JP5167541B2 - Diagnosis method of gastric cancer using blood concentration of apolipoprotein CIII0 as an index - Google Patents

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a method for diagnosing gastric cancer and a kit for use in the method.

BACKGROUND ART Gastric cancer is cancer that originates in the stomach, and mainly originates from secretory cells in the mucous membrane and cells of the conduit that guides the secretion into the stomach. Due to recent advances in diagnosis and treatment, gastric cancer is said to be one of the most curable cancers. However, early detection of gastric cancer remains important because gastric cancer tends to take a long time to become detectable after it occurs and is often difficult to treat if it is detected in an advanced situation It is.

  In general, tumor markers that enable diagnosis by blood tests are useful for early detection of cancer. As tumor markers for gastric cancer, ACT, CA72-4, CA130, CEA, Dupan-2, IAP, KMO-1, NCC-ST-439, NSE, sICAM-1, SLX, SOD, Span-1, STN, TPA YH-206, Shifra 21-1, and the like are known. However, using these tumor markers cannot be sufficient for early detection of gastric cancer. Thus, development of new tumor markers for gastric cancer remains desirable.

  Apolipoproteins are a group of proteins that are specifically present on plasma lipoproteins. More than 10 types of apolipoprotein are known, and their main functions are stabilization of lipoprotein structure, activation of enzymes involved in lipoprotein metabolism, and lipoprotein receptor present on the cell surface. It acts as a ligand for. In recent years, in addition to lipoprotein metabolism, new functions for nerves are being discovered, and an association with Alzheimer's disease has also been shown.

Apolipoprotein CIII, a member of the apolipoprotein family, consists of 79 amino acids, and the 74th threonine residue is a sugar chain binding site (FIG. 1A). Apolipoprotein CIII in which no sugar chain is added to this sugar chain binding site is called apolipoprotein CIII ₀ (FIG. 1B). Furthermore, the apolipoprotein CIII, Apolipoprotein CIII ₁ is a glycosylated body (Fig. 1D) and CIII ₂ (Fig. 1E), and CIII 1 single alanine from the C-terminus of apolipoprotein CIII ₀ are deleted ₀ ' (FIG. 1C) and the like have been reported (Non-patent Document 1: Bondarenko et al. J. Lipid Res. 40: 543.1999).

Bondarenko et al. J. Lipid Res. 40: 543.1999

Summary of the Invention

The present inventors have found that the ionic strength of a peptide peak having a mass of 8765 m / z corresponding to apolipoprotein CIII ₀ in the blood of a stomach cancer patient is significantly lower than that of a healthy person. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide a method for diagnosing gastric cancer.

The diagnostic method according to the first aspect of the present invention is a method for detecting gastric cancer in a mammal, and (a) measures the concentration of apolipoprotein CIII ₀ in a test blood sample obtained from the mammal. step, and the concentration obtained in (b) step (a), the a method comprising the step of comparing the concentration of apolipoprotein CIII ₀ in a normal control blood sample. In this method, the concentration of apolipoprotein CIII ₀ in said test blood samples for the control blood sample if you were reduced, the presence of gastric cancer in the mammal is shown.

  The diagnostic method according to the second aspect of the present invention is a method for detecting gastric cancer in a mammal, and (i) a protein showing a peptide peak with a mass of 8765 m / z in a test blood sample obtained from the mammal. And (ii) comparing the concentration obtained in step (i) with the concentration of the protein in a normal control blood sample. The method indicates that gastric cancer is present in the mammal when the protein concentration in the test blood sample has decreased relative to the control blood sample.

  According to the present invention, gastric cancer can be detected accurately and early.

Detailed description of the invention

  In the present invention, the stomach cancer to be diagnosed (detected) includes all cancers formed in the stomach. That is, the stomach cancer to be diagnosed may have originated from any part of the gastric cardia, body and pylorus, and tubular adenocarcinoma (highly differentiated and moderately differentiated), Any type of poorly differentiated adenocarcinoma, signet ring cell carcinoma and the like may be used.

An indicator of the diagnosis in the present invention, "Apolipoprotein CIII _0" or "Apo CIII _0" is the apolipoprotein CIII ₀ from mammalian as a subject of diagnosis. Apolipoprotein CIII ₀ is a kind of apolipoprotein CIII, and a sugar chain is not added to the 74th threonine residue of apolipoprotein CIII (FIG. 1B). Apolipoprotein CIII ₀ is the only protein that shows a peptide peak with a mass of 8765 m / z in plasma. Therefore, it is understood that the diagnostic method according to the present invention uses a protein showing a peptide peak having a mass of 8765 m / z as an index. Hereinafter, “apolipoprotein CIII ₀ ” and “protein showing a peptide peak with a mass of 8765 m / z” may be collectively referred to as “marker protein”.

  The sequence of the apolipoprotein CIII gene and the amino acid sequence encoded thereby are well known in the art, and examples thereof include the sequences of human-derived apolipoprotein CIII shown in SEQ ID NO: 1 and SEQ ID NO: 2. The sequences of the human-derived apolipoprotein CIII precursor in which a signal peptide of 20 residues is added to these sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 (NCBI access number: X01388). For mammals other than humans, it is possible to use similar genes and proteins corresponding to human-derived apolipoprotein CIII.

In addition to CIII ₀ , apolipoprotein CIII includes CIII ₁ (FIG. 1D) and CIII ₂ (FIG. 1E), which are sugar chain modifiers, and CIII ₀ ′ in which alanine is deleted from the C-terminus of CIII ₀ (FIG. 1C). ) Etc. are known. As shown in FIG. 1D, D-galactosyl (1-3) -N-acetyl D-galactosamine is added to the apolipoprotein CIII ₁ , and sialic acid is added to the C3 position of the D-galactosyl residue. ing. In addition, as shown in FIG. 1E, D-galactosyl (1-3) -N-acetyl D-galactosamine is added to the apolipoprotein CIII ₂ , and the C3 position of the D-galactosyl residue and N-acetyl D -Sialic acid is added at the C6 position of the galactosamine residue. The exact mass of each protein is as shown in FIG. FIG. 2 is a mass spectrum measured using a plasma sample. According to this figure, apolipoprotein CIII ₁ shows a peptide peak with a mass of 9421 m / z, and CIII ₂ shows a peptide peak with a mass of 9712 m / z. Show.

The diagnostic method according to the present invention, rather than the total amount of apolipoprotein CIII, blood levels of only apolipoprotein CIII ₀ is used as an index of diagnosis. Therefore, in the diagnostic method according to the present invention, the blood concentration of apolipoprotein CIII ₀ alone is specifically measured, and CIII ₁ , CIII ₂ and CIII ₀ ′ are excluded from the concentration measurement targets. In other words, in the diagnostic method according to the present invention, the blood concentration of only a protein showing a peptide peak with a mass of 8765 m / z is specifically measured, and peptide peaks with masses of 9421 m / z, 9712 m / z and 8893 m / z, respectively. Are excluded from the target of concentration measurement.

  In the present invention, the “test blood sample” means a blood sample obtained from a mammal to be diagnosed. Also, “control blood sample” means a blood sample obtained from the same type of mammal not suffering from gastric cancer, preferably from a mammal having no history of gastric cancer. The blood sample used for diagnosis may be blood collected from a mammal, but is preferably a plasma sample obtained by removing solids from the blood.

  The mammal to be diagnosed in the present invention may be any mammal such as a human, monkey, dog, cat, rat, mouse, etc., but is preferably a human.

Standard methods known in the art can be used to measure the concentration of the marker protein. For example, there may be mentioned those represented by SEQ ID NO: 2 as the amino acid sequence of apolipoprotein CIII _0, The chemical structures containing this sequence is shown in Figure 1B. Therefore, those skilled in the art can appropriately determine the amount of apolipoprotein CIII ₀ by referring to this sequence and chemical structure.

  The protein concentration measured in the present invention does not have to be expressed as an absolute numerical value such as the weight or the number of moles of protein contained in a predetermined amount of sample, but between the test blood sample and the control blood sample. It may be expressed by a relative numerical value that enables comparison with.

  The concentration of the marker protein can be suitably measured by, for example, an assay using an antibody that binds to the protein. Such an antibody is preferably an antibody specific for the marker protein. The antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. The specific antibody may be a binding fragment of a monoclonal antibody, ScFv (single chain Fv fragment), dAb (single domain antibody) or minimal recognition unit. In the concentration measurement using an antibody, a signal generating means reflecting the concentration of the complex between the marker protein and the antibody, for example, a fluorescent dye bound to the primary antibody or the secondary antibody is used to control the test blood sample and the control. A comparable measurement with a blood sample can be obtained.

Antibodies used in the assays as described above can be made by standard methods known in the art. For example, as a method for producing a monoclonal antibody, a method described in “Monoclonal Antibodies: A manual of techniques”, H. Zola (CRC Press, 1988) and “Monoclonal Hybridoma Antibodies: Techniques and Applications”, JGRHurrell (CRC Press, 1982). Is mentioned. Furthermore, appropriately prepared non-human antibodies can also be “humanized” by known techniques, for example, by inserting the CDR regions of mouse antibodies into the framework of human antibodies. The specificity of an antibody that binds to apolipoprotein CIII ₀ is that it does not bind to other proteins present in the blood, in particular that it does not bind to any of apolipoproteins CIII ₁ , CIII ₂ and CIII ₀ '. It can be evaluated by checking. Also, the specificity of an antibody that binds to a protein showing a peptide peak with a mass of 8765 m / z is that the antibody does not bind to other proteins present in the blood, in particular, 9421 m / z, 9712 m / z and 8893 m / z. It can be evaluated by confirming that it does not bind to any protein showing a peptide peak with a mass of z.

  According to another embodiment, the concentration of the marker protein can be measured using its mass as an index. For example, the marker protein concentration is measured as the ionic strength of a peptide peak having a mass of 8765 m / z.

  The concentration of the marker protein in a normal control blood sample serving as a comparative control can be measured in advance before measuring the concentration of the test blood sample. Further, the concentration of the marker protein may be measured for normal control blood samples from a plurality of different individuals, and the average value may be used as a comparison control. Furthermore, an appropriate value such as an average value-standard deviation is set from data of marker protein concentrations in a plurality of normal control blood samples measured in advance, and the marker protein concentration in the test blood sample is lower than the appropriate value In some cases, it may be determined that there is a decrease in the marker protein concentration. In addition, the lower limit (threshold value) of the appropriate value can be appropriately determined by creating an ROC curve for the marker protein.

  In the diagnostic method according to the present invention, when the marker protein concentration in the test blood sample is decreased with respect to the control blood sample, it can be determined that gastric cancer is present in the mammal.

  In order to carry out the diagnostic method according to the present invention as described above, necessary reagents can be put together into a kit. Therefore, according to the present invention, a kit for diagnosing gastric cancer in a mammal is provided, which kit comprises a reagent for measuring the concentration of a marker protein in a blood sample. Such reagents are selected according to the specific method to be performed.

  Examples of the diagnostic reagent contained in the kit according to the present invention include those that specifically bind to a marker protein. In particular, a specific antibody used in ELISA or the like is preferable. Such a specific antibody may be a monoclonal antibody or a binding fragment thereof, ScFv (single chain Fv fragment), dAb (single domain antibody) or the minimum recognition unit of an antibody.

  The kit according to the present invention may further contain other reagents, reaction containers, instructions, etc., depending on the specific method to be carried out.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention.

Example 1: Relationship between plasma concentration of protein having a mass of 8765 m / z and gastric cancer Plasma samples were obtained from 58 gastric cancer patients and 41 healthy individuals (Table 1). The clinical background of these plasma samples is shown in Table 1.

The protein in the plasma sample was then purified using a c8 reverse phase magnetic bead column and the ionic strength of the peptide peak having a mass of 8765 m / z was examined using mass spectrometry. This 8765 m / z peptide peak is characteristic of apolipoprotein CIII ₀ at least in human plasma.

  FIG. 3 is a mass spectrum showing ionic strength (arrow) of 8765 m / z in 2 healthy subjects and 2 gastric cancer patients. According to FIG. 3, it can be seen that the peak of 8765 m / z observed in healthy subjects is greatly reduced in gastric cancer patients.

FIG. 4 is a graph showing ionic strength of 8765 m / z and ionic strength of 9422 m / z and 9712 m / z corresponding to apolipoproteins CIII ₁ and CIII ₂ in 41 healthy subjects and 58 gastric cancer patients. In FIG. 4, the solid line in the graph indicates the average value, and the value is shown below the graph. The numerical value on the upper side of the graph is a P value by Mann-whitney U test. According to FIG. 4, it can be seen that the ionic strength of apolipoprotein CIII ₀ in the plasma sample from the stomach cancer patient is significantly lower than that in the plasma sample from the healthy subject (U test p <1.11 × 10 6). ^-15 ). Moreover, the significance is remarkably remarkable compared with other apolipoprotein CIII.

FIG. 5 is a diagram showing the ROC curves (receiver operating characteristic curves) and the area under the curve (AUC) of apolipoproteins CIII ₀ , CIII ₁ , and CIII ₂ . The ROC curve is a graph in which the horizontal axis is a false positive rate of 0 to 1, the vertical axis is a sensitivity of 0 to 1, and various cutoff values of a certain diagnostic marker are plotted. In the ROC curve, the closer the area under the curve is to 1, the higher the diagnostic accuracy of the diagnostic marker. According to FIG. 5, the area under the curve (AUC) of apolipoprotein CIII ₀ is a surprising value of 0.974, which is significantly higher than other apolipoprotein CIII.

  From the results shown in FIG. 4 and FIG. 5, the average value ± standard deviation and P value of ionic strength and the area under the curve (AUC) of the ROC curve for each apolipoprotein CIII in healthy subjects and gastric cancer patients are shown in Table 2. Summarized.

According to Table 2, in order to accurately diagnose gastric cancer patients, not the total amount of apolipoprotein CIII but the amount of apolipoprotein CIII ₀ that has not undergone sugar chain modification and has no amino acid deletion is used as an index. Is considered important. That is, by examining the decrease in the plasma concentration of apolipoprotein CIII ₀ , it becomes possible to accurately diagnose gastric cancer.

FIG. 1 is a diagram showing the chemical structures of four types of apolipoprotein CIII. FIG. 2 is a mass spectrum showing three plasma apolipoprotein CIII peak spectra (CIII ₀ , CIII ₁ and CIII ₂ ) predicted from plasma mass. FIG. 3 is a mass spectrum showing ionic strength (arrow) of 8765 m / z in 2 healthy subjects and 2 gastric cancer patients. FIG. 4 is a graph showing ionic strength of 8765 m / z and ionic strength of 9422 m / z and 9712 m / z corresponding to apolipoproteins CIII ₁ and CIII ₂ in 41 healthy subjects and 58 gastric cancer patients. FIG. 5 is a diagram showing the ROC curves of the apolipoproteins CIII ₀ , CIII ₁ and CIII ₂ and the area under the curve (AUC).

Claims (20)

A method for detecting gastric cancer in a mammal, comprising:
(A) measuring the concentration of apolipoprotein CIII ₀ in the test blood sample obtained from the mammal; and (b) determining the concentration obtained in step (a) from the apolipoprotein CIII in a normal control blood sample. Comparing to a concentration of ₀ , said gastric cancer being present in said mammal when the concentration of apolipoprotein CIII ₀ in said test blood sample is reduced relative to said control blood sample ,Method. The method according to claim 1, wherein the apolipoprotein CIII ₀ comprises the amino acid sequence represented by SEQ ID NO: 2.   The method of claim 1, wherein the blood sample is a plasma sample.   The method of claim 1, wherein the mammal is a human. The method of claim 1, wherein the concentration of apolipoprotein CIII ₀ is measured by an assay using a specific antibody. The method of claim 1, wherein the concentration of apolipoprotein CIII ₀ is measured as the ionic strength of a peptide peak having a mass of 8765 m / z. The method of claim 1, wherein the concentration of apolipoprotein CIII ₀ in a normal control blood sample has been determined in advance. A kit for diagnosis of gastric cancer in a mammal, comprising a reagent for measuring the concentration of apolipoprotein CIII _{0 in} a blood sample.   The kit according to claim 8, wherein the blood sample is a plasma sample.   The kit according to claim 8, wherein the mammal is a human. Reagents for measuring the concentration of apolipoprotein CIII ₀ is an antibody that specifically binds to apolipoprotein CIII _0, kit of claim 8. A method for detecting gastric cancer in a mammal, comprising:
(I) a step of measuring the concentration of a protein showing a peptide peak having a mass of 8765 m / z in the test blood sample obtained from the mammal, and (ii) the concentration obtained in step (i) is normal. Comparing the protein concentration in the control blood sample with gastric cancer in the mammal if the protein concentration in the test blood sample is reduced relative to the control blood sample. Will be shown, the method.   The method of claim 12, wherein the blood sample is a plasma sample.   The method of claim 12, wherein the mammal is a human.   13. The method of claim 12, wherein the protein concentration is measured as the ionic strength of a peptide peak having a mass of 8765 m / z.   13. The method of claim 12, wherein the protein concentration in a normal control blood sample is pre-measured.   A kit for the diagnosis of gastric cancer in a mammal comprising a reagent for measuring the concentration of a protein exhibiting a peptide peak with a mass of 8765 m / z in a blood sample.   The kit according to claim 17, wherein the blood sample is a plasma sample.   The kit according to claim 17, wherein the mammal is a human.   The kit according to claim 17, wherein the reagent for measuring the concentration of the protein is an antibody that specifically binds to the protein.

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