JP2010139293A - Early cancer tumor marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an extracorporeal diagnosis method for an early cancer by a new polypeptide which is useful for an early cancer marker. <P>SOLUTION: It is found that MK (midkine) appears in an early stage in blood or in urine of a patient having a cancer of various kinds, and a detection method of an early cancer including a process for measuring MK in blood or in urine based on the knowledge is completed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a tumor marker for detecting early cancer.

The degree of progression or expansion of cancer is expressed as early cancer, advanced cancer, or end cancer. Among these, early cancer is generally regarded as “small cancer, few metastases, and advanced cancer that can be permanently or long-term cured by treatment”.
Early cancer is basically asymptomatic. Therefore, in order to detect early cancer completely close, it is a test for asymptomatic subjects. A test for finding a patient suffering from a specific disease in a wide range of asymptomatic subjects or a test for narrowing down subjects who need a more advanced secondary test is generally called screening. In screening, the number of test objects is generally extremely large. In order to inspect many test subjects, it must first be simple and economical. The tumor marker test is a suitable test method with less invasion to the patient, but at present, it is usually impossible to detect early cancer by measuring the tumor marker.

Midkine (hereinafter referred to as “MK”) is a growth and differentiation factor discovered as a product of a retinoic acid responsive gene, and is a 13 kDa polypeptide rich in basic amino acids and cysteine (Kadomatsu, K. et al. : Biochem. Biophys. Res. Commun., 151: 1312-1318; Tomomura, M. et al .: J. Biol. Chem., 265: 10765-10770, 1990). Since MK is enhanced in various malignant tumors as compared to the surrounding normal tissues, it has been suggested that MK plays an important role in carcinogenesis. Thus, a cancer diagnostic method using Northern blotting using the MK gene as a probe (Japanese Patent Laid-Open No. 6-113898) and a cancer diagnostic agent containing an anti-MK protein antibody (Japanese Patent Laid-Open No. 6-172218) have been proposed. However, these published patents do not describe or suggest that the MK gene or MK protein is useful for early cancer detection. Ye et al. Then reported that MK expression was elevated in precancerous tissues in the adenoma stage of human colon cancer (Ye C. et al .: Br. J Cancer., 79: 179-183). 1999). However, this paper does not describe or suggest the detection of early cancer.
Therefore, discovery of a tumor marker that can be detected from an early cancer state and development of a test method are desired.

  An object of the present invention is to provide a novel polypeptide useful as a marker for early cancer. Another object of the present invention is to provide a method for detecting early cancer using the polypeptide as an index. Furthermore, this invention makes it a subject to provide the in-vitro diagnostic agent of the early stage cancer which can detect this polypeptide.

In the hepatocellular carcinoma patient, the present inventors have found that the MK level detected by the anti-MK antibody is not only in the tissue of hepatocellular carcinoma but also in the blood and urine as compared with the healthy subject and hepatitis patient. We found that it increased significantly at an early stage of cell carcinoma. In addition, for gastric cancer, it was found that MK levels in blood and urine increased significantly at an early stage as in liver cancer. Furthermore, in many cancers such as esophageal cancer, duodenal cancer, colon cancer, bile duct and gallbladder cancer, pancreatic cancer, thyroid cancer, lung cancer, and breast cancer, serum MK levels are significantly increased at an early stage of cancer. I found it. That is, it shows that MK has a very broad spectrum that is not found in known tumor markers for the detection of unspecified cancer.
Based on these findings, the present inventors have revealed that MK is useful as a marker for early cancer. And, by using the simple and sensitive one-step sandwich method developed by the present inventors, the MK level that appears in the body fluid of patients at an early stage for any cancer is detected with high sensitivity by EIA. It enabled screening diagnosis of early cancer. EIA can be fully automated and is very useful as a method for measuring MK for the purpose of early cancer detection.

That is, the present invention relates to the following early cancer detection methods or early cancer diagnosis methods, and diagnostic agents and kits for these methods.
[1] Early cancer detection method including the following steps:
a) the process of measuring midkine and / or fragments thereof in a biological sample;
b) a process of comparing the measured value obtained by the process a) with a measured value of a normal person;
[2] The method according to [1], wherein the early cancer is gastric cancer.
[3] The method according to [2], wherein the gastric cancer is stage 1.
[4] The method according to [1], wherein the early cancer is hepatocellular carcinoma.
[5] The method according to [4], wherein the hepatocellular carcinoma is stage I.
[6] The method according to [1], wherein the early cancer is lung cancer.
[7] The method according to [6], wherein the lung cancer is stage I.
[8] The method according to [1], wherein the biological sample is serum or urine.
[9] Use of an antibody that recognizes midkine and / or a fragment thereof in the detection of early cancer.
[10]
A diagnostic agent for early cancer comprising an antibody recognizing midkine and / or a fragment thereof.
[11]
An early cancer detection kit for determining the presence of midkine and / or a fragment thereof in a biological sample, comprising an antibody that specifically binds to at least one epitope of midkine and / or a fragment thereof An early cancer detection kit comprising a container.
[12]
The early cancer detection kit according to [11], wherein the antibody is adsorbed on a solid phase.
[13]
A method for determining the prognosis of cancer, comprising the following steps.
a) the process of measuring midkine and / or fragments thereof in a biological sample;
b) associating the measurement obtained in step a) with the prognosis of cancer;
[14]
The method according to [13], wherein the cancer is gastric cancer, hepatocellular carcinoma, or lung cancer.

Definitions As used herein, the following terms have the following meanings unless otherwise indicated.
“Early cancer” refers to a cancer that is localized in the tumor site (in the mucous membrane) and has no invasion of surrounding tissues, or has an invasion that is locally limited. In particular, those in which no infiltration is observed are important detection targets in the present invention because it is difficult to detect their presence with known tumor markers. This definition can be applied to most cancers such as skin, oral cavity, respiratory tract, gastrointestinal tract, cervix, ovary, gallbladder, and bladder. Early cancers include TNM classification stage 0 (carcinoma in situ) and stage I. In this stage, there is no intravascular invasion or distant metastasis, and it can be completely cured by local tumor resection.
In the present invention, a “tumor marker” is a substance produced by a tumor cell or a cell that has responded to its presence, and is found in tissues, body fluids, excreta, etc. It is defined as a substance that can be used as an indicator of some property of a tumor.
“MK” includes full-length MK protein and fragments having an amino acid sequence of any length having the biological activity of MK. Mutant MKs lacking the N domain are expressed specifically in cancer (Kaname T. et al .: Biochem. Biophys. Res. Commun., 219: 256-260, 1996), including such mutant MKs . MKs produced by genetic engineering techniques and chemically synthesized MKs are used interchangeably herein. The DNA sequence encoding human full-length MK is known (US patent: 5,461,029). The biological activity of MK includes not only the physiological effect of MK on cells but also immunological reactivity with anti-MK antibodies.
“Sensitivity” is a ratio at which a measured value in a group in which a tumor exists is positive, and is also referred to as a positive rate.
“Specificity” is a ratio at which a measured value in a group having no tumor is negative, and is also referred to as a negative rate.
“Biological sample” means a sample that can be obtained from an organism. More specifically, examples include blood, serum, urine, and other secretions. Among these biological samples, urine is useful as a less invasive sample. Since the urine volume varies widely, it is desirable to perform urine volume correction in order to compare the concentrations of urine components more accurately. As a technique for correcting the urine volume, a technique such as creatinine correction is known.
As the “stage classification”, the TNM classification (tumor-node-metastasis staging) is generally used internationally as a classification based on the degree of macroscopic progression of cancer. The “stage classification” used in the present invention corresponds to the TNM classification [clinical / pathological primary hepatoma handling rules: 22p. Edited by the Japanese Society for Hepatocarcinology (3rd revised edition), Kanbara Publishing, 1992].
In the present invention, “detection of cancer” means that it is determined that there is a high possibility that cancer exists in the body of the subject. For detection, the term screening is a term that refers to a test performed with the aim of narrowing down subjects who are likely to have cancer, particularly in any population. On the other hand, in contrast to screening for an arbitrary group, detecting cancer for a specific subject is particularly called diagnosis. In the present invention, screening and diagnosis differ only in their objects, and both are included in the method for detecting cancer according to the present invention.

Markers made by cancer cells do not change the blood level from the standard value of healthy people until the cancer grows to some extent, so it is usually impossible to detect early cancer from the increase in serum markers as described above. Has been.
The inventors show that MK is highly expressed at the mRNA and protein levels in the precancerous stage of colon cancer (Ye C. et al .: Br. J Cancer., 79: 179-183, 1999). Therefore, when the blood MK levels of patients with various types of cancer were examined, in most patients (87%), the blood MK level was significantly higher than that of healthy individuals. I found it rising. This value of 87% is a very high value compared with the existing tumor marker. MK expressed in cancer tissue is probably secreted into the blood, which is thought to increase blood MK levels. Furthermore, in hepatocellular carcinoma, gastric cancer, lung cancer, etc., the blood MK level was increased early in the cancer.

The level of MK in the blood of patients with hepatocellular carcinoma or lung cancer has already increased significantly at stage I as compared to the reference value for healthy individuals, and further increases were observed at stages II to IV. On the other hand, in gastric cancer patients, the level was significantly increased at stage 1 compared to the reference value for healthy individuals, but the MK level remained at the same value regardless of the stage after stage 2. From these facts, it is clear that measurement of MK makes it possible to detect early stage cancer classified as stage I in a wide range of cancers including hepatocellular carcinoma, lung cancer, and gastric cancer. Furthermore, by measuring MK, it can be said that not only early cancer but also all stages of cancer can be detected regardless of cancer increase or MK accumulation. This feature is important as a tumor marker. This is because tumor markers found only in early cancer may overlook advanced cancer.
In general, the usefulness of a tumor marker is evaluated by “sensitivity” that determines cancer patients as positive, “specificity” that determines non-cancer patients as negative, and the like. However, the sensitivity and specificity of each known tumor marker is limited.

In the case of MK, as shown in the Examples, in various types of unspecified cancers, the MK level in blood and urine rises earlier than the reference value for healthy people. That is, it can be said that it has a broad spectrum for unspecified cancer and has high sensitivity. In order to perform screening of early cancers widely regardless of organs, a wide spectrum for detection of unspecified cancers is required in addition to high detection sensitivity and specificity for specific cancers. MK can be said to have the sensitivity and specificity necessary for such screening.
Furthermore, the sensitivity and specificity limits of known tumor markers can be compensated for by the early cancer detection method based on the present invention. It is known that by combining a plurality of tumor markers, sensitivity may be increased while maintaining a certain specificity. A screening method that combines a plurality of tumor markers that leads to an improvement in sensitivity and specificity is generally called a combination assay.

In the present invention, in the screening for asymptomatic subjects, the presence of various types of malignant tumors can be detected at an early stage by measuring the MK level in the blood or urine of the subject with EIA or the like. . Furthermore, sensitivity and specificity may be improved by combining measurement of other tumor markers.
There are two points to consider when performing a combination assay. One is which tumor marker to select for the combination. Then, once the tumor marker to be combined has been determined, the next point to consider is how to determine its cut off value.

The point of selection of tumor markers is to select combinations with as low a correlation as possible. For example, AFP and PIVKA-II, which are liver cancer markers, have a low correlation, and show a specificity of almost 100% when the cut-off value of PIVKA-II is 0.1 Au / ml. On the other hand, when CA19-9 and CA-50, which are pancreatic cancer markers, are combined, true positive cases are the same, false positive cases are non-overlapping combinations, and combinations are inefficient.
Once the combined tumor marker is selected, the next step is to set the cut off value. The cut off value is an important factor that affects sensitivity and specificity. In general, sensitivity and specificity of tumor markers are in a trade-off relationship, but those skilled in the art can find appropriate cut-off values in the relevant literature (eg Takeshi Kawamura: Tumor markers. Japanese clinical 54: 1649-1653, 1996).

The ideal tumor marker does not overlap the measurements in the tumor and non-tumor groups, and the measurements can determine the presence of the tumor. However, such an ideal tumor marker has not been developed so far. For this reason, it is necessary to set the most appropriate cut-off value for discriminating and distinguishing between the tumor group and the non-tumor group.
In one preferred embodiment, the cut off value is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without cancer. Usually, a sample that produces a signal that is three times the standard deviation of the predetermined cut off value is considered positive for cancer. For the cut off value, the upper and lower limits of the 95% confidence interval (reference range) of the distribution of measured values of healthy subjects are often used. However, the setting of the reference range does not take into account any pathological distribution. Therefore, the cut-off value clearly defines the pathological condition to be identified by the examination, and based on that, a certain number of the disease group and non-disease group are collected and examined, and the degree of separation of the measurement values of the two groups (test It is desirable to decide in consideration of the classification ability and prevalence (the situation in which the test is applied). Samples that produce a signal higher than the cut off value determined by this method are considered positive for cancer.

Furthermore, in patients with hepatocellular carcinoma, the level of MK in the blood decreased significantly after the tumor was surgically removed. This means that MK is useful not only for diagnosis of cancer, but also as an index for monitoring the progress of disease state, a factor for prognosis determination, or a monitor for recurrence. Prognosis means the patient's response to treatment. Therefore, if MK is measured before and after tumor treatment and a decrease in the measured value of MK is observed, it can be assumed that effective treatment is being performed. Furthermore, if the MK measurement value drops to that of a healthy person, it is assumed that the tumor treatment was successful. Examples of tumor treatment include surgical removal, radiation therapy, immunotherapy, or chemotherapy.
In yet other embodiments, MK can be used as a marker for the progression of certain cancers, such as hepatocellular carcinoma. In hepatocellular carcinoma, MK levels in blood increase as the stage progresses. Assays as described above for cancer diagnosis are performed multiple times over time (overtime) to assess changes in MK levels. For example, the assay is performed every 24-72 hours for a period of 6 months to 1 year, and thereafter as necessary. In general, cancer has progressed in patients whose MK detected by antibodies increases over time over time.

In the present invention, the MK level in a biological sample is measured using a latex agglutination method, EIA or RIA method using a specific polyclonal antibody or monoclonal antibody against MK, FIA, chemiluminescence immunoassay, or ECLIA method. be able to. Of these, EIA is suitable as a method for measuring MK in the present invention. Since EIA uses an enzyme as a label, it can be carried out more easily than RIA with radioactive waste and half-life problems. Theoretically, the sensitivity of the measurement method can be made higher than that of RIA.
In addition, for EIA, various assay systems are known to those skilled in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In particular, for the measurement of MK, an excellent EIA method has been developed (Japanese Patent Laid-Open No. 10-160735), and those skilled in the art can appropriately improve this known method in accordance with the purpose of detection.

  In a related embodiment, the assay can be performed using a flow-through or strip test mold in which the antibody is immobilized on a membrane such as nitrocellulose. In the flow-through test, MK in the sample binds to the immobilized antibody when the sample passes through the membrane. Next, when the solution containing the second antibody passes through the membrane, the labeled second antibody binds to the antibody-polypeptide complex. The bound second antibody is then detected as described above. In the strip test type, one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. The sample moves through the membrane so as to pass through the region containing the second antibody and moves toward the area of the immobilized antibody. The concentration of the second antibody in the area of the immobilized antibody indicates the presence of cancer. Typically, the concentration of the second antibody at that site produces a pattern such as a line that can be read visually. If no such pattern exists, a negative result is indicated. In general, the amount of antibody immobilized on the membrane is selected to produce a visually distinguishable pattern. In that case, the biological sample contains a level of MK sufficient to produce a positive signal in a two-antibody sandwich assay. Preferably, the amount of antibody immobilized on the membrane is approximately 25 ng to 1 μg, more preferably approximately 50 ng to 500 ng. Such tests are usually performed on very small quantities of biological samples.

In a flow-through or strip test type assay format, quantitative measurement is possible by mechanically reading the signal intensity. Alternatively, the sensitivity can be adjusted so as to produce a positive result only when a certain concentration or more of MK is present. By adjusting the sensitivity, a positive result can be visually determined without using a special device. Methods for adjusting sensitivity in this type of assay format are techniques well known to those skilled in the art. For example, the sensitivity can be changed by adjusting the amount of antibody used.
Of course, numerous other assay protocols are convenient for use with the antigens or antibodies of the invention. The above description is intended to be exemplary only.

  Anti-MK antibodies necessary for various assay protocols as described above and MK used as a standard sample are useful as early-stage cancer detection kits. The early cancer detection kit according to the present invention comprises at least an anti-MK antibody and MK used as a standard sample. In addition to this, the detection kit of the present invention can be combined with an enzyme substrate, a negative control, a handling instruction, and the like necessary for the detection of the labeled component. When the detection kit is used in a technique such as EIA, the anti-MK antibody can be bound to a solid phase carrier in advance. Generally, a reaction vessel, beads, magnetic particles, or the like is used for the solid phase. By binding the anti-MK antibody to the solid phase in advance, not only can the EIA be operated easily, but it also contributes to automation.

The anti-MK antibody used in the above method can be prepared by various techniques known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). The anti-MK antibody may be a polyclonal antibody or a monoclonal antibody. For example, a monoclonal antibody that specifically recognizes MK can be used in the present invention as described in Patent Application No. 2000-272199 (filed on Sep. 7, 2000) by the present applicant. The anti-MK antibody can also be used as a fragment containing the antigen binding site. Furthermore, the antibody may be single chain, chimeric, CDR-grafted, or humanized. Antibodies can be made by the methods described herein and other methods well known to those skilled in the art.
MK is used as an immunogen for preparing the anti-MK antibody of the present invention or as a standard sample. MK used for these includes living body-derived MK, recombinant MK, or chemically synthesized MK, and a fragment having the biological activity of MK. A method for obtaining MK as a recombinant is known (Japanese Patent Laid-Open No. 9-95454).
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described with reference to examples and test examples, but the present invention is not limited to these specific examples.

Preparation of anti-human MK polyclonal antibody MK protein used for immunization and recombinant MK protein used as a standard material were prepared according to the method described in Example 1 of JP-A-9-95454.
CDNA covering the ORF of human MK was introduced into the expression vector PHIL-D4 using Pichia yeast as a host. This MK expression vector was transfected into Pichia yeast G115 (Pichia pastoris G115; Research Corporation Technologies). MK expression clones were obtained by histidine and G418 selection. MK was purified by ion exchange chromatography and affinity chromatography using a heparin column. The neurotrophic activity of the purified MK protein was similar to that of mouse MK produced by recombinant L cells.
Rabbits and chickens were injected with a total of 6 injections every 2 weeks for rabbits and 8 injections every 2 weeks for chickens.
That is, in the case of rabbits, the same amount of 400 μg of MK mixed with Freund's complete adjuvant is injected into the rabbit subcutaneously for the first time, and after the second time, 400 μg of MK is mixed with Freund's incomplete adjuvant. Was injected subcutaneously. In the case of chickens, it was the same as in rabbits except that 100 μg MK was used to inject chickens.
Antiserum obtained from rabbits was salted out with ammonium sulfate, IgG was isolated with a protein A column, and affinity purified with an MK affinity column in which MK was immobilized on Affigel-10 ^™ (Bio Rad). A purified rabbit anti-human MK specific antibody was obtained. On the other hand, the antiserum obtained from the chicken was salted out with ammonium sulfate and then affinity purified with an MK affinity column to obtain a purified chicken anti-human MK-specific antibody. These antibodies specifically detected human MK by Western blot analysis.

Measurement of MK by 1-step sandwich method (1) Rabbit anti-human MK antibody was dissolved (5.5 μg / ml) in 50 mM PBS (pH 7.2) containing 0.1% NaN _3, and 50 μl thereof was microtitered. It dispensed to each well of a plate (Polysorp plates, Nunc). The plate was left at room temperature for 16 hours to allow the antibody to adsorb to the wells. After washing the wells with PBS containing 0.1% Tween 20, 150 μl of PBS containing 0.5% bovine serum albumin (BSA) was added to each well and incubated at 37 ° C. for 2 hours for blocking.
On the other hand, 10 μl of each stage of liver cancer serum sample, gastric cancer stage serum sample, or healthy human serum sample (control) is 0.5 M KCl, 0.5% BSA, and 0.01% Microcide I (aMReSCO). , Solon, Ohio) was reacted with 100 μl of perosidase-labeled chicken anti-human MK antibody (0.1 μg / ml) dissolved in 50 mM Tris-HCl (pH 8.4). 50 μl of this reaction solution was added to each well of the plate and incubated at room temperature for 1 hour. Each well was washed 5 times with PBS containing 1% Tween20. 100 μl of substrate solution (0.5 mg / ml tetrametylbenzidine) was added to each well and incubated for 30 minutes at room temperature. 2N—H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm / 655 nm was measured with a multiplate reader (Model 3550, BioRad). At the time of measurement, an MK standard substance having a known concentration was also subjected to the same operation to prepare a standard curve.
(2) Rabbit anti-human MK antibody lysis buffer uses 50 mM Tris-HCl (pH 8.1-8.3) instead of 50 mM PBS, and further contains 0.15 M NaCl in the buffer. The concentration was 5 μg / ml, 2) the blocking solution was replaced with 0.5% BSA, and PBS containing 0.1% casein was used, and 3) the perosidase (POD) -labeled chicken anti-human MK antibody. Lysis buffer was 50 mM Tris-HCl (0.5 M KCl, 0.1% casein, 0.5% BSA, 1 mg / ml rabbit gamma globulin, and 0.01% Microcide I (aMReSCO, Solon, Ohio) ( pH 8.2-8.4) is different from the above (1), except that MK was measured by the 1-step sandwich method in the same manner as (1) above.

[Test Example 1] Correlation between each stage of hepatocellular carcinoma and MK level in serum (1) Preparation of serum sample 135 healthy persons (male: 94, female: 41, age 21-75), hepatocytes 76 patients with cancer (hepatocellular carcinoma: HCC) (stage I: 7 patients, stage II: 19 patients, stage III: 23 patients, and IV: 27 patients), and 7 patients with viral hepatitis as liver disease controls, adenocarcinoma patients Blood was collected from 72 people (Stage 1:23, Stage 2: 9, Stage 3: 7, Stage 4: 9, Stage 5: 5, Stage 6: 9, and Stage 7:10) Serum was prepared. Serum was immediately frozen and stored at −20 ° C. until MK measurement.
Thereafter, blood was further collected from 376 healthy individuals (152 men and 224 women) to prepare serum.
(2) Measurement of serum MK levels The serum MK levels of HCC patients, viral hepatitis patients, and 135 healthy individuals were measured by the method described in Example 2 (1) (FIG. 1). ). The bar in the figure indicates the standard deviation. From stage II, it was observed that the MK level in the serum of HCC patients was significantly higher than that of healthy individuals.
Using the one-step sandwich method of Example 2 (2) with enhanced EIA sensitivity, the MK levels in the sera of the above HCC patients, viral hepatitis patients, and 376 healthy individuals were measured (FIG. 2). ). The bar in the figure indicates the standard deviation.
The serum MK level in stage I of HCC patients is 0.22 ng / mL (mean value) and significant for healthy individuals 0.02 ng / mL (mean value) ( ^** p <0.01; Mann Whiteney It was expensive. That is, it was revealed that MK is extremely useful as a serum tumor marker for early HCC.

[Test Example 2] Comparison test with PIVKA-II and AFP in HCC
PIVKA-II and AFP are currently widely used as tumor markers for HCC. PIVKA-II and AFP are in a complementary relationship, and the combination of PIVKA-II and AFP increases the diagnosis rate. Therefore, AFP and PIVKA-II were used as comparative tumor markers.
Serum PIVKA-II was measured using E-test PIVKA-II (Sanko Junyaku Co., Ltd.) (FIG. 3). The measuring method is EIA. It was suggested that HCC may be positive from stage III.
Serum AFP was measured using α-feto rear beads (Dynabot) (FIG. 4). The measurement method is an immunoradiometric assay (IRMA). It was suggested that AFP is not detected in stage I of HCC but detected in stage II.
That is, MK was able to detect HCC early, and a strong correlation was observed between the progression of the HCC stage and the increase in serum MK concentration.

[Test Example 3] Correlation between each stage of gastric cancer and MK level in serum (1) Preparation of serum sample 72 gastric cancer patients 72 (stage 1:23, stage 2: 9, stage 3: 7, stage 4: Serum was prepared by collecting blood from 9 people, stage 5: 5, stage 6: 9, and stage 7:10). Serum was immediately frozen and stored at −20 ° C. until MK measurement. The values of Test Example 1 were used for the MK levels in the serum of 135 healthy people or 376 healthy people, respectively.
(2) Measurement of serum MK level The serum MK level in each stage (1-7) of gastric cancer and that of 135 healthy individuals are shown in FIG. 5 (the bar indicates the standard deviation), and healthy. The case of 376 persons is shown in FIG. 6 ( ^** p <0.01; Mann-Whitney U test; bars in the figure indicate standard deviation).
From FIG. 6, it was recognized that the MK level in the serum of the stomach cancer patient was significantly higher than that of the healthy person from stage 1. That is, it was revealed that MK is useful as a tumor marker for early diagnosis of gastric cancer.

[Test Example 4] Comparative test with CEA and CA19-9 in gastric cancer CEA and CA19-9 were selected as comparative tumor markers. CEA has an increased serum level in various cancers including various digestive organ cancers, and is widely clinically applied. On the other hand, CA19-9 was found to show a high positive rate value in pancreatic and biliary cancers. Currently, CA19-9, along with CEA, is the most popular in daily practice as a tumor marker for digestive organ cancer.
Serum CEA was measured using CEA rear beads (Dynabot) to measure MK levels in the serum of gastric cancer patients (FIG. 7; bars in the figure indicate standard errors). The measuring method is IRMA. Although it is suggested that CEA may be positive from stage 7, there is a large standard deviation between each stage, and no significant difference is observed between stages.
Serum CA19-9 was measured using a Centocor CA19-9 RIA kit (Centocor; normal value: 37 U / ml or less) (FIG. 8; bars in the figure indicate standard errors). The measuring method is IRMA. CA19-9 shows a high value only in stage 5, and it is estimated that there is no correlation with the stage.
That is, it was revealed that MK is useful as a serum tumor marker for early gastric cancer.

[Test Example 5] MK levels in serum of stomach cancer patients and lung cancer patients MK levels in serum were examined in the early stage of stomach cancer patients and lung cancer patients (Table 1). In both gastric cancer patients and lung cancer patients, the mean value of serum MK levels in stage I was lower than that in stages II to IV, but the difference was statistical in both gastric cancer patients and lung cancer patients. Was not significant.

[Test Example 6] Measurement of MK level in urine of cancer patients 72 samples of urine of cancer patients (gastric cancer: 24 people, hepatocellular carcinoma: 24 people, colon cancer: 24 people), and 50 samples of early morning urine at the time of a human health checkup The MK level in the medium was measured by the method of Example 2 (2). Also, the creatinine value in the same urine was measured, and the MK value was corrected with the creatinine value. The results are shown in FIG. A significant difference (p <0.01; using Mann-Whitney U test) was found between the urinary MK values of healthy individuals and cancer patients.

[Test Example 7] Correlation between urinary MK value of cancer patient and cancer stage The correlation between urinary MK value of cancer patient and cancer stage was examined. 3 bile duct cancers, 3 breast cancers, 6 colon cancers, 3 esophageal cancers, 1 gallbladder cancer, 10 hepatocellular carcinomas, 3 pancreatic cancers, 7 rectal cancers, 28 stomach cancers, and 1 thyroid cancer, A total of 65 patients were staged (stages 1 to 7), and urinary MK levels were measured. The MK levels in each stage are shown in FIG. 10 as urinary MK correction values ( ^* p <0.05, ^** p <0.01; Mann-Whitney U test). It was revealed that urinary MK levels were significantly increased in healthy people at stage I of cancer. So far, no tumor marker in the urine that rises to the early stage of cancer has been reported. That is, the urinary MK level is useful for early screening of various cancers.

[Test Example 8] Effect of tumor removal in serum on MK level in serum For five HCC patients, the effect of removal of tumor by surgery on MK level in serum was examined (FIG. 11). Four patients had a significant decrease in serum MK levels 7 days after tumor removal. That is, the serum MK level reflects the HCC therapeutic effect.

[Test Example 9] Serum MK levels in various cancers 18 esophageal cancers, 30 gastric cancers, 2 duodenal cancers, 25 colons, 25 hepatocellular carcinomas, 12 bile duct cancers and 12 gallbladder cancers, 9 pancreatic cancers, Table 2 shows the breakdown of the stages of a total of 150 cancer patients: 5 thyroid cancers, 19 lung cancers, and 5 breast cancers. FIG. 12 shows the results of measuring the MK level in the serum of these 150 cancer patients by EIA. MK levels in the serum of 150 cancer patients showed a significant difference (p <0.001, Mann-Whiteny U-test) from that of healthy individuals. 87% of patients showed serum MK levels greater than the cut-off value of 0.5 ng / mL.

According to the present invention, a tumor marker useful for diagnosis of early cancer is provided. The tumor marker is useful for screening early cancer, estimating the stage and prognosis of certain cancers, and monitoring the course of treatment.
According to the present invention, a tumor marker useful for diagnosis of early cancer is provided. The tumor marker is useful for screening early cancer, estimating the stage and prognosis of certain cancers, and monitoring the course of treatment.

76 patients with stage I-IV hepatocellular carcinoma (stage I: 7, stage II: 19, stage III: 23, and IV: 27), as a comparative control, 7 patients with viral hepatitis, and as a control It is a figure which shows the result of having measured the MK level in serum of 135 healthy persons by the 1-step sandwich method as described in (2) of Example 2 ( ^** p <0.01). Error bars indicate standard deviation. Similarly, the MK levels in the serum of 76 patients with stage I to IV hepatocellular carcinoma, 7 patients with viral hepatitis as comparative controls, and 376 healthy individuals as controls were compared with those described in (2) of Example 2. It is a figure which shows the result measured by the step sandwich method ( ^** p <0.01, Mann-Whitney U test). Error bars indicate standard deviation. It is a figure which similarly shows the result of having measured PIVKA-II in the serum of the patient of stage I-IV hepatocellular carcinoma using Eitest PIVKA-II (Sanko Junyaku Co., Ltd.). Error bars indicate standard error. It is a figure which similarly shows the result of having measured AFP in the serum of the patient of stage I-IV hepatocellular carcinoma using (alpha) -feto rear beads (Dynabot) ( ^* p <0.01). Error bars indicate standard error. It is a figure which shows the result of having measured the MK level in each serum of 72 gastric cancer patients of stage 1-7, and 135 healthy persons by the 1-step sandwich method described in (1) of Example 2. FIG. Error bars indicate standard deviation Similarly, it is a figure which shows the result of having measured MK in serum of 72 patients with gastric cancer of stage 1-7, and 376 healthy persons by the 1-step sandwich method described in (2) of Example 2 ( ^** p <0.01, Mann-Whitney U test). Error bars indicate standard deviation. It is a figure which similarly shows the result of having measured CEA in the serum of 72 patients with gastric cancer of stage 1-7 using CEA rear beads (IRMA method) (Dynabot). Error bars indicate standard error. It is a figure which shows the result of having measured CA19-9 in the serum of 72 gastric cancer patients of the same stage 1-7 using the Centocor CA19-9 kit (IRMA method) (Centocor). Error bars indicate standard error. The distribution of values obtained by correcting MK levels in urine of urine of 72 cancer patients (gastric cancer: 24, hepatocellular carcinoma: 24, and colorectal cancer: 24) and 50 healthy persons with creatinine values is shown. It is a figure (p <0.01; statistical software StatView-J5.0; using Mann-Whitney U test). 3 bile duct cancers, 3 breast cancers, 6 colorectal cancers, 3 esophageal cancers, 1 gallbladder cancer, 10 hepatocellular carcinomas, 3 pancreatic cancers, 7 rectal cancers, 28 stomach cancers, and 1 thyroid cancer It is a figure which shows the result of having corrected the urinary MK level in the stage 1-7 of a total of 65 persons with the creatinine value ( ^* p <0.05, ^** p <0.01). Error bars indicate standard deviation. It is a figure which shows the influence which hepatocellular carcinoma resection has on the serum MK value. Bars with dots indicate before tumor excision (pre-op), black bars indicate after tumor excision (Day 7). FIG. 3 compares serum MK levels in various types of cancer. The thick line shows the mean value of the MK level in the serum of 135 healthy subjects, and the dotted line shows a cut-off value of 0.50 ng / ml. The numbers shown below the name of each type of cancer indicate the number of patients, the median, and the 25-75% confidence interval. The asterisk indicates a significant difference (Mann-Whitney U-test) with respect to healthy individuals. ^*: p <0.05, ^**: p <0.01

Claims (2)

A method for determining the prognosis of cancer, comprising the following steps.
a) the process of measuring midkine and / or fragments thereof in a biological sample;
b) associating the measurement obtained in step a) with the prognosis of cancer; The method according to claim 1, wherein the cancer is gastric cancer, hepatocellular carcinoma, or lung cancer.

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