WO1999039002A1

WO1999039002A1 - Method for examining expression pattern of vascular endothelial growth factor isoform

Info

Publication number: WO1999039002A1
Application number: PCT/JP1999/000344
Authority: WO
Inventors: Masato Nakamura
Original assignee: Masato Nakamura
Priority date: 1998-01-30
Filing date: 1999-01-27
Publication date: 1999-08-05
Also published as: JP4362225B2; AU2183799A

Abstract

A method for examining the expression pattern of a vascular endothelial growth factor isoform in a specimen containing RNA or cDNA, characterized by effecting a nucleic acid amplification reaction by using at least a pair of nucleotide primers hybridizable with mRNA or cDNA of vascular endothelial growth factor; and a reagent kit for examining the expression pattern of a vascular endothelial frowth factor isoform, characterized by containing at least a pair of nucleotide primers hybridizable with mRNA or cDNA of vascular endothelial growth factor.

Description

Description Method for examining the expression pattern of vascular endothelial growth factor isoform

The present invention provides a method for examining the expression pattern of a vascular endothelial growth factor isoform, a method for examining the expression of VEGF189, a method for detecting VEGF189, and a reagent kit that can be used in those methods. About. Background art

Many patients with advanced colon cancer die from liver metastases even after surgical treatment. The important properties and events leading to the formation of metastatic colonies are not well understood. Much research has been directed to the angiogenesis of substrates associated with distant metastasis of cancer. Basic fibroblast growth factor, transforming growth factor and tumor necrosis factor alpha are well known as angiogenic factors.

Recently, vascular endothelial growth factor (VEGF) has been studied as an angiogenic factor (L eung et al., 1989; Keck et al., 1989). VEGF was discovered due to its ability to increase the permeability of the microvasculature to circulating macromolecules (Senger et al., 1983). VEGF is an endothelial cell-specific antigen and angiogenic factor in vivo, which plays an important role in the neovascularization of various types of neoplasms (neoplasnu tumors). Overexpression of VEGF compared to normal tissues (Berse et al., 1992) was detected in the colon (Brown et al., 1993; Takahashi et al., 1995), breast (Brown et al., 1995), brain (Berk dust et al., 1993), ovaries (Boocock et al., 1995) and liver (Suzuki et al., 1996)

Four different isoforms of the VEGF transcript encoding polypeptides of 206, 189, 165 and 121 amino acids may be expressed in human cells. It has been reported (Houck et al., 1991) that these VEGF isoforms retain different biological activities (Park et al., 1993; Houck et al., 1992). VEGF121 and VEGF165 are secreted in soluble form, but the two larger isoforms (VEGF189 and VEGF206) have a stronger affinity for their cell surface proteoglycin. And remains associated with the cell. VEG F121 is not a heparin binding protein, but other isoforms possess heparin binding activity (Cohen et al., 1995). Mitogen activity of VEGF122 and VEGF165 is inhibited by platelet factor 4 (Gengrinovitch et al., 1995).

VEGF165 is expressed in most tissues (Dvorak et al., 1995), but the exact expression patterns of other VEGF transcript isoforms are not well understood. Alternating expression of VEGF isoforms is regulated by cell density in colon cancer cell lines (Koura et al., 1996). Expression of VEGF isoforms is different in tumor and non-tumor tissues of the liver (Suzuki et al., 1996). The clinicopathological significance of the different patterns of VEGF isoform expression in colon cancer is not well understood.

An object of the present invention is to provide a method for examining the expression pattern of VEGF isoforms and a reagent kit that can be used for the method.

Another object of the present invention is to provide a method for examining the expression of VEGF189 in a sample, a method for detecting VEGF189 in a sample, and a reagent kit usable therefor. Disclosure of the invention

The present inventors analyzed the expression pattern of VEGF isoform in colon cancer using the technique of reverse transcription-polymerase chain reaction (hereinafter, referred to as “RT-PCR”), and found that the isoform expression pattern was We found that it was classified into three groups. Furthermore, the correlation between the expression pattern of VEGF isoforms and clinical characteristics was analyzed, and the simultaneous expression of three isoforms of VEGF 121, VEGF 165, and VEGF 189 (type 3) Was found to correlate with liver metastasis, M stage and poor prognosis in colon cancer. The present invention has been made based on these findings. That is, the present invention is characterized in that a nucleic acid amplification reaction is performed on a sample containing RNA or cDNA using at least one pair of nucleotide primers that hybridize to mRNA or cDNA of vascular endothelial growth factor. A method for examining the expression pattern of a vascular endothelial growth factor isoform in a sample is provided. In the above method, at least the presence or absence of VEGF189 expression should be examined. At least one pair of nucleotide primers is designed so that the expression pattern of the VEGF isoform in the sample can be determined. For example, one of a pair of nucleotide primers contains a part of the base sequence of one exon of the VEGF gene or a sequence complementary thereto, and the other part of the base sequence of a different exon of the VEGF gene. Alternatively, a sequence complementary thereto may be included. FIGS. 5 and 6 show the entire nucleotide sequence of the VEGF 189 gene (including exons 1 to 8) derived from human and the amino acid sequence encoded thereby. Specifically, one of a pair of nucleotide primers contains a part of the nucleotide sequence of exons 1 to 5 of the VEGF gene or a sequence complementary thereto, and the other one of the nucleotide sequences of exon 8 of the VEGF gene. It is preferable to include a part or a sequence complementary thereto. As a part of the nucleotide sequence of exons 1 to 5 of the VEGF gene, the nucleotide sequence of SEQ ID NO: 1 can be mentioned. As a sequence complementary to a part of the nucleotide sequence of exon 8 of the VEGF gene, the nucleotide sequence of SEQ ID NO: 3 can be mentioned. Alternatively, one of the pair of nucleotide primers contains a part of the base sequence of exon 4 to 5 of the VEGF gene or a sequence complementary thereto, and the other part of the base sequence of exon 7 of the VEGF gene or a part thereof. It may contain a complementary sequence. As a part of the nucleotide sequence of exons 4 to 5 of the VEGF gene, the nucleotide sequence of SEQ ID NO: 2 can be mentioned. As a sequence complementary to a part of the exon 7 base sequence of the VEGF gene, the base sequence of SEQ ID NO: 4 can be mentioned. The nucleotide primer may have 5 to 70 bases, preferably 10 to 50 bases.

The present invention also provides a reagent kit for examining the expression pattern of a VEGF isoform, comprising at least one pair of nucleotide primers that hybridize to mRNA or cDNA of VEGF. Based on the results of tests performed using this reagent kit, cancer metastasis or prognosis can be diagnosed. Wear. The reagent kit may contain, in addition to primers, amplification reagents (including, for example, enzymes, buffers, and nucleotide triphosphates), buffers, and the like. One or more container means may include compartmentalized carrier means for containing together, each of which comprises one of the elements to be used in the method of the present invention. .

Furthermore, the present invention provides a method for preparing VEGF 18 in a sample containing RNA or cDNA using a nucleic acid probe that specifically binds to the RNA or DNA of VEGF 189, which is one of isoforms of VEGF. Methods for testing the expression of 9 are provided. That is, as shown in Examples described later, VEGF189 is specifically expressed in type 3 as compared with other types. Therefore, by examining at least the presence or absence of VEGF189, metastasis or prognosis in cancer can be diagnosed in the same manner as in the method of examining the expression pattern described above. The nucleic acid probe may be DNA having a part of the base sequence of the region including the boundary between exon 6 and exon 7 of VEGF189 or a sequence complementary thereto. For example, the region containing the boundary between exon 6 and exon 7 of VEGF 189 includes the base sequence of base numbers 15 to 185 in the sequence of SEQ ID NO: 11. More preferably, a base sequence of base numbers 58 to 87 can be mentioned. The part of the base sequence including the boundary between exon 6 and exon 7 of VEGF 189 or a nucleic acid probe sequence complementary thereto preferably has 10 to 400 bases, more preferably 15 to 30 bases. The nucleic acid probe may be labeled with, for example, radioisotope, a fluorescent dye, a chemiluminescent substance, an enzyme, or biotin.

In the above method of the present invention, the specimen containing RNA or cDNA may be derived from a cancer patient. The cancer can be selected from the group consisting of colon cancer (colon cancer), liver cancer, breast cancer, lung cancer, glioma, osteosarcoma and kidney cancer. The specimen containing RNA or cDNA may be tumor tissue.

Furthermore, the present invention provides a method for detecting VEGF189 in a specimen using an antibody or aptamer that specifically binds to VEGF189. Antibodies may be polyclonal antibodies or monoclonal antibodies against a protein antigen consisting of the amino acid sequence encoded at the boundary between exon 6 and exon 7 of VEGF189. Antibody. The protein antigen is encoded in a region including the boundary between exon 6 and exon 7 of VEGF189, preferably 5 or more, more preferably 5 to 100 amino acids. It is good to consist of the amino acid sequence containing. The amino acid sequence encoded in the region including the boundary between exon 6 and exon 7 of VEGF 189 includes the amino acid sequence of amino acids 6 to 62 in the sequence of SEQ ID NO: 12. it can. More preferably, an amino acid sequence of amino acids 15 to 34 can be mentioned. The specimen may be from a cancer patient. The cancer can be selected from the group consisting of colon cancer (colon cancer), liver cancer, breast cancer, lung cancer, glioma, osteosarcoma and kidney cancer. The sample may be serum, cerebrospinal fluid, or bodily fluids (eg, pleural effusion, ascites, etc.).

The present invention also provides a reagent kit for testing the expression of VEGF189, comprising a nucleic acid probe that specifically binds to VEGF189 RNA or DNA, and VEGF189. Providing a reagent kit for detecting VEGF 189, characterized in that it contains an antibody or an abtamer that specifically binds to _{c. C} The results of tests performed with these reagent kits Diagnosis of cancer metastasis or prognosis. The reagent kit may further include primers, amplification reagents, or appropriate buffers for the reaction. The kit may include a carrier means compartmentalized to collectively contain one or more container means, such as vials, tubes, etc., each of which comprises a container of the present invention. Includes one of the elements to be used in the method. Hereinafter, the present invention will be described in detail.

In the method of the present invention, the gene amplification method (for example, RT-PCR, TMA (Japanese Patent Application Laid-Open No. 4-500759), etc.) and the direct method (for example, using the DNA probe to obtain the mRNA of VEG F189 A direct detection method, a method of detecting a VEGF189 protein using a binder that specifically binds to VEGF189, and the like. Hereinafter, these techniques will be described.

1. Gene amplification method

Using the gene amplification method, the expression pattern of VEGF isoforms can be examined, Specific VEGF isoforms can be detected.

RT- P C R

Extract RNA from cancer tissue and perform RT-PCR. First, cDNA is synthesized from RNA extracted from cancer tissue using random primers and RTase. Next, the cDNA is amplified with primers V—S and V—A or V—S 4 and V—A 7.

(refer graph1) . The combination of these primers makes it possible to distinguish the amplified DNA from the VEGF isoform based on its length. See Figure 2 for primers V—S and V—A (447 bp band from VEGF189, 375 bp band from VEGF165, 243 bp band from VEGF12 1)). See Figure 3 for primers V—S4 and V—A7 (204 bp band from VEGF 189, 132 bp band from VEGF 165). Liver metastases were observed more frequently in patients expressing VEGF 189

(Table 1: Type 3: 12/32 = 37.5%; Type 1, 2: 4/29 = 13.8%). Also, patients expressing VEGF189 had a worse prognosis than patients not expressing it

(See Figure 4). Thus, for patients of type 3 (ie, expressing VEGF189), the cancer can be diagnosed as highly metastatic or have a poor prognosis.

The detection method here uses gel electrophoresis, and the difference

189 was detected, but other methods such as Hybridization Protection

Assay (hereinafter referred to as “HPA”) (Arnold LJ, et al, Clin Chem 35: 1588-1594, 1989). After completion of the amplification, the double-stranded DNA, which is the amplification product, is denatured using, for example, thermal denaturation. Add a probe labeled with acridinium ester (hereinafter referred to as “AE”) to perform HPA. The probe has a part of the nucleotide sequence of the region including the boundary between exons 6 and 7 of VEGF (FIG. 1) or a sequence complementary thereto, and is labeled with AE. Therefore, if a higher level of chemiluminescence is observed as compared with the control, it can be known that the mRNA of VEGF189 is present in the sample.

TMA

VEGF isoform mRNA can also be amplified using TMA. For example, the amplification can be performed with a combination of VS and VA primers. However, at least one of the primers has, for example, a T7 RNA promoter sequence on its 5 'side. The product amplified by TMA is RNA, but the presence of VEGF189 can be known by the same HPA as RT-PCR. The AE-labeled probe used here has a nucleotide sequence complementary to the nucleotide sequence guided by the T7 promoter.

2. Direct method

Direct methods can be used to detect a particular VEGF isoform (eg, VEGF189) or its expression.

HP A

In some cases, the presence of VEGF189 mRNA can be determined by applying RNA extracted from a sample to a material, such as HPA, directly without using RT-PCR or TMA, etc. . The AE-labeled probe used here is a part of the region containing the boundary between exons 6 and 7 of VEGF189 mRNA (for example, a part of the nucleotide sequence of nucleotides 58 to 87 of the sequence of SEQ ID NO: 11) or It should have a complementary sequence to it and be bound by the chemiluminescent AE (see RT-PCR). Binder

The presence of VEGF189 in a sample can also be known by detecting the protein with a binder. For example polyclonal antibody that specifically binds to the protein as a binder one mentioned here, here _c refer to monochromatic one monoclonal antibody or Abutama first class, will be described as the monoclonal antibody as an example. A polypeptide (hereinafter referred to as polypeptide 67) corresponding to the region containing the boundary between exons 6 and 7 of VEGF 189 mRNA (FIG. 1) (for example, amino acid number 15 in the sequence of SEQ ID NO: 12) To 34 amino acids), and spleen cells of the mouse are fused with myeloma cells to obtain a hybridoma. Then, by selecting a monoclonal antibody that specifically binds to the polypeptide 67 used as the antigen, a desired monoclonal antibody can be obtained. The polypeptide 67, which is an antigen used here, has a boundary between exons 6 and 7 at its center, and has at least 5 amino acids, more preferably around 20 amino acids. The antigen alone or, for example, Keyhole Lymp et Hemocyanin (hereinafter, referred to as "KLH".) to be coupled, also used when it _c for immunization respect Aputama one can be prepared by Droiet's method (Nature Biotechnology, 14, p. 1021, 1997) .

The binder prepared as described above can be used for RIA (Radio Imnoassy Biochemistry Laboratory Method 12 Tokyo Kagaku Dojin) and EIA (Enzyme Immunoassay 3rd Edition Medical College) to convert VEGF 189 It can be specifically detected and quantified.

An atsey using abutama can be constructed, for example, using the method of Droiet et al.

This description includes all the contents described in the description and drawings of Japanese Patent Application No. 10-185354, which is the basis of the priority of the present application. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows primers for detecting the isoform of VEGF. In FIG. 1, arrows indicate the sites of primers V—S, V—A, V—S4 and V—A7. VEGF 12 1 (24 3 bp), VEGF 16 5 (37 5 bp), VEGF 18 9 (4 47 bp) and VEGF 206 (4 A 98 bp) fragment was obtained. VEGF165, VEGF189 and VEGF206 fragments were obtained by PCR with V—S4 and V—A7.

FIG. 2 shows VEGF expression in tumors determined by RT-PCR (primer set; VS, VA). Lane 1: Patient number 1, type 2. Lane 2: Patient number 4, type 3. Lane 3: patient number 38, type 3. Lane 4: Patient number 5, Type 1. Lane 5: Patient number 26, type 2. Lane 6: patient number 42, type 2. Lane 7: Patient number 45, type 2. Lane 8: patient number 58, type 2.

FIG. 3 shows VEGF expression in tumors determined by RT-PCR (primer set; VS4, VA7). Lane 1: Patient number 1, type 2. Lane 2: Patient number 4, type 3. Lane 3: Patient number 38, type 3. Lane 4: Patient number 60, type 2. Lane 5: Patient number 26, type 2. Lane 6: Patient No. 42, Type 2. Lane 7: Patient number 45, type 2. Lane 8: Patient Number 5 8, Type 2.

FIG. 4 shows the overall viability based on the VEGF isoform pattern. Patients with type 3 isoform expression had a worse prognosis than patients with type 1 or 2 isoform expression patterns (generalized Cox-Mantel test, P <0.0

1) o

FIG. 5 shows the nucleotide sequence and a part of the amino acid sequence (base numbers 1 to 702) of the VEGF189 gene derived from human.

FIG. 6 shows the nucleotide sequence and part of the amino acid sequence (base numbers: 733-1119) of the VEGF189 gene derived from human. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be specifically described by the following examples. These examples are illustrative and do not limit the scope of the invention.

(Example 1)

Materials and methods

Targets and tissue samples

The subjects in this study were 61 colon cancer patients who underwent resection surgery at Tokai University Hospital between October 1998 and October 1999. All patients were assessed by TNM score (UICC, 1978). Patient characteristics are summarized in Table 1. Surgical specimens were snap frozen and stored at 180 ° C until analysis. Whole cell RNA was prepared from frozen samples.

RT-PCR analysis to detect γ E G F_ isoform transcripts

The VEGF isoform was converted to the following primers: V_S 5-AAGCCATCCTGTGTGCCCCT GATG-3 (SEQ ID NO: 1), V-S4 5-CGGATCAAACCTCACCAAGGCC-3 (SEQ ID NO: 2), V-A 5-GCGAATTCCTCCTGCCCGGCTCAC-3 (SEQ ID NO: 3) ) And V-A7 5-CTTTCTCCGCTCTGAGCAA GGC-3 (SEQ ID NO: 4) (FIG. 1) were evaluated by RT-PCR. A probe (378 bp) was prepared by PCR amplification using primers—V—S and V—A, and the sequence was confirmed by an automatic sequencer (ABI PRISM 310, Perkin Elmer, Calif.). Reverse transcription was performed at 42 ° C for 60 minutes (1 g of total cellular RNA, 100 pM Random primer (Boehringer Mannheim) _s reverse transcriptase (GIBCO-BRL)). PCR using a Gene Amp PCR System 9600 (Perkin Elmer) and Taq DNA polymerase (TOYOBO, Japan) consisting of 1 minute at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C. The VEGF cDNA fragment was amplified 0 times. The product block (Zeta-Probe, BI0-RAD) was hybridized with a photochemically labeled probe (ECL; Amersham) and exposed to Kodak AR film. The quality of this RNA was assessed by RT-PCR on / 32 microglobulin.

Histological examination of colon cancer

Colon cancer specimens were fixed in 10% formalin and embedded in paraffin according to the usual method. Histological sections were cut from the center of each colon tumor and stained with Haematoxylin and Eo sin (HkE) and Victoria blue-HkE to determine venous invasion of the colon wall. Histological examinations were examined separately by two pathologists. The degree of venous invasion was classified into the following four groups. ν θ has no vein penetration. v l + has minimal venous invasion, ie one or two foci of venous invasion in the histological section. v 2+ is moderate vein invasion, ie 3 or 4 foci of vein invasion. V3 + is a severe vein invasion with more than 5 invasive foci, and lymphatic invasion is 1 y1 + with a mild lymphatic invasion and 1y2 + with a moderate lymphatic invasion Ly3 + is a severe lymphatic intrusion.

^ Zan and Nozhan Blot analysis

A block of cells (13 g) digested with Ec0RI (13 g) (20 h at 37 ° C, Boehringer Mannheim; Nytran, MSI) was hybridized with VEGF cDNA labeled with ³² P, and KODAK RP The film was exposed at —80 for one week. A whole cell RNA block (15 // g, GeneScreen Plus, New England Nuclear) was hybridized with a ³² P-labeled VEGF cDNA probe (see above). The expression level of the VEGF gene was evaluated by densitometry (Interactive Build Analysis System, Zeiss).

Expression of VEGF receptor (f1t-1 and KDR) and TGF; 9-1

The expression of the VEGF receptor gene (f 1 t-1, KDR) was determined by the following primers: fl tl-S 5-ATGAGCAGTGTGAGCGGCTCCC-3 (2669-2690) (SEQ ID NO: 5), flta-A 5-AAG

-1 o- CTTTCGCTGCTGGTGACGC-3 (3125-3146) (SEQ ID NO: 6), KDR-S 5-CGTCATGGATCCAG ATGAACTCCC-3 (2406-2429) (SEQ ID NO: 7), KDR-A 5-CTTGACGGAATCGTGCCCCTTTGG -3 (2813-2836) (SEQ ID NO: 8) was evaluated by RT-PCR under the same conditions as above. The expression of TGF S-1 gene is also determined by RT-PCR using the following primers: TG FyS-1-S 5-GCCCTGGACACCAACTATTGC-3 (1679-1699) (SEQ ID NO: 9), TGF / S-1-A 5-GTTATGCTGGTTGTACAGGGCC -3 (1864-1885) (SEQ ID NO: 10). Activation of c-K-ras oncogene

Point mutations in the c—K_ras oncogene were assessed by the previously reported enriched PCR method (Ando et al., 1991). Direct sequence analysis confirmed a point mutation in the c-K-ras oncogene.

Statistical analysis

Survival differences between patient subgroups were compared using log-rank tests, and survival curves were plotted according to the method of Kaplan and Meier. ^Two tests were applied to compare between group frequencies.

VEGF isoform pattern

All colon cancer specimens expressed VEGF 12 1 (6 1/6 1). The isoform patterns were divided into three groups: type 1 VEGF 121, type 2 VEGF 121 + VEGF 165, type 3 VEGF 121 + VEGF 165 + VEGF 189 . Of the 61 colon cancers tested, three showed type 1 expression. 6 Of the 1 patient, 26 had type 2 expression and 32 had type 3 pattern. None of the tumors examined showed VEGF 206 expression. These isoform patterns of VEGF were confirmed by amplifying various parts of the VEGF cDNA with two pairs of primers (a-primer) (FIGS. 2 and 3).

Of the 25 normal colon tissues, 22 did not express VEGF_mRNA. One of the three normal colonic specimens expressing VEGF showed the type 1 isoform VEGF121, while the other normal mucosal specimens showed type 3 isoform expression. Histological characteristics and VEGF isoform pattern

Colon cancer specimens showing type 3 isoform expression showed a significantly higher incidence of venous vascular involvement (V2 + and V3 +) (P = 0.006, ² tests, Table 1) _c However, there was no clear correlation between this isoform expression pattern and lymphatic involvement. Histological grade colon cancer showed no significant association with VEGF isoform pattern (Table 1).

Correlation between VEGF isoform and clinical properties

12 Patients had liver metastases at the time of surgery and 4 patients had liver metastases during the follow-up period. Only one patient had lung metastases at the time of surgery, but such metastases were detected in four patients during the follow-up period. Twenty-five patients died from colon cancer during the follow-up period.

32 patients with colon cancer showing a type 3 isoform expression pattern had a significantly worse prognosis than the remaining 29 patients showing type 1 and type 2 expression patterns (P <0.0). 1, Cox-ManteK Figure 4).

Colon cancer patients with a type 3 isoform expression pattern had a significantly higher incidence of liver metastases than patients with a type 1 and type 2 isoform pattern (429, 13.8%). 1 2/3 2, 37.5%) (P <0.05, ² tests, Table 1). Of the 16 patients with liver metastasis injury, 12 (75.0%) showed a type 3 pattern, but 20 of the 45 patients without liver metastases (4 4 . 4%) showed this isoform expression pattern. Abnormal VEGF189 expression (type 3) was significantly correlated with Ml stage colon cancer (P <0.05, ² tests, Table 1). Thirteen of seventeen patients (76.5) with stage TnNnM1 also showed VEGF189 expression, whereas nineteen of fourteen patients with stage TnNnMO (43.5) 2%) expressed this isoform. There was no correlation between VEGF isoform pattern and tumor size (T-stage) or lymph node (N-stage) status.

VEGF gene expression level

Northern plot analysis showed overexpression of VEGF in all tested materials (20/20). The level of VEGF upregulation varied. Five normal colonic mucosa specimens showed no detectable expression of the VEGF gene. This expression level did not correlate with the VEGF isoform pattern. VEGF expression levels did not significantly correlate with metastasis, survival, or any other histological factors examined. Southern blot analysis showed no amplification or rearrangement of the VEGF gene (data not shown).

Expression of VEGF receptor and TGF3-1

Of the 61 colon cancers examined, 30 strongly expressed f1t-11 mRNA, and 43 of 61 colon cancers expressed KDR mRNA. It did not significantly correlate with the VEGF isoform pattern (Table 1). Six of the 61 patients showed expression of TGF / 3-1 mRNA.

Activation of c-K-ras oncogene

Activation of the c-K-ras oncogene was detected in 27 of the 61 colon cancers tested. Eighty-eight (67%) of this 27 colon cancers. —1: A point mutation (GGT—GAT) was found in codons 12 of the & 3 gene. No homozygous point mutations were detected. Activation of the c—K-ras oncogene was not correlated with the VEGF isoform pattern. Many studies have been directed to angiogenesis for distant metastasis of colon cancer. VEGF has been studied as an angiogenic factor (Leung et al., 1989; Keck et al., 1989). VEGF transcripts were detected in all primary human colon cancer specimens, but VEGF expression was detectable by RT-PCR in three of the 25 normal colon tissues. Colon cancer generally overexpresses the VEGF gene, but VEGF transcripts are weak in Northern blots in normal colon specimens. A higher incidence of VEGF-expressing cells has been reported in gastrointestinal cancer than in normal mucosa (Brown et al., 1993). Overexpression of VEGF compared to normal colonic mucosal levels has also been demonstrated in colon cancer (Takahashi et al., 1995). These results support the overexpression of VEGF in colon cancer.

In these bulk studies, variable amounts of the substrate RNA may have been co-purified from the tumor. The present inventor has proposed that the colon element in which the substrate element is replaced by mouse tissue Several cancer xenografts were established and the corresponding primary colon cancer material was preserved. These colon cancer xenografts showed the same VEGF isoform expression pattern as the primary tumor. These results indicate that VEGF was expressed on the tumor cells but not on the substrate. Therefore, the potential for contamination with substrate RNA has little effect.

Four different isoforms of human VEGF have been identified. They all result from alternative splicing of the primary transcript of a single gene. The pattern of the VEGF isoform was accurately analyzed using two different sets of primers. In this study, RT-PCR analysis was used to confirm VEGF isoform expression patterns. The expression pattern of the VEGF isoform was unaffected by increasing the amount of type III of the PCR cycle. In this study, all colon cancer patients expressed VEGF 121, and most patients expressed VEGF 165. However, VEGF189 was expressed in almost half the patients, and none of the patients expressed VEGF165 and VEGF189 alone. Of these isoforms, VEGF121 and VEGF165 were abundantly expressed in most hepatocellular carcinomas (Suzuki et al., 1996). All neoplasms of the central nervous system expressed VEGF 121, VEGF 165 and VEGF 189 (Berkman et al., 1993). The colon carcinoma cell line expressed three isoforms of VEGF in vitro (Koura et al., 1996). It is not clear what percentage of colon cancers express these isoforms. The results of this study indicated that less colon cancer than brain and hepatocellular carcinomas expressed these abnormal isoforms of VEGF. The expression of VEGF 206 (Houck et al., 1991) detected in the human fetal liver DNA library was not expressed in any of the inventors' samples.

It is not known what factors affect VEGF isoforms in colon cancer, but all colon cancers overexpress the VEGF gene compared to normal tissues. In this study, VEGF isoform patterns did not correlate significantly with gene expression levels. c- Activation of the K / H-ras oncogene is correlated with overexpression of the VEGF gene in rat intestinal cell lines (Rak et al., 1995). Therefore, the present inventors examined activation of the Κ-ras oncogene in colon cancer. 45% of colon cancers showed activation of the K-ras oncogene, There was no correlation between ras oncogene activation and VEGF isoform patterns. Expression of VEGF receptor KDR has been correlated with liver metastasis in colon cancer (Tak ahashi et al., 1995). The present inventors also examined the expression of the VEGF receptor (f1t-11, KDR) gene, but found no correlation with the VEGF isoform pattern. It has been reported that transforming growth factor / 3 (TGF 3) up-regulates VEGF gene expression (Dolecki et al., 1991). No correlation with VEGF isoform was found.

Little is known about the angiogenic properties of the four different isoforms of VEGF. Among the various isoforms, VEGF189 protein has the strongest ability to bind to extracellular matrix (ECM) components. Proliferation was strongly stimulated in endothelial cells cultured with ECM derived from VEGF189-expressing cells (Park et al., 1993, Houck et al., 1992). The VEGF 189 isoform was specifically upregulated in vitro in a colon cancer cell line cultured in confluent cells (Koura et al., 1996). The results suggest that angiogenesis, tumor growth and metastasis may be increased. Overexpression of VEGF has been suggested to be associated with the vascularity of colon cancer (Takahashi et al., 1995). However, no clear correlation between the clinical characteristics of colon cancer and the VEGF isoform expression pattern has been reported. The present inventors have shown that the expression of type 3 isoform patterns (VEGF189) correlates strongly with venous involvement in colon cancer. In this study, we also demonstrated that aberrant expression of VEGF 189 isoform correlated with liver metastasis and M1 stage in colon cancer.

Recent studies have suggested a correlation between total VEGF upregulation and liver metastasis in colon cancer (Warren et al., 1995). However, the results presented here do not only examine the level of VEGF expression, but also which isoforms are expressed in order to discuss the prognostic or malignant characteristics of colon cancer. Is important. Examination of the VEGF isoform pattern may help predict the prognosis of patients with colon cancer. Table 1. Single dose analysis of patient characteristics and association of VEGF isoform pattern with patient or tumor characteristics Type 1 Type 3 Ρ value

Total 2 9 4 7.5% 3 2 5 25%

Gender 0.9.1 7 Male 1 6 26.2% 1 8 2 9 5%

Female 1 3 2 1.3% 1 4 2 3 0%

Age 0.5 6 6

<6 0 1 3 2 1.3% 1 2 1 9 7%

≥ 6 0 1 6 2 6.2% 2 0 3 2 8%

Histology 0.96 0 Adenocarcinoma 2 7 4 4.3% 3 1 5 0 8%

Mucinous cancer 2 3.3% 1 16%

V—factor 0.06 0 *

V 1 + ≥ 2 0 3 2, 8% 1 1 1 8 0%

V 2 + ≤ 9 1 4.8% 2 1 3 4 4%

1 y—factor 0.2 8 0 1 y 1 + ≥ 1 2 1 9.7% 9 1 4 8%

1 y 2 + ≤ 17 27, 9% 2 3 3 7 7%

T-stage 0.8 4 7 TO, Tl, Τ2 6 9 · 8% 6 9 8%

Τ3, Τ4 2 3 3 7.7% 2 6 4 2 7%

Ν—Stage 0.6 8 1 NO 1 6 2 6.2% 1 6 2 6 2%

Ν1, Ν2, Ν3 1 3 2 1.3% 1 6 2 6 2%

Μ—Stage 0.0 1 9 * Μ 0 2 5 4 1.0% 1 9 3 1 1%

Μ 1 46.6% 1 3 2 1 3% 1 l 6 Liver metastasis 0.0 3 6 * Yes 46.6% 1 2 19.7%

None 2 5 4 1. 0% 2 0 3 2.7%

f 1 t — 1 expression 0.1 6 6 Yes 1 7 2 7.9% 1 3 2 1.3%

None 1 2 1 9.1% 1 9 3 1.1%

KDR expression 0.1 4 4 Yes 2 3 3 7.1% 2 0 3 2.8%

None 69.8% 1 2 19.7%

K-ras mutation 0.25 6 Yes 1 5 24.6% 1 2 19.7%

None 1 4 2 2.9% 2 0 3 2.8%

* Abnormal VEGF189 expression (type 3) was significantly correlated with colon cancer V-factor, Ml-stage and liver metastases (P <0.05, ² test).

(Embodiment 1)

Amplification method by TMA method and detection method of VEGF189 by HPA method>

1) The expression of VEGF189 can be examined using total cellular RNA prepared from the frozen specimen described in Example 1.

To amplify VEGF189 mRNA, for example, the polymerase chain reaction (PCR) method or the TMA method (RNA synthesis method) can be used.

3) The PCR method was described in Example 1, but here, an example of the TMA method using primers V-S4 (SEQ ID NO: 2) and V-A7 (SEQ ID NO: 4) as primers _c 4 ) When amplifying VEGF 189 mRNA using the TMA method, add a promoter sequence to the 5 'side of primer VA-7. Promoters include T7 polymerase promoter, T3 polymerase promoter and the like.

5) Add the amplification reagent containing the primer set to the whole cell RNA described in Example 1, and incubate at 42 ° C for 2 hours. 6) After amplification, add probe solution and measure chemiluminescence of acridinium ester (hereinafter referred to as “AE”) by HPA method to check for the presence of VEG F189 mRNA. .

[Synthesis of primer]

1) Using a commercially available DNA synthesizer, a primer V-A7 (SEQ ID NO: 4) with a T7 promoter sequence added to the 5 'end and a primer V-S4 (SEQ ID NO: 2) were used. Combine.

[Preparation of probe]

1) As a probe, a part of the region including the boundary between exon 6 and exon 7 shown in SEQ ID NO: 11, for example, the following having a sequence from base number 63 to base number 82:

5-CCT GGA GCG TTC CCT GTG GG-3 (SEQ ID NO: 13) An oligonucleotide is synthesized using a commercially available synthesizer.

2) During synthesis, an amino linker is introduced between base number 72 and base number 73 (arrow) in order to label AE later. AE is a PC of Arnold etc.

Prepare a DNA probe labeled AE and labeled as described in T / US 88/03361.

[Width of VE G FmRNA by TMA method]

1) Using Primer V-A7 to which the T7 promoter sequence has been added and another Primer V-S4, perform TMA targeting VEGF 189 mRNA, and synthesize a large amount of RNA.

2) For example, heat the whole cell RNA solution prepared from the frozen sample described in Example 1 at 60 ° C for 10 minutes and incubate at 42 ° C for 5 minutes.

3) To this, add an amplification reagent containing the above pair of primers (including 800 U of MMLV reverse transcriptase, 400 U of T7 RNA polymerase, buffer, nucleotide triphosphate, etc.) Incubate at 42 ° C for 2 hours.

4) Reagents used for amplification should be 70 mM Tris HC 1 (pH 8), 40 mM KC 1, 6 mM r GTP, 4 mM r CTP, 4 mM r ATP, 4 mM r UTP, 1 mM each in 1 mM — d TT P, d AT P, d CTP and d GT P, and 22mM Mg C 1 _c 5 is a) As will be appreciated by those skilled in the art, it is possible to perform a modification of the method. For example, assays can be performed using different amounts of reagents and incubation temperatures / hours.

[Detection of VEGF RNA by HPA method]

1) An AE-labeled oligonucleotide probe is added to the amplification product (RNA) containing the boundary between exon 6 and exon 7 by the TMA method, hybridization is performed, and the presence or absence of VEGF mRNA is examined.

2) AE labeled with an oligonucleotide probe that does not hybridize (bind) to the amplification product (target RNA) is susceptible to hydrolysis and does not emit chemiluminescence even when hydrogen peroxide is added in the presence of alkali.

3) The AE labeled on the hybridized oligonucleotide probe is resistant to hydrolysis and emits chemiluminescence.

4) Examples of hybridization and hydrolysis conditions include lOOmM Succinic Acid, 230m L OH, 20mM EDTA, 20mM EGTA, 15mM Aldri thiol-2.

, 1.2 ML iC 2 ¾L i L S. 3 ¾Et〇H in solution (pH 4.7), 60 ° C. for 20 minutes, followed by 600 mM sodium borate, Add a solution (pH 8.5) containing 182 mM Na〇H and 1% Triton X-100, and hydrolyze at 60 ° C for 7 to 15 minutes.

5) Thereafter, the binding between the AE-labeled DNA probe and the amplification product (RNA) can be examined by measuring the chemiluminescence using a luminometer.

6) That is, the AE-labeled probe shows a high chemiluminescence value only for VEGF 189 having the boundary between exon 6 and exon 7, and other isoforms having no boundary, such as VEGF 1 21 and a significant difference from the chemiluminescence value of VEGF165.

(Embodiment 2)

[Preparation of monoclonal antibody]

Here, a mouse monoclonal antibody will be described as an example. VEGF 1 8 9 special In order to obtain a monoclonal antibody that binds differentially, there is a method that uses the full-length polypeptide chain of VEGF189 as an antigen.This method includes the boundary between exon 6 and exon 7 of VEGF189. When a peptide is used as an antigen, a desired monoclonal antibody can be obtained more efficiently. In other words, five amino acids with the border almost at the center

(Eg, amino acid sequence 22 to 26), more preferably about 20 amino acids (eg, amino acid sequence 15 to 34) (hereinafter referred to as “peptide 67”) ) Is used for the antigen. Peptide 67 is singly used or conjugated to, for example, Keyhole lepto hemocynan (hereinafter referred to as “KLH”) to immunize mice to produce hybridomas (monoclonal antibody Kodansha Sentifik, immunological experiment procedure). Law I, II Nankodo). Screening of the monoclonal antibody can be performed, for example, using a 96-well plate on which peptide 67 or VEGF206 is adsorbed. That is, a monoclonal antibody that binds to the plate on which peptide 67 is adsorbed and does not bind to the plate on which VEGF206 is adsorbed is selected. It can be determined that the monoclonal antibody thus obtained specifically binds only to VEGF189.

[Polyclonal antibody]

Here we will describe the example of a heron (Immunological Experiment Procedure I, II Nankodo). Immunize a heron three to four times at monthly intervals with the peptide 67 alone or as an antigen, for example, bound to KLH, as described in the section on monoclonal antibodies. Blood is collected 7 to 10 days after the final immunization to obtain antiserum. The antiserum is applied to a protein G column (Pharmacia # 17-0618-01) to purify IgG. The IgG is applied to a column (Pharmacia # 17-0430-01) to which VEGF206 has been bound, and the portion eluted without adsorption to the column is collected. Further, the eluate is applied to a column to which peptide 67 is bound. By collecting the antibody adsorbed on the column, a polyclonal antibody that specifically binds only to VEGF189 can be obtained.

Measurement method of C V E G F 18 9)

Here, an example of an enzyme immunoassay (enzyme immunoassay method medical school) using a heron polyclonal antibody (hereinafter, referred to as "polyclonal antibody") and a mouse monoclonal antibody (hereinafter, referred to as "monoclonal antibody") is given. I will explain. ABS beads

(Sekisui Chemical Co., Ltd. # 80) to adsorb a monoclonal antibody (hereinafter referred to as ). On the other hand, the polyclonal antibody is labeled with, for example, Horse radish peroxdase (hereinafter, referred to as “labeled antibody”). Add 300 mL of phosphate buffer to a tube (Soken RIT-2), and gently put the beads into this. Add 50111 standard samples (for preparing a calibration curve) or samples (eg, serum, plasma, tissue extract) and incubate at room temperature for 1 to 2 hours. After removing the reaction solution from the tube by suction, add 4 mL of washing solution to wash the beads. Repeat this washing operation three times in total. Add 30 OmL of the labeled antibody to the washed beads, and incubate at room temperature for 1 to 2 hours. After removing the reaction solution from the tube by suction, add 4 mL of washing solution to wash the beads. This washing operation is repeated a total of three times. Add 50 OmL of the substrate solution (eg, 1,2-phenylenediamine solution, 3, 3 ', 5, 5'-tetramethylbenzidine solution) to the washed beads, and allow to stand at room temperature for 30 minutes to 1 minute. Incubate for hours. Add 2111 sulfuric acid solution to stop the enzyme reaction. If the substrate solution is 1,2-phenylenediamine, measure the absorbance at 491 nm. If the substrate solution is 3,3 ', 5,5'-tetramethylbenzidine, measure the absorbance at 45Οηιη. A calibration curve is created based on the measured values of each VEGF 189 standard, and the VEGF 189 concentration in the sample can be known from this. Industrial applicability

A method for examining the expression pattern of the VEGF isoform and a reagent kit usable for the method were provided. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

References

ANDO M, MARUYAMA M, OTO M, TAKEMURA, ENDO M AND YUASA Y. (1991) .Higher frequency of point mutations in the cK-ras 2 gene in human colorectal adenomas with severe atypia than in carcinomas. Jpn. J. Cancer. Res., 82,245-249

BER MAN RA, MERRIL MJ, REINHOLD WC, MONACCI WT, SAXENA A, CLARK WC, ROBERTSON JT, ALIE U AND OLDFIELD E H. (1993) .Expression of the vascular permeability factor / vascular endothelial growth gene in central nervous system neoplasms -J. Clin. Invest., 91, 153-159

BERSE B, BROWN L F, VAV DE WATER L, DVORAK H F AND SENGER D R.

Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors .Mol.Biol.Cell, 3, 211-220 (1992).

BOOCOCK AC, CHAKNOCK-JONES SD, SHARKEY MA, MCLAREN J, BAKER JP, WRIGHTAK, WEN YMANRP, ANDSMITHKS. (1995) .Expression of vascular endothelial growth factor and its receptors fit and KDR in ovarian carcinoma. JNC. 87, 506-516

BROWN LF, BERSE B, JAC MAN RW, TOGNAZZ ェ K, GUILDI A, DVORAK H, SENGER D, CONNONLLY J, AND SCH ITT S. (1995) .Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer .Hum.Pathol., 26, 86-91

BROWN LF, BERSE B, JACKMAN RW, TOGNAZZ ェ K, MANSEAU EJ, SENGER D, AND DVORAK H. (1993) .Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract .Cancer Res., 53, 4727-4735

COHEN T, GITAY-GOREN H, SHARON R, SHIBUYA M, HALABAN R, LEVI B Z, AND NEUFELD G. (1995) .VEGF121, a vascular endothelial gtowth factor (VEGF) isof orm lacking heparin binding

ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells .J Biol Chem., 270, 11322-11326

DOLECKI G J, AND CONNOLLY D T. (1991) .Effects of a variety of cytokines and inducing agents on vascular permeability factor mRNA levels in U937 cells .Biochem.Biophys.Res.Conimun., 180, 572-578

DVORAK H F, BROWN L F, DETMAR M, AND DVORAK AM. (1995) .Vascular permeability factor / vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.Am. J. Pathol., 146, 1029-1038

GENGRINOVITCH S, GREENBERG SM, COHEN, GITAY-GOREN H, ROCKWELL P, MAIONESTE ,: LEVI B, AND NEUFELD G. (1995). Platelet f actor-4 inhibi s the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms .J . Biol Chem., 270, 15059- 15065

HOUCK KA, FERRARAN, WINER J, CACHIA ES G, LI B, AND LEUNG DW (1991) .The vascular endothelial growth factor family: identificaion of a fourth molecular species and characterization of alternative splicing of RNA.Mol.Endocrinol., 5, 1806-1814

HOUCK K A, LEUNG D W, ROWLAND A M, WINER J, AND FERRARA N (1992) .Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms .J. Biol. Chem. 267, 26031-26037

KECK P J, HAUSER S D, RIVI G, SANZO K, WARREN T, FEDER J AND CONNOLY D T. (1989) .Vascular permeabili y .factor, an endothelial cell mitogen related to PDGF .Science., 26, 1309-1312

KEISER A, WEICH HA, BRAND ER G, MARME D, AND OLCH W. (1994).

Mutant p53 potentiates protein kinase-C induction of vascular endothelial growth factor expression.Oncogene, 9, 963-969 KOURA A N, LIU W, KITADAI Y, SINGH R RADINSKY R AND ELLIS

M L. (1996) .Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by cell densi y.

Cancer Res., 56, 3891-3894

LEUNG D W, CACHIANES G, KUANG W J, GOEDDEL D V, AND FERRARA N.

(1989) .Vascular endothelial growth factor is a secreted angiogenic mitogen-Science, 246, 1306-1309

PARK J, KELLER GA, AND FERRARA N. (1993) .The vascular endothelial growth factor (VEGF) isof orms: differential disposition into the subepithelial extacellular matrix and bioactivity of extacellular matrix-bound VEGF.Mol.Biol.Cell., 4, 131 -1326 RAK J, MITSUHASH Y, BAYKO L, FILMUS J, SHIRASAWA S, SASAZU IT, AND KERBEL RS. (1995). Mutant ras oncogenes upr.egulate VEGF / VPF expression: Implications for induction and inhibition of tumor angiogenesis. Cancer Res. , 55,4575-4580

SAMBROO J, FRITSH E F, AND MA IATIS T. (1989). Molecular cloning: A laboratory manual, 2nd edn, p.7. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY.

SE GER D R, GALLE S J, DVORAK A M, PERUZZI CA, HARVEY V S, AND DVORAK H F. (1983) .Tumor cells secrete a vascular permeability- factor that promotes accumulation of ascites fluid. Science, 219, 983-985

SUZUKI K, HAYASHIN, MIYAMOTO Y, ΥΑΜΆΜΟΤΟΜ, OHKAWA K, D TO Y, SASAKI Y, YAMAGUCHI Y, NA ASE H'NODA K, ENOMOTO N, ARAI, YAMADA Y, YOSHIHARA H, TUJIMURA T, AWANO K, YOSHI AWA, AND AMADA T. (1996) .Expression of vascular permeability factor / vascular endothelial growth factor in human hepatocellular carcinoma .Cancer Res-, 56, 3004-3009

TAKAHASHI Y, KITADAI Y, BUCANA CD, CLEARY R K, AND ELLIS L M.

(1995) .Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer .Cancer Res., 55, 3964-3968

WARREN SR, YUANH'MATLI RM, GILLE AN, AND FERRARA N. (1995) .Regulation by vascular endothelial growth factor of human colon cancer tumorgenesis in a mouse model of experimental liver metastasis .J. Clin. Invest., 95, 1789- 1797

Claims

The scope of the claims

1. A nucleic acid amplification reaction is performed on a sample containing RNA or cDNA using at least one pair of nucleotide primers that hybridizes to mRNA or cDNA of vascular endothelial growth factor. A method for examining the expression pattern of the vascular endothelial growth factor isoform.

2. The method according to claim 1, wherein the presence or absence of expression of VEGF189, which is at least one isoform of vascular endothelial growth factor, is examined.

3. One of the pair of nucleotide primers contains a part of the nucleotide sequence of one exon of the vascular endothelial growth factor gene or a sequence complementary thereto, and the other nucleotide sequence contains a nucleotide sequence of a different exon of the vascular endothelial growth factor gene. The method according to claim 1, which comprises a part or a sequence complementary thereto.

4. One of the pair of nucleotide primers contains a part of the nucleotide sequence of exons 1 to 5 of the vascular endothelial growth factor gene or a sequence complementary thereto, and the other nucleotide sequence contains the nucleotide sequence of exon 8 of the vascular endothelial growth factor gene. 4. The method according to claim 3, wherein the method comprises a part or a sequence complementary thereto.

5. One of the pair of nucleotide primers contains a part of the nucleotide sequence of exon 4 to 5 of the vascular endothelial growth factor gene or a sequence complementary thereto, and the other nucleotide sequence contains the nucleotide sequence of exon 7 of the vascular endothelial growth factor gene. 4. The method according to claim 3, wherein the method comprises a part or a sequence complementary thereto.

6. The method according to any one of claims 1 to 5, wherein the nucleotide primer has 5 to 70 bases.

7. The method of claim 4, wherein one of the pair of nucleotide primers comprises the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto, and the other comprises the nucleotide sequence of SEQ ID NO: 3 or a sequence complementary thereto.

8. The method of claim 5, wherein one of the pair of nucleotide primers comprises the nucleotide sequence of SEQ ID NO: 2 or a sequence complementary thereto, and the other comprises the nucleotide sequence of SEQ ID NO: 4 or a sequence complementary thereto.

9. The method according to any one of claims 1 to 8, wherein the sample containing RNA or cDNA is derived from a cancer patient.

10. The method according to claim 9, wherein the cancer is selected from the group consisting of colon cancer, liver cancer, breast cancer, lung cancer, glioma, osteosarcoma and kidney cancer.

11. The method according to claim 9 or 10, wherein the specimen containing RNA or cDNA is a tumor tissue.

12. A reagent kit for examining the expression pattern of a vascular endothelial growth factor isoform, comprising at least one nucleotide primer that hybridizes to the vascular endothelial growth factor mRNA or cDNA. G.

13. To test the expression of VEGF189 in a sample containing RNA or cDNA using a nucleic acid probe that specifically binds to the RNA or DNA of VEGF189.

14. The method according to claim 13, wherein the nucleic acid probe is DNA having a part of a base sequence of a region including a boundary between exon 6 and exon 7 of VEGF189 or a sequence complementary thereto.

15. A part of the base sequence of the region including the boundary between exon 6 and exon 7 of VEGF 189 or a sequence complementary thereto has 10 to 1000, preferably 10 to 400 bases. the method of.

16. The method according to any one of claims 13 to 15, wherein the nucleic acid probe is labeled.

17. The method according to any one of claims 13 to 16, wherein the specimen containing RNA or cDNA is derived from a cancer patient.

18. The method according to claim 17, wherein the cancer is selected from the group consisting of colon cancer, liver cancer, breast cancer, lung cancer, glioma, osteosarcoma, and kidney cancer.

19. The method according to claim 17 or 18, wherein the specimen containing RNA or cDNA is a tumor tissue.

20. A reagent kit for testing VEGF 189 expression, comprising a nucleic acid probe that specifically binds to VEGF 189 RNA or DNA.

21. A method for detecting VEGF 189 in a sample using an antibody or an Abmer that specifically binds to VEGF 189.

22. The antibody reacts against a protein antigen consisting of the amino acid sequence encoded by the nucleotide sequence of the region including the boundary between exon 6 and exon 7 of VEGF189. 22. The method according to claim 21, which is a clonal antibody or a monoclonal antibody.

23. The method according to claim 22, wherein the amino acid sequence comprises 5 or more amino acids.

24. The method according to any one of claims 21 to 23, wherein the specimen is derived from a cancer patient.

25. The method according to claim 24, wherein the cancer is selected from the group consisting of colon cancer, liver cancer, breast cancer, lung cancer, glioma, osteosarcoma and kidney cancer.

26. The method according to claim 24 or 25, wherein the specimen is serum, cerebrospinal fluid or body cavity fluid.

27. A reagent kit for detecting VEGF189, comprising an antibody or abtamer that specifically binds VEGF189.

28. The reagent kit according to claim 12, which is used for diagnosing metastasis or prognosis of cancer.