidic and rich in cysteine; osteonectin) (ON); basement membrane protein BM-40 (Genebank registration number: J03040)) gene,

 Cthrombospondin 2 precursor (Genebank registration number: L12350)] gene,

Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large f ibroblast proteoglycan; chondroit in sulfate proteogl yean core protein 2; glial hyaluronate-binding protein (GHAP) Link registration number: U16306)] gene,

Envoplakin (gene bank accession number: U53786) gene, integrin 4 chain Untegrin, beta 4 (gene bank accession number: X53587) gene,

Vimentin (gene bank accession number: Z19554) gene,

c-fms proto-oncogene [c-fms proto-oncogene] (Genebank registration number:

X03663),

Proliferating cell nuclear antigen (Genebank registration number: M15796) gene,

Leukocyte antigen related protein (Genebank registration number: Y00815) gene,

Interleukin 8] (Genebank registration number: M26383)

 GDP dissociation inhibitor (GDI) (Genebank registration number: L20688) gene,

Brain-specific tubulin chain CTubulin, alpha, brain-specific) (Genebank registration number: X01703) gene,

Cell cycle regulatory gene cdc2 (cell division cycle 2, Gl to S and G2 to M) (Gene division registration number: X05360),

Tissue-type plasminogen activator (tissue gene bank registration number: M15518) gene,

Stromal cell-derived factor 1 (Genebank registration number: U16752) gene,

Matrix metal loprot einase 10 (stroraelysin 2) (Genebank accession number: X07820) Matrix metalloproteinase 15 (membran e-inserted)] (Genebank accession number: Z48482) gene,

Cantioxidant protein 1] (Genebank accession number: D49396) gene,

Laminin α-4 chain Claminin, alpha 4] (Genebank registration number: S78569)

Cubiquitin-conjugat in g enzyme E2M (homologous to yeast UBC12)] Gene bank accession number: AF075599 gene,

Retinoblastoma-binding protein 6 gene (Genebank accession number: X85133) gene,

 K-sam (gene bank registration number: M87770) gene,

Cathepsin B] (Genebank accession number: L22569) gene, signaling and activator of transcription 1, 91kD (Genebank accession number: M97935) gene, carcinoembryonic Antigen-related cell adhesion molecule 6 (non-specific cross-reactive antigen) [carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting ant igen)] (Genebank accession number: M18728) gene,

Heparan sulfate proteoglycan (gene bank registration number: M85289) gene,

v-i-ras2 Kirs ten rat sarcoma 2 viral oncogene homolog (v-Ki-ras2 Kirs ten rat sarcoma 2 viral oncogene homolog)

Ras homolog gene family, member D] (Genebank accession number: D85815)

Hexabrachion (tenascin C)

1 δ , Cytotact in)] (Gene Bank accession number: X78565) gene,

Cyclin D 2 Ccycl in D2 (Genebank registration number: D13639) gene, meta-oral proteinase 3 tissue inhibitor [tissue inhibitor of metal loproteinase 3 (Sorsby fundus dystrophy, pseudoinf lammatory)] (Jinnokunk registration number: AL023282) gene,

Laminin receptor 1, liposomal protein S A Claminin receptor 1 (67kD, ribosomal protein SA)] (Genebank registry number: U43901) gene, lactate dehydrogenase (lactate dehydrogenase A) (Genebank registry number: X02152) gene

Keratin 18 [keratin 18] (Genebank accession number: M26326) gene,

 Ia-associated invariant gamma-chain] (Genbank accession number: AH001484) gene,

 CD72 antigen CCD72 antigen) (Genebank accession number: M54992) gene, connective t issue growth factor (Genebank accession number: X78947) gene,

Matrix meta-oral proteinase 3 (stromelysin 1, rogelatinase)] (Genenok registration number: X05232) gene,

Cyclin-dependent kinase 4] (Genebank registration number: Oki 22)

Human SB class I histocompatibility antigen (Human mRNA for SB class l l histocompatibi lity ant igen alpha-chain) (Genebank accession number: X03100) gene,

 Transforming growth factor; 8-3 chain Ctransforming growth factor, beta 3] (Genebank accession number: X14885) gene,

Thyroid hormone receptor interacting protein 6 (Genebank registration number: AJ001902) gene, Platelet endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 antigen) (Genebank accession number: L34657) Gene, profilin 1 (profil in 1) (Genebank accession number: J03191) Gene, GTP-binding protein [GTP-binding protein] (Genebank accession number: AF120334)

Homo sapiens clone # 24703 beta tube,) m R NA CHorao sapiens clone ne 24703 be-tubul in mRNA, complete cds] (Genebank registration number: AF0706 00) gene,

 Rho-associated, coi led -coil containing protein kinase 1 (Gene bank registration number: U43195)

 Type IV collagen, α-1 chain (col lagen, type IV, alpha 1) (Genebank registration number: 26576) gene,

 Topoisomerase (DNA) I Ctopoisomerase (DNA) I] (Genebank accession number: M60706) gene,

 Kunitz type 1 serine proteinase inhibitor, Cserine protease inhibitor, Kunitz type 1] (Genebank registration number: AB000095) gene,

Rho GTP ace activating protein 1 [Rho GTPase act ivating protein 1] (Genbank accession number: U02570) gene,

Nidogen (Genebank accession number: M30269) gene,

Cyclin-dependent kinase 5, regulatory factor 1 Ccycl in-dependent kinase 5. regul atory subunit 1 (p35)] (Genebank accession number: X80343) gene,

 CD9 antigen [CD9 antigen] (Genebank accession number: M38690) gene,

Aortic-type smooth muscle actin gene, exon 9 Caortic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216)],

Bone morphogenetic protein 1 (BMpl) (Genebank

2 o (Record number: U50330)] Gene,

 Type VI collagen α- 3 chain Ccol lagen type VI alpha-3 (Genebank registration number: X52022)] gene,

Interferon-gamma-induced monokine (MIG) (Genebank registration number: X72755)] gene,

Integrin alpha-3 chain Cintegrin alpha-3 chain (Genebank accession number: M599 11)] gene,

Interferon inducible gene family (Genebank accession number: X57351)] gene,

Metastasis-associated MTA1 (genebank accession number: U351 13) gene

Notch2 Notch homolog 3 (Genebank accession number: U97669)]

Proto-oncogene rh0A multidrug resistant tannoII. GTP-binding protein (rhoA) (Geneno Kunk registration number: L25080)] at least 8 species selected from the group consisting of the respective genes, and more preferably And at least 20 genes. Specific examples of at least eight genes, and at least 20 genes, are not particularly limited, but may be any of the genes described in Table 5 of Example 3 and the genes described in Table 4 of Example 2 below. Genes.

 In the method of the present invention for evaluating the malignancy of cancer, particularly gastric cancer, the expression level of the gene can be measured from the amount of mRNA transcribed from the gene or the amount of polypeptide encoded by the gene. it can.

For the measurement of the mRNA amount, various known methods such as a Northern hybridization method, an in situ hybridization method, a dot blot hybridization method, and a method using a DNA array are used. C represented by Ibridization method; nucleic acid amplification method represented by RT-PCR method can be used. From the viewpoint of obtaining sufficient mRNA detection sensitivity, a hybridization method or a nucleic acid amplification method is preferable.More specifically, a nucleic acid or a fragment thereof corresponding to a gene whose expression level is to be measured is deposited on a support. The hybridization method and the RT-PCR method using an array (eg, a DNA array) immobilized in each defined region are suitable. From the viewpoint of simultaneously measuring the expression of a large number of genes, a hybridization method using a DNA array is particularly suitable.

 As used herein, the term "DNA array" refers to a DNA array in which a gene or a DNA fragment derived from the gene is fixed to a predetermined region on a support, and includes, for example, an array called a DNA chip. I do. A DNA or a DNA fragment derived from a gene fixed on a support at a high density is also called a DNA microarray.

The support of the DNA array is not particularly limited as long as it can be used for hybridization, and a slide glass, a silicon chip, a nitrocellulose nylon membrane or the like is usually used. The gene or fragment thereof immobilized on the support is not particularly limited, and may be, for example, a genomic DNA library or a cDNA library, or a gene amplified by a type II PCR. DNA is listed.

 By using such a DNA array, the amounts of various types of nucleic acid molecules contained in a nucleic acid sample can be measured simultaneously. Another advantage is that even small amounts of nucleic acid samples can be measured. For example, by expressing mRNA in a sample or preparing a cDNA labeled with mRNA as a type II, and performing hybridization between this and a DNA array, expression in the sample is performed. MRNA detected at the same time, and its expression level can be measured.

As a labeling substance used for labeling, a substance such as a radioisotope, a fluorescent substance, a chemiluminescent substance, and a substance having a luminophore can be used. For example, a fluorescent substance Examples include Cy2, FluorX, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein isothiocyanate (FITC :), Texas red, rhodamine and the like. In addition, it is desirable to use two or more fluorescent substances and label the test sample and the sample used as a control with different fluorescent substances from the viewpoint of simultaneous detection. Here, the labeling of the sample is carried out by labeling the mRNA in the sample, the cDNA derived from the mRNA, or the nucleic acid transcribed or amplified from the cDNA.

 The method for detecting the label can be appropriately selected depending on the type of the labeling substance used. For example, when Cy 3 and Cy 5 are used as labeling substances, Cy 3 532 nm and Cy 5 can be detected by scanning at a wavelength of 635 nm. The strength of the label is used as an index of the expression level of the gene.

Examples of the nucleic acid amplification method include a PCR (Polymerase Chain Reaction) method (US Pat. No. 4,683,202), an ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acid) method [International Publication No. / 56877 pamphlet], Strand Displacement Amplification (SDA) method (Walker, GT., Et al., Nucleic Acids Res., Volume 20) , Pp. 1691-1696 (1992)], the Nucleic Acid Sequence-Based Amplification (NASBA) method (Compton, J.) Vol. 350, pp. 91-92 (1991)] and the like. When measuring mRNA amount by nucleic acid amplification method, especially RT-PCR method, for example, competitive PCR method, TaqMan method (for example, Linda G. Lee, Nucleic Acids Research) , Vol. 21, p. 376 1-3766 (1993)] and the like]. What The RT-PCR method refers to a method in which mRNA is type III, cDNA is synthesized by a reverse transcription reaction, and nucleic acid amplification is performed by PCR.

 When measuring gene expression level based on mRNA level, (1) Primer, Z or probe for detecting mRNA or its fragment expressed from at least eight genes whose expression level changes depending on the malignancy of cancer A contained kit can be suitably used.

 The amount of polypeptide encoded by the gene can be measured by an enzyme immunoassay, a fluorescent immunoassay, a luminescence immunoassay, or the like using an antibody against the polypeptide or a fragment thereof. it can. Specifically, for example, it can be carried out by a conventional ELISA method using a labeled antibody. Such antibodies may be humanized antibodies, Fab fragments, single-chain antibodies, antibody fragments, etc., as long as they specifically bind to the polypeptide encoded by the gene or a fragment thereof.

 When measuring the gene expression level based on the amount of polypeptide, (2) Antibodies against polypeptides or fragments thereof encoded by at least eight genes whose expression levels change depending on the malignancy of cancer or fragments thereof A kit containing is preferably used.

 In the evaluation method of the present invention, in step (2), the expression level of the gene is analyzed by comparing the expression level of the gene in the test sample with the expression level of the gene in the control sample. The malignancy of gastric cancer can be evaluated. Here, the control sample refers to a sample derived from a healthy person or a sample derived from a non-lesion site.

 Analysis of gene expression can be performed by pattern analysis. Such a pattern analysis can be performed by a class analysis using the expression intensity signal of each gene in the test sample or the relative expression ratio of each gene in the test sample relative to the control sample.

That is, class analysis is performed by computer using the obtained expression intensity of each gene. The data used for analysis include the expression intensity signal of each gene in the test sample or the phase of each gene in the test sample relative to the control sample. To expression ratio.

 In step (2), a correction process of the measured value of the expression level of each gene obtained in step (1) can be further performed.

 The relative expression ratio refers to a value represented by [gene expression level in test sample Z gene expression level in control sample]. Examples of the clustering method include, but are not limited to, forces such as Hierarchical clustering and Neural network clustering.

 The following describes the Hierarchical clustering that is relatively frequently used in the field of biotechnology.

 Hierarchical clustering means that when the data measured in each sample is arranged in multidimensional coordinates, the distance between the data is found in order from the closest sample, and finally similar samples are arranged next to each other. The feature is that the results can be represented by a tree diagram. There are various methods for measuring the distance between samples, such as the Euclidean distance and the Mahalanobis' generalized distance. In the process of creating a dendrogram, there are various ways of defining the mutual distance of a set of individual samples, and the method is not particularly limited, but mainly includes a shortest distance method, a longest distance method, a group average method, and a center of gravity. Law and Ward law.

When using the expression intensity signal of each gene in a test sample, for example, cancer tissue, the value obtained by subtracting the background signal from the spot signal of each gene (this value is referred to as the expression intensity signal) is input to the computer. At this time, the distribution of the signal intensity differs for each array, so it is preferable to perform correction (1100 ^ 112 & 11011). This normalization method has Z-score, Mean deviation ^ Mean abs olute deviation, Median absolute deviation, etc., and is selected as appropriate. Genes to be subjected to the class ring are selected from the genes shown in Tables 1 and 2, and the larger the number, the more accurate the classification. Specifically, even a combination of at least eight limited genes can determine limited malignancy factors Next, by performing Hierarchical cluster analysis on the expression intensity signal after normalization, the class of the specimen, for example, a stomach cancer specimen, and the clustering of genes are performed. As a result, classification between specimens on a dendrogram, that is, between specimens with similar patterns of gene expression, showed the degree of cancer infiltration in pathological diagnosis, malignancy such as lymph node metastasis, peritoneal metastasis, and liver metastasis. Metastatic specimens with high degree of commonality and high-grade metastasis specimens and early-stage cancer specimens with low invasiveness were placed at a distance from each other. By using this method, it is possible to determine the degree of malignancy of an unknown specimen by collecting a small amount of cancer tissue by biopsy before surgery. In other words, mRNA is purified from cancer tissue, and subjected to hybridization using, for example, a probe that has been subjected to one-color fluorescence labeling, thereby obtaining an expression intensity signal of each gene. By performing a clustering analysis together with the expression intensity signal and the expression intensity signal of a gastric cancer sample that has become a standard whose malignancy has already been determined, it is possible to determine which aggressiveness level the unknown sample is closer to the standard sample. In the conventional pathological diagnosis after surgery, if the invasion of the cancer has spread from the mucosa to the muscular layer, the cancer has a relatively good prognosis or a malignant cancer that has the possibility of relapse, Z or metastasis in the future. Although the prognosis could not be determined because it was located at the bifurcation point, by performing the clustering analysis of the present invention, the specimen with unknown malignancy was an early cancer with low malignancy or a high malignancy with metastasis It is clear which of the cancer specimens is closer in distance, and the prognosis can be predicted.

On the other hand, the relative expression ratio of each gene in a test sample (eg, gastric cancer tissue) with respect to a control sample (eg, control normal tissue), that is, [gene expression amount in test sample / gene expression amount in control sample] (specific Specifically, even when [the gene expression level in cancer tissues and the gene expression level in control normal tissues]) are used, clustering of stomach cancer specimens yields almost the same results as when the cancer tissue expression intensity signal is used. Can be When the relative expression ratio is used, there is an advantage that a gene expression profile excluding individual differences in gene expression can be viewed. Gene clusterin Are classified into genes whose expression fluctuations due to canceration and malignancy are similar.By analyzing the correlation between the expression fluctuations and malignancy factors in pathological diagnosis, genes involved in malignancy are identified. It is possible to specify. As a result, the relationship between the 105 genes shown in Tables 1 and 2, the 51 genes shown in Tables 6 and 7, and the malignancy, that is, the invasion level indicating the degree of cancer invasion, Was found.

 The result obtained by the analysis described above is output to another software package such as a printer, a display, or a graphic software for a display. Such an output is particularly advantageous when the result obtained by the evaluation method of the present invention is used for diagnosis or the like.

 Furthermore, the present inventors have found that a simpler diagnosis of malignancy is possible by applying the classification of genes correlated with malignancy found by the above method. In other words, by evaluating the level of gene expression fluctuation using the number of genes whose expression fluctuates and the percentage of expression fluctuation as an index without performing complex clustering analysis using a computer, it is possible to evaluate the malignancy of gastric cancer, especially Was found to be possible. Such an embodiment is also included in the present invention.

Here, the “gene expression fluctuation level” is 1) [indicated by the relative expression ratio of the gene whose expression level is increased in the test sample as compared to the control sample or the sum of the absolute values of the logarithms of the relative expression ratio. 2) [The value of the gene whose expression level is higher in the test sample compared to the control sample is taken as 1 point, and the value indicated by the sum of the points of each gene in the test sample] Can be evaluated. Specifically, for example, when the relative expression ratio of the gene whose expression level is increased in the test sample compared to the control sample is 2 or more, one point is set, and the sum of the points of each gene in the test sample is set as one point. Based on the value indicated by, the expression fluctuation level of the gene can be evaluated. As used herein, the term “fluctuation in gene expression” refers to a fluctuation of two times or more, that is, a relative expression ratio ((gene expression level in a test sample, gene expression level in a control sample)) of 2 or more or 12 or less. Say the case. Here, the relative expression ratio is 2 The above cases are genes with increased expression, and 1Z2 or less are indicators of genes with decreased expression. In 2) above, the number of expression-variable genes in a group consisting of eight or more genes selected from a gene group that greatly correlates with each factor of malignancy is a standard for which the malignancy and prognosis are already known. It can be evaluated by comparing with the number of genes whose expression is fluctuated in cancer tissues.

In the value of 1) above, the value of (the gene expression level in the test sample, the gene expression level in the control sample) is expressed as a logarithm, and the absolute value is used to evaluate the rate of increase and decrease in expression equally. can do. The logarithm of time is the natural logarithm log _2, the number absolute versus logi. May be represented by any of

 When assessing the degree of malignancy using the number of genes whose expression fluctuates and the percentage of expression fluctuation as indices, it is highly reliable to judge based on the intensity signal (background value) of the negative control gene mounted on the same chip. It is possible to That is, when both the expression intensity signal in the test sample and the expression intensity signal in the control sample are smaller than twice the background value, the reliability of the fluctuation is low, but when either of them is 2 times or more and smaller than 4 times, The reliability is high, and if any of them is four times or more, the reliability is even higher. The reliability of the evaluation increases when these reliability factors are added to the determination of malignancy. That is, when either the expression intensity signal of the cancer or the expression intensity signal of the control normal tissue is more than twice the intensity signal of the negative control gene, the evaluation is made based on the number of genes whose expression fluctuates and the sum of the ratio of the expression fluctuation Is more desirable.

 In the present invention, the gene expression level is analyzed by comparing the expression levels of at least eight genes whose expression levels change depending on the malignancy of the cancer and the expression level of the housekeeping gene in the test sample. Can do it.

In the method of the present invention for evaluating the degree of malignancy of gastric cancer, in particular, in step (2), the gene expression level is analyzed by comparing the gene expression level in the test sample with the gene expression level in the control sample. Then, the malignancy of cancer, particularly gastric cancer, may be evaluated based on the obtained analysis result of the gene expression amount and the weighting coefficient for each gene. You In other words, the evaluation is made by summing the value expressed by the logarithm of the relative expression ratio of the gene in the test sample with respect to the control sample and a weighting coefficient in consideration of the contribution of each gene to malignant transformation. Can be performed. In the present invention, since the degree to which each gene expression variation contributes to malignancy is not uniform, it is possible to weight the gene expression variation based on the degree to perform evaluation that reflects malignancy more. Become.

 In the above embodiment, the weighting coefficient of a gene having a large contribution to malignancy is set high, and the weighting coefficient of a gene having a small contribution is set small. If the gene whose expression is increased in the test sample is inversely correlated with malignancy as compared with the control sample, the weighting coefficient is a negative value.

 The stage of assigning the weighting coefficients is not particularly limited, but is usually assigned in 2 to 10 stages.

 For example, in Tables 6 and 7 described in Examples described later, for 70 types of genes, a weighting coefficient consisting of 7 levels of 1, 3, 1, 1, 2, 3, 4, 5 is exemplified. However, this may be changed to 5 or 3 levels.

 As a specific evaluation method using a weighting factor, for example, in a test sample, for a gene having a weighting factor of 5 whose expression level is twice as high as that of a control sample, a natural logarithmic value of 2 as an expression ratio is used. A value obtained by multiplying a certain 0.693 by a weighting coefficient of 5.64.65 becomes the point of the gene. Similarly, the points of all genes are determined, and the malignancy is evaluated by the sum of the points.

 The above-mentioned weighting coefficient is set to a value that can examine the level of gene expression fluctuation using clinicopathological data and a specimen with a known prognosis and appropriately indicate the degree of malignancy.

2. The array of the present invention

Used to evaluate malignant transformation of cancer, especially gastric cancer, The array of the present invention for evaluating the degree of sex includes nucleic acids (for example, DNA) corresponding to at least eight genes whose expression levels change according to the malignancy of cancer, particularly gastric cancer, or fragments thereof. An array immobilized on a defined area on a support. In the array of the present invention, nucleic acids or fragments thereof corresponding to at least eight genes whose expression levels change according to the malignancy of cancer, particularly gastric cancer, as described in the above section, are fixed. Therefore, it has an excellent property that the malignancy of cancer, particularly gastric cancer, can be evaluated by a simple operation.

 Here, "immobilized at a predetermined position" means that the position where the nucleic acid corresponding to each gene or the fragment thereof is immobilized is determined in advance on the support. means. That is, when such an array is used, it is possible to know from the position of the detected signal which nucleic acid of the gene or its fragment is derived from.

The support used in the array of the present invention is not particularly limited as long as it can be used for hybridization, and usually a slide glass, a silicon chip, a nitrocellulose-nylon membrane or the like is used. More preferably, any material may be used as long as it is nonporous and has a structure with a smooth surface, and is not particularly limited. For example, glass such as a slide glass can be suitably used. The surface of the support may be any as long as it can immobilize single-stranded DNA by covalent or non-covalent bonds, and has a hydrophilic or hydrophobic functional group on the surface of the support. Can be suitably used, and there is no particular limitation. For example, those having a hydroxyl group, an amino group, a thiol group, an aldehyde group, a carboxyl group, an acyl group or the like can be preferably used. These functional groups may be present as surface characteristics of the support itself, or may be introduced by surface treatment. Examples of such a surface-treated product include a product obtained by treating glass with a commercially available silane coupling agent such as an aminoalkylon orane, and a product obtained by treating a glass with a polycation such as polylysine or polyethyleneimine. Some of the slide glasses that have undergone these treatments are commercially available. In the array of the present invention, the nucleic acid or a fragment thereof may be fixed in either single-stranded or double-stranded form. For example, a DNA array in which the nucleic acid or a fragment thereof is aligned and immobilized as two denatured strands on a support, or a DNA in which at least a part of the immobilized DNA is single-stranded DNA An NA array may be used. Further, the array of the present invention may be a DNA array in which double-stranded DNA is denatured and aligned and spotted on the same support.

Further, in the array of the present invention, there is no particular limitation on the density of the support of the nucleic acid or fragment thereof of a gene to be immobilized, for example, may be a high density § les I, 1 0 0 dot Z cm ² An array having nucleic acids, particularly DNA, immobilized thereon at the above density can be suitably used.

 The nucleic acid or the fragment thereof immobilized on the support is not particularly limited, and may be any of a polynucleotide and an oligonucleotide. There is no particular limitation on the production method, and it may be chemically synthesized, isolated and purified from natural nucleic acids, enzymatically synthesized, or a combination thereof.

 The nucleic acid or its fragment immobilized on the support is not particularly limited. For example, a double-stranded polynucleotide having a chain length of 50 bases or more or a derivative thereof, and a PCR (polymerase chain reaction) ) The DNA prepared by enzymatic amplification by the method or the like is denatured at the time of immobilizing the DNA on the immobilization support, and a single-stranded DNA or a derivative thereof can be suitably used. The derivative may be modified as long as it can be immobilized on the support surface, and is not particularly limited. Examples thereof include an amino group and a thiol group at the 5 ′ end of DNA. And DNA into which a functional group such as has been introduced.

For example, a DNA amplified by PCR or the like using a genomic DNA library or a cDNA library as a type III can be used. The array can be prepared by immobilizing a nucleic acid corresponding to the above gene or a fragment thereof on a support having an amino group introduced therein, for example. In addition, the above operation of immobilization This is performed using a DNA array production device, for example, a DNA chip production device manufactured by Affymetrix Co., Ltd., whereby an array of the present invention in which nucleic acids corresponding to genes are aligned and immobilized can be produced.

 When a nucleic acid fragment is immobilized on an array, the length of the fragment is not particularly limited, and is preferably, for example, about 100 bases to about 1 kilobase in length. Even if it is shorter or longer than the length, it may be any as long as it specifically hybridizes with the nucleic acid derived from the test sample in the hybridization.

 The gene used in the array of the present invention may be any gene whose expression level changes depending on the degree of malignancy of cancer, particularly gastric cancer, and is not particularly limited, but the genes listed in the above section, Examples thereof include oncogenes, tumor suppressor genes, growth factors, metastasis, invasion factors, angiogenic factors, and genes encoding proteins involved in energy metabolism. Furthermore, for example, differential expression (DD) is used to express the expression of malignant tumors from the total RNA extracted from cancer tissues and control normal tissues of cancer patients of various grades of cancer whose functions are unknown. You may find genes that decrease or increase and immobilize them. Furthermore, all the genes whose expression is fluctuated in the course of malignant transformation of gastric cancer, which are found by the gene selection method according to the above item 1, are all available, and cancers in which all nucleic acids corresponding to such genes are immobilized, In particular, a DNA array for evaluating the degree of malignancy of gastric cancer can be prepared.

 Specifically, at least eight, preferably at least eight, selected from the group consisting of the genes listed in the specific examples of the `` gene whose expression level changes in accordance with the degree of malignancy of cancer '' described in the above section, preferably At least 20 genes are included.

 The array of the present invention can also be used to detect cancer cells in cancer, especially gastric cancer.

3. Kit for evaluating the malignancy of cancer of the present invention, particularly gastric cancer

The kit for evaluating the malignancy of the cancer of the present invention, in particular, gastric cancer, is a detection target (1) a kit containing primers and / or probes for detecting mRNA or a fragment thereof expressed from at least eight genes whose expression level changes depending on the malignancy of the cancer, and (2) malignancy of the cancer And a kit containing an antibody against a polypeptide or a fragment thereof coded for at least eight kinds of genes whose expression level varies depending on the expression.

 The kit of the present invention comprises: (1) a primer and / or a probe for detecting mRNA or a fragment thereof expressed from at least eight genes whose expression level changes depending on the malignancy of the cancer; or Since it contains an antibody or a fragment thereof against a polypeptide or a fragment thereof encoded by at least eight kinds of genes whose expression level varies depending on the expression level, it can be used in the method for evaluating the malignancy of the cancer, particularly gastric cancer. It enables more convenient and quick operation. Further, the kit of the present invention can also be used for detecting cancer cells in cancer, particularly in gastric cancer.

 The kit of the above (1) is a nucleic acid amplification method, specifically, RT-PCR, of mRNA or a fragment thereof expressed from at least eight genes whose expression level changes according to the malignancy of cancer, particularly gastric cancer. It contains primers and Z or probe for detection by the method.

 The primer and / or probe may be used under conditions that are stringent to at least eight genes whose expression levels change depending on the malignancy of cancer, particularly gastric cancer, or to a nucleic acid having a nucleotide sequence complementary to the gene. Any material can be used as long as it can be pre-hybridized. The `` stringent conditions '' are not particularly limited, but include, for example, a solution containing 6 XSSC, 0.5% SDS, 5 x Denhardt, 100 gZm1 two-sperm sperm DNA, (the primer and / or This refers to conditions for keeping the temperature at the probe Tm—25 ° C].

The base sequence of the above primer may be any sequence that can specifically amplify the nucleic acid corresponding to the gene under the reaction conditions of a usual nucleic acid amplification method, particularly RT-PCR. On the other hand, the nucleotide sequence of the probe may be any nucleic acid sequence that can hybridize to the nucleic acid corresponding to the gene under the stringent conditions.

 The Tm of the probe or primer is, for example, represented by the following formula:

Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ]) +0.41 (¾G + C)-(600 / N)

 (Where N is the length of the probe or primer and% G + C is the content of guanine and cytosine residues in the probe or primer)

Required by

 When the length of the probe or primer is shorter than 18 bases, Tm is, for example, the product of the content of A + T (adenine + thymine) residue and 2 and the content of G + C residue. The chain length of the probe, which can be estimated by the sum of the product of the weight and the product of 4 ° C [(A + T) X 2 + CG + C) X 4], is not particularly limited. From the viewpoint of preventing excessive hybridization, the length is preferably 15 bases or more, and more preferably 18 bases or more.

 The chain length of the primer is not particularly limited, but is, for example, 15 to 40 bases, and preferably 17 to 30 bases.

 As the “at least eight genes whose expression level changes according to the malignancy of 瘙”, preferably, specific examples of the “gene whose expression level changes according to the malignancy of cancer” according to the above section And at least 20 genes, more preferably at least 20 genes selected from the group consisting of the genes listed above.

In the kit of (1), the polypeptide may be a polypeptide encoded by at least eight genes whose expression level changes depending on the malignancy of the cancer. More specifically, it may be selected from the group consisting of polypeptides encoded by the genes listed in the specific examples of the “gene whose expression level changes depending on the malignancy of cancer” described in the above section. At least 8, and more preferably, at least 20 selected polypeptides are included. The antibody is not particularly limited as long as it has an ability to specifically bind to the polypeptide, and may be either a polyclonal antibody or a monoclonal antibody. Furthermore, an antibody or an antibody derivative modified by a known technique, for example, a humanized antibody, a Fab fragment, a single-chain antibody, or the like can also be used. The antibody is described, for example, in 1992 by John Wiely & Sons, Inc., edited by John E. Coligan, current 'Protocols'in' Immunology. It can be easily prepared by immunizing a heron rat, a mouse or the like with all or a part of the polypeptide by the method described in Current Protocols in Ionology. The antibody thus obtained is purified and then treated with peptidase or the like to obtain an antibody fragment. Antibodies can also be produced by genetic engineering. Furthermore, the antibody or a fragment thereof of the present invention may be variously modified to facilitate detection by an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, or the like.

 The above-mentioned antibodies or fragments thereof include those which can specifically bind to certain partial fragments of the polypeptide.

 The kit of the present invention may appropriately contain a detection reagent and the like.

 In addition, the present invention analyzes the results obtained by the evaluation method and the like by an analysis means (for example, an image processing method using a computer) and records the results on a recording medium such as paper; It is also possible to provide a diagnostic method which is provided by recording or displaying on a medium or the like. Such a diagnostic method is also included in the present invention.

4. Cancer detection method of the present invention, particularly gastric cancer, and kit used therefor

The “gene whose expression level changes according to the malignancy of the cancer” is also useful as an index for detecting cancer or cancer cells. Therefore, the method for evaluating the malignancy of cancer, particularly gastric cancer, according to the present invention also enables detection of cancer cells, particularly cancer gastric cancer. Heel A method for detecting cancer or cancer cells is also included in the scope of the present invention.

 In the method of detecting pain or cancer cells, the expression of many of the selected genes fluctuates due to malignancy, and the pattern of fluctuation varies due to malignancy.Therefore, the same technique is used to detect cancer or cancer cells. Simultaneously determine the malignancy of the cancer

0

 Specifically, the method for detecting cancer according to the present invention comprises a nucleic acid corresponding to at least eight genes whose expression levels change depending on the degree of malignancy of a test sample and a control sample, or The expression of the expressed polypeptide is examined, and when there is a difference in the expression of the gene or polypeptide between the test sample and the control sample, the test sample is used as an indicator that the test sample contains cancer cells. One major feature of detecting cancer cells is o

 As the test sample, the same sample as the test sample in the above evaluation method can be used. Examples of the control sample include a sample derived from a healthy person and a non-lesion site, that is, cells derived from a normal tissue.

 In the method for detecting cancer according to the present invention, the expression level is changed according to the malignancy of the cancer. In order to examine the expression of nucleic acids corresponding to at least eight kinds of genes or polypeptides encoded by the genes, It has an excellent effect that cancer can be detected simply and with high reliability.

 According to the method for detecting cancer of the present invention, cancer, particularly gastric cancer, more specifically, cancer cells, particularly gastric cancer cells, can be detected.

 In the detection method of the present invention, the cancer, in particular, a nucleic acid amplification method, a hybridization method, an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, etc. It is possible to measure the expression level of at least eight kinds of genes whose expression level changes according to the degree of malignancy or the polypeptide encoded by the gene

O

In the detection method of the present invention, it is preferable that the expression level varies according to the malignancy of the cancer. It is preferable to measure at least 8 types of expression of the nucleic acid corresponding to the "gene" or the polypeptide encoded by the gene, and more preferably to measure at least 20 types of expression.

 When measuring the expression of a nucleic acid corresponding to the “gene whose expression level changes according to the malignancy of the cancer”, various types of “genes whose expression level changes according to the malignancy of the cancer” in the test sample are used. Therefore, it is more preferable to use the array to examine the expression of the gene.

 In the detection method of the present invention, the above-described evaluation method is performed, whereby cancer, particularly gastric cancer, and malignancy can be simultaneously evaluated.

 In the detection method of the present invention, the expression level of at least eight genes whose expression levels change in accordance with the malignancy of the cancer varies in the test sample as compared to the control sample. It serves as an indicator of the presence of cancer, especially gastric cancer cells, in the test sample.

 The result obtained by the above analysis is output to another software package such as a printer, a display, or a graphic software for a display. Such an output is particularly advantageous when the result obtained by the detection method of the present invention is used for diagnosis or the like.

 The method for detecting cancer, particularly gastric cancer, of the present invention includes a primer for detecting mRNA or a fragment thereof expressed from at least eight genes whose expression level changes depending on the malignancy of the pain. And a kit containing a Z or a probe, and a kit containing an antibody against a polypeptide or a fragment thereof encoded by at least eight genes whose expression level changes depending on the malignancy of the cancer or a kit thereof. It can be used for Such a kit can be used as a kit for detecting cancer, particularly gastric cancer. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the scope of the examples.

Example 1 (1) Creating a DNA array

 The DNA fragment found in the pB1uescript phagemid clone of the cancer-related gene described in International Publication No. WO98 / 371187, which was found by the differential display (DD) method, was designated as type III, and The cDNA fragment of interest was amplified and recovered by the PCR method using primers set at both ends of the multicloning site of the cDNA. In addition, glyceraldehyde 3-phosphate dehydrogenase (GAPD) and cyclophilin genes were prepared as housekeeping genes and plasmid pUC18 was prepared in the same manner as a negative control. These DNAs were recovered by ethanol precipitation. 0 OmM Carbonate buffer-Dissolve to 1 czM with (pH 9.5), and spot on amino group-introduced slide glass (Sigma) using DNA chip making equipment (Affilimes). It was fixed by UV irradiation. The slide was washed with 0.2% SDS and then with distilled water and dried to obtain a DNA array.

(2) Preparation of fluorescently labeled cDNA

As shown in Table 3, gastric cancer tissues and control normal stomach tissues were separated from the tissues removed at the time of cancer removal surgery from 10 gastric cancer patients with different degrees of cancer invasion, metastasis, or progression. Next, all RNAs were individually extracted from each tissue by the AGPC (Acid Guanidium Phenol-Chloroform) method. MRNA was purified from each of these total RNAs using Oligotex-MAG mRNA Purification kit (Takara Shuzo). Table 3

なお、 表 3中の各項目は以下のような所見を示す：

Each item in Table 3 shows the following findings:

Degree of progress:

The stage I is an early stage cancer in which the degree of invasion of the cancer is shallow and no metastasis is observed. The stage I I refers to a state in which the cancer has infiltrated and spread to nearby lymph nodes.

Progression I I I refers to a condition that has spread to distant lymph nodes.

Grade IV refers to a state in which the disease has progressed further, spread to more distant lymph nodes, and has metastasized to the peritoneum and liver.

Differentiation degree:

As general types, papillary adenocarcinoma (pap), well differentiated adenocarcinoma (tub 1), moderately differentiated adenocarcinoma (tub 2), poorly differentiated adenocarcinoma (por), signet ring cell carcinoma (sig), mucinous carcinoma (muc) are categorized Depth:

It is the deepest layer of the stomach wall that has invaded the cancer. Is expressed.

 Lymph node metastasis (Indicates the degree of metastasis of the lymph node group found histologically): No metastasis (No)

Metastasis only in group 1 lymph nodes

Metastasis in lymph nodes of group 2 but no metastasis in groups 3 and 4 (N ₂ ) Metastasis in lymph nodes of group 3 but no metastasis in group 4 (N ₃ )

Metastasis to lymph nodes in group ₄ (N ₄ ) Lymphatic invasion:

No invasion found (0)

Mildly invasive (1)

Moderate invasion (2)

Highly invasive (3) Venous invasion:

No invasion found (0)

Mildly invasive (1)

Moderate invasion (2)

Highly invasive (3) Peritoneal metastasis:

No sowing at all (0)

Seeding in the adjacent peritoneum (1)

Few metastases to distant peritoneum (2) Numerous metastases to the distant peritoneum (3) Liver metastases:

No liver metastasis (0)

Liver metastasis is observed only in one lobe (1)

Few sporadic metastases on both leaves (2)

Many scattered metastases on both leaves (3) Invasive growth pattern:

The cancerous layer shows a swelling growth and is distinguished from its surroundings.

The lesion shows invasive growth and the boundary with surrounding tissue is unclear (τ

Intermediate () The above mRNA is defined as type II, and Cy 3—dUTP (manufactured by Amersham Moorf Almatia) for the control normal gastric cancer tissue group, and Cy 5—dUTP (manufactured by Amersham) for the gastric cancer tissue group. (Pharmacia Co., Ltd.), respectively, to prepare fluorescently labeled cDNA.

 The composition of the reaction solution is shown below.

Reaction solution A: oligo dT primer (manufactured by Takara Shuzo Co., Ltd.) of about 1 µg of mRNA and 300 pmo 1 and getyl pyrocarbonate (DEPC, manufactured by Nacalai Tesque, Inc.) Addition;

Reaction solution B: 5 xAMV RTase buffer (manufactured by Life Science) 4〃1, 0. ImM dATP, dCTP, dGTP and 0.065 mM dTTP, 6 OU RNase inhibitor (manufactured by Takara Shuzo), 0.0351111 ^ 73- or 5-y5 labeled dUTP (manufactured by Amersham Pharmacia) was mixed to obtain a solution having a final volume of 6.51. The reaction solution A was kept at 70 ° C for 5 minutes, and then cooled on an ice bath. Then, add reaction solution B (6.561) and AMV RTase (manufactured by Life Science) to reaction solution A.

30 U was added to obtain an RT reaction solution. After keeping the RT reaction solution at 55 for 30 minutes, the temperature was adjusted to 42. To the RT reaction solution, 30 U of AMV RTase was further added to adjust the solution volume to 201, and the obtained mixture was again subjected to 6 by pressing at 42.

Hold for 0 minutes. To the obtained reaction solution, 50 OmM EDTA solution 2.51 and 1 M sodium hydroxide (5-1) were added, and the mixture was kept at 37 ° C for 10 minutes to degrade type I RNA. After cooling the reaction solution to room temperature, 12.5 1M Tris-hydrochloric acid (pH 7.5) was added. This solution was subjected to gel filtration using a Centri-Sep spin column (Applied 'Biosystems). As a result, a Cy3-labeled cDNA solution (about 351) and a Cy5-labeled cDNA (about 35〃1) were obtained.

 The Cy3-labeled cDNA and Cy5-labeled cDNA obtained in this manner are mixed in pairs for each patient, concentrated by ethanol precipitation, and then subjected to hybridization buffer (6XSSCZ0.2% SDSZ5 X denhardt). Solution ZO. Lmg / ml salmon sperm DNA) was dissolved in 8/1 to prepare fluorescently labeled cDNA.

(3) Hybridization of labeled cDNA and DNA array

 Prehybridization buffer (6 x SSC / 0.2% SDSZ5 X Denhardt's solution Zl mg / m 1) was added to the DNA array prepared in (1) above and a commercially available DNA array (Intelligene Cancer Chip, manufactured by Takara Shuzo). Salmon sperm DNA) was dropped, covered with a cover glass, and the periphery was sealed with a film. After keeping this at room temperature for 2 hours, the cover glass was removed, washed with 2 × SSC, then with 0.2 × SSC, and air-dried.

After heat denaturation (94 ° C, 3 minutes) of the fluorescent-labeled cDNA prepared in (2), 81 was dropped on the prehybridized DNA array, covered with a cover glass, and sealed with a film. After keeping the obtained array at 65 ° C for 16 hours, cover With the exception of the glass, 2XSSCZ 0.2% SDS was washed twice at 55 ° C for 30 minutes, then once at 65 ° C for 5 minutes, and then washed at room temperature for 10 minutes in 0.05XSSC and air-dried. did. The obtained array was applied to a microarray scanner (manufactured by GMS) to measure the fluorescent signal of each spot. The measured signals were analyzed by an expression data analysis software Imagenage (manufactured by BioDiscovery), and the expression levels of each gene in cancer tissues and control normal tissues were examined.

 The relative expression ratio of the individual genes immobilized on the DNA array used in the cancer tissue and the control normal tissue ([expression level signal of cancer tissue Z expression level signal of control normal tissue]), ie, [gene in test sample The expression level of the gene in the Z control sample] was complicatedly different among the 10 gastric cancer patients. However, some genes showed expression patterns depending on the degree of cancer invasion, the presence or absence of metastasis, or the degree of progression. Through the following steps, genes related to the malignancy of gastric cancer were found from these genes.

 First, Hierarchical clustering using the clustering software GeneSight (manufactured by BioDiscovery) was performed on the [natural logarithmic conversion value of the relative expression ratio] between the cancer tissues of all the genes used on the DNA array and the control normal tissues. The distance between the samples was measured using the Euclid distance, and the distance was defined using the centroid method. In the dendrogram obtained by this analysis, the vertical axis indicates the classification of the specimen, which is large, and indicates that the initial gastric cancer specimen, and that the cancer has invaded into the muscularis propria or the subscleral tissue but metastasized. Stomach cancer specimens without metastasis) and [metastatic specimens]. In addition, the horizontal axis of the dendrogram indicates the classification of the genes, which are [gene groups having a low expression intensity ratio in all cancer tissues of gastric cancer patients], [genes having low expression intensity ratio in all cancer tissues of gastric cancer patients]. It can be seen that the genes are classified into three classes: a high gene group] and a gene group having a different expression pattern for each gastric cancer patient.

Genes related to the malignancy of gastric cancer were considered to belong to [genes with different expression patterns for each gastric cancer patient] among the above three classifications. First selected as a candidate. From these candidate genes, genes whose cancer expression intensity signal and control normal tissue expression intensity signal were smaller than twice the background value were excluded because their expression was low and the fluctuation evaluation reliability was low. The candidate genes with different expression patterns for each gastric cancer patient are sub-clustered with about 3 to 10 genes as one group.Next, the selected genes are excluded from each sub-cluster gene group. The class was repeated in the same manner using the remaining genes. As a result, we found a sub-clustering group in which the correlation between the classification between specimens and the malignancy was lost. It is thought that the expression of such a gene is closely related to the malignancy of gastric cancer.

 The 105 gene names selected through the above steps and their accession numbers are the same as those shown in Tables 1 and 2 above.

 Tree diagrams obtained by Hierarchical clusterin using these 105 genes are shown in FIGS. 1 and 2. FIG. 1 shows the classification of gastric cancer patients. FIG. 2 shows a tree diagram of the expression pattern of each gene in each gastric cancer patient. As shown in Fig. 1, patients with early gastric cancer (MK38 and MK168), who have low cancer penetration and no lymphatic invasion, and those with moderate to advanced advanced gastric cancer with invasive disease I understand. In addition, it can be seen that the latter includes a group of specimens (MK153, MK44 and MK143) in which the cancer has deeply invaded and metastasized to the lymph nodes, and subclusters of MK144, MK80 and MK96. Of these, MK96 had relatively low cancer penetration and showed no metastases, but microscopic examination of surgical sections revealed that the cancer was mildly invading the lymphatic vessels. Currently, 4 years and 10 months have passed since the operation, but he is still alive. Although no metastasis was seen during surgery for MK80, the cancer had deeply infiltrated, and the lymphatic vessels were highly invaded by microscopic observation of the excised surgical section. Later, the cancer recurred and died. MK144 was a high-grade, cancer patient with metastases to both the lymph nodes and the peritoneum.

Hierarchic al clustering was also performed using the expression intensity of the gene on the DNA array in cancer tissues as an index. Z-sc for normalization of expression intensity in cancer tissue The ore method was used, the city block distance was used to measure the distance between samples, and the centroid method was used to define the distance. The obtained dendrograms are shown in FIGS. 3 and 4. Patient classification on the vertical axis (Fig. 3) Although the classification of specimens is slightly different from that using the expression ratio, 10 gastric cancer patients still have low cancer penetration and early gastric cancer without lymphatic invasion. There were two major groups of patients with moderate to advanced advanced gastric cancer with lymphatic invasion and Z or dislocation. In addition, the latter contained a subcluster of MK80 and MK96 where metastasis was not observed, and metastasis specimens were intricately entangled with this subcluster (Fig. 4). Hierarchical clustering when using the relative expression ratio of control normal tissues of the above was similar and correlated with the malignancy of gastric cancer. Instead, it was determined to be related to malignant transformation of gastric cancer. Example 2

 (1) Preparation of DNA array

 For the 105 genes selected in Example 1, a cDNA fragment of about 100 nucleotides to about 1 kilonucleotide in length) was prepared by the method described in Example 1, and the DNA fragment was prepared. A DNA array spotted was prepared.

(2) Hybridization of labeled cDNA and DNA array

 Using the fluorescence-labeled cDNA prepared in Example 2, hybridization with the DNA array prepared in (1) above was performed. The conditions for the hybridization were the same as in Example 1.

Based on the results of the hybridization, out of the fluorescent signals obtained for the 105 genes, 20 types of genes shown in Table 4 were selected, and for the 20 types of genes, Examples 1 and 2 were used. Hierarchical clustering was also conducted. Gene name GenBank registration number alpha-2-macro globulin precursor (alpha-2-M) M1 1313 aortic-type smooth muscle alpha-actin gene, exon 9 M33216 beta platelet-derived growth factor receptor precursor (PDGFR-beta): CD 140B antigen J03278

B1GH3 M77349 bone morphogenetic protein 1 (BMpl) U50330 collagen type V [alpha-3 X52022 collagen type XVI11 alpha L22548 cytokine humig; interferon-gamma-induced monokine (MIG) X72755 growth hormone-dependent insulin-like growth factor-binding protein M35878 hepatoma-- derived growth factor (HDGF) D 16431 insulin-like growth factor binding protein 2 (IGFBp2) XI 6302 integrin alpha-3 chain M5991 1 leukocyte interferon-inducible peptide X57351 metastasis-associated MTA1 U351 13

MMP-2; gelatinase A Z48482

Notch2 Notch homolog 3 U97669 proto-oncogene rhoA multidrug resistance protein; GTP—binding protein (rhoA) 25080 spare precursor (secreted protein acidic and rich in cysteine; osteonectin) (ON); basement membrane protein BM-40 J03040 thrombospondin 2 precursor L12350 versican core protein precursor; large fibroblast proteoglycan; chondroitin sulfate proteoglycan core protein 2; J02814 glial hyaluronate-binding protein (GHAP)

The normal logarithm of the relative expression ratio was used for each gene relative to the control normal tissue of the cancer tissue, no normalization was used, the distance between samples was measured using the Euclidean distance, and the definition of the distance was analyzed using the centroid method did. The obtained dendrograms are shown in Fig. 5 and Fig. 6. As a result, it can be seen that the 10 gastric cancer patients are divided into two groups, a non-metastatic group and a metastatic group (Fig. 5). In addition, the non-metastatic group was found to be subclustered into patients with early gastric cancer (MK38 and MK168) and MK80 and MK96 with lymphatic invasion. It can be seen that all the samples constituting the sub-cluster of the metastatic group were samples of highly advanced cancer in which metastasis was observed in a wide range of lymph nodes. Thus, the combination of the 20 genes shown in Table 4 indicates that clustering closely related to malignancy is possible. This experiment reconfirmed the involvement of these 20 genes in the malignancy of gastric cancer. Example 3

 (1) Hybridization of labeled cDNA and DNA array

 The clinicopathological data at the time of surgery indicate that the malignancy level, whose prognosis is difficult to predict, is gastric cancer with a depth of mp in which the cancer has reached the muscle layer of the stomach without metastasis. MRNA was purified from the gastric cancer tissue of stomach cancer patient MK115 classified as clinicopathologically at this level and the control normal gastric tissue by the method described in Example 1, and further fluorescent-labeled cDNA was prepared. Example using this fluorescently labeled cDNA and the fluorescently labeled cDNA prepared in Example 1

Hybridization with the DNA microarray prepared in 2 (1) was performed. The conditions for hybridization were the same as in Example 1. The clinicopathological data of the gastric cancer patient MK115 is as follows:

Progression I, differentiation mod, invasion mp, lymph node metastasis 0, lymph node invasion 0, vein invasion 0, peritoneal metastasis 0, liver metastasis 0, invasive growth mode.

As a result of the hybridization, fluorescent signals for the 105 genes obtained were obtained. Of the genes, the eight genes shown in Table 5 were selected, and the relative expression ratio of the 瘙 tissue to the control normal tissue was determined. The malignancy was determined based on the gene whose expression in cancer tissue is more than twice as high as that in the control normal tissue, that is, the sum of the relative expression ratios of genes with relative expression ratios of 2 or more or the number of genes with relative expression ratios of 2 or more. .

The above-mentioned samples were compared with the 10 cancer samples used in Examples 1 and 2 as standard cancer samples. Table 5 shows the results.

g i§ Patient ¾ No. 38 ΜΚΊ 68 MK96 MK80 K30 M1 53

 GenBank climb

apoptosis inhibitor survivin U75285 1.66 0.69 1.87 2.27 1.27 3.82 collagen type I, J03464 0.64 0.56 1.20 1.60 5.01 2.57 collagen type ΠΙ pro-alpha-1 X14420 0.67 0.95 1.12 t.45 10.0Ϊ 3.72 fibronectin precursor (FN) X02761 0.47 0.45 1.33 1.44 8.71 2.28

 MMP-1; collagenase-1 X54925 0.84 0.44 1.53 2.29 1.99 0.94

 WP-7; matrilysin X07819 0.36 0.32 0.70 6.57 2.84 3.73 urokinase-type plasminogen activator precursor X02419 0.92 0.44 1.35 1.25 2.05 2.71 uroKmase-type plasminogen activator receptor 09937 0.60 0.60 0.87 1.42 1.31 1.07 合計 total expression ratio 6.17 4.45 9.97 18.29 20.83

Relative expression ratio of expressed child Total of ιε 0 0 0 1 1.13 28.62 18.82

 遺 伝 子 0 ひ 0 3 5 6

 Patient code MK44 ΜΚΊ 43 ΜΚ Ί 44 MK 1 74 ΜΚΠ 5 m en

 GenBank registration $ | number

apoptosis inhibitor survivin U75265 1.6t 2.97 2.28 1.42 1.36

collagen type I J03464 3.17 3.38 1.79 3.96 6.76

collagen type III pro-alpha-1 X14420 3.81 3.1 1 2.59 3.76 7.37

fibronectin precursor (FN) X02761 1.73 2.72 3.60 4.34 8.58

 MMP-1; collagenase-1 X54925 2.89 0.62 8.69 0.61 2.24

 MP-7; matrilysin X07819 1.84, 1.85 3.97 1.09 0.96

urokinase— type plasminogen activator precursor X02419 3.34 1.29 3.47 1.29 1.74

urokinase-type plasminogen activator receptor U09937 2.76 1.55 2.46 1.87 1.21

 21.15 17.48 28.84 18.34 30.21

Increased expression ^ Total relative expression ratio of gene 15.97 12.18 27.05 12.06 24.95

5 4 7 3 4

Gastric cancer patients MK115 Pama lymphatic invasion, no vascular invasion No progression Ib Despite progression Ib, the sum of the relative expression ratios of the 8 genes selected and the number of genes with relative expression ratios of 2 or more are both Largely at the same level as advanced cancer. Similarly, the sum of the relative expression ratios of genes having a relative expression ratio of 2 or more was also at the same level as in advanced cancer. Examining the prognosis of this patient, the cancer recurred and died three years and nine months later. Patients with stage I who do not usually have lymphatic invasion or vascular invasion rarely recur in this early stage, and recurrence of cancer three years later is already predicted by changes in gene expression at the time of surgery What you can do is significant.

 Of the eight genes selected here, many genes have been suggested to correlate with metastasis or malignancy, but even from the viewpoint of metastasis and non-metastasis, the expression of these genes differs from sample to sample and is not uniform. Absent. For example, matrilysin (MMP-7) has been reported to be highly associated with gastric cancer malignancy (Senoyu A. et al., Clinical & Experimental Metastasis, 16th Edition). Vol., Pp. 313–3221 (19998)], but only 4 of 11 patients measured this time overexpressed. Moreover, even in metastatic specimens with a high degree of malignancy, only half increase the expression. As is evident from these results, highly accurate determination of malignancy is not possible with only a single gene, and prognosis can be determined by multiple combinations of genes associated with malignancy.

 Thus, it has been clarified that the present invention is effective for diagnosis of cancer malignancy ゃ prognosis, which cannot be predicted by conventional pathological diagnosis. Example 4 Search for Genes That Are Indicators of Malignancy

Example 11 Using eight of the samples (stomach cancer tissue and control normal stomach tissue) from the gastric cancer patient described in (2) and a sample newly obtained from 14 patients, MRNA was extracted and purified in the same manner as described above. Further, using the obtained mRNA as type III, a fluorescently labeled cDNA was prepared. These labeled cDNAs were prepared in Example 2 (1). Hybridization was performed using the DNA array thus prepared and a commercially available DNA array (Intelligene Cancer Chip, manufactured by Takara Shuzo Co., Ltd.), and the results were analyzed in the same manner as in Example 11 (3).

 As a result of the above hybridization, 70 types of genes belonging to a subclass ring group involved in the malignancy of gastric cancer were found. A closer look at the genes belonging to this sub-clustering group reveals that genes whose expression increases significantly with increasing malignancy; there is a tendency for expression to increase with increasing malignancy, but there are variations between samples at the same progression level. It was found that some genes were included; genes whose gene expression decreased with increasing malignancy were included. Therefore, by weighting these genes using weighting factors, we attempted to make point diagnosis that reflects the contribution rate to malignancy.

 For genes whose expression level increases in correlation with malignancy, a weighting factor of 5 to 1 is assigned according to the degree of increase, and conversely, gene expression level decreases as malignancy increases The genes were assigned 11 or 13 scoring. The logarithm of the relative expression ratio of the gene in the test sample with respect to the control sample was multiplied by the above-mentioned weighting coefficient to obtain the sum of the points of the 70 genes.

 The sum of the points increases in proportion to the grade of malignancy determined by the degree of invasion or progression of the cancer, and more than 100 points of relapse, Z or death within 5 years The re-assignment of the weighting factor of each gene was repeated until.

Tables 6 and 7 show the names of the above 70 genes and the weighting factors finally obtained as a result of the above work.

Table 8 shows the clinicopathological data of the 22 types of samples used in the measurement of the gene expression level, and the total points of the changes in the gene expression level of each sample, taking the above weighting factors into consideration. Each item in Table 8 is the same as in Table 3 above.

Table 8 Patient number of gastric cancer MK38 MK1 68 MK96 MK34 MK1 75 MK22 Progression la la la II II Differentiation type tubl tubl tub2 por tubl por Depth depth mm sm sm mp mp Lymph node metastasis 0 0 0 0 0 0 Lymphatic invasion 0 0 1 1 0 0 Venous invasion 0 0 0 0 0 0 Peritoneal metastasis 0 0 0 0 0 0 Liver metastasis 0 0 0 0 0 0

Survival 5 years Survival Survival Unknown Unknown Survival point -46.7 23.3 53.9 31.1 20.1 66 Gastric cancer patient number K46 MK1 58 MK92 MK80 MK48 MK 1 37 Progression II II II II II Differentiated por por sig pap tub2 tub2 Depth mp ssse se se se Lymph node metastasis 0 0 0 0 1 1 Lymph vessel invasion 2 0 0 3 1 2 Venous invasion 1 0 0 0 0 0 Peritoneal metastasis 0 0 0 0 0 0 Liver metastasis 0 0 0 0 0 0

Survival 5 years Unknown Survival Survival Death at 4 years Survival Survival point 65.9 39.3 93.3 107.2 45.6 31.8 Stomach IS Patient ID K1 6 MK44 MK1 43 MK1 5 MK42 MK1 09 Progression III IV IV IV IV IV Differentiated tub2 sig por por por por por depth Degree se se ss se se ss Lymph node metastasis 2 3 3 4 4 1 Lymph vessel invasion 3 3 3 3 3 1 Venous invasion 0 0 0 0 0 _ 1 Peritoneal metastasis 0 0 0 0 0 0

 0 0 0 0 0 0

5 years survival Died in 4 months Died in less than 2 years Died in less than 1 year Died in 4 months Died in 2 months Died in 1 year Points 147.5 124.1 1 18.4 137 128.5 128.3 3B3 Churan MK27 MK1 74 MK1 44 M1 1 1

Progress Illb IV IV IV

Differentiated por tub2 tub2 tub2

Depth of se se se se

Lymph node metastasis 2 2 2 2

Lymph vessel invasion 1 3 2 2

Venous invasion 1 1 2 3

Peritoneal metastasis 0 2 2 3

Liver metastasis 0 1 0 0

 5 years survival 2 months died 7 months died 3.5 years died 9 months died

Point 122 126.4 161.9 164.8 As shown in Table 8, there is a clear correlation between the points calculated by the above method and the cancer malignancy determined from the pathological findings of the sample. It can be seen that the degree can be evaluated.

 Furthermore, the samples derived from the gastric cancer patient MK115 described in Example 3 were examined for changes in the expression levels of the above 70 genes, and the points were calculated. As a result, 138.6 points were obtained. In other words, it has become clear that even in gastric cancer that is clinically diagnosed with low malignancy, the malignancy can be accurately determined by the above method. Industrial applicability

 The cancer of the present invention, in particular, the method for evaluating the malignancy of gastric cancer, the method for detecting cancer, particularly gastric cancer, the cancer, the array for evaluating the malignancy of gastric cancer, particularly the cancer, particularly the gastric cancer, and the cancer, particularly the gastric cancer A kit for assessing malignancy or detecting cancer, especially gastric cancer, enables rapid, high-sensitivity and systematic measurement of changes in the expression of many genes involved in cancer, especially gastric cancer aggravation.

By analyzing the pattern of these gene expression fluctuations, it is possible to determine the degree of malignancy of cancer, especially gastric cancer, or to detect cancer, particularly gastric cancer.

Claims

The scope of the claims 1. The following steps:  (1) measuring the expression level of at least eight genes whose expression level changes in accordance with the malignancy of the cancer in the test sample; and  (2) Step of evaluating the malignancy of cancer based on the expression level of each gene obtained in (1) A method of evaluating the malignancy of cancer. 2. apoptosis inhibitor survivin (gene bank registration number: U75285) gene,  Type I collagen-2 (collagen type I «-2 (Genebank accession number: J03464))  Type III Collagen Pro-One Chain! : collagen type III pro-alpha- 1 (Gene Bank registration number: X14420)] Fibronectin precursor (FN) Cfibronectin precursor (FN) (Genebank registration number: X02761))  MMP-1; the gene for matrix metalloproteinase 1; intestinal collagenase (Genebank registration number: M13509) MMP-7; uterine matrilysin Cmatrix metalloproteinase 7; matrilysin, ut erine (Genebank accession number: Z11887)] gene,  U-plasminogen activator (urokinase-type plasminogen activator pre cursor (EC 3.4.21.73); U-plasminogen activator (UP A); plasminogen activa tor urokinase (Genebank registration number: X02419) Perkinase-type plasminogen activator receptor [urokinase-type plasminogen activator receptor; GPI-anchored from precursor (U-PAR); monocyte act ivation antigen M03; CD87 antigen (Genebank accession number: U09937)) Alpha-2-macroglobulin in (aIpha-2-Μ) (Genbank accession number: M11313)  Type 5 platelet-derived growth factor receptor precursor Cbeta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank registration number: J03278)] gene,  BIGH3 (Genebank accession number: M77349) gene,  XVI I type I collagen 1 chain [col lagen type XVI I I alpha-1 (Genebank registration number: AF018081)] Growth hormone-dependent insulin-like growth factor-binding protein (gene NK registration number: 35878)] gene, Hepatoma-derived growth factor (HDGF) (Genebank registration number: Dl 6431)] Insulin-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: XI 6302)] gene, IV type collagenase [type IV col lagenase (Genebank accession number: M55593)] gene, sparte B'J precursor (osteonectin) Csparc precursor (secreted protein acidic and rich in cysteine; osteonect in) (ON); basement membrane protein BM-40 (Genebank registration number: J03040)  Thrombospondin 2 precursor (gene bank registration number: L12350) gene, Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large f ibroblast proteoglycan; chondroi tin sulfate proteogl yean core protein 2; glial hyaluronate-binding protein (GHAP) (Genenok registration number: Ul 6306)] gene, Enpoplakin (gene bank accession number: U53786) gene, integrin yS—4 chain Cintegrin, beta 4 (gene bank accession number: X53587) gene, Vimentin (gene bank accession number: Z19554) gene, c-fms proto-oncogene (c-fms proto-oncogene) (Genebank registration number: X03663), Proliferating cell nuclear antigen Cprol iferating cel l nuclear ant igen) (Genebank accession number: M15796) gene, Leukocyte antigen-related evening. Cleukocyte antigen related protein] (Gene nok registration number: Y00815) gene, Interleukin 8 (Genebank registration number: M26383) Gene,  GDP dissociation inhibitor (GDI)] (Genebank registration number: L20688) Brain-specific tubulin chain (Tubul in, alpha, brain-specific) (Genebank registration number: X01703) gene, Cell cycle control gene cdc2 (cell division cycle 2, G1 to S phase & G2 to M phase) Ccell division cycle 2, Gl to S and G2 to M] (Genebank registration number: X05360), Tissue-type plasminogen activator Cplasminogen activator, t issue Gene bank accession number: M15518] gene, Cstromal cell l-derived factor 1] (Genebank accession number: U16752) gene, Matrix metal mouth proteinase 10 (stromelysin) (matrix metal loprot einase 10 (stromelysin 2)] (gene bank accession number: X07820) gene, matrix metal loproteinase 15 (membran e-inserted)] (gene bank accession number: Z48482) gene, Antioxidant protein 1 (Genebank registration number: D49396 ) Gene, Laminin Λ— 4 chain Uaminin, alpha 4] (Genebank registration number: S78569) Ubiquitin-conjugating enzyme E2M (homologous to yeast UBC12) Gene Bank Accession No .: AF075599] gene, Cretinoblastoma-binding protein 6) (Genebank accession number: X85133) gene,  K-sam (gene bank registration number: M87770) gene, Cathepsin B] (Genebank accession number: L22569) Gene, signal transduction & activator of transcription 1, 91kD (Genebank accession number: M97935) Gene, carcinoembryonic antigen-related Cell adhesion molecule 6 (non-specific cross-reactive antigen) [carcinoembryonic ant igen-related cell adhesion molecule 6 (non-specific cross reacting antigen)] (Genebank accession number: M18728) gene, Heparan sulfate proteoglycan (gene bank registration number: 85289) gene, v-Ki-ras2 Kirsten rat sarcoma virus oncogene homolog [v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homology (Genenokunk registration number: M54968) gene, Ras homolog gene family, member D] (Genebank accession number: D85815) gene, 6 ο Hexabrachion (tenascin C, cytotactin)] (Genebank registration number: Χ78565) gene, Cyclin D 2 Ccycl in D2] (Genebank registration number: D13639) Gene, meta-oral proteinase 3 tissue inhibitory factor ": AL023282) gene, Laminin receptor 1, liposomal protein S A Claminin receptor 1 (67kD, ribosomal protein SA)] (Genebank registration number: U43901) gene, Lactate dehydrogenase A (Genebank registration number: X02152) gene Keratin 18 Ckeratin 18] (Genebank accession number: M26326) gene,  I a-related inno, 'lianto 7 chain C la-associated invariant gamma-chain] (Genbank accession number: AH001484) gene,  CD72 antigen CCD72 antigen] (Genebank accession number: M54992) gene, Connective t issue growth factor (Genebank accession number: X78947) gene, Matrix metaoral proteinase 3 (stromelysin 1, progelatinase)] (Geneno's registration number: X05232) gene, Cyclin-dependent kinase 4] (Genebank registration number: U37022) Human SB class II histocompatibility antigen CHuman mRNA for SB class I histocompat ibi l i ty ant igen alpha-chain (Genebank accession number: X03100) gene,  Transforming growth factor, beta 3] (Genebank accession number: X14885) gene, Thyroid hormone receptor interacting factor 6 Cthyroid hormone receptor interacting protein 6] (Genebank accession number: AJ001902) gene, Platelet endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 anti en)) (Genebank accession number: L34657) Gene, profilin 1 [profil in 1] (Genebank accession number: J03191 ) Gene, GTP-binding factor [GTP-Mnding protein] (Genebank accession number: AF120334) Homo sapiens clone 24703/8 tubulin m R NA CHomo sapiens clone 24703 beta-tubul in mRNA, complete cds] (Genebank accession number: AF0706 00)  Rho-associated, coil-containing protein kinase 1 (Gene bank registration number: U43195)  Type IV collagen, one chain Ccol lagen, type IV, alpha 1] (Genebank registration number: M26576) gene,  Topoisomerase (DNA) I Ctopoisomerase (DNA) I] (Genebank accession number: M60706) gene,  Kuni tz type 1 serine proteinase inhibitor Cserine protease inhibitor, Kuni tz type 1] (Genebank registration number: AB000095)  Rho GTP ace activating protein 1 [Gene bank registration number: U02570] gene, Nidogen (gene bank accession number: M30269) gene, Cyclin-dependent kinase 5, regulatory subunit 1 (p35)] (Genebank accession number: X80343) gene, CD9 antigen (CD9 antigen) (Genebank registration number: M38690) gene, aortic-type smooth muscle actin gene, exon 9 Caort ic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216) , Bone morphogenetic protein 1 (BMpl) (Genebank registration number: U50330)] gene,  Collagen VI type 1 3 chain Ccol lagen type VI alpha-3 (Genebank accession number: X52022)] Cytokin humig (cytokine humig; interferon-gamraa-induced monokine (MIG) (Genebank accession number: X72755)) Integrin << — 3 chain Cintegrin alpha-3 chain (Genebank accession number: M599 11)] gene, Interferon inducible gene family (Genebank accession number: X57351)] gene, Metastasis-associated MT A 1 Cmetastasis-associated MTA1 (Genebank accession number: U351 13)] gene Notch2 Notch homolog 3 (Genebank accession number: U97669)] Proto-oncogene rh0A multidrug resistant tannoII. Gene [proto-oncogene rhoA mult idrug resistance protein; GTP-binding protein (rhoA) (Gene No. registration number: L25080) gene The evaluation method according to claim 1, wherein the expression levels of at least eight genes selected from the group consisting of: 3. The evaluation method according to claim 2, wherein the expression levels of at least 20 genes are measured. 4. The method according to any one of claims 1 to 3, wherein the expression level of the gene is measured from the amount of mRNA transcribed from the gene or the amount of polypeptide encoded in the gene. 5. The evaluation method according to claim 4, wherein the amount of mRNA is measured by a hybridization method or a nucleic acid amplification method. 6. The hybridization method according to claim 5, wherein an nucleic acid or a fragment thereof corresponding to a gene whose expression level is to be measured is immobilized in a predetermined region on a support. Evaluation method. 7. The evaluation method according to claim 5, wherein the nucleic acid amplification method is an RT-PCR method. 8. The evaluation method according to claim 4, wherein the amount of the polypeptide encoded by the gene is measured using an antibody against the polypeptide or a fragment thereof. 9. In step (2), the gene expression level is analyzed by comparing the gene expression level in the test sample with the gene expression level in the control sample, thereby evaluating the malignancy of the cancer. Evaluation method according to any one of 1 to 8. 10. In step (2), the gene expression level was analyzed by comparing the gene expression level in the test sample with the gene expression level in the control sample, and the obtained gene expression level analysis results and each gene were analyzed. 10. The evaluation method according to claim 9, wherein the degree of malignancy of the cancer is evaluated by using a weighting coefficient for the cancer. 1 1. The evaluation method according to claim 9 or 10, wherein the control sample is a sample derived from a healthy person or a sample derived from a non-lesion site. 1 2. The expression level of a gene is analyzed by pattern analysis. 1 Evaluation method described in any one of 1. 13. The gene expression pattern analysis according to claim 12, wherein the pattern analysis of the gene expression is performed by a clustering analysis using an expression intensity signal of each gene in the test sample or a relative expression ratio of each gene in the test sample with respect to the control sample. Evaluation method. 14. The evaluation method according to claim 13, wherein the relative expression ratio is a value represented by [gene expression amount in test sample / gene expression amount in control sample]. 15. The expression variation level of the gene is evaluated based on the value indicated by the absolute value of the logarithm of the relative expression ratio of the gene whose expression level is increased in the test sample as compared to the control sample. Or the evaluation method described in 14. 1 6. The value indicated by the sum of the points of each gene in the test sample when the relative expression ratio of the gene whose expression level is higher than 2 in the test sample compared to the control sample is 2 or more. The evaluation method according to claim 13 or 14, wherein the expression fluctuation level of the gene is evaluated by the following. 17. Gene expression analysis is performed by comparing the expression levels of at least eight genes whose expression levels vary depending on the malignancy of the cancer with the expression level of the housekeeping gene in the test sample. The evaluation method according to any one of claims 1 to 7. 18. The evaluation method according to any one of claims 1 to 17, wherein in the step (2), the measured value of the expression level of each gene obtained in the step (1) is further corrected. 1 9. The cancer malignancy, in which nucleic acids or fragments thereof corresponding to at least eight genes whose expression levels change according to the malignancy of the cancer are immobilized in defined regions on the support, respectively. Array to evaluate. 20. The array of claim 19, wherein the support is a glass slide. 2 1. Apoptosis inhibitor survivin (Gene bank registration number: U75285) gene  Collagen type I hy-2 (Genebank accession number: J03464)] gene,  Collagen type I I pro-alpha-1 (Gene Bank accession number: XI 4420)] gene, Fibronectin precursor (FN) (Genebank registration number: X02761)] gene,  MM P-1; Matrix raetal loproteinase 1; intestinal collagenase (Genebank registration number: M13509)] gene, MM P-7; uterine matrilysin (matrix metal loproteinase 7; raatri lysin, ut erine (Genebank registration number: Z11887)) gene,  U—plasminogen activator [urokinase-type plasminogen activator pre cursor (EC 3. 4.21.73); U-plasminogen activator (UP A); plasminogen activa tor, urokinase (Genebank registration number: X02419)] gene, Perokinase-type plasminogen activator receptor [urokinase-type plasrainog en activator receptor; GPI-anchored from precursor (U-PAR); monocyte act ivation anti en M03; CD87 antigen (Genebank registration number: U09937)] Child, Alpha-2-macroglobulin (alpha-2-Μ) Bank registration number: M11313)] gene,  / 8 type platelet-derived growth factor receptor precursor Cbeta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank registration number: J03278)] gene,  BIGH3 (Genebank accession number: M77349) gene,  XVI I I α- 1 chain Ccollagen type XVI I I alpha-1 (Genebank registration number: AF018081)] Growth hormone-dependent insulin-like growth factor-binding protein (growth hormone-dep-endent insul in-like growth factor-binding protein (registered accession number: M35878)] gene, Oiepatoma-derived growth factor (HDGF) (Genebank registration number: D16431)] gene, Insulin-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: X16302)] gene, C type IV col lagenase (Genebank accession number: M55593)] gene, s p arc precursor (osteonectin) Csparc precursor (secreted protein acidic and rich in cysteine; osteonectin) (ON); basement membrane protein BM-40 (Genebank registration number: J03040)]  Cthrombospondin 2 precursor (Genebank registration number: L12350)] gene, Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large fibroblast proteoglycan; chondroi tin sulfate proteogl yean core protein 2; glial hyaluronate-binding protein (GHAP) Enpoplakin (gene bank accession number: U53786) gene, Integrin y8—4 chain Untegrin, beta 4] (Genebank accession number: X53587) gene, Vimentin (gene bank accession number: Z19554) gene, c-fms proto-oncogene (c-fms proto-oncogene) (Genebank registration number: X03663), Proliferating cell nuclear antigen (Genebank registration number: 15796) gene,  C l ^ Seki ^ Evening. (Leukocyte antigen related protein) (Genenok registration number: Y00815) gene, Interleukin 8 (Genebank registration number: M26383) Gene,  GDP dissociation inhibitor (GDI) CGDP dissociation inhibitor (GDI)) (Genebank registration number: 20688) Brain-specific tubulin alpha chain! : Tubulin, alpha, brain-specific) (Genebank registration number: X01703) gene, Cell cycle control gene cdc2 (cell division cycle 2, G1 to S phase & G2 to M phase) [cell division cycle 2, Gl to S and G2 to M] (Genebank registration number: X05360), Tissue-type plasminogen activator, tissue] Gene Bank accession number: M15518] gene, Stromal cell l-derived factor 1 (Genebank accession number: U16752) gene, Matrix metal lipoproteinase 10 (stromelysin 2) (Genebank accession number: X07820) Gene, Matrix metal lipoproteinase 15 (membran e-inserted) Gene bank registration number: Z48482) gene, Antioxidant protein 1] (Genebank accession number: D49396) gene, Laminin 4 chain Uaminin, alpha 4] (Gene bank registration number: S78569) Ubiquitin conjugating enzyme E2M (homologous to yeast UBC12)] Genebank Accession No .: AF075599] gene, Retinoblastoma binding tanno ,. Gene 6 (retinoblastoma-binding protein 6) (Genebank accession number: X85133) gene,  K-sam (gene bank registration number: M87770) gene, Cathepsin B] (Genebank accession number: L22569) Gene, signal transduction & transcriptional activator 1; Sleep Csignal transducer and activator of transcription 1, 91kD] (Genebank accession number: M97935) Gene, carcinoembryonic antigen Related cell adhesion molecule 6 (non-specific cross-reactive antigen) [carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting ant igen)] (Genebank accession number: M18728) gene, Heparan sulfate proteoglycan (gene bank registration number: M85289) gene, v-Ki-ras2 Kirsten rat sarcoma 2 virus oncogene homolog (v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog) Ras homolog gene family, member D] (Genebank accession number: D85815) gene, Hexabrachion (tenascin C) , cytotact in)] (Gene bank accession number: Χ78565) gene, Cyclin D 2 Ccycl in D2) (Genebank accession number: D13639) gene, Meta-oral proteinase 3 tissue inhibitor Ctissue inhibitor to metal loproteinase 3 (Sorsby fundus dystrophy, pseudoinf lamraatory)] (Jinnokunk registration number: AL023282) Laminin receptor 1, Ribosomal protein S A Claminin receptor 1 (67kD, ribosomal protein SA)] (Genebank registration number: U43901) gene, Lactate dehydrogenase A (Genebank accession number: X02152) gene, Keratin 18 Ckeratin 18) (Genebank accession number: M26326) gene,  Ia-associated invariant gamma-chain (Genbank accession number: AH001484) gene,  CD72 antigen CCD72 antigen) (Genbank accession number: M54992) gene, Connective tissue growth factor] (Genebank accession number: X78947) gene, Matrix meta-oral proteinase 3 (stromelysin 1, gelatinase precursor) Lmatrix metal loproteinase 3 stromelysin 1, progelatinase] (Geneno's link registration number: X05232) gene, Cyclin-dependent kinase 4] (Genebank registration number: U37022) Human SB class II histocompatibility antigen CHuman mRNA for SB class l histocompatibi lity ant igen alpha-chain (Genebank accession number: X03100) gene,  Transforming growth factor, beta 3] (Genebank accession number: X14885) gene, Thyroid hormone receptor interacting protein 6] (Genebank registration number: AJ001902) gene, Platelet Z endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 antigen) (Genebank accession number: L34657) gene, Profilin 1 (Genebank accession number: J03191) gene,  GTP-binding protein (Gene Bank accession number: AF120334) Homo sapiens clone No. 24703; 8 tubulin m R NA CHorao sapiens clone 24703 beta-tubul in mRNA, complete cds] (Genebank accession number: AF0706 00) gene,  Rho-associated, coil-containing protein kinase 1 (Gene bank registration number: U43195)  Type IV collagen, α-l chain Ccol lagen, type IV, alpha 1] (Genebank registration number: M26576) gene,  Topoisomerase (DNA) I Ctopoisomerase (DNA) I] (Genebank accession number: M60706) gene,  Kuni tz type 1 serine protease inhibitor, Kunitz type 1 (Genebank registration number: AB000095) gene, Rho GTP ace activating protein 1 [Gene bank registration number: U02570] gene, Nidogen (gene bank accession number: M30269) gene, Cyclin-dependent kinase 5. regulatory subunit 1 (p35) (Genebank accession number: X80343) gene,  CD9 antigen [CD9 ant igen] (Genebank registration number: M38690) gene, aortic-type smooth muscle actin gene, exon 9 Caortic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216)] , Bone morphogenetic protein 1 (BMpl) (Genebank registration number: 圆 30)] Type VI collagen α- 3 chain Ccol lagen type VI alpha- 3 (Genebank registration number: X52022)] gene, Interferon-ga marauder a- induced monokine (MIG) (Genebank registration number: X72755)] gene, Integrin alpha-3 chain (Genebank accession number: M599 11)] gene, Interferon inducible gene family (Genebank accession number: X57351)] gene, Metastasis-associated MT A 1 (metastasis-associated MTA1 (Genebank accession number: U351 13)) gene, Notch 2 Notch homolog 3 (Genebank accession number: U97669)] gene, and Proto-oncogene rh0A multidrug resistance protein Cproto-oncogene rhoA mul tidrug resistance protein; GTP-binding protein (rhoA) (Geneno, 'NK registration number: L25080)] gene, The array according to claim 22, wherein nucleic acids corresponding to at least eight kinds of genes selected from the group consisting of are fixed. 22. The array according to claim 21, wherein at least 20 genes are fixed. 23. A primer containing a primer and a probe for detecting mRNA or a fragment thereof expressed from at least eight genes whose expression level changes depending on the grade of cancer malignancy, Kit for evaluation or detection of cancer. 24. The kit according to claim 23, wherein the primer and / or the probe is for detection by a nucleic acid amplification method. 25. The kit according to claim 24, wherein the nucleic acid amplification method is an RT-PCR method. 26. A gene whose expression level changes depending on the grade of cancer Apoptosis inhibitor survivin (Gene bank registration number: U75285) gene,  Collagen type Ia-2 (Genebank accession number: J03464)] gene,  ΙΠ type collagen procollagen 1 chain Ccollagen type III pro-alpha-1 (Gene bank registration number: XI 4420)] gene, Fibronectin precursor (FN) Cfibronectin precursor (FN) (Genebank registration number: X02761)]  MMP-1; Cmatrix metalloproteinase 1; intestinal collagenase (Genebank registration number: M13509)] gene, MMP-7; uterine matrilysin Cmatrix metalloproteinase 7; matrilysin, ut erine (Genebank registration number: Ζ118δ7)] gene,  U-plasminogen activator (Urokinase—type plasminogen activator pre cursor (EC 3.4.21.73); U-plasminogen activator (UP A); plasminogen activa tor, urokinase (Genebank registration number: X02419)) gene, Perokinase-type plasminogen activator receptor Curokinase-type plasminogen activator receptor; GP anchored from precursor (U-PAR); monocyte act ivation antigen M03; CD87 antigen (Genebank registration number: U09937)] Alpha-2-macroglobulin (alpha-2-M) (Genbank accession number: M11313)] gene, Platelet-derived growth factor receptor precursor Cbeta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank accession number: J03278)] gene,  BIGH3 (Genebank accession number: M77349) gene,  XVI I type collagen α-1 鏆 Ccol lagen type XVI I I alpha-1 (Genebank registration number: AF018081)] Growth hormone-dependent insulin-like growth factor-binding protein (growth hormone-dep endent insul in-like growth factor-binding protein (GeneNon registration number ^: M35878)] gene, Hepatoma-derived growth factor (HDGF) (Genebank registration number: D16431)] gene, Insulin-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: X16302)] gene, C type IV col lagenase (Genebank accession number: M55593)] gene, sparc precursor (osteonectin) Csparc precursor (secreted protein acidic and rich in cysteine; osteonect in) (ON); basement membrane protein B-40 (Genebank registration number: J03040)] gene,  Cthrombospondin 2 precursor (Genebank registration number: L12350)] gene, Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large fibroblast proteoglycan; chondroi tin sulfate proteogl yean core protein 2; glial hyaluronate-binding protein (GHAP) (Genenok registration number: U16306) Envoplakin (gene bank accession number: U53786) gene, integrin / 8—4 chain (integrin, beta 4) (gene bank accession number: X53587) gene, Vimentin (gene bank accession number: Z19554) gene, c-fms proto-oncogene (c-fms proto-oncogene) (Genebank registration number: X03663), Proliferating cell nuclear antigen (Genebank registration number: M15796) gene, Leukocyte antigen-related Tannoヽ⁰ click quality (leukocyte antigen related protein] (Jin'noku link registration number: Y00815) gene, Interleukin 8] (Genebank registration number: M26383)  GDP dissociation inhibitor (GDI) (Genebank registration number: L20688) gene, Brain-specific tubulin alpha chain (Tubulin, alpha, brain-specific) (Genebank registration number: X01703) gene, Cell cycle control gene cdc2 (cell division cycle 2, G1 to S phase & G2 to M phase) [cell division cycle 2, Gl to S and G2 to M] (Genebank registration number: X05360) Tissue-type plasminogen activator (tissue) Gene bank registration number: M15518] gene, Stromal cell-derived factor 1 (Genebank accession number: U16752) gene, Matrix metalloproteinase 10 (stromelysin 2) (Genebank accession number: X07820) Gene, matrix metalloproteinase 15 (membran e-inserted) (Gene bank registration number: Z48482) gene, Antioxidant protein 1 (Genebank registration number: D49396 ) Gene, Lamininna 4 chain Claminin, alpha 4] (Genebank registration number: S78569) Cubiqui tin-conjugatin g enzyme E2M (homologous to yeast UBC12)] Gene bank accession number: AF075599] gene, Retinoblastoma binding tanno ,. Cretinoblastoma-binding protein 6) (Genebank accession number: X85133) gene,  K-sam (gene bank registration number: M87770) gene, (Cathepsin B) (Genebank registration number: L22569) gene, signal transduction and activator of transcription 1, ■ (signal transducer and activator of transcription 1, edition) (Genebank registration number: M97935) gene, carcinoembryonic antigen Related cell adhesion molecule 6 (non-specific cross-reacting antigen) [carcinoembryonic ant igen-related cell adhesion molecule 6 (non-specific cross-reacting antigen)] (Genebank accession number: M18728) gene, Heparan sulfate proteoglycan (gene bank registration number: M85289) gene, v-Ki-ras2 irsten rat sarcoma virus oncogene homolog [v-Ki-ras2 Kirs ten rat sarcoma 2 viral oncogene homology (genenokun registration number: M54968) gene, Ras homolog gene family, member D] (Genebank accession number: D85815) gene, Hexabrachion (tenascin C, cytotact in)) (Genebank accession number: Χ78565) gene, Cyclin D 2 Ccycl in D2] (Genebank registration number: D13639) Gene, meta-oral proteinase 3 tissue inhibitor [tissue inhibitor of metal loproteinase 3 (Sorsby fundus dystrophy, pseudoinf lamraatory)] (Geneno registration number : AL023282) gene, Laminin receptor 1, Ribosomal protein S A Clarainin receptor 1 (67kD, ribosomal protein SA)] (Genebank accession number: U43901) gene, Lactate dehydrogenase A (Genebank accession number: X02152) gene, Keratin 18 Ckeratin 18] (Genebank accession number: M26326) gene,  Ia-associated invariant gamma-chain (Genbank accession number: AH001484) gene,  CD72 antigen CCD72 ant igen) (Genebank accession number: M54992) gene, Connective t issue growth factor (Genebank accession number: X78947) gene, Matrix meta-oral proteinase 3 (stromelysin 1, progelatinase) j (Geneno's registration number: X05232) gene, Cyclin-dependent kinase 4] (Genebank registration number: U37022) Human SB class II histocompatibility antigen CHuman mRNA for SB class 11 histocompatibility antigen alpha-chain (Genebank accession number: X03100) gene,  Transforming growth factor 8—3 chain Ctransforming growth factor, beta 3] (Genebank accession number: X14885) gene, Thyroid hormone receptor interacting protein 6 (Genebank registration number: AJ001902) gene, Platelet endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 antigen) (Genebank accession number: L34657) gene, profiling 1 Cprofilin 1] (Genebank accession number: J03191) gene , GTP-binding protein (Genebank accession number: AF120334) Kid, Homo sapiens clone 24703 S tubulin m R NA CHomo sapiens clone 24703 beta-tubul in mRNA, complete cds] (Genebank accession number: AF0706 00)  Rho-associated, coi led -coil containing protein kinase 1 (Gene bank registration number: U43195)  Type IV collagen, Hiichi 1 chain Ccol lagen, type IV, alpha 1] (Genebank registration number: M26576) gene,  Topoisomerase (DNA) I Ctopoisomerase (DNA) I] (Genebank accession number: M60706) gene,  Kunitz type 1 serine proteinase inhibitor, Cserine protease inhibitor, Kunitz type 1] (Genebank registration number: AB000095) gene, Rho GTP ace activating protein 1 [Gene bank registration number: U02570] gene, Nidogen (gene bank accession number: M30269) gene, Cyclin-dependent kinase 5, regulatory factor 1 (p35)] (Genebank accession number: X80343) gene,  CD9 antigen [CD9 antigen] (Genebank registration number: M38690) gene, aortic-type smooth muscle actin gene, exon 9 Caortic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216) ], Bone morphogenetic protein 1 (Bpl) (Gene Bank registration number: U50330)] gene,  Collagen VI type 1 3 chain Ccol lagen type VI alpha-3 (Genebank accession number: X52022)] Humig cytokine humig; interferon-gamma-induced monokin e (MIG) (Genebank accession number: X72755)] gene, Integrin alpha-3 chain (Genebank registration number: M599 11)] gene, Interferon inducible gene family (Genebank registration number: X57351)] gene, Metastasis-associated MT A 1 One stasis-associated MTA1 (Genebank accession number: U351 13)] gene, Notch2 Notch homolog 3 (Notch2 Notch homolog 3 (Genebank accession number: U97669)) gene, and Proto-oncogene r h 0 A Multidrug resistant tannoII. [Proto-oncogene rhoA mult idrug resistance protein; GTP-binding protein (rhoA) (GeneNonk registration number: 25080)] The kit according to any one of claims 23 to 25, wherein the kit is at least 8 kinds selected from the group consisting of: 27. The kit according to claim 26, wherein the gene whose expression level changes according to the malignancy of the cancer is at least 20 genes. 28. An antibody or a fragment thereof against a polypeptide or a fragment thereof encoded by at least eight genes whose expression level changes depending on the malignancy of the cancer, for evaluating the malignancy of the cancer or for cancer. Detection kit. 29. The polypeptide is apoptotic inhibitor survivin (apoptosi s inhibitor survivin (Genebank accession number: U75285)) gene, Type I collagen α-2 Ccollagen type I-2 (Genebank accession number: J03464)] gene, Collagen type III pro-alpha-1 (gene bank registration number: XI 4420)] gene, Fibronectin precursor (FN) (Genebank registration number: X02761)] gene,  MM P-1; Cannatrix metal loproteinase 1; intestinal col lagenase (Genebank registration number: M13509)] gene, MM P-7; child matrilysin Cmatrix metal loproteinase 7; matri lysin, ut erine (Genebank registration number: Z11887)] gene,  U-plasminogen activator (urokinase-type plasminogen activator pre cursor (EC 3.4.21.73); U-plasminogen activator (UP A); plasminogen activa tor, urokinase (Genebank registration number: X02419)) gene, Perokinase-type plasminogen activator receptor [urokinase-type plasminogen activator receptor; GP anchored from precursor (U-PAR); monocyte activat ion antigen M03; CD87 antigen (Genebank registration number: U09937)] Child, Alpha-1-macroglobulin (alpha-2-macroglobulin (alpha-2-Μ) (Genbank accession number: Ml 1313)) gene,  A type 8 platelet-derived growth factor receptor precursor (beta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank registration number: J03278)) gene;  BIGH3 (Genebank accession number: M77349) gene,  XVI II type I collagen 1 type [collagen type XVI I alpha-1 (Genebank registration number: AF018081)] gene, Growth hormone-dependent insulin-like growth factor-binding protein (growth hormone-dep endent insul in-like growth factor-binding protein (gene no. Registration number: 35878)] gene, Liver cancer-derived growth factor C epatoma-derived growth factor (HDGF) (Genebank registration number: D16431)) Insulin-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: X16302)] gene, C type IV collagenase (Genebank registration number: M55593)] gene, sparc precursor (osteonectin) Csparc precursor (secreted protein acidic and rich in cysteine; osteonectin) (ON); basement membrane protein B-40 (Genebank registration number: J03040)] gene,  Cthrombospondin 2 precursor (Genebank registration number: L12350)] gene, Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large fibroblast proteoglycan; chondroi tin sulfate proteoglyan core protein 2; glial hyaluronate-binding protein (GHAP) Envoplakin (gene bank registration number: U53786) Gene, integrin 8-4 chains! : integrin, beta 4] (Genebank accession number: X53587) gene, Vimentin (gene bank accession number: Z19554) gene,  C-fms proto-oncogene [c-fms proto-oncogene] (Genebank registration number: X03663), Proliferating cell nuclear antigen (Genebank registration number: Ml 5796) gene, Leukocyte antigen-related evening. (Leukocyte antigen related protein) (Genenok registration number: Y00815) gene, Interleukin 8 Cinterleukin 8) (Genebank accession number: M26383) Child,  GDP Dissociation Inhibitor (GDI) CGDP dissociation inhibitor (GDI)] (Genebank registration number: L20688) Brain-specific tubulin chain! : Tubul in, alpha, brain-specific] (Genebank registration number: X01703) gene, Cell cycle control gene cdc2 (cell division cycle 2, G1 to S & G2 to M) (cel division cycle 2, Gl to S and G2 to M) (Genebank registration number: X05360), Tissue-type plasminogen activator (plasrainogen act ivator, t issue) Gene Bank accession number: M15518 Cstromal cell l-derived factor 1] (Genebank accession number: U16752) gene, Matrix metal lipoproteinase 10 (stromelysin 2) (Genebank registration number: X07820) Gene, Matrix metal liproteinase 15 (membran e-inserted) )] (Gene Bank accession number: Z48482) gene, Antioxidant protein 1 (Genebank registration number: D49396 ) Gene, Laminin-4 chain Uaminin, alpha 4] (Genebank registration number: S78569) Ubiquitin conjugating enzyme E2M (homologous to yeast UBC12)] Genebank Accession No .: AF075599] gene, Cretinoblastoma-binding protein 6) (Genebank accession number: X85133) gene, K-Sam [Gene bank accession number: M87770] gene, (Cathepsin B) (Genebank accession number: L22569) gene, signal transduction & activator of transcription 1, prototyping (Genebank accession number: M97935) gene, Carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross-reacting antigen) (Genebank accession number: M18728) gene, Heparan sulfate proteoglycan (heparan s fate proteoglycan) (Genebank accession number: M85289) gene, v-Ki-ras2 irsten rat sarcoma virus oncogene homolog [v-Ki-ras2 Kirs ten rat sarcoma 2 viral oncogene homolog] (Geneno Kunk registration number: M54968) Cras homolog gene family, member D] (Genebank accession number: D85815) Hexabrachion (tenascin C, cytotactin)] (Genebank registration number: X78565) Cyclin D 2 Ccycl in D2] (Genebank Accession Number: D13639) gene, tissue inhibitor of raetalloproteinase 3 (Sorsby fundus dystrophy, pseudoinf lammatory) (GeneNon Accession Number ^ ": AL023282 ) Gene, Laminin receptor 1, liposomal protein S A Claminin receptor 1 (67kD, ribosomal protein SA)] (Genebank registration number: U43901) gene, Lactate dehydrogenase Clactate dehydrogenase A] (Genebank registration number: X02152) gene, Keratin 18 Ckeratin W (Genebank accession number: M26326) gene, Ia-associated invariant gamma-chain (Genbank accession number: AH001484) gene, CD72 antigen CCD72 ant igen] (Genebank accession number: M54992) gene, Connective tissue growth factor (genebank accession number: X78947) gene, Matrix meta-oral proteinase 3 (stromelysin 1, progelatinase)] Gene code: X05232) gene, Cyclin-dependent kinase 4) (Genebank registration number: U37022) Human SB class II histocompatibility antigen CHuman mRNA for SB classl l histocorapatibi liti ant igen alpha-chain ^ (Genebank accession number: X03100) gene,  Transforming growth factor 5 _ 3 chain [transforming growth factor, beta 3] (Genebank accession number: X14885) gene, Thyroid hormone receptor interacting protein 6 (Genebank registration number: AJ001902) gene, Platelet endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 antigen) (Genebank accession number: L34657) gene, profilin 1 Cprofil in 1] (Genebank accession number: J03191) gene, GTP-binding protein [GTP-binding protein] (Genebank accession number: AF120334) Homo sapiens clone No. 24703 β tube,) m R NA CHomo sapiens clone 24703 beta-tubul in mRNA, complete cds] (Genebank registration number: AF0706 00) gene,  Rho-associated, coil-coil-containing protein kinase 1 (Gene bank registration number: 95) Type IV collagen, α-1 chain (col lagen, type IV, alpha 1) No .: M26576) gene,  Topoisomerase (DNA) I Ctopoisomerase (DNA) I] (Genebank accession number: M60706) gene,  Kuni tz type 1 serine proteinase inhibitor Cserine protease inhibitor, Kuni tz type 1] (Genebank registration number: AB000095)  Rho GTP Ace activating protein 1 [Rho GTPase activating protein 1] (Genbank accession number: U02570) gene, Nidogen (gene bank accession number: M30269) gene, Cyclin-dependent kinase 5, regulatory subunit 1 (p35) (Genebank accession number: X80343) gene,  CD9 antigen [CD9 antigen] (Genebank registration number: M38690) gene, aortic-type smooth muscle —actin gene, exon 9 Caort ic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216)] , Bone morphogenetic protein 1 (BMpl) (Genebank registration number: U50330)]  Collagen type VI, 3 chains Ccol lagen type VI alpha-3 (Genebank accession number: X52022)] Cytokine humig (cytokine humig; interferon-gamma-induced monokine (MIG) (Genebank accession number: X72755)) Integrin one-chain Cintegrin alpha-3 chain (Genebank accession number: M599 11)] gene, Interferon inducible gene family (Genebank accession number: X57351)] gene, Metastasis-associated MT A 1 Cmetastasis-associated MTA1 (Genebank accession number: U351 13)] gene, Notch 2 Notch homo log 3 (Genebank registration number No .: U97669)] gene, and Proto-oncogene rhoA multidrug resistance protein [proto-oncogene rhoA multi-drug resistance protein; GTP-binding protein rhoA) GeneNo'nk registration number: L25080)] 29. The kit according to claim 28, wherein the kit is at least eight kinds of polypeptides. 30. The kit of claim 29, wherein the polypeptides are at least 20 polypeptides. 31. With respect to the test sample and the control sample, the expression levels of at least eight genes or the polypeptides coded for the genes whose expression levels change depending on the degree of malignancy of the cancer are examined. Detecting a cancer cell as an indicator that the test sample contains cancer cells when there is a difference in the expression of the gene or polypeptide between the test sample and a control sample. Method. 3 2. A gene whose expression level changes depending on the grade of cancer malignancy Apoptosis inhibi tor survivin page —Bank registration number: U75285)] gene,  Type I collagen α-2 (collagen type I-2 (Genebank accession number: J03464)) gene,  Collagen type I I pro-alpha-1 (Genebank accession number: X14420)] gene, Fibronectin precursor (FN) (Genebank registration number: X02761)] gene, MM P-1; gastrointestinal collagenase Cmatrix raetal loproteinase 1; intestinal col lagenase (Genebank registration number: M13509)] gene, MM P-7; uterine matrilysin (matrix metal loproteinase 7; matri lysin, ut erine (Genebank accession number: Z11887)) gene,  U-plasminogen activator Curokinase-type plasminogen activator pre cursor (EC 3. 4. 21. 73); U-plasminogen activator (UP A); plasminogen activa tor urokinase (Genebank registration number: X02419)] gene , Perokinase-type plasminogen activator receptor [urokinase-type plasminogen activator receptor; GPI-anchored from precursor (U-PAR); monocyte act ivat ion antigen 03; CD87 antigen (Genebank registration number: U09937)] Genes, Alpha-2-macroglobulin (alpha-2-M) (Genbank accession number: Ml 1313)  Platelet-derived growth factor receptor precursor Cbeta platelet-derived growth factor receptor precursor (PDGFR-beta); CD140B antigen (Genebank registration number: J03278)] gene,  BIGH3 (Genebank accession number: M77349) gene,  XVI I type collagen α-1 chain (collagen type XVI I I alpha-1 (Genebank registration number: AF018081)) Growth hormone-dependent insulin-like growth factor-binding protein C-growth hormone-dep endent insul in-like growth factor-binding protein (gene registration number: M35878)] gene, Hepatoma-derived growth factor (HDGF) (Genebank registration number: D16431)] gene, Cinsul in-like growth factor binding protein 2 (IGFBp2) (Genebank accession number: X16302)] gene, C type IV col lagenase (Genebank accession number: M55593)] gene, Csparc precursor (secreted protein acidic and rich in cysteine; osteonectin) (ON); basement membrane protein B-40 (Genebank registration number: J03040)] gene,  Cthrombospondin 2 precursor (Genebank registration number: L12350)] gene, Chondroitin sulfate proteoglycan core protein 2 Cversican core protein precursor; large f ibroblast proteoglycan; chondroi tin sulfate proteogl yean core protein 2; glial hyaluronate-binding protein (GHAP) (Geneno; Envoplakin (gene bank accession number: U53786) gene, integrin — 4 chains (integrin, beta 4) (gene bank accession number: X53587) gene, Vimentin (gene bank accession number: Z19554) gene, c-fms proto-oncogene (gene bank registration number: X03663), Proliferating cell nuclear antigen ^ (Genebank registration number: M15796) gene, Cleukocyte antigen-related protein] (Genebank registration number: Y00815) gene, Interleukin 8 Unterleukin 8] (Genebank registration number: M26383)  GDP dissociation inhibitor (GDI)] (Genebank registration number: L20688) gene, Brain-specific tubulin alpha chain CTubul in, alpha, brain-specific) (Genebank registration number: X01703) gene, Cell cycle regulatory gene cdc 2 (cell division cycle 2, from G1 to S & G2 M period) Ccel l division cycle 2, Gl to S and G2 to M] (Genebank registration number: X05360), Tissue-type plasminogen activator (t issue) Gene bank accession number: M15518] gene, Stromal cel l-derived factor 1 (Genebank accession number: U16752) gene, Matrix metal lipoproteinase 10 (stromelysin 2) (Genebank accession number: X07820) Gene, matrix metal loproteinase 15 (membran e-inserted) (Gene bank registration number: Z48482) gene, Antioxidant protein 1 (Genebank accession number: D49396) gene, Laminin — 4 chains [laminin, alpha 4] (Gene Bank Registry Number: S78569) Gene, Ubiquitin-conjugating enzyme E2M (homologous to yeast UBC12) Gene bank accession number: AF075599 Retinoblastoma-binding protein 6 (Gene bank accession number: X85133) gene,  K-sam (gene bank registration number: M87770) gene, Cathepsin B] (Genebank accession number: L22569) gene, signal transduction & activator of transcription 1, 91kD (Genebank accession number: M97935) gene, Carcinoembryonic ant igen-related cell adhesion molecule 6 (non-specific cross-reacting antigen) (gene bank registration number: M18728) gene, To Nono ⁰ run sulfate proteoglycans (heparan sulfate proteoglycan) (Jinban click registration number: 85289) gene, v-Ki-ras2 Kirs ten rat sarcoma virus oncogene homolog (v-Ki-ras2 Kirs ten rat sarcoma 2 viral oncogene homolog) (Jinnokun registration number: M54968) Ras homolog gene family, member D] (Genebank accession number: D85815) gene, Hexabrachion (tenascin C, cytotact in) (gene bank registration number: Χ78565) gene, Cyclin D 2 Ccycl in D2) (Gene bank accession number: D13639) gene, meta-oral proteinase 3 tissue inhibitor Ctissue inhibitor of metal loproteinase 3 (Sorsby fundus dystrophy, pseudoinf lammatory) , Laminin receptor 1, liposomal protein SA Claminin receptor 1 (67kD, ribosomal protein SA)] (Gene bank accession number: U43901) gene, lactate dehydrogenase (lactate dehydrogenase A) (Gene bank accession number: X02152) gene, Keratin 18 Ckeratin 18) (Genebank accession number: M26326) gene,  I a-related invariant 7 chain C la-associated invariant gamma-chain] (Genbank accession number: AH001484) gene,  CD72 antigen CCD72 ant igen) (Genebank accession number: M54992) gene, connective t issue growth factor] (Genebank accession number: X78947) gene, Matrix meta-oral proteinase 3 (stromelysin 1, rogelatinase)] (Genenok registration number: X05232) gene, Cyclin-dependent kinase 4] (Genebank registration number: U37022) Human SB class II histocompatibility antigen CHuman mRNA for SB class l histocompatibi lity ant igen alpha-chain (Genebank accession number: X03100) gene,  Transforming growth factor S—3 chain Ctransforming growth factor, beta 3] (Genebank accession number: XI 4885) gene, Thyroid hormone receptor interacting protein 6 (Genebank registration number: AJ001902) gene, Platelet Z endothelial cell adhesion molecule (CD31 antigen) Cplatelet / endothelial cell adhesion molecule (CD31 antigen) (Genebank accession number: L34657) Gene, profilin 1 (Genebank accession number: J03191) Gene, GTP-binding protein (Gene Bank accession number: AF120334) Homo sapiens clone # 24703 β-thousand, mRNA NA CHomo sapiens clone 24703 beta-tubul in mRNA, complete cds] (Genebank accession number: AF0706 00) gene,  Rho-associated, coi led -coil containing protein kinase 1 (Genebank registration number: U43195)  Type IV collagen, one chain [col lagen, type IV, alpha 1] (Genebank registration number: M26576) gene,  Topoisomerase (DNA) I (genebank accession number: M60706) gene,  Kimi tz type 1 serine proteinase inhibitor, [serine protease inhibitor, Kunitz type 1] (Genebank registration number: AB000095) gene, Rho GTPase activating protein 1 —Bank registration number: U02570) gene, Nidogen (Genebank accession number: M30269) gene, Cyclin-dependent kinase 5, regulatory subunit 1 (p35) (Genebank accession number: X80343) gene,  CD9 antigen [CD9 antigen] (Genebank registration number: M38690) gene, aortic-type smooth muscle a-actin gene, exon 9 Caort ic-type smooth muscle alpha-act in gene, exon 9 (Genebank registration number: M33216) ], Bone morphogenetic protein 1 (BMpl) (Genebank registration number: U50330)] gene,  Collagen type VI alpha-3 (col lagen type VI alpha-3 (Genebank registration number: X52022)) gene, Humig cytokine huraig; interferon-gamma-induced monokine (IG) (Genebank accession number: X72755)] gene, Integrin 1-3 chain Cintegrin alpha-3 chain (Genebank accession number: M59911)] gene, Interferon inducible gene family (Genebank accession number: X57351)] gene, Metastasis-associated MTA 1 Cmetastasis-associated MTA1 (Genebank accession number: U351 13)] gene, Notch2 Notch homolog 3 (Notch2 Notch homolog 3 (Genebank accession number: U97669)) gene, and Proto-oncogene rhoA multidrug resistance protein; GTP-binding protein (rhoA) (Genenokunk registration number: 25080)] gene, 31. The method for detecting cancer according to claim 31, wherein the method is at least 8 types selected from the group consisting of: 33. The method for detecting cancer according to claim 31 or 32, wherein expression of at least 20 genes or a polypeptide encoded by the gene is examined.  34. The method for detecting cancer according to any one of claims 31 to 33, wherein the expression of a gene is examined using the array according to any one of claims 19 to 22.  35. The method for detecting cancer according to any one of claims 31 to 34, comprising a step of performing the evaluation method according to any one of claims 1 to 18.