JP3295261B2 - Cancer grade kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a kit for determining the degree of malignancy of cancer.

[0002]

2. Description of the Related Art In the present age, cancer is an extremely difficult disease to be treated. For the treatment, various anticancer drugs have been developed, new therapeutic methods using biotechnology such as gene therapy and hyperthermia have been developed. Therapies are being developed. However, these new drugs and new therapies have not been able to fully exert their effects. This is because cancer has various stages in which the ease of treatment and the degree of malignancy are different, and a sufficient effect cannot be obtained unless a treatment method suitable for the stage is taken. For example, even if chemotherapy should be given very early to patients with stage IV terminal cancer, no effect can be obtained, but radiation therapy or radiation combined with hypoxic cell radiosensitizers represented by misonidazole etc. It is known that the effect is high if a therapy is performed. Also, if you treat patients with mild cancer to patients with severe cancer,
The treatment does not work because it produces only side effects unnecessarily. That is, it is considered that the cancer treatment effect can be improved if the stage of the cancer, that is, the degree of malignancy can be known. However, at present, no means has been found to know the stage of the cancer, in other words, the degree of malignancy in a short time and clearly. For example, a detailed judgment of cancer can be made by various pathological examinations of tissues obtained by biopsy. Only after various biochemical tests have been carried out can this be clarified, and all of these take a considerable number of hours, which can be said to have reduced the therapeutic effect. In particular, in the case of esophageal cancer, there are cancers that are extremely easy to treat and cancers that are difficult to treat, and it is difficult to discriminate between the two. In the past, the malignancy of cancer had to wait for histopathological analysis of samples taken by surgery, biopsy, etc., and the only thing that revealed the malignancy was that treatment had already started. There were many. That is, there has been a demand for a method for easily and quickly knowing the malignancy of cancer and, in particular, the malignancy of esophageal cancer.

[0003]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and it is an object of the present invention to provide a method which can easily and quickly determine the degree of malignancy of cancer.

[0004]

Means for Solving the Problems To solve the above problems, the present inventors have conducted intensive studies for a means for easily and quickly determining the degree of malignancy of cancer. When an immune complex was allowed to act on a sphere in the presence of a chemiluminescent substance and the amount of generated chemiluminescence was measured, the inventors found that the amount of chemiluminescence increased in accordance with the degree of malignancy of the cancer, and completed the invention. Hereinafter, the present invention will be described in detail.

(1) Disease and Chemiluminescence Peripheral blood monocytes are the main players in the immune system, which is a defense mechanism on the living body side in the battle between cancer and the living body. Granulocytes are also a major player in host defense against pathogenic microorganisms of other diseases regardless of cancer. In the inflammation caused by pathogenic microorganisms such as pharyngitis, the present inventors investigated the expression of FcγRI receptor on the cell surface of granulocytes by immunizing peripheral blood of sheep treated with trinitrophenol with anti-TNP-mouse IgG2a. The complex (hereinafter, mIgG2a-TN)
It is called P-SRBC. ) Was allowed to act as a chemiluminescent substance (hereinafter referred to as CL) by acting in the presence of a chemiluminescent substance. Furthermore, it was also found that by examining the expression level, the presence or severity of inflammation can be distinguished. The human (h) Fcγ receptor is roughly divided into FcγRI, FcγRII, FcγR
III is known, but mIgG2a is
It has been elucidated by the present inventors that mIgG2b specifically binds to hFcγRII. These F
As a result of intensive studies for the relationship between the cγ receptor and the disease, a causal relationship was found between the CL expression level of FcγRII and the malignancy of cancer. The index of determination in the present invention is FcyRII on monocytes. This is because FcγRI or FcγRIII cannot determine the malignancy of cancer as described below, and it is related to FcγRII
Because it is unique to

[0006] The inventors of the present invention obtained a hint from these facts and considered and considered whether the malignancy of cancer could be determined by measuring the expression level of Fcγ receptor using this method even in cancer patients. And mIgG2b-TNP
-The inventors have found that there is a causal relationship between the amount of chemiluminescence indicating the degree of activation of monocytes in SRBC and the malignancy of cancer, and have completed the invention.

(2) Specific Judgment Method In order to carry out the judgment method of the present invention, it is necessary to first collect peripheral blood and separate monocytes therefrom. Any method can be used for separating monocytes as long as it is a usual method. For example, EDTA blood or heparin blood may be overlaid on a monopoly separated solution and centrifuged. The monocytes thus separated are cultured in a medium such as RPMI-1640 in a mixed gas stream of carbon dioxide, and then reacted with fresh sheep erythrocytes coated with trinitrobenzenesulfonic acid and anti-TNPmIgG2b (mIgG2b-TNP-SRBC). ), A chemiluminescent substance is added thereto, and the luminescence intensity may be measured immediately with a luminescence intensity meter. The chemiluminescent substance is not particularly limited as long as it is commonly used, and examples thereof include luminol, luciferin, and lucigenin. If the luminescence intensity value thus obtained is high, the malignancy of the cancer is high, and such a patient needs immediate intensive treatment with some danger. If the light emission intensity is weak, there is no need for an emergency evacuation measure that involves danger, and treatment may be performed according to each medical condition. Here, the determination of the intensity of the light emission intensity may be made by setting an average value of a normal person or the like as a normal value as in a normal clinical test value. According to the determination method of the present invention, such a determination can be made immediately by merely measuring the luminescence intensity of a mixture of peripheral blood monocytes and an immune complex.

(3) Significance of Judgment of Cancer Malignancy Judgment of cancer malignancy is very useful in treating cancer. In particular, in the case of esophageal cancer, it is known that there are high-grade cancers and low-grade cancers.
This decision was important in setting treatment guidelines. This means that the treatment method differs depending on the stage of the cancer, that is, the degree of malignancy, and if the treatment method is incorrect, the life of the patient may be jeopardized or the quality of life of the patient may be significantly impaired. For example, cancer with low initial malignancy can be treated with surgery or mild radiation therapy.For severe cancer, hypoxic cell radiation typified by misonidazole has been increased from the beginning. It is possible to overcome cancer by using radiation therapy using sensitizers, radiation therapy using heavy ion beams, combined chemotherapy, etc., and considering the quality of life. Hospice can also be used. In any case, it is essential to determine what kind of cancer the patient has, and it is understood that the significance of the method for determining cancer of the present invention is significant. In particular, in the case of esophageal cancer, it is difficult to distinguish between a cancer having a good prognosis and a cancer having a bad prognosis, so that the effect of the determination method of the present invention is great. Incidentally, the mortality within 6 months after treatment for esophageal cancer classified histologically in the early stage is 0%, whereas that for esophageal cancer classified in the late stage is 47%. Have been obtained by the present inventors. Therefore, the ability to easily distinguish the malignancy of esophageal cancer is very useful for cancer treatment.

(4) Determination kit of the present invention The determination kit of the present invention comprises a culture part (1) for culturing blood cells obtained from peripheral blood, a buffer solution of mIgG-TNP-SRBC (2), a chemical It consists of a luminescent substance buffer solution (3). The requirements in the culture section are that they do not adversely affect the monocyte culture and do not absorb the luminescence due to chemiluminescence. Examples of such a material include ordinary 96-well culture dishes and CL measurement tubes. In this kit, the blood cells are put into (1) together with the culture solution, (2) and (3) are sequentially added, and the luminescence intensity may be immediately measured with a luminescence intensity meter. The cancer of a patient with a high luminescence intensity thus obtained can be determined to have a high malignancy, and the cancer of a low patient can be determined to have a low malignancy.

[0010]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to only these Examples.

Example 1 Judgment Example in Esophageal Cancer Patients Blood was collected from 28 patients who were determined to have esophageal cancer and were scheduled for surgery, monocytes were separated from this blood, and mIgG2
b-TNP-SRBC was added and emission intensity was measured. That is,
5 ml of EDTA blood was layered on the monopoly separated solution, and the mononuclear cells separated by centrifugation (400 G, 30 minutes) were subjected to RPMI-1.
CL tube with 640 medium (diameter 10mm, length 47m)
m, manufactured by Berthold), and cultured at 37 ° C. for 2 hours in a mixed gas stream of carbon dioxide gas. Cells that did not adhere to the tube were removed by counting using a hemocytometer, and fresh 5% fetal calf serum-supplemented RPMI-1640 medium was added to the CL tube and retained. Immediately before the measurement of the luminescence intensity, the medium was replaced with the same amount of Hanks' solution of the medium, and the luminol solution 10 μm adjusted as described below was used.
l and mIgG2b-T, also adjusted as described below
The luminescence intensity was measured immediately after adding 10 μl of the NP-SRBC solution. The emission intensity was measured using Biolmart LB9505 emission intensity meter (manufactured by Berthold). Table 1 shows the results. In order to create a normal value, the luminescence intensity was measured similarly using 38 normal persons. The normal value was determined to be 0.9 cpm / monocyte or less from these average values. Referring to this value, the emission intensity is apparently reduced from this value to ２2 × normal value (2
N), 2 × normal value (2N) to 3 × normal value (3N), 3 ×
Normal value (3N) to 4 × normal value (4N), 4 × normal value (4
N) to 4 groups. Follow-up of each group, histopathological determination of cancer tissue removed by surgery,
The deaths due to cancer within 3 years were examined. Table 1 shows the results
And Table 2. Here, the least squares correlation coefficient between the average CL value (average luminescence intensity; cpm / monocyte) of each group and the incidence of death cases is 0.99 in the interval of 0 to 4N or more.
It is 0.910 in the interval of 9, 2N to 4N or more, and the existence of a strong causal relationship between the CL value and the death rate is apparent. The least squares correlation coefficient between the incidence of malignant cancer and the CL value is 0.671, 2N-4
A good correlation coefficient of 0.849 is shown in the section N or more. That is, if these are summarized, it can be said that the group with high luminescence intensity has many histologically malignant cancers, and the group with low luminescence intensity has many histologically early cancers. In addition, the number of deaths caused by cancer increased in the group with higher emission intensity, and it was revealed that the determination method of the present invention can accurately determine the malignancy of cancer. In particular, C more than twice the normal value
It is clear from these results that cancer patients showing L values need attention, and if the value is three times or more, considerable attention is required.

[0012]

[Table 1]

[0013]

[Table 2]

(Preparation of mIgG2b-TNP-SRBC solution) Fresh sheep erythrocytes (SRBC, Denka Seikaken) 0.5
The suspension was centrifugally washed with phosphate buffered saline (PBS) (2,500 rpm, 5 minutes) and suspended in 1 ml of PBS.
Next, 1 ml of a 3 mg / ml PBS solution of trinitrobenzene sulfonic acid (hereinafter abbreviated as TNP) was added to 1 ml of SRBC, and reacted by shaking at 37 ° C. for 15 minutes. Then, gelatin veronal solution (containing Ca ²⁺ and Mg ²⁺ ; GVB
Say ) Twice and suspended with GVB at 2 × 10 ⁹ cells / ml. After adding 4 ml of GVB to this 0.2 ml, an anti-TNP-mIgG2b monoclonal antibody was sensitized at a sub-aggregation concentration. This was washed three times with GVB (37 ° C., 30 minutes) and finally suspended in GVB to a concentration of 2 × 10 ⁸ . This was used as mIgG2b-TNP-SRBC solution.

(Preparation of Luminol Solution) Luminol is converted to dimethyl sulfoxide (hereinafter abbreviated as DMSO) 1.8.
It was dissolved to a concentration of mg / μl. To this, PBS was added to a concentration of 1 × 10 −3 M to obtain a luminol solution. This was stored at -20 ° C, protected from light, until immediately before use.

As a control for the experiment, anti-TNP-mIgG2
bAnti-TNP / mIgG2 instead of monoclonal antibody
a Using a monoclonal antibody, FcγR
Examination of I-related chemiluminescence revealed no causal relationship with cancer malignancy as shown in FIG.

[0017]

According to the determination method of the present invention, the malignancy of cancer can be determined easily and in a short time, which is very useful for setting a guideline for cancer treatment.

[Brief description of the drawings]

FIG. 1 is a diagram showing FcγRI-related chemiluminescence of a normal person and a cancer patient.

Claims (4)

(57) [Claims] 1. Malignancy of cancer containing (A) a physiologically acceptable buffer solution of a chemiluminescent substance, (B) a physiologically acceptable buffer solution of an immune complex, and (C) a culture part. Judgment kit. 2. The kit according to claim 1, wherein (B) the immune complex is a complex containing mIgG2b. 3. The cancer according to claim 1, wherein (A) the immune complex is reacted with peripheral blood monocytes of a cancer patient in the presence of a chemical substance, and the amount of generated chemiluminescence is measured. Malignancy determination kit. 4. The kit for determining a cancer malignancy according to claim 1, wherein the cancer type is esophageal cancer.