Novel cancer biomarkers in pancreatic cancer or malignant intraductal papillary mucinous neoplasms

Abstract

On the basis of the findings that a fucosylated α1-acidic glycoprotein (fAGP) is useful for the diagnosis of pancreatic cancer and malignant intraductal papillary mucinous carcinoma (IPMC), found is a novel biomarker for pancreatic cancer or malignant IPMC, particularly, a novel biomarker which is useful for rapidly diagnosing IPMC and improves a proper diagnosis rate of malignant IPMC and assists a conventional image diagnosis, by performing a preoperative diagnosis on whether an intraductal papillary mucinous neoplasm (IPMN) is benign or malignant.

Description

[Technical field]

[0001] The present invention relates to novel cancer biomarkers in pancreatic cancer or malignant intraductal papillary mucinous neoplasms. [Background technology]

[0002] Pancreatic cancer is the third most common cause of death in Japan after lung cancer, stomach cancer, and colorectal cancer, and because it is difficult to detect early and has a poor prognosis, while the number of deaths from many cancers has been decreasing in recent years, the mortality rate from pancreatic cancer has been increasing. Therefore, along with new treatments, there is a strong need to develop new biomarkers that enable early diagnosis. On the other hand, intraductal papillary mucinous neoplasm (IPMN) is the most common cystic tumor in the pancreas and is a disease that may become cancerous. It is known that IPMN progresses from adenoma (benign IPMN, IPMA) to cancer (malignant IPMN, IPMC). Although the diagnosis of IPMN has become relatively easy with the development of testing methods, problems remain regarding the resection of lesions, which is the basis of treatment. That is, for IPMN patients, the surgical indication is usually determined based on the IPMN International Consensus Guidelines by examining the presence or absence of carcinogenesis risk from each image finding. However, in addition to IPMC that has become cancerous, this may include IPMA that is not originally suitable for surgery, and the accuracy rate of the diagnosis has been reported to be 64.3% (Non-Patent Document 1). Furthermore, the preoperative diagnosis of pancreatic cancer using serum cancer markers, which has been used conventionally, has been difficult to apply for diagnosis due to its low positive rate. Therefore, considering the risks associated with pancreatic resection, there is an urgent need to establish new biomarkers that are useful for early diagnosis of cancer in IPMN and for improving the accuracy of diagnosis of IPMC.

[0003] In addition, α 1-Acid glycoprotein (hereinafter, also referred to as AGP) is an inflammatory marker whose blood concentration increases during inflammation, and the present inventors have so far elucidated cancer-related changes in the sugar chains of AGP molecules and demonstrated that quantitative changes in fucosylated AGP (hereinafter, also referred to as fAGP) are useful not only for the malignancy of cancer, but also for predicting cancer prognosis and assessing the effectiveness of treatment (Patent Document 1, Non-Patent Document 2). Furthermore, the present inventors have demonstrated the possibility of application to early detection of recurrence and metastasis and assessment of treatment effectiveness in various chemotherapy cases (Non-Patent Document 3), and further the possibility of application to assessment of the effectiveness of immunotherapy, which has recently achieved revolutionary therapeutic effects (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-11978 A [Non-patent literature]

[0005] [Non-Patent Document 1] Dig. Dis. Sci., 63:860-867, 2018 [Non-Patent Document 2] Cancer, 101:2825-2836, 2004 [Non-Patent Document 3] PLoS ONE, 11(6)e0156277, 2016 [Non-Patent Document 4] Sci. Reports, doi.org:10.1038, 2019 [Non-Patent Document 5] BioMed. Res. Inter., Vol 2013, Article ID 834790, 2013 [Non-Patent Document 6] Clinica Chimica Acta, 478:120-128, 2018 [Non-Patent Document 7] Jpn. J. Cancer Res. (Gann), 79:538-543, 1988 [Non-Patent Document 8] Cancer Res., 55:1473-1478, 1995 Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of the present invention is to provide a new biomarker for pancreatic cancer or IPMC, in particular, to provide a new biomarker that is useful for the rapid diagnosis of IPMC, thereby increasing the accuracy of IPMC diagnosis and supplementing conventional imaging diagnosis, by performing appropriate preoperative diagnosis of whether IPMN is a benign or malignant tumor. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above problems, the present inventors discovered that fAGP is a useful biomarker for diagnosing pancreatic cancer or IPMC, and thus arrived at the present invention.

[0008] That is, the present invention is as follows. [1] Fucosylated α in the specimen 1 - A method for obtaining data for diagnosing pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC), comprising measuring the amount of fAGP. [2] The method according to [1], wherein the sample is plasma or serum. [3] The measurement is α 1 -Measuring the amount of fucosyl glycans in acidic glycoprotein (AGP), Desialylation of AGP in the sample; and The method according to [1] or [2], comprising a step of measuring the amount of fucosyl glycan by enzyme immunoassay (EIA) using an anti-AGP antibody immobilized on a substrate and having a defucosylated glycan, and a lectin that recognizes the fucosyl group of the desialylated AGP. [4] The method according to [3], wherein the desialylation is carried out by sialidase treatment and the defucosylation is carried out by periodate oxidation. [5] The method according to [3] or [4], wherein the lectin is Acanthopanax lectin (AAL). [6] The method according to any one of [3] to [5], further comprising the steps of: Quantifying AGP in the sample; and A step of normalizing the amount of the fucosyl sugar chain with the amount of the AGP. [7] The method according to any of [1] to [6], wherein the diagnosis is based on the criterion that the diagnosis is pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC) if the amount of fAGP in the sample is higher than the reference value or higher than the amount of fAGP in a sample taken from a healthy subject or a patient with benign intraductal papillary mucinous neoplasm (IPMA). [8] The method according to any one of [1] to [7], wherein the diagnosis is a diagnosis of prognosis of cancer treatment. [9] A diagnostic kit for pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC), comprising: (A) Desialylation reagent, (B) An EIA substrate on which an anti-AGP antibody is immobilized, and (C) A kit comprising a reagent for defucosylation of an anti-AGP antibody immobilized on an EIA substrate.

[10] The kit according to [9], further comprising (D) a labeled lectin and / or (E) an avidin-labeled enzyme.

[11] Furthermore, (F) a sandwich ELISA substrate having an anti-AGP antibody immobilized thereon for quantifying AGP in a sample; and (G) A solution containing an enzyme-labeled anti-AGP antibody; The kit according to [9] or

[10] , Effect of the Invention

[0009] According to the present invention, by measuring the amount of fAGP in a sample, it is possible to provide a novel biomarker useful for diagnosing pancreatic cancer or IPMC. By using this biomarker as an indicator, IPMC can be distinguished from IPMA, and it is also useful for predicting the prognosis of IPMN. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the results of measuring serum fAGP levels in pancreatic cancer patients and IPMN patients. Each horizontal line in the figure represents the average value for each group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One embodiment of the present invention is a method for obtaining data for diagnosing pancreatic cancer or IPMC, comprising measuring the amount of fAGP in a sample. The data acquisition method of this embodiment is intended to assist in the diagnosis of pancreatic cancer or IPMC. This method is useful for distinguishing between IPMC and IPMA, and is useful for predicting the prognosis of IPMN, particularly for diagnosing the occurrence of pancreatic cancer in IPMN. In the present invention, IPMC, which is a malignant tumor, may be included in pancreatic cancer, but IPMA, which is a benign tumor, is not included in pancreatic cancer.

[0012] The animal from which the specimen is derived is preferably a mammal such as a human or a rat, and more preferably a human. The specimen is preferably a body fluid derived from a human, such as blood, lymph, cerebrospinal fluid, urine, ascites, etc. Collection and storage can be performed according to conventional methods. In the case of blood, plasma or serum is preferred, and preparation and storage can be performed according to conventional methods. The present invention may include a step of collecting a specimen from the animal to be diagnosed.

[0013] The amount of fAGP can be measured by measuring the amount of fucosyl sugar chains in AGP, although there is no particular limitation. For example, the amount of fAGP can be measured based on the method described in Patent Document 1 as shown below. This method allows the amount of fAGP to be measured simply and easily from a very small amount of serum sample (e.g., one drop, 25 μl).

[0014] [Step of desialylating AGP in the sample] The measurement method may include a step of desialylating AGP in a sample. If the sugar chain of AGP in a sample remains highly sialylated, the capture of AGP by anti-AGP antibody and the detection of fucosyl groups by lectin in the subsequent EIA method are inhibited.

[0015] (Desialylation) The desialylation is not particularly limited as long as it can release α2,3 and α2,6 sialic acid residues, which are sialic acid bonds at the non-reducing end of the AGP sugar chain, and is preferably carried out using a desialylation enzyme, such as sialidase (also known as neuraminidase). There are no particular limitations on the sialidase, so long as it is an exo-type enzyme that has specificity for the α2,3 and α2,6 sialyl groups present at the non-reducing end of the AGP sugar chain. Sialidases derived from various organisms can also be used. Although one type of sialidase may be used alone or two or more types may be used in combination, it is preferable to use sialidase derived from Arthrobacter ureafaciens, which efficiently hydrolyzes both sialyl bonds.

[0016] The amount of sialidase used in the desialylation treatment with sialidase is not particularly limited as long as it can sufficiently release sialic acid residues from the non-reducing ends of the sugar chains, but is preferably 0.005 mU or more, more preferably 0.01 mU or more, and even more preferably 0.02 mU or more per 1 μg of AGP, while it is preferably 2 mU or less, more preferably 1.5 mU or less, and even more preferably 1 mU or less. Sialyl lactose is used as a substrate, and the amount of enzyme required to decompose 1 μmol of sialic acid per minute at pH 5.0 and 37° C. is defined as 1 U (unit).

[0017] The treatment time for desialylation with sialidase is not particularly limited as long as it can sufficiently release sialic acid residues from the non-reducing terminals of sugar chains, but is preferably 20 minutes or more, more preferably 30 minutes or more, and even more preferably 1 hour or more. On the other hand, the upper limit is not particularly limited, but may be, for example, 2 hours or less.

[0018] The treatment temperature for desialylation with sialidase is not particularly limited as long as it can sufficiently release sialic acid residues from the non-reducing ends of sugar chains, but is preferably room temperature (e.g., 25° C.) or higher, more preferably 30° C. or higher, and even more preferably 35° C. or higher. On the other hand, the upper limit is the temperature at which sialidase maintains its activity, and is preferably 38° C. or lower, and particularly preferably 37° C.

[0019] As a buffer for desialylation with sialidase, a buffer commonly used in biochemical techniques can be used, such as acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, phosphate buffered saline (PBS), etc. The buffer concentration can also be a common concentration used in biochemical techniques.

[0020] The pH of the buffer solution is not particularly limited as long as desialylation proceeds, but is preferably 6.0 or higher, more preferably 6.5 or higher, and even more preferably 6.8 or higher, while it is preferably 8.0 or lower, more preferably 7.5 or lower, and even more preferably 7.2 or lower.

[0021] After desialylation with sialidase, it is preferable to inactivate the sialidase. The inactivation can be performed according to a conventional method, for example, by treating the serum at 90°C for 5 minutes in a state where the serum is diluted with 490 μl of PBS per 10 μl of serum.

[0022] That is, the desialylation process may include the steps of mixing the specimen with sialidase, treating the mixed solution of the specimen and sialidase for an arbitrary period of time at a temperature at which the sialidase maintains its activity, and inactivating the sialidase.

[0023] [Step of measuring the amount of fucosyl glycan by EIA method] The measurement method includes, after the step of desialylating AGP in the sample, a step of measuring the amount of fucosyl glycan by enzyme immunoassay (EIA) using an anti-AGP antibody immobilized on a substrate and having a defucosylated glycan and a lectin that recognizes the fucosyl group of the desialylated AGP. By reacting the glycan on the substrate with the defucosylated anti-AGP antibody after the step of desialylating AGP, a larger amount of AGP including various fucosyl glycan in the sample can be measured compared to the case where the glycan on the substrate is reacted with the defucosylated anti-AGP antibody before the step of desialylating AGP.

[0024] This process may include, for example, the steps of defucosylating the glycan of an anti-AGP antibody, immobilizing the anti-AGP antibody with its glycan defucosylated on a substrate, adding a sample to the substrate to bind the anti-AGP antibody with its glycan defucosylated immobilized on the substrate to the AGP in the sample, binding a lectin that recognizes the fucosyl group possessed by the AGP bound to the anti-AGP antibody, and measuring the amount of fucosyl glycan by measuring the lectin using an EIA method.

[0025] In this step, the anti-AGP antibody immobilized on the substrate and used in the EIA method has its sugar chains defucosylated.

[0026] If the glycan of the anti-AGP antibody (Fc region) remains fucosylated, when EIA is performed, a lectin that recognizes the fucosyl groups of the desialylated AGP will bind not only to the fucosyl groups of the AGP but also to the fucosyl groups of the anti-AGP antibody immobilized on the substrate, making it impossible to accurately measure the amount of fucosyl groups of the AGP glycan.

[0027] Examples of the substrate include wells in a plate and beads. The anti-AGP antibody immobilized on a substrate and defucosylated with its glycan may be one obtained by immobilizing an anti-AGP antibody before defucosylation on a substrate and then defucosylated on the substrate, or one whose glycan has been defucosylated in advance at the stage of immobilization on the substrate. The method of immobilizing an anti-AGP antibody before defucosylation or an anti-AGP antibody whose glycan has been defucosylated in advance on a substrate can be a method used in conventional EIA methods.

[0028] (Defucosylation) When defucosylation is performed on an anti-AGP antibody that has not yet been defucosylated and is fixed to a substrate used in EIA, the method is not particularly limited, but is preferably performed by treatment with a defucosylation solution, for example, periodate oxidation using a sodium periodate solution.

[0029] When periodic acid is used, its concentration is not particularly limited as long as sufficient defucosylation is achieved, but is preferably 5 mM or more, more preferably 7 mM or more, and even more preferably 8 mM or more, while it is preferably 20 mM or less, more preferably 15 mM or less, and even more preferably 12 mM or less.

[0030] The treatment time for defucosylation using periodic acid is not particularly limited as long as sufficient defucosylation is achieved, but is preferably 20 minutes or more, more preferably 30 minutes or more, and even more preferably 50 minutes or more. On the other hand, the upper limit is preferably 2 hours or less in order to minimize damage to the antibody used by the defucosylation treatment.

[0031] The treatment temperature in defucosylation using periodic acid is not particularly limited as long as sufficient defucosylation is achieved, but is preferably 15° C. or higher, more preferably 20° C. or higher, and even more preferably 25° C. or higher, while it is preferably 40° C. or lower, more preferably 35° C. or lower, and even more preferably 30° C. or lower.

[0032] In addition, as a buffer solution when periodic acid is used, a buffer solution generally used when a biochemical technique is used can be used. For example, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, phosphate buffered saline (PBS), etc. can be mentioned. In addition, the buffer solution may contain a surfactant such as Tween 20 or Tween 80 as appropriate. The concentrations of the buffer solution and the surfactant can be the same as those generally used when a biochemical technique is used.

[0033] The pH of the buffer solution is not particularly limited as long as defucosylation proceeds, but is preferably 6.0 or higher, more preferably 6.5 or higher, and even more preferably 6.8 or higher, while it is preferably 8.0 or lower, more preferably 7.5 or lower, and even more preferably 7.2 or lower.

[0034] Defucosylation using periodic acid is preferably carried out in the dark. In addition, after defucosylation using periodic acid, the substrate can be washed with a buffer solution, incubated in PBS containing 1% BSA at room temperature for 2 hours or overnight at 4°C, and then washed with a washing solution (e.g., PBS containing 0.05% Tween 20) before EIA, in a manner similar to the preparation of substrates used in conventional EIA.

[0035] In addition, antibody activity is generally protected by a reduction treatment after defucosylation using periodic acid, but in the measurement method of the present invention, since there is no significant difference in the quantitative results of fucosyl glycans in AGP whether or not the reduction treatment is performed, it is preferable not to include a reduction treatment step after defucosylation.

[0036] The process of defucosylation of the glycan of an anti-AGP antibody may include the steps of contacting a defucosylation solution with the anti-AGP antibody on the substrate after immobilizing the anti-AGP antibody on the substrate, treating the substrate at any temperature for any time to defucosylate the glycan of the anti-AGP antibody immobilized on the substrate, and washing the substrate. The step of immobilizing the defucosylated anti-AGP antibody on a substrate can be carried out by a method known to those skilled in the art, and may include the steps of contacting the substrate with the anti-AGP antibody, treating the substrate at any temperature for any period of time to immobilize the anti-AGP antibody on the substrate, and washing the substrate. The process of adding a sample to a substrate to bind the AGP in the sample to the anti-AGP antibody that has defucosylated the glycans immobilized on the substrate is not particularly limited as long as the antigen-antibody reaction between AGP and anti-AGP is sufficiently carried out, and can be carried out for any time and at any temperature. The step of binding a lectin that recognizes the fucosyl group on the AGP bound to the anti-AGP antibody is not particularly limited as long as the lectin can recognize the fucosyl group on the AGP, and can be carried out at any time and temperature. Also, a step of labeling the lectin with biotin in advance may be included.

[0037] (Lectin that recognizes the fucosyl group of desialylated AGP) The lectin may be any lectin that binds to the Fucα1,3GlcNAc sugar chain present in the AGP sugar chain, and is preferably A. difficile lectin (AAL). These may be natural or artificially produced. They may also be recombinant proteins that are highly homologous to natural AAL, where high homology means, for example, 80% or more homology. The AAL may be labeled or modified as used in conventional EIA methods.

[0038] (detection) A conventional EIA method can be used to detect the binding of a lectin that recognizes the fucosyl group of desialylated AGP to the fucosyl group. The lectin itself may be labeled, or, for example, the lectin may be pre-labeled with biotin, and the fucosyl group of AGP may be bound to the biotin-labeled lectin, followed by binding to an avidin-labeled enzyme, which is then detected by the color or luminescence produced by the reaction of the enzyme.

[0039] In either case, the enzyme may be any of the enzymes commonly used in conventional EIA methods, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP).

[0040] The substrate may be any that corresponds to the enzyme used, for example, HRP substrates include 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), etc. ALP substrates include paranitrophenyl phosphate (PNPP), etc.

[0041] (EIA Act) The EIA method in this step can be carried out in the same manner as in conventional methods. The reaction temperature, reaction time, types of reagents, reagent concentrations, detection methods for fluorescence or color development, and the like can all be in accordance with conventional methods. As an example, a case will be described in which a lectin that recognizes and binds to the fucosyl group of desialylated AGP is used as a biotin-labeled AAL, and the binding is detected by adding the avidin-labeled enzyme HRP and detecting the color development by the substrate TMB.

[0042] First, as described above, an EIA substrate is prepared on which anti-AGP antibodies defucosylated by periodate oxidation are immobilized. To prevent nonspecific adsorption to the substrate, blocking may be performed with a blocking buffer solution used in the conventional method, such as PBS containing 10% sorbitol and 1% BlockAce or PBS containing 2% bovine serum albumin (BSA), preferably PBS containing 10% sorbitol and 1% BlockAce. Next, a sample containing desialylated AGP is used to bind the defucosylated anti-AGP antibody immobilized on a substrate to the desialylated AGP, and unbound components are then removed by washing with a washing solution (e.g., PBS containing 0.05% Tween 20). Next, the biotin-labeled AAL is used to bind to the fucosyl glycan in the desialylated AGP bound to the anti-AGP antibody, and the unbound biotin-labeled AAL is removed by washing with a washing solution. The concentration of the biotin-labeled AAL can be adjusted using PBS containing no BlockAce, and the concentrations of the other antibodies can be adjusted using PBS containing 0.4% BlockAce. Next, the biotin-labeled AAL is bound to the avidin-labeled enzyme HRP using the avidin-labeled enzyme HRP, and unbound avidin-labeled enzyme HRP is removed by washing with a washing solution. Next, TMB, a substrate for the enzyme HRP, is added, and color development is detected in the same manner as in the conventional antibody-lectin EIA method. It is preferable that the measurement method according to the present invention further comprises a step of converting the amount of color developed into the amount of fucosylated AGP. The method used in this step includes converting the amount of fucosylated AGP in the sample from the amount of color development or luminescence measured by the above-mentioned EIA using a calibration curve. It is preferable to prepare a calibration curve by, for example, desialylating highly fucosylated AGP obtained from serum or ascites fluid by the method described in Non-Patent Document 5, and then using a similar EIA method to obtain the amount of fucosylated AGP and the relationship between the amount of color development or luminescence (U / ml). The α in the sample thus obtained 1 - The method may include a step of correlating the amount of fucosyl glycans in acid glycoprotein (AGP) or the converted amount of fucosylated AGP as an indicator of pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC).

[0043] It is preferable that the measurement method further comprises the steps of quantifying AGP in the sample and normalizing the amount of fucosyl sugar chain with the amount of AGP.

[0044] [Step of quantifying AGP in a sample] The measurement method preferably includes a step of quantifying AGP in a sample. The method for quantifying AGP in a sample is not limited, but examples include a method in which AGP in a sample is desialylated, and then the resulting solution is diluted and used to perform a sandwich ELISA using an anti-AGP antibody immobilized on a substrate and an enzyme-labeled anti-AGP antibody, and a calibration curve is used to convert the amount of color or light produced by the enzyme reaction into the concentration of AGP in the sample. A calibration curve is preferably prepared, for example, by desialylating a commercially available human plasma AGP (specifically, Sigma-Aldrich) (concentration known) as a standard sample, and using this to perform a sandwich ELISA using an anti-AGP antibody immobilized on a substrate and an enzyme-labeled anti-AGP antibody, and then from the relationship between the amount of AGP (concentration) and the amount of color development or luminescence (μg / ml).

[0045] [Step of normalizing the amount of fucosyl glycan with the amount of AGP] It is preferable that the measurement method further includes a step of normalizing the amount of fucosyl glycan in the sample obtained above with the amount of AGP (U / μg) obtained in the above "step of quantifying AGP in the sample." In conventional methods, analysis of fucose attached to N-glycans was possible using the CAIE method or MALDI-TOF-MS, but the procedures were complicated, results could not be obtained quickly, and quantification was difficult. In contrast, this process makes it possible to simply and quickly calculate the amount of fucosyl glycan per unit amount of AGP in a sample.

[0046] [Diagnostic criteria for pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC)] In this embodiment, the diagnostic criteria for whether or not a patient has pancreatic cancer or IPMC are not particularly limited, but may be, for example, a diagnostic criterion for indicating that a patient has pancreatic cancer or IPMC when the amount of fAGP in a sample is greater than the standard value or greater than the amount of fAGP in a sample taken from a healthy subject or an IPMA patient, or a diagnostic criterion for indicating that a patient has pancreatic cancer or IPMC when the amount of fAGP in a sample is greater than the standard value or greater than the amount of fAGP in a sample taken from an IPMA patient. The reference value may be a cutoff value predetermined based on the measurement values ​​of healthy subjects and / or IPMA patients by a method known to those skilled in the art. For example, the cutoff value may be the mean of the measurement values ​​of healthy subjects + 3 × standard deviation (SD). In the present invention, a healthy subject means a subject who does not have cancer or a disease accompanied by acute inflammation or chronic inflammation.

[0047] In the present invention, the diagnosis may be a diagnosis of treatment prognosis of pancreatic cancer or IPMC. The treatment of pancreatic cancer may be one or more selected from the group consisting of surgery, chemotherapy, radiation therapy, antibody therapy, and immunotherapy. Preferably, the treatment is one or more selected from the group consisting of surgery, chemotherapy, and radiation therapy. The immunotherapy may be an immunotherapy using an antibody drug or a molecular targeted drug. The treatment of IPMC may be surgery. Without being particularly limited, for example, a sample taken from a patient after treatment may be measured according to the criteria described in the above-mentioned section on diagnostic criteria for pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC), and if the patient is determined to have pancreatic cancer or IPMC, it may be determined that the treatment is ineffective or the prognosis is poor.

[0048] Another embodiment of the present invention is a kit for diagnosing pancreatic cancer or IPMC. The kit includes the following elements (A) to (C), preferably (A) to (D), and more preferably (A) to (E). (A) Desialylation reagent, (B) EIA substrate with immobilized anti-AGP antibody (C) a reagent for defucosylation of anti-AGP antibodies immobilized on an EIA substrate; (D) a labeled lectin, such as a biotin-labeled lectin (e.g., AAL), and (E) Avidin-labeled enzyme (e.g., HRP).

[0049] It is preferable that the kit further comprises (F) a sandwich ELISA substrate on which an anti-AGP antibody is immobilized for quantifying AGP in a sample, and (G) a solution containing an enzyme (e.g., HRP)-labeled anti-AGP antibody. The kit preferably further comprises a dilution buffer, a blocking buffer, and a fAGP standard, which may be, for example, a partially purified fAGP standard from ascites or serum derived from a human cancer patient, or a culture medium of human-derived cultured cells, such as a hepatoma cell line.

[0050] The preferred types, concentrations, labels, and conditions of use of (A), (C) to (E), and (G) are described in the data acquisition method according to the present invention. Each solution may be appropriately concentrated before use, or may be appropriately diluted with water or the like just before use. Furthermore, when any of (A), (C) to (E), and (G) contains a plurality of solvents or solutions, they may be mixed and contained in one container, or may be contained in separate containers. Preferred embodiments of the above (B) and (F) include the embodiments described in the description of the measurement method according to the present invention.

[0051] The kit is for diagnosing pancreatic cancer or IPMN, and the diagnostic criteria for whether or not a patient has pancreatic cancer or IPMN can be the same as those described in the data acquisition method according to the present invention.

[0052] Furthermore, the kit may further include an instruction manual or the like that describes a method for measuring the amount of fAGP included in the data acquisition method according to the present invention described above. EXAMPLES

[0053] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0054] <Example 1> [Comparison of serum fAGP levels in IPMN patients, pancreatic cancer patients, and healthy subjects] Using a specific method for measuring fucosyl glycans on AGP molecules that the present inventors have already established (Patent Document 1 and Non-Patent Document 6), the amount of fAGP was measured in preoperative sera of IPMN patients and pancreatic cancer patients, and in sera of healthy subjects as negative controls. Only a small amount of serum sample (one drop, 25 μl) was used for each specimen. In addition, IPMN patients were classified as IPMC or IPMA by postoperative lesion tissue examination, a method known to those skilled in the art. The amount of fAGP in serum was measured by first measuring the amount of AGP in serum by sandwich ELISA using anti-AGP antibody for standardization, then measuring the amount of fAGP in serum by EIA using anti-AGP antibody / fucose-binding lectin (AAL), and calculating the amount of fucosyl glycan per unit amount of AGP in serum from these measurement results. IPMN patients were divided into IPMC and IPMA, and compared with pancreatic cancer patients and healthy subjects as negative controls (Figure 1). The serum fAGP levels of IPMC (n=14) and IPMA (n=7) were 118.15±113.01 and 11.63±8.47U / μg, respectively, and were significantly higher in IPMC than in IPMA (P<0.05). IPMA was also significantly higher than healthy subjects (indicated as "Healthy control" in the figure, n=53) (4.07±3.49U / μg) (P<0.05), and pancreatic cancer patients (indicated as "Ca. Pancreas" in the figure, n=40) (904.37±1708.32U / μg) than IPMC (P<0.05). In addition, a cutoff value (mean+3S.D.=14.54U / μg) was determined from the measured values ​​of fAGP in healthy subjects, and the positive rate in each sample group was confirmed. The results were: pancreatic cancer patients: 97.5% (39 / 40), IPMC patients: 92.86% (13 / 14), IPMA patients: 28.6% (2 / 7), and healthy subjects: 1.9% (1 / 53). In addition, the accuracy rate of IPMC was calculated based on this cutoff value and was 85.7%, which was significantly higher than the accuracy rate of conventional imaging diagnosis (58.8%). It was also shown that the positive rate in pancreatic cancer was extremely high.

[0055] <Example 2> [Comparison of preoperative blood concentrations of fAGP and other pancreatic cancer markers in IPMN patients] The amount of fAGP in the serum of each IPMN patient measured in Example 1 is shown in Table 1. In addition, the results of measuring four antigens (CEA, CA19-9, SPan-1, and DUPAN-2) that are conventionally used as markers for pancreatic cancer by a method known to those skilled in the art using serum samples from the same patients as those used in Example 1 are also shown in Table 1. Regarding CEA, CA19-9, SPan-1, and DUPAN-2, which have traditionally been used as cancer markers for pancreatic cancer, the positivity rates of each in the preoperative serum of 14 IPMC cases were CEA (1 / 14 = 7.1%), CA19-9 (5 / 14 = 35.7%), SPan-1 (1 / 14 = 7.1%), and DUPAN-2 (1 / 14 = 7.1%), and all markers other than CA19-9 were low (Table 1). The antigenic determinant of CA19-9 is sialyl Le a (NANAα2,3Galβ1,3[Fucα1,4]GlcNAc), which is the common antigen of human Lewis blood group antigens. a (Galβ1,3[Fucα1,4]GlcNAc) antigen and the same key enzyme involved in the synthesis of Fucα1,4 glycan are present in both. In Japan, approximately 20% of people are negative for Lewis blood group antigens and do not synthesize Fucα1,4 glycan. Therefore, sialyl Le a In these cases, CA19-9 cannot be used as a cancer marker because it is not genetically synthesized. Lewis blood group typing is difficult using normal serological methods, and type conversion often occurs in association with cancer, making accurate typing impossible, as has already been clarified by the inventors (Non-Patent Document 7). For accurate typing of Lewis blood group, FUT6 enzyme activity measurement or FUT6 gene typing is essential (Non-Patent Document 8). In addition to these limitations of CA19-9, the results in Table 1 clearly show that the sensitivity of CA19-9 measurement for diagnosing IPMC is low, and that fAGP measurement is superior for diagnosing IPMN and distinguishing IPMC. [Table 1]

[0056] From the above, serum fAGP levels measured using the specific measurement method for fucosyl glycans on AGP molecules that the inventors have already established are significantly elevated in pancreatic cancer, and IPMC can be identified with a higher positive rate in preoperative diagnosis of IPMN patients than with conventional cancer markers. This indicates the possibility of significantly improving the accuracy of diagnostic imaging for IPMN patients who are candidates for surgery (IPMC). In addition, since the incidence of pancreatic cancer is significantly elevated in IPMC patients, it is expected that this method can be applied to the diagnosis of pancreatic cancer in IPMN patients. Therefore, it is considered that this method could be a useful biomarker for malignant tumors in IPMN patients.

Claims

1. Fucosylated α in the specimen １ - Pancreatic cancer, including measuring the amount of fAGP (fAcid Glycoprotein) or a data acquisition method for diagnosing malignant intraductal papillary mucinous neoplasm (IPMC), comprising: The measurement is to measure the amount of fucosyl glycan in α 1 -acid glycoprotein (AGP). So, Desialylation of AGP in the sample; and The fucosylated AGP was detected by enzyme immunoassay (EIA) using an anti-AGP antibody immobilized on a substrate and having a defucosylated glycan, and a lectin that recognizes the fucosylated group of the desialylated AGP. Measuring the amount of sugar chains, The method is based on the criterion that the diagnosis is pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC) if the amount of fAGP in the sample is greater than the reference value or greater than the amount of fAGP in a sample taken from a healthy subject or a patient with benign intraductal papillary mucinous neoplasm (IPMA).

2. The method of claim 1 , wherein the sample is plasma or serum.

3. The method of claim 1 or 2, wherein the desialylation is carried out by sialidase treatment and the defucosylation is carried out by periodate oxidation.

4. The method according to any one of claims 1 to 3, wherein the lectin is Acanthologus lectin (AAL). The method according to claim 5.

5. The method according to any one of claims 1 to 4, further comprising the steps of: Quantifying AGP in the sample; and A step of normalizing the amount of the fucosyl sugar chain with the amount of the AGP.

6. The method according to any one of claims 1 to 5, wherein the diagnosis is a prognosis diagnosis of cancer treatment.

7. A diagnostic kit for pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC) for aiding in the diagnosis of pancreatic cancer or malignant intraductal papillary mucinous neoplasm (IPMC) by the method according to any one of claims 1 to 6, comprising: (A) a desialylation reagent; (B) an EIA substrate having an anti-AGP antibody immobilized thereon; and (C) A kit comprising a reagent for defucosylation of an anti-AGP antibody immobilized on an EIA substrate.

8. The method according to claim 7, further comprising: (D) labeling lectin and / or (E) avidin-labeling enzyme. Kit included.

9. moreover, (F) A sandwich ELISA substrate with immobilized anti-AGP antibody for quantifying AGP in a sample. Materials, and (G) a solution containing an enzyme-labeled anti-AGP antibody; 9. The kit according to claim 7 or 8, comprising:

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