JP5133842B2 - Method for discriminating between cancerous part and non-cancerous part of patient suspected of cancer or tissue derived from cancer patient, and discrimination reagent used therefor - Google Patents

Description

  The present invention relates to a method for discriminating between a cancerous part and a non-cancerous part of a patient suspected of having cancer or tissue derived from a cancer patient, and a discrimination reagent used therefor. More particularly, expression of clathrin heavy chain and / or formiminotransferase cyclodeaminase in tissue specimens from patients suspected of cancer such as primary hepatocellular carcinoma or cancer patients such as patients with primary hepatocellular carcinoma A method for discriminating cancerous and non-cancerous parts by measuring levels, and cancer of a patient suspected of cancer or tissue derived from a cancer patient, comprising a clathrin heavy chain antibody and / or a formiminotransferase cyclodeaminase antibody The present invention relates to a discrimination reagent for discriminating between a part and a non-cancer part.

Primary hepatocellular carcinoma (HCC) is a serious disease worldwide and ranks third in cancer mortality in Japan. In general, early diagnosis of hepatocellular carcinoma improves prognosis. However, it is often difficult to diagnose benign or malignant tumors.
Usually, detection of HCC is divided into serum markers and diagnostic imaging. Serum markers include AFP-L3 and PIVKA-II, which are widely used but are difficult to detect at an early stage (Non-patent Documents 1 and 2). Ultrasonography is used for image diagnosis, and a nodule of 2 cm or less can be detected, but definitive diagnosis with HCC is difficult. For nodules of 2 cm or less, pathological diagnosis of biopsy tissue is recommended. A pathologist must diagnose this only with information in a small section, but it is often difficult even for an experienced pathologist to diagnose such a small nodule as good or malignant.
In recent years, pathological diagnosis using Grypican-3 etc. as a marker has been reported to be useful as an examination to distinguish benign nodules from primary primary hepatocellular carcinoma (eHCC) in cirrhosis (Non-patent Document 3 and Non-patent Document 4). . However, according to the study by the present inventors, in these diagnoses, it cannot be said that cancer can be determined as cancer with high probability, and it cannot be said that non-cancer can be determined as non-cancer with high probability. Development is desired.
By the way, in recent years, a genome database has been announced by the Human Genome Project, and the development of MS / MS technology has enabled highly efficient identification of proteins. These developments in analysis and analysis technology have enabled comprehensive protein expression analysis, that is, proteome analysis.
In HCC, proteome analysis using two-dimensional electrophoresis, multiple fluorescent labeling two-dimensional electrophoresis, and two-dimensional high-performance liquid chromatography was reported by comparing the pathological condition with normal expression. As a result, many proteins were identified as eHCC marker candidates. However, there are almost no examples of clinical application.
Lancet 2003; 362: 1907-1917 Am J Gastroenterol 1999; 94: 650-654 Gastroenterology 2003; 125: 89-97 HEPATOLOGY 2007; 45: 725-734

  Therefore, an object of the present invention is to distinguish between cancerous and non-cancerous parts of tissues derived from patients suspected of cancer such as primary primary hepatocellular carcinoma or cancer patients such as primary primary hepatocellular carcinoma with high sensitivity and specificity. It is an object of the present invention to provide a discriminating method and a discriminating reagent used therefor.

  The present inventors considered that a broader and more sophisticated method is necessary to develop a method for detecting a new protein related to carcinogenesis of liver cancer. In order to discover a new marker useful for the diagnosis of HCC, the present inventors have made a 2D-DIGE method (two-dimensional fluorescence difference gel electrophrosis) in order to provide agarose two-dimensional electrophoresis with higher reproducibility and a wider dynamic range. Modified agarose two-dimensional electrophoresis (Clin Cancer Res 2004; 10: 2007-2014; Proteomics 2006; 6: 1-11-1018). The present inventors compared the protein expression levels of HCC cancerous sites and surrounding non-cancerous tissues by proteome analysis. As a result, at least 127 substances differed in the protein expression levels of the cancerous part and the surrounding non-cancerous tissues, and 83 proteins were identified. Of these, two proteins were found to be effective in discriminating cancerous and non-cancerous parts of eHCC. The present invention has been achieved through this process.

That is, the present invention measures the expression level of clathrin heavy chain and / or formiminotransferase cyclodeaminase in a patient suspected of cancer or a tissue sample derived from a cancer patient to discriminate between a cancerous part and a non-cancerous part. The present invention relates to a method for discriminating between a cancerous part and a non-cancerous part of a patient suspected of cancer or a tissue derived from a cancer patient.
Furthermore, the present invention relates to a discrimination reagent for discriminating cancerous and non-cancerous parts of a patient suspected of cancer or a tissue derived from a cancer patient, comprising an anti-clathrin heavy chain antibody and an anti-formiminotransferase cyclodeaminase antibody. .

  Distinguishing between cancerous and non-cancerous parts of a tissue derived from a patient suspected of cancer such as primary primary hepatocellular carcinoma or a cancer patient such as primary primary hepatocellular carcinoma by the discrimination method of the present invention with high sensitivity and specificity. I can do it. In addition, benign nodules and malignant nodules can be distinguished with high sensitivity and specificity from nodules derived from patients suspected of cancer such as primary primary hepatocellular carcinoma or cancer patients such as primary primary hepatocellular carcinoma. Furthermore, the degree of differentiation of cancer can also be determined.

In the present invention, a cancerous part and a non-cancerous part are discriminated by measuring the expression level of at least one of clathrin heavy chain and formiminotransferase cyclodeaminase in a patient suspected of cancer or a tissue sample derived from a cancer patient. can do.
In the present invention, clathrin heavy chain (Clathrin heavy chain) is a protein composed of CHC and a molecular weight of 192 kDa, 1675 amino acids encoded by Swiss-Protentry No, Q00610, Hc; CHC; CHC17; CLH-17; CLTCL2; KIAA0034; A protein that is involved in phagocytosis of receptors for intracellular transport and various macromolecular substances on the surface of cell membranes and organelles, such as CLTC.
In the present invention, formiminotransferase cyclodeaminase (formiminotransferase cyclodeaminase) is a protein composed of a molecular weight of 59 kDa, 541 amino acids, which is denoted as FTCD and encoded by Swiss-Protent O95954. It is an enzyme expressed specifically in the liver that has two functions: folate metabolism (formiminotransferase) and glutamate metabolism (cyclodeaminase).

In the present invention, a cancerous part and a non-cancerous part can be discriminated by measuring the expression level of CHC and / or FTCD in a tissue sample collected from a patient suspected of cancer or a cancer patient. Specifically, when the expression level of CHC is higher than the normal level, it can be determined as a cancerous part, and when the expression level of FTCD is lower than the normal level, it can be similarly determined as a cancerous part. On the other hand, when the expression level of CHC is lower than the normal level, it can be determined as a non-cancerous part, and when the expression level of FTCD is higher than the normal level, it can be similarly determined as a non-cancerous part.
Here, sensitivity means the probability that cancer can be determined as cancer, and specificity means the probability that non-cancer can be determined as non-cancer. Increasing sensitivity as well as specificity is necessary for more accurate discrimination.
Therefore, by measuring the expression levels of both CHC and FTCD and discriminating them based on the expression levels of both, it is possible to discriminate cancerous parts from non-cancerous parts more specifically. That is, when the expression level of CHC is higher than the normal level or the expression level of FTCD is lower than the normal level, it can be determined more sensitively as a cancerous part, while the expression level of CHC is lower than the normal level. When the expression level of FTCD is higher than the normal level, it can be determined more specifically as a non-cancerous part.

According to the discrimination method of the present invention, the cancerous part and the non-cancerous part are accurately identified for patients with suspected cancers such as liver cancer, bladder cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, thyroid cancer, skin cancer, or tissues of cancer patients. In particular, with regard to tissues of liver cancer, hepatocellular carcinoma, and primary hepatocellular carcinoma, it is possible to discriminate between cancerous and non-cancerous parts with high sensitivity, and in particular, Thus, cancerous and non-cancerous parts can be discriminated sensitively and specifically with respect to the primary primary hepatocellular carcinoma tissue.
In the present invention, benign nodules and malignant nodules are discriminated sensitively and specifically by measuring the expression level of at least one of CHC and FTCD for patients suspected of having cancer or tissue of nodules of cancer patients. can do. Furthermore, it is possible to determine the degree of differentiation of cancer.

In order to measure the expression level of CHC and FTCD in a patient suspected of cancer or a tissue sample derived from a cancer patient, a measurement method usually used for measuring the expression level of protein in the tissue can be employed. Examples include tissue immunostaining, immunoblotting, and two-dimensional electrophoresis.
Here, the expression level may be the amount of mRNA of the transcription product of the CHC and FTCD genes, the protein amount of the translation product, and in the case of FTCD, it can also be indicated by the enzyme activity value. For example, if it is protein mass, it can also be displayed by the protein mass measured by a normal method with the antibody which recognizes it. In addition, for example, in the tissue immunostaining method using a labeled antibody, which will be described in detail below, it is also possible to detect the expression level as the staining intensity.
In the present invention, the tissue immunostaining method refers to a method of staining so that tissues, cells, etc. can be observed with a microscope or the like by an immune reaction using a labeled antibody. When detecting the expression level of the CHC and FTCD proteins by the tissue immunostaining method, for example, the following is performed. A part of the tissue of a patient suspected of having cancer or the like is taken, fixed in formalin by a conventional method, embedded in paraffin, sliced with a microtome, and used as a tissue specimen. The tissue section is completely treated with xylene to remove paraffin, passed through an alcohol solution, and washed with water. Subsequently, in a citrate buffer solution, microwave treatment is performed 2 to 5 times at 80 ° C. to reflux for 2 to 5 times to activate the antigen. After natural cooling, it is washed with running water, and then the endogenous peroxidase is inactivated with a hydrogen peroxide solution. After rinsing with purified water, 1% bovine albumin solution is applied for 10 minutes. Drop the 1% bovine albumin solution on the specimen and place the primary antibody as it is. As the primary antibody, a diluted anti-CHC antibody or anti-FTCD antibody is used. In this state, the reaction is allowed to proceed overnight at 4 ° C., followed by washing with PBS for 2 to 10 minutes, 1 to 5 times. An enzyme-labeled antibody (CHC: mouse antibody, FTCD: rabbit antibody) is placed thereon and reacted at 1-30 ° C. for 0.5-10 hours. After washing with PBS for 5 minutes × 3 times, the mixture is reacted with a coloring reagent such as DAB for 1 to 30 minutes, washed with PBS and then permeated into purified water for 2 minutes. Hematoxylin nuclear staining is performed as a counterstaining and washed with water. After passing through the alcohol solution and xylene, the encapsulant is dropped and covered with a cover glass. By observing the stained tissue specimen with a microscope, the expression level of each protein of CHC or FTCD in the tissue can be detected as the staining intensity.

  As an immunoblot method, for example, a protein extract from a patient suspected of cancer or a tissue sample derived from a cancer patient is separated with a polyacrylamide concentration gradient gel, transferred to a film such as a vinylidene fluoride film, The membrane is reacted with an anti-CHC antibody or anti-FTCD antibody as a primary antibody, and then, for example, an anti-mouse IgG horseradish-derived peroxidase / goat antibody is reacted as a secondary antibody, and the antigen on the membrane is reacted with a chemiluminescent reagent. There is a method of detecting the expression level of CHC or FTCD protein as luminescence intensity by emitting light.

The anti-CHC antibody or anti-FTCD antibody used in the tissue immunostaining method or immunoblot method described above may be a polyclonal antibody such as antiserum, a monoclonal antibody, or a fragment thereof.
In this invention, the discrimination reagent for discriminating the cancer part and the non-cancer part of the patient suspected of cancer or the tissue derived from a cancer patient containing at least one of these anti-CHC antibody and anti-FTCD antibody is provided. This discriminating reagent may contain other reagents such as a secondary antibody usually used in tissue immunostaining or immunoblotting as necessary.

  In order to measure the expression level of CHC and / or FTCD in a patient suspected of cancer or a tissue sample derived from a cancer patient by a two-dimensional electro-peristaltic method, isoelectric focusing (first dimension), SDS- It can be performed by a normal two-dimensional electrophoresis method combined with PAGE (second dimension).

EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited at all by these Examples.
Example 1
Discrimination between cancerous part and non-cancerous part by tissue immunostaining method The expression level of CHC and FTCD was measured for tissue specimens collected from primary hepatocellular carcinoma by the discrimination method of the present invention.

(1) Methods Primary hepatocellular carcinoma (HCC) tissue was obtained from excised cases after obtaining written informed consent from all patients prior to surgery. Excised samples were obtained within 1 hour after surgery for both cancerous and non-cancerous tissues. All excised tissues were immediately frozen in liquid nitrogen and stored at −80 ° C. until used for analysis.
Detection of the expression level of CHC and FTCD proteins by tissue immunostaining was performed as follows. That is, an HCC tissue obtained from a patient was used as a specimen, the specimen was fixed in formalin by a conventional method, embedded in paraffin, sliced with a microtome, and used as a tissue specimen. The tissue section was completely treated with xylene to remove paraffin, passed through an alcohol solution, and washed with water. Subsequently, a microwave treatment at 100 ° C. for 5 minutes in a citrate buffer was performed 3 times to activate the antigen. After natural cooling for 20 minutes, it was washed with running water, and then the endogenous peroxidase was inactivated with a hydrogen peroxide solution. After rinsing with purified water, a 1% bovine albumin solution was placed for 10 minutes. The 1% bovine albumin solution on the specimen was dropped and the primary antibody was placed as it was. As a primary antibody, an anti-CHC monoclonal antibody (manufactured by BD Bioscience) or an anti-FTCD monoclonal antibody (manufactured by Abcam) diluted 200-fold with an antibody diluent (manufactured by DAKO) was used. The reaction was allowed to proceed overnight at 4 ° C. in this state, followed by washing with PBS for 5 minutes × 3 times. The ENVISION polymer reagent (CHC: mouse antibody, FTCD: rabbit antibody manufactured by DAKO) was placed on this and allowed to react at room temperature for 2 hours or at 4 ° C. for 5-6 hours. After washing with PBS for 5 minutes × 3 times, the mixture was reacted with a coloring reagent (DAB) for 10 minutes, washed with PBS and then permeated into purified water for 2 minutes. Hematoxylin nuclear staining was performed as counterstaining and washed with water. After passing through the alcohol solution and xylene, the encapsulant was dropped and the cover glass was covered. By observing the stained tissue specimen with a microscope, the expression level of each protein of CHC or FTCD in the tissue specimen was detected as the staining intensity.

(2) Results 1) The subjects of implementation are 83 cases of primary hepatocellular carcinoma and 68 cases of non-cancerous part. A part of the tissue of each part was taken out, and by the above method, after staining, the staining was observed with a microscope, and the staining intensity was classified into four levels: negative, weak positive, positive, and strong positive. The results are shown in Table 1.

From the results shown in Table 1, it was found that the cancerous part of HCC is more stainable than that of the non-cancerous part for CHC, while it is weaker for FTCD.
If the staining of CHC is strongly positive, if it is a primary hepatocellular carcinoma, the sensitivity (probability that cancer can be determined as cancer) is 51.8% (43 cases / 83 cases), and the specificity (non-cancer is The probability of being able to determine non-cancer was 95.6% (65 cases / 68 cases). In addition, if the staining of FTCD is less than a weak positive, it is assumed that the primary hepatocellular carcinoma, the sensitivity is 61.4% (51 cases / 83 cases), the specificity is 98.5% (67 cases / 68 cases) )Met. All proteins showed a high determination accuracy with a sensitivity of 50% or more and a specificity of 95% or more. Thus, the protein expression levels of CHC and FTCD were confirmed to be useful for the diagnosis of primary hepatocellular carcinoma. . It was found that the boundary between the cancerous part and the non-cancerous part can be determined by this method by staining with CHC and FTCD.

  2) The degree of differentiation of cancer is related to the growth rate and drug reactivity of the cancer, and generally the lower the degree of differentiation, the worse the prognosis. Therefore, pathological determination of the degree of differentiation of primary hepatocellular carcinoma is important. Of the above 83 cases of primary hepatocellular carcinoma, the relationship was investigated for 65 cases whose differentiation degree could be determined. The results are shown in Table 2.

  From the results shown in Table 2, in CHC, the lower the degree of differentiation, the higher the ratio of strong positives, whereas in FTCD, the lower the degree of differentiation, the lower the degree of strong positives. Therefore, it was confirmed that the protein expression levels of CHC and FTCD are useful for determining the degree of differentiation.

Example 2
Discrimination between malignant and benign nodules by tissue immunostaining Primary hepatocellular carcinoma often develops from a few centimeters of the nodule, but many benign nodules also occur in the liver, and these should be correctly differentiated and diagnosed. Is very important. In particular, it is difficult for pathologists to diagnose early hepatocellular carcinoma, which is the initial stage of primary hepatocellular carcinoma, only from the shape of the cells.
17 or 18 cases of early hepatocellular carcinoma, 8 cases of benign nodules and 2 cases of benign tumors diagnosed by comprehensive judgments such as cell infiltration and capsule state by experienced pathologists, It dye | stained similarly to the method of (1) of Example 1. If the staining of CHC is strongly expressed as compared with non-cancerous part (or non-nodule part), the sensitivity (probability that cancer can be determined as cancer) is 41.2% 7 cases / 17 cases) and specificity (probability that non-cancer was judged to be non-cancer) was 77.8% (7 cases / 9 cases). Further, if the staining of FTCD is weakly expressed as compared with non-cancerous part (or non-nodule part), the sensitivity is 44.4% (8 cases / 18 cases) and the specificity is assumed to be early hepatocellular carcinoma. Was 80.0% (8 cases / 10 cases). In addition, combining both proteins increased the sensitivity to 72.2%. Therefore, the expression level of CHC and FTCD proteins at the nodal margin of the liver, that is, the intensity of staining, was useful in determining whether the nodule was an early hepatocellular carcinoma or a benign nodule.

Example 3
Discrimination in various cancers by CHC expression level Since the expression of CHC increases in other cancers, the usefulness of tissue diagnosis other than liver cancer was confirmed. Subjects were bladder cancer 20 cases and bladder non-cancerous part 5 cases, breast cancer 34 cases and breast non-cancerous part 5 cases, lung cancer 30 cases and lung non-cancerous part 5 cases, ovarian cancer 34 cases and ovarian non-cancerous part 5 cases, prostate cancer 20 cases and 5 cases of non-cancerous prostate, 20 cases of skin cancer and 5 non-cancerous parts of skin, 20 cases of thyroid cancer and 5 non-cancerous parts of thyroid.
Assuming that the strong positive in the bladder is bladder cancer, the sensitivity (probability that cancer could be determined as cancer) was 75.0%, and the specificity (probability that non-cancer could be determined as non-cancer) was 100%. Similarly, if it is assumed that positive or strong positive in the breast is breast cancer, the sensitivity is 76.4% and specificity is 100%. If strong positive in the lung is assumed to be lung cancer, the sensitivity is 86.6% and specificity is 100. %, Sensitivity assumed to be ovarian cancer in the ovary, sensitivity 58.8%, specificity 80.0%, sensitivity assumed in the prostate to be prostate cancer, sensitivity 36.8%, specific 100% sensitivity, high sensitivity in skin is assumed to be skin cancer, sensitivity is 55.2%, specificity is 70.0%, positive in thyroid is positive or strong positive is assumed to be bladder cancer, sensitivity is 100%. , Specificity was 100%.
Therefore, CHC was useful for histological diagnosis of at least bladder cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, skin cancer, and thyroid cancer. It was also shown that it may be useful for histological diagnosis of any malignant tumor.

  By the discrimination method of the present invention, cancerous and non-cancerous parts of tissues derived from patients suspected of cancer or cancer patients such as primary primary hepatocellular carcinoma can be well discriminated. In addition, benign nodules and malignant nodules can be distinguished with good sensitivity and specificity from nodules derived from patients suspected of cancer or cancer patients such as primary primary hepatocellular carcinoma. Furthermore, the degree of differentiation of cancer can also be determined.

Claims (10)

  It is characterized by measuring the expression level of clathrin heavy chain and / or formiminotransferase cyclodeaminase in a patient suspected of cancer or a tissue sample derived from a cancer patient to distinguish between a cancerous part and a non-cancerous part A method for discriminating cancerous and non-cancerous parts of a patient suspected of having cancer or tissue derived from a cancer patient.   The method according to claim 1, wherein the cancer is primary hepatocellular carcinoma.   The discrimination method according to claim 1 or 2, wherein the expression levels of clathrin heavy chain and formiminotransferase cyclodeaminase in a tissue sample are measured.   The clathrin heavy chain and / or formiminotransferase cyclodeaminase in a tissue specimen is measured by a tissue immunostaining method using an anti-clathrin heavy chain antibody and / or an anti-formiminotransferase cyclodeaminase antibody. The discrimination method according to any one of the above.   The malignant nodule and the benign nodule are discriminated by measuring the expression level of clathrin heavy chain and / or formiminotransferase cyclodeaminase in a tissue sample derived from a patient suspected of cancer or a nodule of the cancer patient. 5. The determination method according to any one of 4 above.   The discrimination method according to any one of claims 1 to 5, wherein the differentiation level of cancer is discriminated by measuring at least one expression level of clathrin heavy chain and formiminotransferase cyclodeaminase in a tissue sample. The expression level of clathrin heavy chain in tissue specimens is measured by tissue immunostaining using anti-clathrin heavy chain antibody, liver cancer, bladder cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer A method for discriminating between a cancerous part and a noncancerous part of a patient suspected of cancer or a tissue derived from a cancer patient in cancer selected from skin cancer and thyroid cancer.   A discrimination reagent for discriminating between cancerous and non-cancerous parts of a patient suspected of cancer or a tissue derived from a cancer patient, comprising an anti-clathrin heavy chain antibody and / or an anti-formiminotransferase cyclodeaminase antibody.   The discrimination reagent according to claim 8, comprising an anti-clathrin heavy chain antibody and / or an anti-formiminotransferase cyclodeaminase antibody.   The discrimination reagent according to claim 8 or 9 for discrimination by tissue immunostaining.