JPS62195557A - Diagnosing agent for degree of progression of gastric cancer - Google Patents

Abstract

PURPOSE:To improve the accuracy of the diagnosis and examination of the gastric cancer by permitting the measurement of the presence or content of the epidermic cell growing factor in the gastrotissue cells. CONSTITUTION:This diagnosing agent for the presence of the gastric cancer or the degree of the progression thereof is constituted to permit the measurement of the presence or content of the epidermic cell growing factor (EGF) in the gastrotissue cells. More specifically, a means for measuring the EGF is optional and is preferably an immunochemical measurement method. Namely, an anti-EGF antibody, for example, preferably antiserum is incorporated into the diagnosing agent. The EGF derived from the animal to be diagnosed is preferably used as the antigen to be used at the time of preparing the antibody and, for example, hEGF is preferably used in the case in which the diagnosis of man is intended.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] 艮子 1. The present invention typically relates to a diagnostic agent for the progression of gastric cancer, which comprises an anti-epithelial cell growth captive antibody.

11 Wataru Mouse epidermal growth factor (hereinafter sometimes abbreviated as mEGF), which was first isolated from the submandibular gland of male mice, is 53
A polypeptide consisting of several amino acids that promotes the proliferation of many cells in vitro and in vivo.

Human β-urogastrone, a potent inhibitor of gastric acid secretion, was subsequently isolated from human urine and is probably the same substance as human epidermal growth factor (hereinafter sometimes abbreviated as hEGF). hEGF has been found in human extracellular body fluids, urine, saliva, and milk, and has been immunohistochemically localized to the submandibular and Brunner's glands.
M was detected9. Q5? On the other hand, gastric and intestinal epithelial cells are known to contain the EGF receptor, which is closely related to the oncogene protein of erythroblastosis virus (erb-B). Kirini recently, EGF
Overproduction of barge pigs is different from humans! 17i2
1. ), whereas no production of EGF by tumor cells has been observed in any tumor cell line or tumor tissue.

On the other hand, among the so-called cancer-related diseases, gastric cancer in particular has a particularly large number of patients and a high mortality rate, so establishing a diagnosis and treatment method is one of the major challenges not only in Japan but worldwide. .

As for this diagnostic method, X-rays, endoscopy, and optionally additional biopsy are currently performed as routine examinations. However, such current methods
At present, good results are not necessarily obtained in terms of early detection of cancer or diagnostic accuracy. Therefore, if some new biological indicators can be used to diagnose gastric cancer, to differentiate the degree of progression, and the degree of biological indicators,
It is expected that it will contribute to improved diagnosis and differential accuracy, early detection of cancer, treatment strategies, etc.

[Summary of the invention]

Summary The present invention aims to provide a solution to the above-mentioned points, and aims to provide a solution to the above-mentioned problems, and to clarify the existence of EGF in gastric cancer tissue and its EGF11.
This was based on the discovery that the degree of cancer changes depending on the degree of progression of gastric cancer.

Therefore, the agent for diagnosing the presence or progression of gastric cancer according to the present invention is configured to be able to measure the presence or content of epidermal growth factor in gastric tissue tsmrm;
It is characterized by:

Effects By providing the diagnostic agent of the present invention, it is possible to improve the accuracy of diagnosis and differentiation of gastric cancer.

It was unexpected that EGF exists in gastric cancer tissue, that its prototype ω changes depending on the stage of progress of gastric cancer, and that the stage of progress of gastric cancer can be diagnosed by detecting these changes. Was that.

[Detailed description of the invention]

Method for measuring the presence or content of EGF in gastric tissue cells The method for measuring the presence or content of EGF in gastric tissue cells is arbitrary, but it is possible to use a biological sample in which an infinite number of contaminants exist; As is clear from the experimental examples below, EG
Considering that the F content is extremely small, it is preferable to use a measurement method that utilizes an immunochemical reaction.

Immunochemical assays include radioimmunoassays and non-radioimmunoassays such as enzyme immunoassay (EIA), all of which are preferably used.

In addition, the agent for diagnosing the presence or progression of gastric cancer according to the present invention is configured to be able to measure the presence or content of EGF in gastric tissue nIIIW&.

The specific h E G F measuring means is optional as long as this purpose is achieved. That is, the means for measuring the presence or content of EGF can be freely selected from known methods. However, as mentioned above, it is preferred to choose an immunochemical assay.

In this preferred embodiment, when using an immunochemical assay, the diagnostic agent of the present invention comprises an anti-EGF antibody. The antibodies in this case include both so-called monoclonal antibodies and polyclonal antibodies (antiserum), and both can be suitably used. An example of a preferred antibody is an antiserum.

EGF used as an antigen in preparing antibodies is
It is preferable to use an antigen derived from the animal to be diagnosed. For example, when the purpose is human diagnosis, it is preferable to use hEGF as the antigen.

Not only when an immunochemical assay method is selected and an anti-EGF antibody is used, other materials such as radioactive isotopes, oxygen, substrates, in-phase carriers, etc. may be added depending on the type of assay method used in the present invention. It goes without saying that it can be freely included as a component of a diagnostic agent. These auxiliary materials are
(2) The diagnostic agent of the present invention can be constructed as a standard product or as a kit.

EXPERIMENTAL EXAMPLE The following is an experimental example that confirmed the presence of EGF in gastric cancer tissue and showed changes in EGFIi degree due to differences in the degree of progression of gastric cancer.

B) Materials and Methods (1.) Test Samples Surgically excised human gastric cancer tissues from a total of 210 cases, including 52 cases of early gastric cancer, 113 cases of advanced gastric cancer, and 45 cases of scirrhous type gastric cancer, were used. All these excised tissues are 10
% neutral formalin and embedded in paraffin. For some cases of advanced cancer, representative portions of tumor tissue can be excised and quickly removed and frozen in liquid nitrogen for radioimmunoassay (RIA) and immunostaining.
Stored at 0°C. The degree of progression of gastric cancer (Sta (18)) and histological classification are according to the Gastric Cancer Handling Standards of the Japan Gastric Cancer Research Society.
Immunohistochemical search was performed using antibodies.

In 41 of 113 cases of advanced gastric cancer, h
EGF content was measured by RIA method. These advanced cancers 41
In 15 of the cases, frozen sections were also examined immunohistochemically for hEGF.

(2) Anti-hEGF antibody Antibodies are obtained by injecting high-purity hEGF obtained from a genetically engineered E. coli host (see specification of Japanese Patent Application No. 60-22630) into New Sealant white rabbits. 31 That is, highly pure hEGF is obtained by transforming Escherichia coli using a chimeric plasmid containing a gene encoding a signal peptide of alkaline phosphatase and a hEGF structural gene directly linked to the gene, for example, and
After culturing it to secrete and produce hEGF, it can be purified and obtained using ion exchange chromatography and high performance liquid chromatography as appropriate. Furthermore, the obtained anti-hEGF rabbit antiserum was purified by hEGF affinity chromatography.

This antibody exhibits hEGF specificity and mEGF.

It has been determined that there is no cross-reactivity with other peptides including FGF, PDGF, ECG51 secretin, insulin, glucagon, cholecystokinin and endorphins.

(3) Immunohistochemistry As the immunohistochemical method, the enzyme antibody avidin-biotin complex W)y>improved method by Hsu et al. was used. For deparaffinized sections (4 μm) and frozen sections (6 μm), the following (1) to (3) were reacted at room temperature for 30 minutes or more. (1) anti-hEGF antiserum, (2) biotinylated anti-heron IQG goat antiserum (-8 dilution 1:100, Vect
or Lab, Inc.

U, S, ^), and (3) avidin, DH-biotinylated horseradish complex (8x1:501Vector
jab, Inc., U, S, A), peroxidase was 30 ay of 3° 3'-diaminobenzidine-tetrahydrochloride solution in 0.05 M Tris-HCl buffer (al 17.6) containing o, ooi% H2O2. for 10 to 20 minutes.

Each section was poststained with 3% methyl green. Reaction specificity was determined by Sternberger's method. That is, (i) the anti-hEGF anti-supernatant is
Absorption for 24 hours at 4°C with EGF, (ii) non-immune rabbit V serum as the first layer, and (iii) 3.3' from the incubation mixture for the peroxidase reaction.
Diaminobenzidine-tetra-chloride or H2O
Omission of 2.

hEGF immunoreactivity in tumor tissue was ranked as follows. That is, 50% or more of tumor cells with immune activity: +10+, 25-50%: ++, 25% or less:
+, and no activity knee.

(4) RIA method of hEGF The hEGF content in tumor tissues was measured by the two-antibody method using anti-hEGF antiserum and anti-Heron IQG goat antiserum. was homogenized with 5 times m of 1M acetic acid, and the extraction was repeated twice. The mixture of first and second supernatants was lyophilized and dissolved in Phosphorus M buffered saline 11d. 1 opq of 125I hEGF (2100 Ci/m
800 μm phosphate buffered saline containing mole) and 100 μl of anti-hEGF blood (dilution 1:10,
000) and incubated at 40°C.
■ was purchased from AllerShal (UK) and hE
GF was labeled using the chloramine-T method. - After overnight incubation, anti-heron IQG goat serum 100 μm (dilution 1:10.881 Co, Japan), polyethylene glycol 100 μm! and 100 µl of normal rabbit serum (dilution 1:100) as a carrier, and then at 4°C.
and incubated for 6 hours. The mixture was centrifuged and the resulting precipitate was collected using Alokaγ-Automated Scintillation 4.
The number of groups was determined by the loser. The measurement was repeated three times, and the average was taken as the measured value.

11! used for hEGF RIA! Frozen sections were prepared from part of the tumor pieces, and the presence of tumor cells was confirmed histologically.

(5) Statistics The obtained data were evaluated by 5tUd13nt t-test, χ2 test and Z-test.

Results (1) Immunohistochemical search results for hEGF A comparison of the immunohistochemical staining results for hEGF in paraffin sections and frozen sections of advanced gastric cancer is shown in Table 1. There was a good correlation between hEGF-immunoreactivity between paraffin sections and frozen sections, with staining results in 14 out of 15 cases (93.3%
) was matched.

Table 2 shows the hEGF immunohistological search results in 52 cases of early gastric cancer, 113 cases of advanced gastric cancer, and 45 cases of scirrhous gastric cancer. While no hEGF immunoreactivity was observed in early gastric cancer, hEGF-positive cases were observed in 24 of 113 advanced cancer cases (21.2%) and 15 of 45 scirrhous cancer cases (33.3%). It was done. The frequency of hEGF immunoreactivity in each stage of advanced cancer is 33.5% in stages I, ■, and ■.
They were 3%, 15.0% and 25.6%. However, in 9 cases of stage I advanced cancer, h
No EGF immunoreactivity was observed.

・In comparison with the histological type of advanced cancer, differentiated adenocarcinoma (
The frequency of hEGF immunoreactivity in papillary adenocarcinomas and tubular adenocarcinomas was significantly higher than in poorly differentiated adenocarcinomas (including middle cell carcinomas and colloid adenocarcinomas). The 45 cases of scirrhous type carcinoma were classified into 3 cases of tubular adenocarcinoma and 42 cases of poorly differentiated adenocarcinoma, and hEGF immunoreactivity was detected in 13 cases (30.4%) of the 42 cases of poorly differentiated scirrhous type adenocarcinoma. Admitted. Its appearance frequency was significantly higher than that of non-scirrhous poorly differentiated adenocarcinoma (1)<0.05).

hEGF immunoreactivity was often observed in fibroblasts, capillary endothelial cells, and smooth muscle cells found within the same sections subjected to experiments. However, it was not observed in normal preepithelial cells.

Table 1 EGF 1■ 1, por ++ ++ 4.32, por
−−3, 3 3, por −+ 3.2 4, tub2 ++ + 2.95, po
r 2.9 6, tub2 ++ ++ 2.77, tu
b2 − − 2.7 8, por 2.6 9, tub2 −−2.3 10, pap 2.3 11, por 2.2 12, DOr + + 1.813, por
1.5 14, por 1, 5 15, por -1, 3 ・Stomach Cancer Handling Regulations edited by Japan Gastric Cancer Study Group'I! It depends.

por: poorly differentiated carcinoma, tub: moderately differentiated tubular adenocarcinoma,
and pap: papillary adenocarcinoma.

Table 2 Well 17 0 (-) Poorly 35 0 (-) Advanced cancer well 46 14 (30.4%)
0poorly 67 10 (14.9%)
Total 113 24 (21.2%)・
According to the Handling of Stomach Cancer edited by Japan Stomach Cancer Study Group ti=='L.

Early cancer: carcinoma in which tumor cell invasion stops at the submucosal layer; well: differentiated adenocarcinoma including papillary adenocarcinoma and tubular adenocarcinoma; and poor: poorly differentiated adenocarcinoma including moderate cell carcinoma and glial adenocarcinoma.

Kasamoto: Significant difference from poorly differentiated adenocarcinoma (p<0.05).

*Significant difference from this non-schirrhous poorly differentiated adenocarcinoma (p<0.
05).

(2) Comparison of hEGF immunohistochemical results and hEGF content Immunohistochemical results and tumor tissue content were compared. hEGF-containing R in tumor tissue was immunohistochemically determined as hEGF-containing R.
In GF-positive cases, the moisture content was 0.9-11.6 ng/Q. In comparison, hEGF-negative cases had 0.1 to 3.2 nq/g wet weight. In 6 cases where hEGF content was 4 ng or more per wet weight,
In all cases, hEGF-positive tumor #All cells were observed. hEGF
The average interval was 3.7 in immunohistochemically hEGF positive cases.
7±0.61 (standard deviation above average) nQ/Q wet weight,
In hEGF-negative cases, 2.19±0, 1sno10 humidity was found, and a difference was observed between both groups (p<0.05).

(3) Prognosis and hEGF immunoactivity of gastric cancer patients When we compared the prognosis of all patients with advanced gastric cancer and scirrhous type gastric cancer and hEGF immunoactivity, we found that hEGF-positive cases had a poorer prognosis than hEGF-negative cases. , a significant difference (p<0.05) was observed after 2 years after helmet removal.Furthermore, 5
A comparison of 81 patients with taae m and ■ gastric cancer (excluding scirrhous gastric cancer) showed that all 16 hEGF-positive patients died within 18 months after surgery, while 28 out of 65 hEGF-negative patients (42 , 7%) were alive at 18 months.

(4) Conclusion From the above results, EGF produced by tumor cells is
It can be assumed that it plays an important role in the proliferation and fibrosis of gastric cancer, and is also useful as a biological indicator of high malignancy in gastric cancer patients.

References The documents mentioned in the detailed description of the invention are as follows. These documents, with the exception of -, were originally published in the English language and, in accordance with bibliographic citation conventions, are presented with their English names and art-recognized abbreviations.

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Claims (1)

[Scope of Claims] 1. An agent for diagnosing the presence or progression of gastric cancer, which is configured to be able to measure the presence or content of epithelial cell growth in gastric tissue cells. 2. The diagnostic agent according to claim 1, which comprises an anti-epidermal growth factor antibody. 3. The diagnostic agent according to claim 2, wherein the epidermal growth factor is of human origin. 4. The diagnostic agent according to claim 2 or 3, wherein the antibody is an antiserum.