JP5548872B2 - Colorectal cancer liver metastasis marker and method for analyzing colorectal cancer liver metastasis marker in a sample - Google Patents

Description

  The present invention relates to clinical diagnosis / examination / follow-up techniques, and in particular, the present invention relates to techniques for diagnosis (follow-up, prognosis prediction, etc.) of liver metastasis of colorectal cancer. Specifically, the present invention relates to a method for analyzing a colorectal cancer liver metastasis marker and a colorectal cancer liver metastasis marker in a sample.

  Colorectal cancer accounts for the third highest morbidity and second mortality in the world among cancer diseases. 70% of patients with colorectal cancer are treated with surgery, of which 30-40% are known to relapse or metastasize. The most common metastasis of colorectal cancer is metastasis to the liver (ie, liver metastasis). For metastatic cases including liver metastasis, chemotherapy such as 5-fluorouracil (5-FU) and administration of molecular targeted therapeutic drugs are generally performed.

  JP 2008-14937 A (Patent Document 1) discloses a marker protein identified from a sample derived from a colon cancer patient without liver metastasis.

  GSN (Gelsolin) (Non-patent document 1: Litwin M et al., Gelsolin in human colon adenocarcinoma cells with different metastatic potential. Acta is a protein that has been shown to be associated with cancer metastasis by experiments using cultured cells. Biochim Pol. 2009; 56: 739-43.); And HSP90AA1 (Heat shock protein HSP 90-alpha) (non-patent document 2: Moser C et al., Blocking heat shock protein-90 inhibits the invasive properties and hepatic growth of Human colon cancer cells and improves the efficacy of oxaliplatin in p53-deficient colon cancer tumors in vivo. Mol Cancer Ther. 2007; 6: 2868-78.) has been reported. However, these proteins have not been suggested to be directly related to liver metastasis.

  Proteasome subunit type alpha 7 (PSMA7) has been reported as a protein that has been shown to be associated with liver metastasis from colorectal cancer by immunohistochemical staining experiments using human colorectal cancer tissue (Non-patent Document 3). : Hu XT et al., The proteasome subunit PSMA7 located on the 20q13 amplicon is overexpressed and asscociated with liver metastasis in colorectalcancer. Oncol Rep 2008; 19 (2): 441-446).

On the other hand, regarding ACTR2 (Actin-related protein 2), it has been reported that co-expression with WAVE2 in the same cancer cell suggests a relationship with liver metastasis from colorectal cancer (Non-patent Document 4). : Iwaya K et al. Correlation between liver metastasis of the colocalization of actin-related protein 2 and 3 complex and WAVE2 in colorectal carcinoma. Cancer Sci 2007; 98: 992-9.).

  In addition, FN1 (Fibronectin), together with integrin receptors (α9β1, α5β1) for TN (Tenascin) and FN1 and TN, has been shown by immunohistochemical experiments to be correlated with the cancer pathology. However, the relationship with liver metastases has not been directly demonstrated (Non-patent document 5: Maya G et al .: Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors α9β1 and α5β1 in gastic and colorectal cancers with lymph node and liver metastases. Acta histochemica 2006; 108: 2-6).

  CEA and CA19-9 have been reported to be upregulated in liver metastasis from colorectal cancer (Non-patent document 6: Bakalakos EA et al .: Is carcino-embryonic antigen useful in the follow-up management of patients with colorectal. Liver metastases? Am J Surg 177: 2-6, 1999).

JP 2008-14937 A

Acta Biochimica Polonica 2009, Volume 56, p. 739-743. Molecular Cancer Therapeutics, 2007, Volume 6, p. 2868-2878. (Oncology Reports, 2008 Volume 19, p. 441-446) Cancer Science, 2007, Vol. 98, p. 992-999. Acta Histochemica, 2006, 108, p. 25-35 American Journal of Surgery, 1999, Vol. 177, p. 2-6

  At present, the chemotherapy and molecular target therapy for metastasis cases including liver metastasis described above do not necessarily have a sufficient inhibitory effect or life prolonging effect on colorectal cancer liver metastasis. In addition, there are problems that impose an economic and mental burden on patients, such as administration of anticancer drugs to patients after surgery regardless of whether they have liver metastases.

  Moreover, the above-mentioned ACTR2, GSN, HSP90AA1 and FN1 have been shown to be related to colorectal cancer liver metastases through experiments using cultured cells as samples, but uncultured cells, ie samples collected from patients ( The association between these proteins in tissues etc. and colon cancer liver metastasis has not been shown. CEA and CA19-9 have been reported as proteins that can serve as an indicator of colorectal cancer liver metastasis, but have specificity for colorectal cancer liver metastasis (ie, determination of the presence or absence of liver metastasis). It is not used as a clinical marker.

  An object of the present invention is to provide a biomarker having specificity for colorectal cancer liver metastasis. Another object of the present invention is to provide an analysis method for identifying the presence or absence of liver metastasis of colorectal cancer using a sample collected from a living body. Furthermore, an object of the present invention is to provide a therapeutic target and drug discovery target for metastatic liver cancer in colorectal cancer.

  As a result of intensive studies, the present inventors have found a protein showing a significant difference in expression between colorectal cancer tissue with liver metastasis and colorectal cancer tissue without liver metastasis, and completed the present invention. It was.

The present invention includes the following inventions.
The following relates to colorectal cancer liver metastasis markers and analysis methods thereof.
(1)
2 6S proteasome subunit p40. 5, V oltage-dependent anion-selective channel protein 1, and at least one protein selected from Paraneoplastic antigen Ma 2 or Ranaru group, colon cancer liver metastasis marker.

The expression level of the above protein is increased compared with the reference level in patients with colorectal cancer liver metastases.
The reference level can be a protein level that is a control of the protein level in a cancerous sample derived from a patient with colorectal cancer liver metastasis.

(2)
In a biological sample, the 2 6S proteasome subunit p40. 5, V oltage-dependent expression level of the anion-selective channel protein 1, and at least one colon cancer liver metastasis markers selected from Paraneoplastic antigen Ma 2 or Ranaru group A method for analyzing a colorectal cancer liver metastasis marker, which is obtained and evaluated for the level of the expression level based on a reference level of the colorectal cancer liver metastasis marker.

(3)
The biological sample is a primary lesion tissue to be immunohistochemically stained, the expression level is a staining intensity of a cancer site in the tissue, and the reference level is a cancer site in a colon tissue of a liver metastasis patient of colorectal cancer The method according to (2), wherein
(4)
The biological sample includes a crushed tissue, the expression level is a measured concentration detected in the sample, and the reference level is a reference concentration of the colon cancer liver metastasis marker, (2) The method described in 1.
In the methods (3) and (4), the tissue may be a biopsy tissue (tissue biopsy material). A biopsy tissue is a tissue collected by examination or surgery. The biopsy tissue can be a resected tissue. The excised tissue is tissue that has been excised by surgery.

The method described above, wherein the biological sample is a blood sample, and the expression level information of the colorectal cancer liver metastasis marker is obtained using a measurement method based on a test based on biospecific affinity.
In the above method, the blood sample is discarded without being returned to the living body.

The protein of (1 ) above becomes a therapeutic target molecule for the treatment of metastatic liver cancer in colorectal cancer or a drug discovery target molecule for the suppression of liver metastasis of colorectal cancer or the treatment of metastatic liver cancer. sell.
Based on the therapeutic target molecule or drug discovery target molecule, the following drug candidate molecules can be provided.

A drug candidate molecule having binding specificity to the therapeutic target molecule or drug discovery target molecule.
A drug candidate molecule comprising an antibody that specifically binds to the therapeutic target molecule or drug discovery target molecule.
When the drug candidate molecule is supplied to colon cancer cells, it can cause a reaction that kills colon cancer cells or suppresses colon cancer cell growth and promotes suppression of liver metastasis.

A drug candidate molecule comprising the therapeutic target molecule or drug discovery target molecule.
The drug candidate molecule is capable of inducing an immune response that suppresses liver metastasis and kills or grows metastatic liver cancer cells by being supplied to the colon cancer cells in an immunostimulatory amount.

The present invention provides a marker having specificity for liver metastasis from colorectal cancer in vivo.
In addition, by providing the colorectal cancer liver metastasis marker of the present invention, it is possible to provide an analysis method for identifying the presence or absence of liver metastasis of colorectal cancer using a sample collected from a living body. Therefore, according to the present invention, it is possible to provide an index for evaluating liver metastasis diagnosis and prognosis diagnosis of colorectal cancer.
In particular, the ability to predict the prognosis of colorectal cancer patients by the liver metastasis marker of the colorectal cancer of the present invention makes it possible to select a treatment method such as surgical operation or chemotherapy from an early stage. Yes, it is thought to reduce the burden on patients and medical costs.

Furthermore, the colorectal cancer liver metastasis marker of the present invention can be provided as a new therapeutic target for metastatic liver cancer in colorectal cancer.
The colorectal cancer liver metastasis marker of the present invention can be provided as a new drug discovery target for more effectively achieving treatment / suppression of colorectal cancer liver metastasis.
In particular, the development of drug discovery aimed at treating cancer metastasis (that is, liver metastasis) leads to the solution of the most difficult problems in cancer treatment, and results in improved therapeutic results.

The main flow of the proteome analysis by the NBS method for the liver metastasis marker search performed in Example 1 is shown. Clinical specimens (cancerous and non-cancerous parts of colonic mucosa epithelial tissue of colon cancer patients without liver metastasis and colonic mucosal epithelial tissue of colon cancer patients with liver metastasis performed in Example 2) The result of the expression confirmation of the liver metastasis related protein by the immunohistochemical staining in a cancer part and a non-cancer part is shown.

<Colorectal cancer liver metastasis marker>
The present invention provides a marker for colorectal cancer liver metastasis.
In the present invention, proteins provided as markers for colorectal cancer liver metastasis include cancerous and non-cancerous parts of colonic mucosal epithelial tissue collected from colorectal cancer patients with liver metastases, and colorectal cancer without liver metastases Proteome analysis by isotopic labeling method (NBS method) using 2-nitrobenzenesulfenyl chloride (NBSCl) from protein samples extracted from cancerous and non-cancerous parts of colonic mucosa epithelial tissue collected from patients Identified by technology. The NBS method modifies one of two types of protein samples (specifically, a sample derived from a normal site and a sample derived from a cancer site) with a heavy reagent (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride). Then, the other is modified with a light reagent (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride), and both NBS-modified protein samples obtained are mixed with each other and subjected to appropriate treatment by a person skilled in the art such as trypsin digestion. In this method, the relative difference in protein content in two types of protein samples can be examined by measuring the difference in peptide amount with an analyzer. The NBS method is described in Rapid Commun. Mass Spectrom., 2003, 17, 1642-1650 and International Publication No. 2004/002950 pamphlet.

  These proteins can be isolated and purified by any protein purification technique selected by one skilled in the art. For example, techniques including chromatography such as ion exchange, affinity, and size exclusion column chromatography, centrifugation, differential solubility, and electrophoresis can be used.

  Specifically, colorectal cancer liver metastasis markers of the present invention are Serotransferrin, Vacuolar protein sorting-associated protein 13B, Zinc finger protein 445, Spectrin beta chain, brain 1, 2, 3, Signal recognition particle 9 kDa protein, GTP: AMP phosphotransferase mitochondrial, Protein disulfide-isomerase A4, Myosin-9, Cytochrome c1 heme protein, mitochondrial, 26S proteasome subunit p40.5, Hyaluronan and proteoglycan link protein 4, Voltage-dependent anion-selective channel protein 1, Staphylococcal nuclease domain-containing protein 1, Choline O-acetyltransferase, Paraneoplastic antigen Ma2, and UBA domain-containing protein; and Heat shock protein HSP 90-alpha, Actin-related protein 2, Gelsolin, and Fibronectin.

  There is a statistically significant difference in the expression level of the above protein between the two groups of cancerous samples from colon cancer patients with liver metastases and those from colon cancer patients without liver metastases. It is done. That is, the expression of the protein is significantly increased in colorectal cancer patients with liver metastasis as compared with colorectal cancer patients without liver metastasis.

  The colon cancer liver metastasis marker of the present invention is used, for example, in the following applications. By measuring the expression level of the marker in the tissue, for example, it can be used for diagnosis of liver metastasis and prognosis of colorectal cancer patients. Moreover, it can be used as a target of a probe used in diagnostic imaging such as PET. Alternatively, it can be used as a therapeutic target for colorectal liver metastasis.

<Analysis method of colorectal cancer liver metastasis marker>
The present invention provides a method for analyzing a sample by using at least one protein selected from the above protein group as a colon cancer liver metastasis marker. In the method of the present invention, the above-mentioned protein is used as a marker whose expression level is increased compared to a reference level in a patient with colorectal cancer liver metastasis. The reference level may be a level that serves as a control for the above-mentioned colorectal cancer liver metastasis marker level in a cancerous sample derived from a colorectal cancer liver metastasis patient. For example, the above-mentioned colorectal cancer liver metastasis marker level in a cancerous sample derived from a colorectal cancer patient without liver metastasis can be mentioned.

  In the method of the present invention, a sample to be subjected to analysis is prepared, and the expression level of at least one colorectal cancer liver metastasis marker selected from the protein group is obtained from the sample. Based on the reference level of the colorectal cancer liver metastasis marker, an evaluation is performed regarding the level of the acquired expression level. An evaluation that the expression level is higher than the reference level can be used as an indicator that the individual from which the sample is derived has a high possibility of suffering from colon cancer liver metastasis.

The sample to be subjected to the analysis may be a biological sample derived from an individual who is to identify a disease of liver metastasis from colon cancer (hepatic metastasis of colon cancer).
The biological sample is preferably a sample collected from a living body. A sample collected from a living body is intended to exclude cultured cell lines. A sample collected from a living body directly reflects a life phenomenon in the living body.
The sample to be subjected to analysis is not particularly limited. Examples include cells, tissues, body fluids, and tissue extracts.
Cells and tissues include tissue biopsy materials. The tissue biopsy material includes a biopsy tissue and can be collected by examination or surgery. In particular, a tissue excised by surgery may be mentioned. These tissues may be provided in the form of tissue sections, tissue fragments or tissue extracts described below. The tissue may be derived from the large intestine which is the primary focus. Examples of the large intestine-derived tissue include mucosal epithelium, lamina propria, mucosal lamina, submucosa, lamina propria and serosa.
Body fluids include blood, urine, and body secretions. The blood includes whole blood, plasma, serum and the like.
Tissue extract refers to tissue homogenated or solubilized by methods known to those skilled in the art.

  Acquisition of the expression level of the protein can be performed, for example, by a test based on biospecific affinity. The test based on biospecific affinity is a method well known to those skilled in the art, and is not particularly limited, but an immunoassay is preferable. Specifically, Western blot, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation method, precipitation reaction, immunodiffusion method, immunoagglutination measurement, complement binding reaction analysis, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, etc. Immunoassays, including competitive and non-competitive assay systems are included. In an immunoassay, the presence of an antibody that binds to a marker in a sample of an individual is detected. Specifically, it is carried out by bringing the sample into contact with the antibody under the conditions that can form an immune complex consisting of the marker protein to be measured and the antibody of the protein in the assay sample. More specific immunoassay protocols can be easily selected by those skilled in the art.

  Or acquisition of the expression level of the said protein can also be measured by protein quantification methods other than the method based on said biospecific affinity. For example, the NBS method already described is an excellent method for quantitativeness. In this case, the measurement can be performed by examining the difference in the abundance of the protein from the sample to be analyzed, using a sample prepared with a known level of the protein or an appropriate sample such as a normal sample as a control sample. it can.

The following is mentioned as an example of the aspect by which the method of this invention is implemented.
In this embodiment, as a sample to be analyzed, a section of cancer tissue (more specifically, primary lesion tissue) excised by surgery or the like is used. This tissue is subjected to immunohistochemical staining, and the intensity of staining produced by immunohistochemical staining and the distribution of staining (staining ratio per unit area) are used as indicators of the expression level of the marker. The reference level can take into account the staining intensity of the cancer site in colorectal cancer patients without liver metastasis, and the distribution of staining (staining ratio per unit area). For example, the region where the staining intensity is stronger than the reference level is distributed in a range of 30% or more of the tissue subjected to analysis, so that the individual from which the sample is derived may suffer from liver metastasis from colorectal cancer It can be used as an indicator of high nature.
Alternatively, in the above example, acquisition and evaluation of the expression level may be performed based on a mass imaging method.

The following is mentioned as another example of the aspect by which the method of this invention is implemented.
In this embodiment, a sample prepared by crushing or extracting a biopsy tissue or the like is used as a sample to be analyzed. This sample is subjected to marker protein concentration measurement, and the obtained measurement value becomes information on the expression level of the marker. The reference level includes, for example, a measurement value of the marker obtained in another sample and a threshold unique to the marker. In this embodiment, the measured value is compared with the reference level, and an indication that the individual from which the sample is derived is likely to have colorectal cancer liver metastasis when the measured value exceeds the reference level It can be.
Alternatively, in another example, the sample may be a blood sample. In that case, the blood sample is discarded without being returned to the living body.

  In the method of the present invention, the colorectal cancer liver metastasis marker of the present invention may be used alone or in combination with any other marker. Therefore, the method of the present invention may include obtaining the expression level information of other markers as well as obtaining the expression level information of the colorectal cancer metastasis marker of the present invention.

<Therapeutic target molecules and drug discovery target molecules>
The colorectal cancer liver metastasis marker of the present invention is a therapeutic target molecule for treating metastatic liver cancer in colorectal cancer, or a drug discovery for inhibiting liver metastasis of colorectal cancer or treating metastatic liver cancer Can be a target molecule.
Treatment of colorectal cancer liver metastasis includes killing colon cancer cells that have metastasized to the liver. Inhibiting liver metastasis of colorectal cancer includes inhibiting liver metastasis of colon cancer cells or killing metastatic liver cancer cells.
By using the colorectal cancer liver metastasis marker of the present invention as a therapeutic target molecule or a drug discovery target molecule, the following drug candidate molecules can be provided.

One embodiment of the drug candidate molecule has specificity for a therapeutic target molecule or a drug discovery target molecule. More specifically, those containing an antibody that immunospecifically binds to a therapeutic target molecule or a drug discovery target molecule are exemplified. Antibodies include polyclonal antibodies, monoclonal antibodies, and antibodies prepared by molecular biological techniques. Here, the antibody may be any substance that binds widely immunospecifically, and an antibody fragment or an antibody fusion protein may also be used. In either case, antibody preparation is carried out by methods well known to those skilled in the art. In addition, the antibody may be subjected to molecular alteration and modification usually performed by antibody engineering techniques in the field of drug discovery.
When the drug candidate molecule is supplied to colon cancer cells, it can induce a reaction that suppresses liver metastasis and kills or grows metastatic liver cancer cells.

  Another embodiment of the drug candidate molecule includes the therapeutic target molecule for colorectal cancer liver metastasis of the present invention. The drug candidate molecule is capable of inducing an immune response that suppresses liver metastasis and kills or grows metastatic liver cancer cells by being supplied to the colon cancer cells in an immunostimulatory amount. Here, the immunostimulatory amount refers to the amount of an antigen capable of eliciting a desired immune response for the treatment of cancer, and is determined by a method well known by those skilled in the art. According to this form, cancer treatment is performed using a method well known to those skilled in the art, known as so-called cancer vaccine therapy.

  The drug candidate molecule can be a pharmaceutical composition by containing itself as an active ingredient and further containing a pharmaceutically acceptable diluent, carrier, excipient and the like. The pharmaceutical composition can be identified as a potential therapeutic agent used for the treatment of colorectal cancer liver metastasis and the suppression of colorectal cancer liver metastasis. Alternatively, the pharmaceutical composition can be used as a therapeutic agent used for the treatment of liver metastasis of colorectal cancer or the suppression of liver metastasis of colorectal cancer.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
Collect colorectal cancer tissues from patients with consent in accordance with the Osaka University School of Medicine ethical regulations (n = 23 colorectal cancer patients, 10 cases with liver metastases and 13 cases without liver metastases) Then, the sample was subjected to quantitative proteome analysis by the NBS (2-nitrobenzenesulfenyl) method by the following method. The main flow of proteome analysis by the NBS method for searching for liver metastasis markers performed in this example is shown in FIG.

  Homogenize each collected tissue in solubilization buffer A (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, protease inhibitor (aprotinin, PMSF, leupepsin) aqueous solution), 100000 × G After centrifugation at 4 ° C for 1 hour, the supernatant was obtained as a soluble fraction. Next, the precipitate obtained by centrifugation was homogenized again in solubilization buffer B (9 M urea, 2 w / v% CHAPS, 10 mM EDTA, protease inhibitor (aprotinin, PMSF, leupepsin aqueous solution)), and 100000 × G ( After centrifugation at 4 ° C. for 1 hour, the supernatant was obtained as an insoluble fraction.

The extracted proteins of these soluble and insoluble fractions were each treated with NBS reagent, and protein expression analysis was performed. Regarding NBS reagent treatment, stable isotope labeling was performed according to the recommended protocol of ¹³ C NBS Stable Isotope Labeling Kit-N (Shimadzu Corporation). Specifically, proteins extracted from normal tissues were labeled with ¹² CNBS (Light NBS), and proteins extracted from cancer tissues were labeled with ¹³ CNBS (Heavy NBS). Each of the obtained labeled proteins was mixed, and desalted, reduced, alkylated, and trypsin digested according to the recommended protocol of the kit. Next, the labeled peptide was concentrated according to the protocol of the kit. Concentrated labeled peptides were subsequently separated by μHPLC using a C18 column and applied to MS plates. The sample thus prepared was measured using a mass spectrometer Axima-CFR plus (manufactured by Shimadzu Corporation), and a relative quantitative analysis of each protein was performed.

  Statistical analysis by Mann-Whiteney U-test using proteome data (Tumor (cancer site) / Normal (non-cancer site): T / N ratio) based on the results of relative quantitative analysis of each case It was. As a result, the mass spectrometer Axima-QIT (p <0.05) obtained a statistically significant difference (p <0.05) between the two groups of liver metastasis cases (n = 10) and no liver metastasis cases (n = 13). The protein was identified by MS / MS analysis using Shimadzu Corporation.

  Among 46 paired peaks from which significant differences were obtained, sequences were identified for 22 paired peaks, and finally 21 proteins were identified as liver metastasis-related proteins. The results are shown in Table 1. Among these identified proteins, Proteasome subunit alpha type 7 has been previously reported to be related to colorectal cancer liver metastasis by immunohistochemical staining experiments using colorectal cancer tissue. there were. The remaining 20 proteins have never been reported for association with liver metastasis in colorectal cancer tissues. In addition, of the remaining 20 proteins, 17 proteins except for heat shock protein HSP 90-alpha, Actin-related protein 2 and Gelsolin have not been reported to be associated with liver metastasis of colorectal cancer. It was not.

[Example 2]
The liver metastasis-related proteins listed in Table 1 were validated by immunohistochemical staining.
<Comparison of primary tissue in patients with colorectal cancer without liver metastasis and colorectal cancer with liver metastasis>
Paraffin-embedded sections (4 μm) of normal tissue (ie, non-cancerous part of colonic mucosa epithelial tissue) and cancer tissue (ie, cancerous part of colonic mucosal epithelial tissue) collected from patients with colorectal cancer with liver metastasis ) Was prepared. The following antibodies were used.

Primary antibody:
Rabbit anti-human PNMA2 monoclonal antibody (antibody dilution ratio 1/200)
Rabbit anti-human SND1 monoclonal antibody (antibody dilution ratio 1/200)
Mouse anti-human GSN monoclonal antibody (antibody dilution ratio 1/200)
Mouse anti-human ACTR2 monoclonal antibody (antibody dilution ratio 1/200)
Rabbit anti-human FN1 monoclonal antibody (antibody dilution ratio 1/100)

Secondary antibody:
Biotin conjugate-anti-rabbit IgG antibody (antibody dilution ratio 1/300)
Biotin conjugate-anti-mouse IgG antibody (antibody dilution ratio 1/300)

  The paraffin section and the primary antibody were reacted at room temperature for 1 hour, washed with PBS buffer, and further reacted with the secondary antibody for 20 minutes. Further, after washing with PBS, the mixture was reacted with streptavidin-biotin peroxidase complex (dilution ratio 1/300) for 20 minutes. Finally, tissue staining was performed by reacting with a coloring solution (3,3'-diaminobenzydynetetrahydrochloride, 0.01% peroxidase, 0.05 M Tris aqueous solution (pH 7.6)) for 5 minutes.

  In this validation, when 30% or more of the cells in the tissue were stained, it was judged as high expression (high), and when not, it was judged as low expression (low). Fig. 2 shows that among the metastasis-related proteins of liver metastasis, it was confirmed that expression in the primary lesion of colon cancer was increased in cases with liver metastasis compared to cases without liver metastasis, GSN, PNMA2, ACTR2, and FN1 are shown. In FIG. 2, the scale of the bar shown in the photograph is 20 μm.

Table 2 shows 16 cases that showed high expression and 7 cases that showed low expression for PNMA2.
・ Average age and range ・ Location of cancer (specifically colon or rectum) and the number of cases
・ Histology (specifically well differentiated adenocarcinoma (well (well differentiated adenocarcinoma)) or moderately differentiated adenocarcinoma (mod), poorly differentiated adenocarcinoma (poorly differentiated adenocarcinoma) )) And the number of applicable cases,
・ Invasion (specifically, cancer invasion only extends to the proper muscle layer (-mp (propria mucosae)) or cancer invasion beyond the proper muscle layer (ss- (subserosa))) and the number of cases,
・ The presence or absence of positive lymph node metastasis (Lymph node) and the corresponding number of cases,
・ Cancer metastasis (positive or negative) and the number of cases
・ With or without lymphatic invasion (ly (lymphovascular invasion)) and the number of affected cases,
・ Indicates whether the vascular invasion (v (vascular invasion)) is positive or negative (number of cases), and the CEA level mean and range, respectively, and the p-value in the chi-square test (p-value) )showed that.
Table 3 shows the age, each finding, and the number of corresponding cases for 9 cases showing high expression and 14 cases showing low expression for Gelsolin. Shown as well.

  As shown in Table 2 and Table 3, it was confirmed that each protein was expressed with a statistically significant difference (chi-square test, p <0.05) in the presence or absence of liver metastasis among various findings in the table. . Specifically, the expression in liver metastasis positive in patients with liver metastasis was statistically significant compared to the expression in liver metastasis negative (liver metastasis negative) (chi-square test, p < 0.05).

<Comparison of colorectal cancer tissues in cases without liver metastasis and cases with liver metastasis>
Paraffin-embedded sections (4 μm) prepared for normal tissue (ie, non-cancerous part of large intestine mucosal epithelial tissue) and cancer tissue (ie, cancerous part of large intestinal mucosal epithelial tissue) collected from patients with colorectal cancer without liver metastasis Except for the above, validation by immunohistochemical staining was performed in the same manner as described above.
The tissue staining results for the tissues of colon cancer with liver metastasis were compared with those for primary lesions and normal tissues of colon cancer cases without liver metastasis. Fig. 2 shows that among the metastasis-related proteins of liver metastasis, it was confirmed that expression in the primary lesion of colon cancer was increased in cases with liver metastasis compared to cases without liver metastasis, GSN, PNMA2, ACTR2, and FN1 are shown. In FIG. 2, the scale of the bar shown in the photograph is 20 μm, T indicates the staining result in the cancerous part, and N indicates the staining result in the non-cancerous part.

<General comments>
As shown in FIG. 2, expression of all five proteins subjected to validation was enhanced in cancer cells (or stromal cells surrounding the cancer cells) in the primary lesion of colorectal cancer with liver metastasis. I confirmed. That is, in the quantification result by the NBS method shown in Example 1 above, the protein that showed increased expression in the primary lesion tissue of the colon cancer case with liver metastasis than in the primary lesion tissue of the colon cancer case without liver metastasis is The results of analysis by the immunohistochemical staining method shown in the present Example also actually showed increased expression. Thus, the quantitative result by the NBS method and the analysis result by the immunohistochemical staining method show a good correlation. Therefore, it was confirmed that these molecules are proteins related to colon cancer liver metastasis.

  From the above, the protein according to claim 1 is expressed more significantly in colon cancer tissue with liver metastasis than in colon cancer tissue without liver metastasis. It is clear that these proteins can be used as markers for colorectal cancer liver metastasis. When these proteins are used as markers for colorectal cancer liver metastasis, and the colorectal cancer specimen whose liver metastasis is unknown is analyzed, the methods described in the above examples can be used.

Claims (4)

2 6S proteasome subunit p40. 5, V oltage-dependent anion-selective channel protein 1, and at least one protein selected from Paraneoplastic antigen Ma 2 or Ranaru group, colon cancer liver metastasis marker. In a biological sample, the 2 6S proteasome subunit p40. 5, V oltage-dependent expression level of the anion-selective channel protein 1, and at least one colon cancer liver metastasis markers selected from Paraneoplastic antigen Ma 2 or Ranaru group A method for analyzing a colorectal cancer liver metastasis marker, which is obtained and evaluated for the level of the expression level based on a reference level of the colorectal cancer liver metastasis marker.   The biological sample is a primary lesion tissue to be immunohistochemically stained, the expression level is a staining intensity of a cancer site in the tissue, and a staining intensity of a cancer site in a colon tissue of a colon cancer patient, The method of claim 2.   The biological sample includes a crushed tissue, the expression level is a measured concentration detected in the sample, and the reference level is a reference concentration of the colon cancer liver metastasis marker. The method described in 1.