WO2010087434A1 - Method for detecting pancreatic cancer - Google Patents

Abstract

Disclosed is a novel diagnosis method for pancreatic cancer.  Specifically disclosed is a method for determining the occurrence of pancreatic cancer in a mammal.  The method comprises the following steps (a) to (b): (a) measuring the concentration of a protein having a mass of 16922 (D) in a test blood sample collected from the mammal; and (b) comparing the concentration obtained in step (a) with the concentration of the protein having a mass of 16922 (D) in a normal control blood sample.  In the method, it is determined that pancreatic cancer occurs in the mammal when the concentration of the protein having a mass of 16922 (D) in the test blood sample is increased compared with that in the control blood sample.

Description

Detection of pancreatic cancer Reference to related applications

This patent application is accompanied by a claim of priority based on Japanese Patent Application No. 2009-19920 filed on January 30, 2009, and the entire disclosure in such earlier patent application is incorporated by reference. It is made a part of this specification.

The present invention relates to a method for detecting pancreatic cancer, a blood protein serving as an index of the detection method, and a kit for measuring the protein.

Pancreatic cancer is one of the intractable cancers, and effective treatment other than surgical operation has not been established, so early treatment is important. It is known that CA19-9, which is an existing serum tumor marker, is used as a method for diagnosing pancreatic cancer (Tumor Marker Clinical Manual, Hisanao Okura, Medical School, p88-89, p124-127). Recently, helical CT, magnetic resonance apparatus (MRI), endoscopic ultrasonography (EUS), and the like are used in the diagnosis of pancreatic cancer. However, pancreatic cancer has few symptoms at an early stage, is an advanced cancer at the time of discovery, and is still often difficult to treat. Therefore, it can be said that development of a diagnostic technique capable of accurately and easily finding pancreatic cancer at an early stage is desired.

On the other hand, apolipoproteins are a group of proteins that exist specifically on plasma lipoproteins. Ten or more types of apolipoproteins 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.

Apolipoprotein AII, a member of the apolipoprotein family, consists of 76 amino acids, and has an alanine residue at its N-terminus, a threonine residue at its 75th, and a glutamine at its 76th C-terminus. It is known as one of the main apolipoproteins of high density lipoprotein (HDL).

In addition, it has been reported that there are dimers of apolipoprotein AII and three types of post-translational modifications with different masses in plasma (Pankhurst, G., et al. Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein. J Lipid Res 44, 349-355 (2003). According to the analysis based on the three-dimensional structure data (PDB ID: 1L6L) of the Apo AII protein registered in the protein structure data bank (PDB, Protein Data Bank; http://www.pdbj.org/index_j.html) The dimer of apolipoprotein AII is said to be formed by binding the N terminus of apolipoprotein AII (FIG. 1A). As post-translational modifications, Apo 片 AII dimer lacking glutamine on one side C-terminal (Fig. 1B: Apo AII ATQ / AT), apolipoprotein AII dimer lacking glutamine on both sides C-terminal ( FIG. 1C: Apo AII AT / AT), there is an apolipoprotein AII dimer (Fig. ID: Apo AII AT / A) lacking glutamine on one side C-terminal and glutamine threonine on the other side C-terminal.

In WO2006 / 098087, the present inventors have found that blood protein having a mass of 17253 ± 9 (m / z) decreases with a statistically significant difference compared to healthy subjects. And reported that this blood protein can be used as a marker for pancreatic cancer. Furthermore, the present inventors have clarified that this blood protein corresponds to Apo AII ATQ / AT shown in FIG. 1B above among the post-translational modifications of the apolipoprotein AII dimer. . However, in WO2006 / 098087, the blood protein corresponding to the post-translational modification of the apolipoprotein AII dimer, except for the description of the blood protein having a mass of 17253 ± 9 (m / z), There is no mention of any association with pancreatic cancer.

Amino acid sequence and theory of apolipoprotein AII dimer (Apo AII ATQ / ATQ) and its post-translational modifications (Apo AII ATQ / AT, Apo AII AT / AT, Apo AII AT / A and Apo AII A / A) It is a figure which shows a mass value. FIG. 1A is an apolipoprotein AII dimer (Apo AII ATQ / ATQ), FIG. 1B is an Apo AII dimer (Apo AII ATQ / AT) with a glutamine deletion at one side C-terminal, and FIG. Apolipoprotein AII dimer lacking bilateral C-terminal glutamine (Apo 両 側 AII AT / AT), Fig. 1D lacks glutamine on one side C-terminal and glutamine threonine on the other side C-terminal FIG. 1E shows an apolipoprotein AII dimer (Apo AII A / A) lacking bilateral C-terminal glutamine / threonine. Apolipoprotein AII dimer (Apo AII ATQ / ATQ) and its post-translational modifications (Apo AII ATQ / AT, Apo AII AT / AT, Apo / AII AT / AT) in 2 healthy subjects and 2 patients with pancreatic cancer It is the graph which expressed those expression levels when A and Apo (AII (A) A / A) () were detected with the SELDI-QqTOF-M method by the gel image. Apolipoprotein AII dimer (Apo AII ATQ / ATQ) and post-translational modifications (Apo AII ATQ / AT, Apo AII AT / AT, Apo AII AT) in plasma of 71 healthy subjects and 80 patients with pancreatic cancer It is a figure which shows those relative amounts when detecting / A and Apo (AII (A) A / A) by the SELDI-QqTOF-MS method. It is a graph which shows the correlation state of the expression of Apo | AII | ATQ / AT and apo | AII | A / A.

The present inventors have recently found that the ionic strength of a peptide peak having a mass of 16922 (D: Dalton) is significantly increased in the blood of pancreatic cancer patients as compared to healthy individuals.
Furthermore, the present inventors have found a novel protein corresponding to the peptide peak. The present invention is based on these findings.

Therefore, an object of the present invention is to provide a method for detecting pancreatic cancer and a novel protein that can be used in this detection method.

The detection method according to the present invention is a method for detecting the presence or absence of pancreatic cancer in a mammal,
(A) measuring the concentration of a protein having a mass of 16922 (D) in the test blood sample obtained from the mammal, and (b) converting the concentration obtained in step (a) to normal control blood. Comparing to the concentration of protein having a mass of 16922 (D) in the sample;
And when the concentration of the protein having a mass of 16922 (D) in the test blood sample is increased relative to the control blood sample, the presence of pancreatic cancer in the mammal is indicated .

The protein according to the present invention is a protein having a mass of 16922 (D) and is a dimer of subunits consisting of the amino acid sequence represented by SEQ ID NO: 1.

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

The “protein having a mass of 16922 (D)” (hereinafter also referred to as “marker protein”), which is an index for diagnosis (detection) of the present invention, has a higher expression level in pancreatic cancer patients than in healthy individuals. One feature. It is a surprising fact that a protein having such a molecular weight serves as an index for detection of pancreatic cancer. Therefore, according to one aspect of the present invention, a pancreatic cancer marker comprising the above protein is provided. Moreover, according to another aspect, use of the said protein as a pancreatic cancer marker is provided.

The marker protein is a novel protein represented as a dimer of subunits consisting of the amino acid sequence represented by SEQ ID NO: 1. The marker protein corresponds to a post-translational modification of the apolipoprotein AII dimer. As shown in Apo AII A / A in Fig. IE, both glutamine and threonine at the C-terminal of AII in both subunits are used. It has been deleted.

Thus, although the marker protein corresponds to the post-translational modification of the apolipoprotein AII dimer, pancreatic cancer is inversely correlated with the known post-translational modification (FIG. 1B: Apo AII ATQ / AT). It is expressed in patients. Under this technical situation, the protein according to the present invention can be particularly advantageously used for detecting pancreatic cancer accurately and early when it is used together with Apo 癌 AII と と も に ATQ / AT for detection of pancreatic cancer.

The pancreatic cancer to be detected in the present invention includes all cancers formed in the pancreas. That is, as a pancreatic cancer to be diagnosed, it may have originated from a site such as an acinus, pancreatic duct, islets of Langerhans, and invasive pancreatic duct cancer, tubular adenocarcinoma in the pancreatic duct, acinar cell cancer, It may be a type such as endocrine cancer.

In the present invention, “test blood sample” means a blood sample obtained from a mammal to be diagnosed. In addition, “control blood sample” means a blood sample obtained from the same type of mammal not suffering from pancreatic cancer, preferably from a mammal having no history of pancreatic 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.

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

Also, standard methods known in the art can be used for measuring the concentration of the marker protein. For example, the chemical structure of the marker protein is as shown in FIG. 1B. Therefore, those skilled in the art can appropriately measure the amount of the marker protein by referring to this chemical structure.

The concentration of the marker protein 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. It may be expressed by a relative numerical value that enables comparison between the two.

The concentration of the marker protein can be suitably 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 16922 (m / z). According to a preferred embodiment of the present invention, the measurement of the marker protein is performed by time-of-flight mass spectrometry.

Also, the concentration of the marker protein can be measured by an assay using an antibody that binds to the protein. Such an antibody is preferably an antibody specific for the marker protein. The antibody against the marker protein 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.

The 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, the 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 the antibody that binds to the marker protein is that the antibody does not bind to other proteins present in the blood, in particular apolipoprotein AII dimer (Apo AII ATQ / ATQ) and its known modifications (Apo It can be evaluated by confirming that it does not bind to any of AII ATQ / AT, Apo AII AT / AT and Apo AII AT / A).
That is, the specificity of an antibody that binds to a protein showing a peptide peak with a mass of 16922 (D) is that the antibody does not bind to other proteins present in the blood, in particular, 17380 m / z, 17252 m / z, 17124 m. It can be evaluated by confirming that it does not bind to any of the proteins showing peptide peaks of / z and 16922 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 marker protein concentration data in a plurality of normal control blood samples measured in advance, and the marker protein concentration in the test blood sample is higher than the appropriate value. In some cases, it may be determined that there is an increase 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 detection method according to the present invention, when the concentration of the marker protein in the test blood sample is increased relative to the control blood sample, it can be determined that pancreatic cancer is present in the mammal.

In order to carry out the detection 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 diagnosis of pancreatic cancer in a mammal is provided, and the 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 detection reagent contained in the kit according to the present invention include those specifically binding to a marker protein, and it is particularly preferable to use a specific antibody used for ELISA or the like. 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.

Hereinafter, the present invention will be specifically described by way of examples, but these examples do not limit the present invention.

Example 1: Association of plasma concentration of protein having a mass of 16922 m / z with pancreatic cancer Plasma samples were obtained from 80 pancreatic cancer patients and 71 healthy individuals.
Next, according to the same method as Experiment 1 of WO2006 / 098087 (Patent Document 1), SELDI-QqTOF-MS (surface-enhanced laser desorption / ionization high-resolution performance hybrid quadrupole time of flight mass spectrometry) method is used. The ionic strength of the peptide peak having a mass of 16922 m / z was investigated. In addition, the ionic strength of peptide peaks having masses of 17380, 17252, 17124 and 17023 was also measured.
These peptide peaks at 17380, 17252, 17124, 17023 and 16922 m / z are characteristic of apolipoprotein AII or its post-translational modification, at least in human plasma.

FIG. 2 is a graph showing the expression of 17380, 17252, 17124, 17023 and 16922 m / z in arbitrarily selected healthy subjects and pancreatic cancer patients as gel images. According to FIG. 2, Apo AII ATQ / ATQ, Apo AII ATQ / AT, and Apo AII AT / AT can be confirmed in the plasma of healthy subjects, but Apo AII A / A is not confirmed. On the other hand, the peak of Apo 癌 AII ATQ / AT is significantly reduced in patients with pancreatic cancer, indicating that Apo AII A / A is highly expressed.

FIG. 3 shows ionic strength of 16922 m / z corresponding to Apo AII A / A and Apo AII ATQ / ATQ, Apo AII ATQ / AT, Apo AII AT / AT Apo in 80 healthy subjects and 71 pancreatic cancer patients. It is a graph which shows the ion intensity of 17380, 17252, 17124, and 17023 m / z corresponding to AII AT / A. In FIG. 3, the solid line in the graph indicates the median value. Moreover, the numerical value on the upper side of the graph is a P value by Student ’s t test. According to FIG. 3, it can be seen that the ionic strength of Apo / AII 血漿 A / A in the plasma sample from the pancreatic cancer patient is significantly higher than that of the plasma sample from the healthy subject (t-test p = 0.0277).

FIG. 4 is a diagram showing the correlation between the ionic strengths of Apo AII ATQ / AT and Apo AII A / A. The ionic strength of Apo AII ATQ / AT is plotted on the vertical axis, and the ionic strength of Apo AII A / A is plotted on the horizontal axis for each case. Here, the healthy person is ◆, and the pancreatic cancer patient is x.
As a result, the ionic strength of Apo AII A / A was found to be inversely correlated with Apo AII ATQ / AT, and the correlation coefficient was -0.2152.

Claims (15)

1. A method for detecting the presence or absence of pancreatic cancer in a mammal, comprising:
   (A) measuring the concentration of a protein having a mass of 16922 (D) in the test blood sample obtained from the mammal, and (b) converting the concentration obtained in step (a) to normal control blood. Comparing to the concentration of protein having a mass of 16922 (D) in the sample;
   And when the concentration of the protein having a mass of 16922 (D) in the test blood sample is increased relative to the control blood sample, the presence of pancreatic cancer in the mammal is indicated ,Method.
2. The method according to claim 1, wherein the protein is a dimer of subunits consisting of the amino acid sequence represented by SEQ ID NO: 1.
3. The method according to claim 1, wherein the concentration of the protein is measured as the ionic strength of the protein.
4. The method according to claim 3, wherein the measurement is performed by time-of-flight mass spectrometry.
5. The method according to claim 2, wherein the concentration of the protein is measured by an assay using a specific antibody.
6. The method according to claim 1, wherein the concentration in a normal control blood sample is measured in advance.
7. The method according to claim 1, wherein the blood sample is a plasma sample.
8. The method according to claim 1, wherein the mammal is a human.
9. A protein having a mass of 16922 (D), which is a dimer of subunits consisting of the amino acid sequence represented by SEQ ID NO: 1.
10. A pancreatic cancer marker comprising the protein according to claim 9.
11. An antibody against the protein according to claim 9.
12. A kit for diagnosing pancreatic cancer in a mammal, comprising a reagent for measuring the concentration of the protein according to claim 9 in a blood sample.
13. The kit according to claim 12, wherein the blood sample is a plasma sample.
14. The kit according to claim 12, wherein the mammal is a human.
15. Use of a protein having a mass of 16922 (D) as a pancreatic cancer marker and a dimer of subunits consisting of the amino acid sequence represented by SEQ ID NO: 1.

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