WO2000058728A1

WO2000058728A1 - Means of early examination of malignant tumor

Info

Publication number: WO2000058728A1
Application number: PCT/JP2000/001775
Authority: WO
Inventors: Katsuaki Uno; George Hosokawa
Original assignee: Uno, Akitane; Sasaki, Hiroshi; Immuni-X Incorporation
Priority date: 1999-03-26
Filing date: 2000-03-23
Publication date: 2000-10-05
Also published as: AU3326000A

Abstract

A means of early examination and prevention of a malignant tumor characterized by comprising comparing data obtained by a means of examining a malignant tumor and data obtained by a means of examining immunological functions with normal values, standards or cutoff values for each marker, and thus predicting or detecting the expression or presence of the malignant tumor on the basis of substantial changes in the immunological functions.

Description

CT / JP00 / 01775

1

Statement

Techniques for early detection of malignant tumors

The present invention relates to provision of a means for early detection and examination of malignant tumors in a comprehensive disease examination means. Background art

A comprehensive disease testing tool, called a medical checkup, is recognized as a useful system for early detection of various diseases, and is widely used today. However, testing of immune function has not been regarded as important in these conventional comprehensive disease testing methods.

In particular, in the examination of malignant tumors, general examinations (blood, urine, diagnostic imaging, etc.) that are not necessarily specialized in detecting cancer, and so-called cancer markers (tumor markers) Testing was being performed as an indicator of disease. Cancers found by conventional examinations and conventional examination methods using tumors were often cancers at an advanced stage, and cancer sites found were limited. . As a result, there was no means for early detection of cancer that could not be detected by general tests or tumor markers. Disclosure of the invention

An object of the present invention is to provide a means capable of predicting and / or detecting the occurrence of cancer that could not be detected by the conventional method at an earlier stage.

That is, the present invention compares the information obtained by the test means relating to the immune function with a normal value, a reference value, or a cut-off value for each of the markers, and 1775

Two

The present invention provides an early examination means for malignant tumors, which comprises predicting malignant tumor expression and / or detecting malignant tumors at an early stage by recognizing a significant change. Further, the present invention can analyze and obtain information obtained by a test means relating to a malignant tumor and information obtained by a test means relating to an immune function using blood or urine of a sample, and list and examine the information. By adjusting to the specifications, it is possible to predict the occurrence of malignant tumors and to detect them early by recognizing substantial changes in immune function by making them comparable to the normal value, reference value, or cut-off value for each marker. Provide a possible, comprehensive means of early detection of malignant tumors.

The comprehensive malignant tumor early examination means of the present invention executes malignant tumor examination means and immune function examination means as comprehensive disease examination items, and obtains information obtained by at least these two examination means. · Characterized by analyzing. In addition, the comprehensive early inspection means for malignant tumors of the present invention includes: means for recognizing information obtained by the examination means for malignant tumor and the examination means for immune function; and a normal value, a reference value, or a power off by each examination means in advance. It is characterized by comprising means for storing or recognizing numerical values specified as values, and means for recognizing a significant difference between these numerical values. In the comprehensive means for early examination of malignant tumors of the present invention, the tests for immune function can be a natural killer (NK) cell activity test and a helper T cell system test (test for cytokines in helper T cells), Judgment can be made based on the fact that this test value is significantly different from the normal value, reference value or cut-off value.

Further, in the comprehensive means for early examination of malignant tumors of the present invention, tests for immune function include natural killer (NK) cell activity test, helper T cell test (helper T cell cytokine test), and killer T cell test. The test value can deviate from the normal value, reference value, or cut-off value with a significant difference. Can be determined by

Furthermore, in the comprehensive means for early examination of malignant tumors of the present invention, a test for immune function is performed in addition to a natural killer (NK) cell activity test and a helper T cell line test (helper—intracellular cytokine test). Site force-in test may include ferrona (IFN-a), tumor necrosis factor (TNF-), interleukin 12 (IL-12), and inuichi leukin 18 (IL-18). It can be judged based on the fact that this test value is significantly different from the normal value, reference value or cut-off value.

Furthermore, in the comprehensive means for early examination of malignant tumors of the present invention, tests for immune function include natural killer (NK) cell activity test, helper T cell line test (helper T cell cytokine test) and killer test. In addition to T cell line testing, interferon (IFN-A), tumor necrosis factor (TNF-), in Yuichi Leukin 12 (IL-12), in Yuichi Leukin 18 (IL- It can be judged from the fact that this test value deviates from the normal value, reference value or cut-off value with a significant difference.

In addition, in the comprehensive means for early examination of malignant tumors of the present invention, an immunological function test is performed in addition to a natural killer (NK) cell activity test and a helper T cell system test (test for cytokines in helper T cells). Cytokine testing and immunosuppressive acid containing Yuichi Feron-a (IFN-a), tumor necrosis factor (TNF-hi), interleukin-12 (IL-12), and interleukin-18 (IL-18) It can be a test for immunosuppressive substances containing protein (IAP), and it can be judged from the fact that this test value deviates from the normal value, the reference value, or the cut-off value with a significant difference.

Furthermore, in the comprehensive means for early examination of malignant tumors of the present invention, tests for immune function include natural killer (NK) cell activity test, helper T cell system test (helper -Intra-T cell cytokine test) and killer T cell line test, plus interferon y (IFN-a), tumor necrosis factor (TNF-), interleukin 12 (IL-12), It can be a cytokine test containing leukin-18 (IL-18) and an immunosuppressive substance test containing immunosuppressive acidic protein (IAP), and this test value is significantly different from the normal value, standard value or cut-off value. Judgment can be made based on the fact that there is a difference.

In the comprehensive means for early examination of malignant tumors of the present invention, the test for immune function is performed by using interleukin 12 (IL-12), interferona (IFN-y), and inuichi leukin 18 (IL-18). It may be a sitekine test that includes at least one of them, and it can be determined based on the fact that this test value deviates from the normal value, the reference value, or the cutoff value with a significant difference.

In the above-mentioned comprehensive early examination of malignant tumors of the present invention, the examination judgment on the immune function may be performed by obtaining the discriminant function coefficient for each immune marker using discriminant analysis. In addition, the test decision on immune function is based on at least the cancer incidence rate (prevalence) adjusted for age and gender, and the risk of cancer is estimated, and the discrimination score obtained by the discriminant is divided into several groups. It may be performed by the following.

In the comprehensive early examination method for malignant tumors of the present invention, the examination means for malignant tumors is performed by calculating the sensitivity and specificity at each cut-off value of each tumor marker and / or various immune markers. Is also good. In addition, the means of examination for malignant tumors is to estimate the risk of cancer based on the known or general cancer incidence or cancer prevalence, and set each cut-off value in several groups. May be done by

The malignant tumor early test means of the present invention described above is characterized in that the cut-off value calculated by the malignant tumor test means is not fixed but variable in each group based on the discrimination score determined by the immune function test judgment. By using, the risk T / JP00 / 01775

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It can also be determined. Here, the means for early inspection of malignant tumor of the present invention can also display the risk as a single score. In the means for early detection of a malignant tumor according to the present invention, the means may be a measuring method. In the means for early detection of a malignant tumor according to the present invention, the means can be a reagent necessary for measurement. In the means for early inspection of a malignant tumor according to the present invention, the means may be a measurement result sheet.

In the means for early examination of malignant tumor of the present invention, the means can be a computer-readable recording medium.

Description of the drawings

Figure 1: It is an explanatory diagram showing the ROC curve (A), curve coordinates (B), and distribution graph (C) of the Th1 test.

Fig. 2: 1 ^ 1 (10 for cell activity test. It is an explanatory diagram showing the curve (A), the coordinates of the curve (B), and the distribution graph (C). FIG. 4 is an explanatory diagram showing a curve (A), coordinates (B) of the curve, and a distribution graph (C).

Fig. 4: It is an explanatory diagram showing 10 (curve (A), curve coordinates (B), and distribution graph (C) of the Ding 12 inspection.

Figure 5: Illustrated diagram showing the ROC curve of the IL-12 test, the coordinates of the curve (Β), and the distribution graph (C).

Figure 6: An explanatory diagram showing the R〇C curve (A), the coordinates of the curve (B), and the distribution graph (C) of the IFN-key test.

Figure 7: The ROC curve (A) and the coordinates (B) of the Th1 / Th2 test are shown. T / 01775

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FIG.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides, in an earlier stage, the information obtained by the conventional malignant tumor examination means in order to achieve a state where the occurrence of the malignant tumor is predicted and / or detectable. The most important feature is that the information obtained by the means for examining immune function is introduced into comprehensive disease examination means. The present inventor has named the whole or immunological test of this means as Cancer Immune Dock ^™ (ImmunoDec ^™ ) or Cancer Immune Judgment ^™ (ImmunoDec ^™ ). Hereinafter, the term ^TM means a trademark. As the information obtained by the examination means for malignant tumors, conventionally known information can be widely used, but is not limited thereto. For example, immunosuppressive acidic protein (IAP) ヽ ferritin, TPA, FP, PI VKA-2, DUPAN-2, eras 1, CA 19-9, CEA, SPan-1, NCC-ST 439, CA 15-3, B CA 225, SLX, PSA, sm, PAP, CA125, CA72-4, STN, SCC, SYFRA, Pro GRP. NSE, BFP, 1 CTP, GAT , CSLEX, NMP22, etc., and cancer-related antigens. A plurality of these inspection items can be selected and used as desired, and can be added as needed.

The information obtained by the inspection means about immune function, tests for immune cells, cytokines (immune physiologically active substance) inspection, major histocompatibility antigen test, § La Kydon acid (prostaglandin E _2, etc.) tests, Guru Information obtained through evening thione inspections, etc., is mentioned. However, it is also possible to add substances that serve as indicators of immune function that have been identified with the advancement of immunology in the future as test items. Examples of tests for immune cells include the helper T (Th) cell test (helper — T-cell site force-in test; CD 4/1 FN—IL / IL—4: Thr eec o 1 or analysis), killer T (Tk) cell test (HL A-DR / CD 8: Two co 1 or analysis), and natural killer (NK) cell activity test.

These immunological function test means can be used in an appropriate combination, but are preferably NK cell activity test and Th cell test, more preferably both Tk cell test and / or cytokine test, more preferably It is desirable to conduct immunosuppressive substance tests including IAP in addition to these. Alternatively, only the cytokine test may be selected as the immune function test.

Site-in (immunophysiologically active substance) tests include IL-12, IFN-, TNF-, IL-4, IL-6, IL-12, IL-18, etc. Is exemplified.

These test methods can be used by applying clinical and biochemical tests known per se. Each test method is listed in Table 1, Table 2-1 and Table 2-2.

(Hereinafter the margin)

Replacement form (Rule 26) 7/1

table 1

Replacement paper (Kaikai IJ26) Table 2 — 1

Replacement paper (26 8/1

Table 2—2

(Below margin) Replacement paper (Rule 26) P00 / 1775

ECLIA (Electrochemi luminescence Immunoassay): Electro Idani luminescence immunoassay EIA (Enzyme Immunoassay): Enzyme immunoassay

ELISA (Enzyme Linked Immunosorbent Assay): Enzyme-linked immunosorbent assay

IEMA (Immunoenzymometric Assay)

I MA (Immunoradiometric Assay): Immunoradiometric method

LPIA (Latex Photometric Immunoassay): Latex optical immunoassay RIA (Radio I tuno uno Assay): Radioimmunoassay

RPHA (Reversed Passive Hemagglutination): Reverse Hemagglutination

SRID (Single Radial Immunodiffusion): Simple radial immunodiffusion

TIA (Turbidimetric Immunoassay): Immunity! : The dangdo method

Each measurement method is known per se, and is described in detail, for example, in the Proposal of Clinical Laboratory Test, September 20, 1999, Edition 31.

Further, the following inspection items can be preferably added.

1) Immune function-related tests

In-Yuichi Feron, Interferon II, Interleukin 1, In-Yuguchi Ikin 5, In-Yuichi Leukin 8, Inter-Ileukin 10, Granulocyte Colony Stimulating Factor (G-CSF), Granulocyte Macrophage Colony-Stimulating Factor ( GM-CSF), soluble VCAM-1, soluble ICAM-1, percentage of T cells, percentage of B cells, lymphocyte transformation by phytohemagglutinin (PHA), lymphocytes by concanapalin-A (Con-A) Immature assay: IL-2 production ability test, LAK (lymphokine activated key cell) activity test, lymphocyte surface marker analysis by monoclonal antibody (CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22, CD24, CD25, CD30, CD33, CD34, CD38, CD56, CD57, CD71, TCR-H, TCR- A5 (Gun Ten

Analysis of lymphocyte surface markers by each test), Two-Co1 or analysis (CD4 / CD45RA, CD4 / CD29, CDllb / CD8, CD4 / HLADR, HLADR / CD8, CD57 / CD16, CD57 / CD8, CD16 / CD56, CD3 / HLADR, CD25 / CD3, CD25 / CD4, CD25 / CD8, CD4 / CD8, TCRd / CD3, CD1 / CD3, CD5 / CD20, CD23 / CD20, B5 / CD20), Erythrocyte surface marker test (CD55, CD59) _C 2) Gene-related test (test for cancer-related genes, etc.)

H-ras _N N-ras, K-ras, c-myc, N-myc, erb-B-2, K-ras codon, N-ras codoru p53 point mutation, p53 DNA sequence, telomerase activity.

In addition, if desired, it is preferable to conduct a state-of-the-art study in the field of cancer immunity, such as a test for the oxidation / reduction state of macrophages, a test for glucanthione in macrophages, a test for immunosuppressive growth factor (TGF-), Oxidation test (lipid peroxide, superoxide dismutase (SOD) activity, 8-OHdG), erythrocyte surface carbohydrate antigen-related test, natural killer T (NKT) cell test, 3 high An index such as a droxiproline test can be added as a judgment index.

The malignant tumor early examination means of the present invention executes the above malignant tumor examination means and the immune function examination means, lists information and data obtained as a result, adjusts them to specifications that can be examined, and The marker can be compared with the normal or reference value or force, and the soot-off value of the marker, and by recognizing substantial changes in immune function, it becomes possible to predict the occurrence of malignant tumors and to detect them at an early stage.

The normal value, reference value, or cut-off value for each marker refers to a scientifically and statistically established value in healthy subjects, and is a normal value for tumor markers and many immune function tests. Alternatively, the reference value or cutoff value is known per se. In addition, for immunological function tests and tumor marker tests, there are cases where normal values are not used as the criteria for test judgment, and the present inventor's own cut-off value is set from the accumulated data of the tests performed. is there.

Substantial changes in immune function are substantial changes in each of the immune function tests listed above. 11

The deviation from the normal value, reference value, or cut-off value. If at least one test item deviates from the normal value or reference value or cut-off value for each test item, and more specifically three or more test items, it is a substantial change in immune function. The percentage of deviation is considered to be a significant deviation when the value exceeds the normal value, the reference value, or the cut-off value, but may change due to the accumulation of data over time.

As exemplified in the examples as specific examples of the substantial change in the immune function, a sample in which no sign of cancer was found by a normal blood test item was found to have a decreased NK cell activity, Single-site T helper (T h) cell line T hreeco 1 or low element values in analytical tests (IFN-a / 1 L-4 / CD4, Thl site force in / Th2 site cytokine ratio ), Low levels of inferior ferrona, low levels of TNF-IL, low levels of IL-112 as cytokine tests, and high levels of IAP (immunosuppressive acidic protein) as immunosuppressant tests. The earliest cancer was found in histological examinations that were later performed for further work-up. Predicting the development of malignant tumors refers to detecting the occurrence of malignant tumors at the very early stage with at least the significant change in immune function as described above, and predicting the tendency of developing constitutional malignant tumors This means obtaining guidance on the treatment policy for malignant tumors and predicting the progress of malignant tumors.

The preparation or analysis of blood or urine as a sample is achieved by means known per se, automatically or manually.

The data obtained by analysis and inspection is adjusted automatically or manually to specifications that can be reviewed and examined. The specifications that can be reviewed by listing information and data mean that doctors and nurses are adjusted to be able to view information and data at a necessary time. It may be a (measurement result sheet) or a list by a computer system or a computer-readable magnetic medium.

The means and system according to the present invention have been introduced as a comprehensive disease testing system. 12

Used. It is sufficient that at least these tests for malignant tumors and those for immune function should be performed at least on immune function, and if desired, these two tests should be performed to obtain relevant information. The timing of the inspection is not particularly significant if they are substantially at the same time. More preferably, the inspection is performed simultaneously or consecutively.

In the present invention, generalization is disclosed as a means for early examination of malignant tumors and / or as a means for predicting expression based on measured values of markers related to malignant tumors and immune functions obtained by the above means. I do.

That is, in the present invention, multivariate analysis is performed on the value of each marker obtained by the test means relating to the immune function based on known data collected at the start of the present invention (Reference: Multivariate Analysis Handbook, First edition, second print 1 98 8 Published on March 20, 1996, Hyundai Mathematics Co., Ltd.), performed discriminant analysis (literature), obtained the discriminant function coefficient (literature), and obtained the results. A linear discriminant (same in the literature) or a nonlinear discriminant (same in the literature) is derived, and the discriminant score of each individual or the Mahalanobis distance (same in the literature) is obtained from this equation. Based on known prevalence or morbidity by age adjustment and clinical experience, factors by age and gender, more preferably by race, past disease, family history, and previous test results To estimate the risk of cancer, and by adding this information, a more appropriate cut-off value is set and discrimination is performed. Depending on the degree of risk estimated from these methods, several groups ranging from `` almost no cancer risk '' to `` significant cancer risk '' (groups 3 to 10, preferably groups 4 to 8, more (Preferably 5 groups). For example, when dividing into five groups, These groups are expressed as estimated prevalence (morbidity). The names of the groups may not be exactly the same, and may be grouped using similar words that can recognize the same meaning. Further, the degree of risk may be displayed as the score 1. 13

Table 3 Almost no cancer risk 1

Mild cancer risk 2

Moderate cancer risk 3

High risk of cancer 4

Cancer risk is remarkable5 In the present invention, the test decision for malignant tumor may be made based on the risk of cancer occurrence estimated from the above-mentioned test for immune function. The determination may be made by adding the obtained information.

In the examination means for malignant tumors, the sensitivity and specificity of each tumor marker at each cut-off value are calculated, and divided into several groups in the same manner as above based on the estimated risk of cancer incidence. Set a cutoff value that is variable (variable). Based on the immunological function test judgment and the estimated risk of cancer, select the tumor group that is deemed appropriate and the appropriate cutoff value to use in the test judgment. In other words, the present invention also provides a means for early examination of malignant tumors and a means for predicting the occurrence of malignant tumors, which select and use an appropriate cutoff value variably (variably) according to the risk of cancer. According to this method, it is possible to obtain an inspection judgment result with higher accuracy than a conventional inspection.

For example, the measured value of IL-12 for a certain 56-year-old man is 7.8 (unit: pg / ml). When discriminating only one type of IL-12, the discriminant formula = Ax _VIL — ₁₂ + K (A: Discriminant analysis is performed on the basic test data group collected in advance, and the obtained canonical discriminant function coefficient, V _IL - ₁₂ : _IL - ₁₂ measured value, K: the discriminant is established Necessary constants All these values can be changed by adding new inspection data to the basic inspection data group. 14

) Is calculated as 0.57. If this calculated score is assigned to the estimated risk group derived from the cancer risk estimated based on the above age and gender, the group will be “significant cancer risk”. In this case, the cut-off value of each test for various tumor markers corresponding to this group can sufficiently fall below the value set as the reference value.

The following table shows the relationship between the discrimination score of patients judged to be cancer by several detailed tests and the values of various immune markers.

(Hereinafter, margin)

Age 7 types 2 types 1 type ΝΚ cells IFN-r IL-12 TNF-a Th2 Th1 Th1 / T 2 Risk classification Post-examination diagnosis Female 55-0.64723-0.64120-0.56660 11 2.4 7.8 1000 3.5 11.3 3.2 4 Hepatocytes Cancer Male 71-0.37551-0.48200-0.56660 18 22.3 7.8 1240 2.8 11.5 4.1 5 Hepatocellular carcinoma it Male 45-0.72408-0.60680-0.56660 56 6.7 7.8 280 1.1 11.5 10.5 4 Malignant lymphoma Female 66-0.19770-0.11980-0.02640 11 5.6 15.2 3820 3.7 11.7 3.2 3 Ovarian cancer Male 76 ― 0.47509-0.20630-0.22350 40 17.4 12.5 2220 3.8 11.7 3.1 4 Pharyngeal cancer

0 \

ROC curves were determined to determine the usefulness of each immune marker in clinical laboratory tests for cancer. : The OC curve (Recommended clinical test method 31st edition: Kanehara Publishing Co., Ltd.) is an evaluation method suitable for the purpose of comprehensively evaluating the sensitivity and specificity of the test method. Sensitivity as used herein refers to the true positive rate (a / (a) when the test results are classified into true positive (a), false negative (b), false positive (c), and true negative (d). + b)), and the specificity means the true negative rate (d / (c + d)). 1—specificity is the false positive rate, that is, a positive result indicates a test subject that has been obtained but has no disease. A test method that satisfies both sensitivity and specificity is an excellent test method. In other words, when the ROC curve is calculated from the sensitivity (vertical axis) and the specificity (horizontal axis) and plotted, the usefulness of the test is that the ROC curve obtained is on a diagonal line from the lower left corner to the upper right corner. An inspection that cannot be performed, or an inspection that shows a curve that is closer to the upper left corner, is a highly capable inspection, and the usefulness of that inspection can be judged. Fig. 1 shows Thl, Fig. 2 shows NK cell activity, Fig. 3 shows TNF-hi, Fig. 4 shows Th2, Fig. 5 shows IL-12, Fig. 6 shows IFN-α, and Fig. 7 shows Thl / Th2. 3 shows the test results. Among them, the test in which IL-12 or IFN- was set as a parameter was considered to be a test with a high ability to detect cancer because the ROC curve was close to the upper left corner.

According to the test evaluation method described above, it was found that a decrease in the test value in a test in which IL-12 or IFN-α was a parameter all at once showed a very close association with cancer, It was recognized that IL-12 or IFN- could be a marker for detecting malignant tumors. In particular, IL-12 is useful as a parameter for measuring the risk of cancer, and as shown in Table 5 in Example 5, there is a significant difference between the value in healthy subjects and the value in cancer patients. Admitted.

As described above, it is clear that malignant tumors can be detected early by the examination means of the present invention. In addition, the IL-12 level was reduced by a 00/01775

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—The car showed abnormal values, but no cancer was detected by various detailed inspections, and there were some subjects whose cancer risk was judged to be high in the future. Administration of a substance with an IL-12 increasing effect to this subject for the purpose of preventing cancer development resulted in an increase in IL-12 levels and the return of tumor markers to normal levels. Laboratory findings indicate that the risk of cancer has been reduced. That is, the examination means of the present invention can not only detect malignant tumors early but also predict their occurrence. In addition, the effect of increasing site-related proteins related to the risk of cancer such as IL-12, IFN-, TNF-, etc., especially IL-12, and these, especially IL-12, is increased. Substance and Z or a composition containing the substance are useful for preventing cancer development, and if the risk of cancer in the future is determined to be high by the testing means of the present invention, Ingestion of these substances for cancer prevention may reduce the risk of cancer. Further, the present invention can also process each of the above information as a computer system (a magnetic medium readable by a computer). It is a means for recognizing and accumulating at least the information obtained by the above-mentioned test means for the immune function, preferably a test means for the malignant tumor and a test means for the immune function, and a normal value, a reference value or a cut-off value by each test means in advance. This is a comprehensive malignant tumor early examination means that includes means for accumulating and recognizing numerical values specified as toe-off values and means for recognizing and accumulating significant differences in these numerical values. It is also possible to add a means for recognizing and accumulating information relating to the significance and the prediction of the disease to the early inspection means. Furthermore, if desired, a means for recognizing and accumulating personal information relating to the specimen and a means for displaying the information relating to the obtained prediction and outputting it as a printout can be added. For example, if the data of an individual obtained by an immune function test is input as information to the combination system of the present invention, the discrimination score of each individual is automatically calculated and obtained, and stored in the computer system. Each individual based on the cancer risk table 18

Can be determined. Furthermore, by inputting the personal data of the tumor marker obtained by the test for malignant tumor, the data is stored in the computer system based on the cancer risk of each individual obtained by the immune function test. A cut-off value suitable for each individual is automatically selected from the list of cut-off values for tumor markers for each cancer risk level, and test results are automatically obtained based on this. Can be Example

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the present invention is not limited to only these Examples.

Example 1

(Cancer immunity dock ^TM inspection Example 1)

0 Female Born in Showa 21

a) —Dental inspection items

(1) Complete blood count: No abnormalities in red blood cells, white blood cells, hemoglobin, hematocrit, or platelet count.

(2) Biochemistry (blood) tests: Total protein, albumin, BUN, Cr, GOT, GPT, ALP, LDH, G-GTP, T-Bil, D-Bil, ChE, CPK, AMY, Na, No abnormalities were observed for K, Cl, CRP, Fe, TG, T-Coll, and BS.

(3) Blood sedimentation test: within normal range

(4) Urinalysis: No abnormalities were observed in all items.

(5) ECG: No abnormalities were observed.

(6) Fundus examination: No abnormalities were observed.

(7) Hearing test: No abnormality was observed.

(8) Pulmonary function test: No abnormality was found. 19

(9) Abdominal ultrasonography: No abnormalities were observed.

(10) Gastroscopy: No abnormalities were observed.

(1 1) Chest X-ray: No abnormal shadow was found.

b) general immunity test items by cancer immunotherapy dock ^TM

(1) Immune cell test

1) NK cell activity test (7%)… decrease

2) Helper T (T h) cell line Thr eeco 1 by flow cytometry or low values of elements in analytical tests (IFN-y / IL-4 / CD4, Thl site force in / Th2 Site force-in ratio) (Low, 3.5)

(2) Sitekine inspection

1) Interferona (low, 3.6)

2) T N F—Hi (Low, 30 1)

3) I L- 1 2 (low value, 7.8 or less)

(3) Testing for immunosuppressive substances

1) IAP (immunosuppressive acidic protein) (high, 970)

(4) Testing for cancer-related antigens (including tumor marker testing)

1) CE A (high, 20)

2) TPA (high, 1 30)

3) BFP (within normal range, 45)

4) S C C (high, 1 1)

5) NSE (within normal range, 10)

6) FP (within normal range, 5.2)

7) P I VKA2 (within normal range, 25)

8) CA 19-9 (within normal range, 6 or less)

9) CA50 (within normal range, 30) 20

10) SPan-1 (within normal range, 30)

11) S YFRA (high, 29)

12) DUPAN2 (within normal range, 101)

13) Eras evening 1 (within normal range, 280)

14) CA 125 (within normal range, 27)

15) CA72 -4 (within normal range, 2.5)

16) NCC-ST-439 (within normal range, 5.5)

(5) Spiral CT inspection (precise lmm scan)

1) Chest: A tumor of about 7 mm in diameter was found in the lower lobe of the right lung.

2) Abdomen: No abnormalities were observed.

c) Inspection judgment

Completely revealed no abnormalities in general checkup inspection items, but in a normal decision been made on a case that is determined to be normal, abnormal immunological examination in the examination of a plurality of items by was performed as cancer immunity dock ^TM test Therefore, there was a strong suspicion that there was cancer of a size that could not be detected by conventional examinations, which were commonly performed as a medical checkup.

A precision spiral CT scan showed a 7 mm diameter tumor in the lower lobe of the right lung, so the dog was considered to have a possibility of lung cancer, and he was ordered to undergo medical treatment in accordance with the health insurance system. A sputum cytology later confirmed a definitive diagnosis of squamous cell carcinoma, and surgery was performed for an early lung cancer patient to remove the tumor. This is an example in which a very early stage of lung cancer that cannot be normally detected is found by the cancer immunodock ^™ system according to the present invention.

Example 2

(Cancer immunity dock ^TM inspection Example 2)

H Boy Born in 1952 twenty one

a) —Dental inspection items

(2) Biochemistry (blood) test: total protein, albumin, BUN, Cr, ALP, LDH, T-Bil, D-Bil, ChE, CPK, AMY, Na, K, Cl, C

No abnormalities were observed in RP, Fe, T-Chol and BS. GOT (47), GPT (56), GTP (80), TG (235) High values (approximately 2 go a day addicts, at this point in general medical judgment alcoholic fatty liver Was most suspected).

(3) Blood sedimentation test: within normal range

(4) Urinalysis: No abnormalities were observed in all items.

(5) ECG: No abnormalities were observed.

(6) Fundus examination: No abnormality was found.

(7) Hearing test: No abnormalities were observed.

(8) Lung function test: No abnormalities were observed.

(9) Abdominal ultrasonography: The brightness of the liver parenchyma was high, and typical findings were found in fatty liver. No mass was observed.

(10) Gastroscopy: No abnormalities were observed.

Chest X-ray: No abnormal shadows.

b) Cancer ^ImmunodockTM Test Items (Immunological Test)

(1) Immune cell test

1) NK cell activity test (4%)… decrease

2) Helper T (Th) cell line Threeco 1 or low element value in analysis test (IFN-y / IL-4 / CD4, Thl cytokine / Th2 cytokine ratio) (low value, 2.7) twenty two

(2) Site strength inspection

1) Inn Yuichi Ferrona (Low, 0.9)

2) TNF-α (low, 387)

3) I L-12 (low value, 7.8 or less)

(3) Immunosuppressive substance test

1) IAP (immunosuppressive acidic protein) (high, 1120)

(4) Testing for cancer-related antigens (including testing for tumor markers)

1) CEA (within normal range, 2.5 or less)

2) TP A (high, 220)

3) BFP (high, 130)

4) S C C (within normal range, 1.5 or less)

5) NSE (within normal range, 10 or less)

6) FP (within normal range, 18)

7) P I VKA2 (within normal range, 32)

8) CA 19-9 (within normal range, 6 or less)

9) CA-50 (within normal range, 24)

10) SP an-1 (within normal range, 30)

11) S YFRA (within normal range, 3.5 or less)

12) DUP AN 2 (High, 330)

13) Elastase 1 (within normal range, 122)

14) CA 125 (within normal range, 31)

15) CA72-4 (within normal range, 4.0)

16) NCC-ST-439 (within normal range, 5.0)

(5) Spiral CT examination (precise lmm scan) 1) Chest: No abnormalities were observed. twenty three

2) Abdomen: In the liver area S6, there was a finding that a low concentration area with a diameter of 1 cm or less was slightly suspected. c) Inspection judgment

Regarding general laboratory tests, mild hepatic dysfunction was detected by biochemical examination, but fatty liver was determined to be due to daily drinking history. At the same time, abdominal ultrasonography showed fatty liver findings to confirm this, but no mass was found. In the normal case, the case was judged to be suspected of alcoholic fatty liver and dietary guidance was given.

But the presence of cancer that can not be found in the general medical checkup inspection carried items found abnormalities in the inspection of a plurality of items by carrying out the immunological examination as cancer immunotherapy dock ^T M inspection was strongly suspected. At the same time, a fine spiral CT scan revealed a mildly low absorption area-like finding with a diameter of less than 10 mm in section S6 of the liver. Normally, such a single, mild low-absorption area-like finding is difficult to distinguish from liver structures such as blood vessels and bile ducts and other liver diseases. He recommended a detailed examination under the insurance medical treatment system.

As a result, early diagnosis of hepatocellular carcinoma was obtained by abdominal angiography, liver biopsy, and the like. The patient was immediately hospitalized, and surgery was performed. The patient was diagnosed with early stage hepatocellular carcinoma with a diameter of about 1.2 cm. She has been discharged and is in good condition. Common general human dock embodiment workup instructions inspection hope is thinner in this example (dietary advice is mostly observation and Do that case, etc.), by the cancer immunity dock ^TM consisting of the present invention was performed I was able to find liver cancer very early.

Example 3

(Relationship between low IL-12 levels and cancer)

6 For a 1-year-old male subject with a low IL-12 of 7.8 (unit: pg / ml), the linear discriminant obtained by discriminant analysis, one of the multivariate analyses, The test result of each immune marker was incorporated, and the discrimination score was calculated to be -0.57. This number is twenty four

The cut-off value for the tumor is reduced because it falls into the group with significantly higher risk of cancer than the estimated risk of cancer. The measured values of the tumor parameters were IAP = 179, 1 CTP = 3.3, CEA = 0.5, DUPAN 2 = 43 ^ S LX = 28. As a result of judging the change of the cut-off value by introducing the measurement method of the immunomarker of the present invention, it was found that DUPAN 2 entered the area of caution at 30 or more, and SLX also entered the area of caution at 25 or more. .

As a result, the subject determined that there was a risk of cancer and, upon a detailed examination, found gastric cancer.

Example 4

(Relationship between low IL-12 and cancer)

In a 64-year-old male subject with a low IL-12 value of 7.8 (unit: pg / ml), a linear discriminant determined by discriminant analysis, one of the multivariate analyses, showed that each immune The marker test results were incorporated and the discrimination score was calculated to be 0.57. This value falls into the group with a higher risk of cancer than the estimated risk of cancer, thus lowering the cut-off value for the tumor. Tumor marker measurements were IAP = 208, 1 CTP = 2.9, CEA = 0.6, STN = 34, CA15-3 = 8.6, SLX = 31. As a result, as a result of the judgment of the change of the cut-off value by introducing the measuring means of the immunomarker of the present invention, 3 is in the area of caution when 25 or more.

Example 5

(R〇C curve and provisional cut-off value)

For each immune marker, an ROC curve diagram and a distribution graph were created, and the discrimination rate (discrimination between cancer patients and healthy subjects) based on the provisional cutoff value was determined from these. Immunomer twenty five

Fig. 1 shows Thl, Fig. 2 shows NK cell activity, Fig. 3 shows TNF-H, Fig. 4 shows Th2, Fig. 5 shows IL-12, Fig. 6 shows IFN- FIG. 7 shows Thl / Th2. In the R〇C curve diagram of each figure A, the vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The curve coordinates indicate the sensitivity and the specificity corresponding to the test value of each marker, and the provisional cut-off value for each marker was calculated. The reason for this provisional is that if the number of known test results to be examined is added, the cutoff value may fluctuate. In the distribution graph in each figure C, the horizontal axis indicates the stage of cancer or the classification of healthy subjects, and the vertical axis indicates the power of each force.

The Thl test showed an ROC curve (a contrast curve between sensitivity and 1-specificity) that was close to the diagonal line connecting the lower left corner and the upper right corner, and it was concluded that this test alone was a test with low cancer detection ability. Based on the coordinates of the curve, it was determined that the provisional cut-off value was 25.45, the sensitivity was 63%, and the specificity was 60%. From the distribution graph, it is difficult to recognize a clear difference between healthy people and cancer patients. However, when the Mann-Whitney test, one of the nonparametric tests, is performed (literature), a significant difference of 1% or less is obtained. Admitted. The unit of Th1 is%, which is the ratio of observed lymphocytes producing Th1 cytokines contained in CD4 + lymphocytes.

The NK cell activity test showed an ROC curve (a contrast curve between sensitivity and 1-specificity) close to the diagonal line connecting the lower left corner and the upper right corner, and it was concluded that this test alone had a low cancer detection ability. . From the coordinates of the curve, a provisional cut-off value of 36.5, a sensitivity of 57%, and a specificity of 55% were determined. From the distribution graph, no clear difference could be obtained between healthy people and cancer patients (N.S .; Not Significant). The unit of NK cell activity is the percentage (%) considered to have damaged the target cells when the NK cells were cultured with the target cells.

The TNF-HI test shows an ROC curve (a contrast curve between sensitivity and 1-specificity) that is close to the diagonal line connecting the lower left and upper right corners. 26

It was determined that there was. From the coordinates of the curve, it was determined that the provisional cut-off value was 2170, the sensitivity was 65%, and the specificity was 65%. The distribution graph shows a clear difference between healthy subjects and cancer patients, and a significant difference of 1% or less was obtained when the Mann-Whitney test, one of the nonparametric tests, was performed. Was. TNF—The unit is pg / ml.

The Th2 test showed an ROC curve close to the diagonal line between the lower left corner and the upper right corner (sensitivity versus 1-specificity curve), and it was concluded that this test alone was a test with low cancer detection ability. Based on the coordinates of the curve, it was determined that the provisional cutoff value was 2.45, the sensitivity was 65%, and the specificity was 65%. The distribution graph did not show a clear difference between healthy and cancer patients. The unit of Th2 is%, which is the ratio of observed lymphocytes producing Th2 cytokines contained in CD4 + lymphocytes.

The IL-12 test shows an R〇C curve (sensitivity versus 1-specificity curve) near the upper left corner, far away from the diagonal of the lower left and upper right corners, indicating the ability to detect cancer alone. The test was judged to be extremely high. Based on the coordinates of the curve, the provisional cutoff value was determined to be 7.85, sensitivity 74%, and specificity 91%. The distribution graph showed a clear difference between healthy subjects and cancer patients, and a Mann-Whitney test, one of the nonparametric tests, showed a significant difference of 1% or less in the risk factor. The unit of I L-12 is pg / ml.

The IFN-a test shows an R〇C curve (a contrast curve between sensitivity and 1-specificity) that is close to the upper left corner, far away from the diagonal of the lower left and upper right corners, and is extremely capable of detecting cancer by itself. It was judged to be a high test. Based on the coordinates of the curve, the provisional cut-off value was determined to be 20, sensitivity 77%, and specificity 77%. The distribution graph showed a significant difference between healthy individuals and cancer patients, and when the Mann-Whitney test, one of the nonparametric tests, was performed, a significant difference of 1% or less was found. The unit of IFN-I is IU / ml. 27

The Thl / Th2 test shows an ROC curve (a contrast curve between sensitivity and 1-specificity) that is close to the diagonal line connecting the lower left corner and the upper right corner, and it is judged that this test alone has low cancer detection ability. did. From the coordinates of the curve, it was determined that the provisional cut-off value was 10.13942, the sensitivity was 61%, and the specificity was 61%. Thl / Th2 is the ratio of the proportion of lymphocytes producing Th1 cytokines to the proportion of lymphocytes producing Th2 cytokines contained in CD4 + lymphocytes.

Example 6

(Discrimination value)

The discrimination value between cancer patients and healthy subjects was specified, and the value of each immune marker was measured. The results are shown in Table 3. The seven types of discrimination are defined as: (AXV _IL - ₁₂ ), which is the immune marker for IL-12, IFN-α, TNF-HI, Thl, Th2, Thl / Th2 ratio, and NK cell activity. + (BXV _{I FN} —a)) + (CXV _TNK _J + (DXV _Thl ) + (ExV _Th2 ) + (FxV _{Thl / Th2} ) + (GXV _NK activity) + K (A to G: Collect in advance Discriminant analysis was performed on the basic test data group obtained, and the canonical discriminant function coefficient for each test item obtained, V: the measured value of each immune marker, K: the constant required to establish the discriminant. All of these values are variable by adding new test data to the basic test data group.) The two types of discriminant values are obtained by IFN- and IL-. Discriminant = AxV _IL — ₁₂ + BxV _IFN — ₇ + K (similar to the note) One kind of discriminant is the discriminant of Ax V _IL _ ₁₂ + K (similar to the note) Units of disease parameters one coater is, NK cells activity and Thl, Th2 is%, I FN- § the IU / ml, IL one 12 and TNF- flight are pg / ml.

s 辇

SZ./.I0/00df/X3d 8 ^ 8S / 00 OAV 29 As a result, IFN-a and IL-12 clearly showed a significant difference between the values in healthy subjects and those in cancer patients. For IFN- ァ, the association with cancer was suggested at 1 IU / ml or less, more specifically at 16 IU / ml or less, and for IL-12, 20 pg / ml or less, more specifically at 1 IU / ml. Opg / ml or less, more specifically 9 pg / ml or less, suggested a link to cancer.

On the other hand, if the IFN-A was 18 IU / ml or more and the IL_12 was 20 pg / m1 or more, it was determined that the group had a low cancer prevalence. Conversely, it is also judged to be a high-health group with a low risk of cancer.

Example 7

51 year old male

Cytokine examination showed a decrease of IL-12: 7.8, indicating an abnormal value of BFP: 7.9, which is the best tumor. No cancer was detected by various detailed tests, and the risk of cancer was determined to be high in the future. Administration of AHCC (Active Hemi Cellulose Compound), a substance with an IL-12-increasing effect, for the prevention of cancer development, resulted in an increase of IL-12: 24.2, and at the same time, BFP returned to a normal value of 60. did. From the laboratory findings, it was concluded that the risk for cancer had been reduced.

CT / JP00 / 01775

30

Industrial applicability

The means of the present invention provides means for early detection of malignant tumors, and makes a great contribution in the field of preventive medicine. Further, the means of the present invention enables the prediction of the transition of a malignant tumor, provides a report on the predisposition to the occurrence of a malignant tumor, enables advance prediction of the onset of a malignant tumor, and allows the recurrence of a malignant tumor to occur. And / or unprecedented means of testing that can predict the risk of metastasis.

Claims

31 Claims

1. Comparing the information obtained by the testing means for immune function with the normal value, reference value or cut-off value for each marker, and recognizing substantial changes in immune function, predicting the onset of malignant tumors and early An early means of testing for malignant tumors, which involves making a discovery

2. Analyze and obtain the information obtained by the test means for malignant tumors and the information obtained by the test means for immune function using the blood or urine of the sample, and list the information to make specifications that can be reviewed. To make a comparison with the normal value, reference value, or cut-off value for each device, and to recognize substantial changes in immune function to predict the occurrence of malignant tumors and detect them early. An early means of comprehensive malignancy testing that can enable

3. As a comprehensive disease test item, a test method for malignant tumors and a test method for immune function are executed, and at least the information obtained by these two test methods is obtained and analyzed. For early examination of typical malignant tumors.

4. Means for recognizing the information obtained by the test means for malignant tumors and the test means for immune function, and means for storing or recognizing numerical values previously specified as normal values, reference values, or cut-off values by each test means, Means for recognizing a significant difference in these numerical values, a comprehensive malignant tumor early inspection means.

5. The tests for immune function are the natural killer (NK) cell activity test and the helper-T cell system test (helper T cell cytokine test), and this test value is significantly different from the normal value, the reference value, or the cut-off value. The comprehensive early examination method for malignant tumors according to any one of claims 1 to 4, which is determined based on a deviation. 32

6. The tests for immune function are natural killer (NK) cell activity test, helper-T cell system test (helper-one T-cell cytokine test) and killer T-cell system test. 5. The comprehensive early examination method for malignant tumors according to any one of claims 1 to 4, wherein the means is determined based on a significant difference from the value or cutoff value.

7. Testing of immune function includes natural killer (NK) cell activity test, helper-T cell system test (helper T-cell cytokine test), interfacial protein (IFN-a), tumor necrosis factor (TNF-hi), interleukin-12 (IL-12), in situ-leukin-18 (IL-18), and the value of this test is significantly different from the normal value, standard value, or cut-off value. The comprehensive malignant tumor early inspection means according to any one of claims 1 to 4, which is determined based on the fact that the deviation has occurred.

8. Testing for immune function includes natural killer (NK) cell activity test, helper-T cell system test (helper T cell cytokine test), and killer T cell system test, plus interferona (IFN-a). This is a cytokine test containing tumor necrosis factor T (TNF), INY-LEUKIN 12 (IL-12), and INY-leukin 18 (IL-18). The comprehensive early stage malignant tumor inspection method according to any one of claims 1 to 4, wherein the determination is made based on a deviation from the cut-off value with a significant difference.

9. Immunological function tests include natural killer cell (NK) cell activity test and helper T cell line test (helper T cell cytokine test), as well as IFN-a and tumor necrosis. Cytokine testing including factor T (HIF), Inuichi Leukin 12 (IL-12), and Inuichi Leukin 18 (IL-18) and immunosuppressive substances including immunosuppressive acidic protein (IAP) This is a test based on the fact that this test value deviates from the normal value, reference value, or cut-off value with a significant difference. 33

The comprehensive malignant tumor early inspection means according to any one of claims 1 to 4, wherein the means for early inspection of malignant tumors is used.

10. Testing for immune function includes natural killer (NK) cell activity testing, helper T-cell testing (helper T-cell cytokine testing), and killer T-cell testing, as well as IFN-A, Site-in test containing tumor necrosis factor (TNF-), interleukin-12 (IL-12), interleukin-18 (IL-18) and immunosuppressive containing immunosuppressive acidic protein (IAP) A comprehensive inspection according to any one of claims 1 to 4, which is a substance inspection and is judged based on the fact that the inspection value is significantly different from a normal value, a reference value, or a cut-off value. For early examination of typical malignant tumors.

11. A test for immune function was performed by a cytokine test that included at least one of Inuichi Leukin 12 (IL-12), Interferona (IFN-a), and Inuichi Leukin 18 (IL-18). The comprehensive value according to any one of claims 1 to 4, which is determined based on the fact that this test value is significantly different from the normal value, the reference value, or the power-off value. An early means of testing for malignant tumors.

12. The method according to any one of claims 1 to 11, wherein the test judgment regarding the immune function is performed by determining a discriminant function coefficient for each immune marker using discriminant analysis. Means for early examination of a comprehensive malignancy.

13. Test decisions on immune function are made by estimating the risk of cancer from at least age and gender-adjusted cancer incidence (prevalence) and assigning the discriminant score obtained by the discriminant formula to several groups. 13. The comprehensive early examination method for malignant tumors according to any one of claims 1 to 12.

14. The scope of the request made by the examination means for malignant tumors by calculating the sensitivity and specificity of each tumor marker and Z or various immunomarkers at each power cut-off value. Early examination of comprehensive malignant tumors described in paragraph 1 34

means.

15 5. Measuring means for malignant tumors should estimate the risk of cancer based on the known or general cancer incidence or cancer prevalence and set each cut-off value in several groups. The comprehensive early examination means for malignant tumors according to any one of claims 2 to 14, comprising:

16. In each group based on the discrimination score determined by the immunological function test judgment, the risk is determined by using the cut-off value calculated by the test means for malignant tumors not fixed but variable. The comprehensive early examination means for malignant tumors according to any one of claims 2 to 15, wherein

17. The comprehensive malignant tumor early inspection means according to any one of claims 16 to 17, wherein the risk is integrated into a score and displayed.

18. The comprehensive early examination means for malignant tumors according to any one of claims 1 to 17, wherein the means is a measuring method.

19. The comprehensive early stage examination of malignant tumors according to any one of claims 1 to 17, wherein the means is a kit comprising reagents necessary for measurement.

20. The comprehensive early examination means for malignant tumors according to any one of claims 1 to 17, wherein the means is a measurement result sheet.

21. The comprehensive early examination of malignant tumors according to any one of claims 1 to 17, wherein the means is a computer-readable recording medium.