Pancreatic cancer testing method

Abstract

To provide a means and a method for noninvasively and simply determining pancreatic cancer.SOLUTION: The present invention relates to a method and a device for determining pancreatic cancer in a subject by measuring a urinary tumor marker (glycochenodeoxycholic acid 3-sulfate and / or δ - valerobetaine) in a subject-derived urine sample.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for diagnosing pancreatic cancer in a subject based on the measurement of a urinary tumor marker. [Background technology]

[0002] Pancreatic cancer is difficult to treat because symptoms are rarely apparent and the cancer is already advanced by the time it is discovered. Therefore, early detection and treatment are desirable. Tumor markers for the early detection of various cancers are being sought. For example, propionylcarnitine has been reported as a blood tumor marker for pancreatic cancer (Patent Document 1). Furthermore, a research group including the present inventor has reported that glycochenodeoxycholic acid 3-sulfate can be used as a urinary tumor marker for the diagnosis of biliary tract cancer (Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2013 / 079594 issue [Patent Document 2] WO2023 / 233945 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a means and method for non-invasively and simply diagnosing pancreatic cancer, which has long been desired. [Means for solving the problem]

[0005] In the course of searching for tumor markers in cancer urine, the inventors identified glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine as markers associated with pancreatic cancer, and discovered that by using these markers alone or in combination, it is possible to easily and non-invasively diagnose pancreatic cancer, predict its risk, and monitor it.

[0006] That is, the present invention relates to a method and apparatus for diagnosing and / or monitoring pancreatic cancer in a subject by measuring urinary metabolites that are urinary tumor markers. Specific embodiments include the following:

[0007] [1] A method for determining pancreatic cancer in a subject, comprising: measuring a urinary tumor marker in a urine sample from a subject, wherein the urinary tumor marker comprises at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; determining whether the subject has pancreatic cancer based on the measurement results; A method comprising:

[0008] [2] A pancreatic cancer diagnosis device, a measurement unit for measuring a urinary tumor marker in a urine sample, the urinary tumor marker comprising at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; a comparison unit that compares the measured value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measured value; a determination unit for determining pancreatic cancer based on the comparison results obtained by the comparison unit; An apparatus comprising:

[0009] [3] A method for evaluating the effectiveness of a treatment for pancreatic cancer, comprising: measuring urinary tumor markers in urine samples from animals with pancreatic cancer treated with a test therapeutic agent or therapy, wherein the urinary tumor marker comprises at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; A step of evaluating the efficacy of the test therapeutic drug or treatment method for pancreatic cancer based on the measurement results. A method comprising: [Effects of the Invention]

[0010] The present invention provides a method and device for diagnosing pancreatic cancer that is minimally invasive, simple, and low-cost. Because the test is based on urine, the collection method in clinical settings is extremely simple, greatly improving convenience for medical professionals. Therefore, the present invention is useful in fields such as pancreatic cancer diagnosis, testing, treatment evaluation, and drug development. [Brief explanation of the drawings]

[0011] [Figure 1] This graph shows the quantitative results of the urinary metabolites glycochenodeoxycholic acid 3-sulfate (A) and δ-valerobetaine (B) in urine samples collected from pancreatic cancer patients (pancreatic cancer), patients who were diagnosed with suspected pancreatic cancer but tested negative in detailed tests (negative), healthy individuals who collected urine at a health checkup center (health check), and patients diagnosed with colorectal cancer (colorectal cancer). Each quantitative value was normalized by the quantitative value of urinary creatinine. [Figure 2] The ROC curve and AUC obtained by performing logistic discriminant analysis for glycochenodeoxycholic acid 3-sulfate alone (A), δ-valerobetaine alone (B), and the combination of glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine (C) are shown. [Figure 3] 1 shows an example of the configuration of an apparatus to which the present invention is applied. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method and device provided by the present invention utilize a novel urinary tumor marker associated with pancreatic cancer. This urinary tumor marker is a metabolite whose urinary level differs depending on the presence or absence of pancreatic cancer, and is therefore useful for detecting pancreatic cancer, predicting the risk of pancreatic cancer, staging pancreatic cancer, determining the prognosis of pancreatic cancer, monitoring pancreatic cancer, and / or monitoring the effectiveness of treatment for pancreatic cancer.

[0013] The method for diagnosing pancreatic cancer according to the present invention includes the steps of measuring a urinary tumor marker in a urine sample derived from a subject and diagnosing pancreatic cancer in the subject based on the measurement results. Alternatively, the method for diagnosing pancreatic cancer according to the present invention includes the step of measuring a urinary tumor marker in a urine sample derived from a subject, and diagnosing pancreatic cancer in the subject based on the measurement results.

[0014] In the present invention, pancreatic cancer is diagnosed. Pancreatic cancer refers to cancer (malignant tumor) that develops in the pancreas (mostly the pancreatic duct). Pancreatic cancer does not show symptoms when it is small, making early detection difficult. After the cancer progresses, symptoms such as abdominal pain, loss of appetite, abdominal distension, jaundice, and lower back and back pain may appear. Pancreatic cancer can be primary, metastatic, or recurrent, and is classified into stages based on the degree of progression and spread. The required treatment (surgery, chemotherapy, radiation therapy, immunotherapy, etc.) varies depending on whether it is primary, metastatic, or recurrent, and on the stage.

[0015] In one embodiment, the step of measuring a urinary tumor marker measures a urinary tumor marker associated with pancreatic cancer. The "urinary metabolite" or "urinary tumor marker" to be measured in the present invention refers to a metabolite containing glycochenodeoxycholate 3-sulfate and / or delta-valerobetaine. Compared to substances in blood, urinary metabolites are less susceptible to enzymes and are structurally stable, making them highly convenient tumor markers. Furthermore, since urine is used as a sample, it can be easily collected from a subject, making it highly useful for cancer screening.

[0016] "Measuring" refers to determining the relative abundance or absolute concentration of a metabolite in a urine sample. Relative abundance refers to the ratio of the measured intensity of a target metabolite to that of an intentionally added standard substance. On the other hand, absolute concentration refers to a method in which a calibration curve (the relationship between the concentration of a metabolite and the measured intensity of the metabolite) is prepared in advance using the same metabolite for the target metabolite, and the absolute concentration is calculated from the measured intensity. Furthermore, in the present invention, "measuring a urinary tumor marker" may refer to measuring a metabolite that is a urinary tumor marker, or to measuring a derivative or derivative thereof. "Derivative" and "derivative" refer to a substance derived from a metabolite that is a urinary tumor marker and a substance derived from the metabolite, respectively. "Derivative" and "derivative" include, for example, metabolite fragments, modified metabolites, etc., but are not limited thereto.

[0017] Urinary tumor markers used in the present invention include glycochenodeoxycholate 3-sulfate and / or delta-valerobetaine.

[0018] In one embodiment, glycochenodeoxycholic acid 3-sulfate is measured using a liquid chromatography mass spectrometer (LC / MS) in negative ion detection mode. When measured in multiple reaction monitoring (MRM) mode, the precursor ion is measured at m / z 528 and the product ion is measured at m / z 448.

[0019] In one embodiment, δ-valerobetaine is measured. That is, detection is performed in the positive ion detection mode of a liquid chromatograph mass spectrometer (LC / MS). In MRM mode, a compound is measured in which the precursor ion is measured at m / z 160 and the production ion is measured at m / z 101. However, the measurement m / z width for the precursor ion and production ion can be set arbitrarily and is generally about 0.5 to 1.0 Da. The measurement m / z width is set to at least a range that includes the above m / z values. Here, the description is based on a triple quadrupole MS as the quantitative MS. Measurement is not limited to this instrument, and high-resolution MS such as a time-of-flight format may also be used. In this case, collision-induced dissociation is not necessarily required.

[0020] The measured m / z is the mass-to-charge ratio of the ionized metabolite, and varies by ±1 from the mass of the original metabolite due to the addition or loss of one proton. However, depending on the measurement conditions, multiple protons or sodium may be added or lost, causing the measured m / z to fluctuate accordingly. In addition, the m / z of the measured product ion may change depending on the conditions of collision-induced dissociation. Both glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine can be analyzed by LC / MS using a reversed-phase column. However, if retention on the column is poor, it can be improved by using an ion pair reagent.

[0021] In one embodiment of the present invention, at least one of urinary tumor markers (glycochenodeoxycholic acid 3-sulfate, δ-valerobetaine) can be used to diagnose pancreatic cancer and monitor the effectiveness of treatment.

[0022] The above two urinary tumor markers can be used alone to determine pancreatic cancer. In one embodiment, the marker includes at least glycochenodeoxycholic acid 3-sulfate. In another embodiment, the marker includes at least δ-valerobetaine.

[0023] Furthermore, in the present invention, the use of two urinary tumor markers in combination enables more accurate and highly precise determination and monitoring of the effectiveness of treatment. Furthermore, known urinary tumor markers known to be associated with pancreatic cancer may be combined.

[0024] The combination of urinary tumor markers can be selected appropriately depending on the type, sex, and age of the subject, as well as the purpose, including the determination of pancreatic cancer, the follow-up of subjects at high risk (e.g., based on family history) or subjects without abnormalities (pancreatic cancer monitoring), or the monitoring of treatment.

[0025] As an example of a method for identifying urinary tumor markers, partial least squares, a type of multivariate analysis, particularly OPLS-DA, can be used. When performing multivariate analysis by combining multiple metabolites that vary between two groups, using multidimensional data as is can make it difficult to understand the characteristics of the data, so it is preferable to visualize the data by reducing it to two- or three-dimensional data. Analytical methods known in the art, such as principal component analysis, can also be used for multivariate analysis.

[0026] A urine sample refers to urine collected from a subject, and samples obtained by treating the urine (for example, urine to which a preservative such as toluene, xylene, or hydrochloric acid has been added).

[0027] The subject is human urine. Because metabolic activity in the human body is expected to differ depending on race, etc., monoglot individuals, including Japanese, are preferred, but are not limited to, the subjects of analysis in the present invention. For example, the subject may be a mass screening such as a health checkup or cancer test, a follow-up test after such mass screening, or before or after surgery at a hospital or during treatment such as chemotherapy or radiation therapy.

[0028] Furthermore, even for the same individual, the concentration of urinary metabolites can easily vary depending on the timing of urine collection, water intake, and other factors. To standardize the content of urinary metabolites in random urine samples, the amount of creatinine or osmolality in the urine is generally measured and normalized by dividing the amount of each metabolite. Furthermore, since the concentration of urinary metabolites can also vary due to variations in the measurement system, corrections can be made, for example, by simultaneously measuring the isotopes of urinary tumor markers and dividing the measured values ​​of the tumor markers by the isotope values. Specifically, isotopes of glycochenodeoxycholic acid 3-sulfate and / or δ-valerobetaine can be simultaneously measured, and the measured values ​​of glycochenodeoxycholic acid 3-sulfate and / or δ-valerobetaine can be corrected using the measured values ​​of the isotopes. When the isotopes of the urinary tumor markers are unavailable, isotopes of urinary metabolites other than the urinary tumor markers can be simultaneously measured, and the measured values ​​of the urinary tumor markers can be corrected using the measured values ​​of the isotopes. For example, since isotopes of δ-valerobetaine are not commercially available, the isotopes of propionylcarnitine, another urinary metabolite, can be simultaneously measured, and the measured values ​​of δ-valerobetaine can be corrected using the measured values ​​of the propionylcarnitine isotope. In the case of glycochenodeoxycholic acid 3-sulfate, the d5 isotope can be measured. In MRM mode for negative ion detection, the precursor ion can be detected at m / z 533, and the product ion can be detected at m / z 453. The d3 isotope of propionylcarnitine can be measured. In MRM mode for positive ion detection, the precursor ion can be detected at m / z 221, and the product ion can be detected at m / z 85.

[0029] Measuring a urinary tumor marker means measuring its amount or concentration in a urine sample, preferably semi-quantitatively or quantitatively, and the amount may be absolute or relative. Measurement can be performed directly or indirectly. Direct measurement involves measuring the amount or concentration based on a signal that directly correlates with the number of molecules of a urinary metabolite present in a sample. Such a signal is based, for example, on specific physical or chemical properties of the urinary metabolite. Indirect measurement is the measurement of a signal obtained from a secondary component (i.e., a component other than a urinary metabolite), such as a ligand, a label, or an enzymatic reaction product.

[0030] In one embodiment of the present invention, urinary tumor markers, i.e., urinary metabolites, are measured. The measurement method can be any method or means known in the art and is not particularly limited. For example, urinary tumor markers can be measured by a means for measuring physical or chemical properties specific to urinary metabolites, such as a means for measuring accurate molecular weight or NMR spectrum. Examples of means for measuring urinary metabolites include analytical devices such as mass spectrometers, NMR analyzers, two-dimensional electrophoresis devices, chromatographs, and liquid chromatography / mass spectrometry (LC / MS). Urinary tumor markers may be measured using a single analytical device, or multiple analytical devices may be used to measure urinary tumor markers.

[0031] Alternatively, if a reagent for detecting the metabolite to be measured, such as an immunoreaction reagent or an enzyme reaction reagent, is available, the metabolite in urine can be measured using such a reagent.

[0032] The above-mentioned urinary metabolites were discovered by LC / MS, and therefore, these urinary metabolites can be measured using LC / MS.

[0033] In this manner, it is possible to measure urinary tumor markers contained in a urine sample collected from a subject and diagnose pancreatic cancer in the subject based on the results. Furthermore, urinary tumor markers may be measured in urine samples collected from the subject at multiple time points.

[0034] In one embodiment, a higher level of glycochenodeoxycholic acid 3-sulfate than a reference value (e.g., a measurement from a healthy or low-risk individual, or a previous measurement) indicates that the subject may have pancreatic cancer or that their pancreatic cancer has worsened or progressed. In one embodiment, a lower level of delta-valerobetaine than a reference value (e.g., a measurement from a healthy or low-risk individual, or a previous measurement) indicates that the subject may have pancreatic cancer or that their pancreatic cancer has worsened or progressed.

[0035] The method for diagnosing pancreatic cancer of the present invention enables early detection of the presence and progression of pancreatic cancer, which is useful for determining detailed testing and treatment strategies. If a simple test can diagnose whether or not a patient has pancreatic cancer, it is expected that the invasive risks associated with testing as well as treatment can be avoided. This allows subjects to receive treatment for pancreatic cancer early, or, if the risk is high, to be monitored for the onset of pancreatic cancer. Furthermore, the effectiveness of pancreatic cancer treatment can be monitored, allowing for the discontinuation, continuation, or modification of treatment depending on its effectiveness. Furthermore, since the method uses a urine sample, it is minimally invasive, has very few psychological barriers, and allows for simple, low-cost detection of pancreatic cancer. Therefore, it is highly suitable for screening purposes for early detection, and since urine can be collected repeatedly, it is also suitable for routine monitoring.

[0036] The method of the present invention for diagnosing pancreatic cancer can be carried out easily and simply by using a kit and / or device equipped with a means for measuring urinary tumor markers, which are urinary metabolites.

[0037] The kit for diagnosing pancreatic cancer according to the present invention comprises at least a means for measuring at least one (preferably two) of the above-mentioned urinary tumor markers.

[0038] One example of the kit of the present invention is a reagent set for mass spectrometry, which may include, for example, an isotope-labeled reagent, a fractionation minicolumn, a buffer solution, etc. Another example of the kit is a reagent set for immune reactions, which may include, for example, a substrate on which a primary antibody is immobilized, a secondary antibody, etc. Another example is a reagent set for enzymatic reactions, which may include, for example, an enzyme, a buffer solution, etc. The kit of the present invention may also include instructions describing the procedures and protocols for carrying out the method of the present invention, a table showing reference values ​​or reference ranges to be used in determining pancreatic cancer, etc.

[0039] The components included in the kit of the present invention may be provided individually or in a single container. Preferably, the kit of the present invention contains all of the components necessary to carry out the method of the present invention, for example, as components in adjusted concentrations, so that they can be used immediately.

[0040] The apparatus for diagnosing pancreatic cancer according to the present invention comprises the following means: a measurement unit for measuring at least one urinary tumor marker selected from the group consisting of glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine (preferably glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine) in a urine sample; a comparison unit that compares the measured value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measured value; A determination unit that determines pancreatic cancer based on the comparison results obtained by the comparison unit.

[0041] The device of the present invention is preferably a system in which the above-mentioned measurement unit, comparison unit, and judgment unit are operably linked to each other so that the method of the present invention can be carried out. An example of the configuration of a device to which the present invention is applied is shown in Figure 3.

[0042] Here, the measurement unit includes a means for measuring urinary tumor markers in a urine sample, as described above, and is equipped with an analytical device such as a mass spectrometer, an NMR analyzer, a two-dimensional electrophoresis device, a chromatograph, or a liquid chromatography mass spectrometry (LC / MS) device.

[0043] The measurement unit is equipped with a data analysis unit consisting of software and a computer that processes measurement values ​​obtained from the above-mentioned analyzer, etc. The data analysis unit calculates the amount or concentration of urinary tumor markers contained in the urine sample by referring to data such as a calibration curve based on the measurement values ​​obtained from the above-mentioned analyzer, etc. The data analysis unit can include, for example, a signal display unit, a unit that analyzes measurement values, a computer unit, etc.

[0044] The comparison unit also reads out reference values ​​for the amount or concentration of urinary tumor markers from a storage device (database) or the like, and compares the measured values ​​of the urinary tumor markers measured by the measurement unit with the reference values. At this time, the comparison unit selects and reads out an appropriate reference value depending on the type of urinary tumor marker. Alternatively, in the case of longitudinal monitoring of the same subject, the comparison unit reads out the previous measured value from a storage device (database) or the like, and compares it with the measured value of the urinary tumor marker measured by the measurement unit.

[0045] Furthermore, the determination unit determines pancreatic cancer based on the results of the comparison unit's comparison of the measured values ​​of the urinary tumor marker with a reference value, or based on the results of the comparison unit's comparison of the measured values ​​of the urinary tumor marker at multiple time points. Here, the determination unit obtains information indicating the presence of pancreatic cancer in the subject, the stage of pancreatic cancer, whether there is any abnormality, etc. Preferred devices are those that can be used without the knowledge of a specialized clinician, such as electronic devices that require only the addition of a sample.

[0046] In one embodiment, the determination unit determines that the subject has pancreatic cancer or that pancreatic cancer may have worsened or progressed if the glycochenodeoxycholic acid 3-sulfate level is higher than a reference value (e.g., a measurement value derived from a healthy or low-risk individual) or a previous measurement, or is equal to or higher than a reference value (e.g., a patient diagnosed with pancreatic cancer or a patient with a specific pancreatic cancer). Alternatively, for example, the determination unit determines that the subject is normal or that pancreatic cancer may have regressed if the glycochenodeoxycholic acid 3-sulfate level is equal to or lower than a reference value (e.g., a measurement value derived from a healthy or low-risk individual) or a previous measurement.

[0047] In one embodiment, the determination unit determines that the subject has pancreatic cancer or that pancreatic cancer may have worsened or progressed if the δ-valerobetaine level is lower than a reference value (e.g., a measurement value derived from a healthy or low-risk individual) or a previous measurement value, or is equal to or lower than a reference value (e.g., a patient diagnosed with pancreatic cancer or a patient with a specific pancreatic cancer). Also, for example, the determination unit determines that the subject is normal or that pancreatic cancer may have regressed if the δ-valerobetaine level is equal to or higher than a reference value (e.g., a measurement value derived from a healthy or low-risk individual) or a previous measurement value.

[0048] In one embodiment, when glycochenodeoxycholic acid 3-sulfate is higher than a reference value (e.g., a measurement value derived from a healthy subject or a low-risk subject) or a previous measurement value, or is equivalent to or higher than a reference value (e.g., a patient diagnosed with pancreatic cancer or a patient with a specific pancreatic cancer), and δ-valerobetaine is lower than a reference value (e.g., a measurement value derived from a healthy subject or a low-risk subject) or a previous measurement value, or is equivalent to or lower than a reference value (e.g., a patient diagnosed with pancreatic cancer or a patient with a specific pancreatic cancer), the determination unit can determine that the subject is more likely to have pancreatic cancer or that pancreatic cancer has worsened or progressed than when each molecule fluctuates independently relative to the reference value.

[0049] The device of the present invention may further include a data storage unit, a data output / display unit, and the like.

[0050] As used herein, "(assistance in) diagnosing pancreatic cancer" refers not only to detecting pancreatic cancer in a subject, but also to predicting the risk of pancreatic cancer in a subject, determining the stage of pancreatic cancer in a subject, determining the prognosis of pancreatic cancer in a subject, monitoring pancreatic cancer in a subject, monitoring the effectiveness of treatment for pancreatic cancer present in a subject, and assisting in the diagnosis of pancreatic cancer. Furthermore, in the present invention, "diagnosis" also encompasses continuous monitoring of pancreatic cancer that has already been detected or diagnosed, and confirmation of an already-detected or diagnosed pancreatic cancer detection or diagnosis.

[0051] The "determination" by the pancreatic cancer determination method, determination kit, and determination device of the present invention is intended to determine a statistically significant proportion of subjects. Therefore, the "determination" by the pancreatic cancer determination method, determination kit, and determination device of the present invention includes cases where the correct result is not necessarily obtained for all subjects (i.e., 100%). A statistically significant proportion can be determined using various well-known statistical evaluation tools, such as determining a confidence interval, a p-value, a Student's t-test, or a Mann-Whitney test. A preferred confidence interval is at least 90%. The p-value is preferably 0.1, 0.01, 0.05, 0.005, or 0.0001. More preferably, at least 60%, at least 80%, or at least 90% of subjects can be appropriately determined by the pancreatic cancer determination method, determination kit, and determination device of the present invention.

[0052] A specific example of how pancreatic cancer can be diagnosed is as follows: In one embodiment, urinary tumor markers are measured in a urine sample from a subject, and the measured values ​​are compared with reference values ​​or previous measured values. When multiple urinary tumor markers are measured, each urinary tumor marker may be compared with its respective reference value or previous measured value, or a calculated value of a response variable obtained by multivariate analysis may be obtained and compared with the reference value or previous measured value.

[0053] The standard (reference value) is the amount or concentration of a urinary tumor marker associated with pancreatic cancer, or the range of the amount or concentration, or the amount or concentration range of a urinary tumor marker that indicates no abnormality. For example, the reference value can be derived from a healthy population or a low-risk population of pancreatic cancer. Alternatively, the reference value can be derived from a patient (patient population) who has pancreatic cancer (e.g., pancreatic cancer at a specific stage) or has pancreatic cancer that indicates a specific prognosis. The reference value applied to an individual subject may vary depending on various physiological parameters such as the type, age, and sex of the subject animal.

[0054] Preferably, the correlation between the amount or concentration of a urinary tumor marker and the presence or specific prognosis of pancreatic cancer is recorded as a database. The measured values ​​of the urinary tumor marker in a measured urine sample can then be compared with the reference values ​​in the database. Such a database is useful as a reference value or reference range that serves as an indicator of the presence or absence of pancreatic cancer (or a specific stage of pancreatic cancer) or as an indicator of prognosis.

[0055] The amount or concentration of the above-mentioned urinary tumor markers varies depending on whether or not a subject has pancreatic cancer, and the presence of pancreatic cancer alters the amount or concentration. Specifically, the amount or concentration of glycochenodeoxycholic acid 3-sulfate is elevated in patients with pancreatic cancer compared to subjects without pancreatic cancer. Therefore, if the glycochenodeoxycholic acid 3-sulfate level is higher than the reference value derived from a normal population (subjects without pancreatic cancer) or is equal to or higher than the reference value derived from a patient population with pancreatic cancer, it can be said that the subject may have pancreatic cancer or is at high risk of pancreatic cancer.

[0056] On the other hand, the amount or concentration of δ-valerobetaine is reduced in patients with pancreatic cancer compared to subjects without pancreatic cancer. Therefore, if δ-valerobetaine is lower than the reference value derived from a normal population (subjects without pancreatic cancer) or is equal to or lower than the reference value derived from a patient population with pancreatic cancer, it can be said that the subject may have pancreatic cancer or is at high risk of having pancreatic cancer.

[0057] In another embodiment, urine samples are collected from a subject at multiple time points, the urinary tumor marker contained in the urine sample at each measurement time point is measured, and the measured values ​​of the urinary tumor marker at each measurement time point are compared. More specifically, the amount or concentration of the urinary tumor marker at a first time point (a) is compared with the amount or concentration of the urinary tumor marker at a second time point (b). Measurements are performed at least two, three, four, five, ten, fifteen, twenty, thirty, or more times over time, for example, at intervals of one day, two days, five days, one week, two weeks, three weeks, one month, two months, three months, six months, one year, two years, three years, five years, or more. This comparison allows for monitoring over time and allows for evaluation of pancreatic cancer progression, pancreatic cancer metastasis or recurrence, malignant transformation of benign tumors, and the development of pancreatic cancer from no abnormality.

[0058] In another embodiment, the urinary tumor markers used in the present invention can be used to monitor the effect of treatment (therapeutic drug or therapy) on pancreatic cancer in a subject. (a) measuring urinary tumor markers in a urine sample from a patient with pancreatic cancer prior to treatment with a therapeutic agent or therapy; (b) measuring urinary tumor markers in urine samples from patients with pancreatic cancer after treatment with a therapeutic agent or therapy; (c) repeating step (b) as necessary; (d) monitoring the effect of the therapeutic drug or treatment method on pancreatic cancer based on the measurement results of (a) to (c). Includes:

[0059] In the above method, a urine sample is collected from a patient with pancreatic cancer before treatment with a therapeutic drug or therapy, and urinary tumor markers in the urine sample are measured. After treatment with a therapeutic drug or therapy, a urine sample is collected at appropriate times to measure the urinary tumor markers in the urine sample. For example, urine samples are collected immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2 to 10 days, 10 to 20 days, 20 to 30 days, and 1 month to 6 months after treatment. Measurement of urinary tumor markers in urine samples can be performed as described above. Measuring urinary tumor markers before and after treatment makes it possible to monitor the effectiveness of the treatment with the therapeutic drug or therapy. Monitoring results can help determine whether to stop, continue, or change treatment.

[0060] Furthermore, the method for diagnosing pancreatic cancer may be performed in combination with other conventionally known methods for diagnosing pancreatic cancer, such as blood tests (measurement of blood cancer markers, pancreatic function tests, etc.), imaging tests (e.g., abdominal ultrasound, computed tomography (CT), MRI, positron emission tomography (PET), etc.), endoscopic tests, and pathological tests such as biopsy or cytology.

[0061] Based on the above-described assessment results, a physician can diagnose the subject's pancreatic cancer and provide appropriate treatment. That is, the present invention also relates to a method for diagnosing and treating pancreatic cancer in a subject. For example, if a subject is diagnosed with pancreatic cancer using the method of the present invention and the subject is assessed as having a high probability of having pancreatic cancer, the subject is treated for pancreatic cancer or treated to prevent the progression of pancreatic cancer. Furthermore, if the subject's pancreatic cancer is assessed as having an advanced stage or a poor prognosis, treatment can be continued or, if necessary, a change in treatment can be considered. Alternatively, if the subject is assessed as being at high risk but has not yet developed pancreatic cancer, urinary tumor markers can be measured over time to monitor pancreatic cancer in order to avoid excessive testing and treatment. Furthermore, if the subject is assessed as having a high probability of having pancreatic cancer, other pancreatic cancer diagnostic methods such as those described above can be used to confirm the presence of pancreatic cancer. Furthermore, based on the pre- and post-treatment assessment results, the effectiveness of treatment can be monitored to determine whether to stop, continue, or change treatment. If no abnormalities are found, the tumor markers in urine can be measured over time to allow for follow-up observation.

[0062] Pancreatic cancer can be treated by surgery (surgical resection), chemotherapy, radiotherapy, immunotherapy, proton therapy, heavy ion therapy, etc., either alone or in appropriate combination. A person skilled in the art can appropriately select a treatment for pancreatic cancer, taking into consideration the type, stage, malignancy, sex, age, and condition of the pancreatic cancer, responsiveness to treatment, and genetic polymorphisms (SNPs) present.

[0063] As an example of application of the present invention, cancer testing at a testing center will be described. The testing center provides information about cancer testing in response to a request from the subject. When applying for the primary test, the subject may select the number of biomarkers to be tested. For example, the number of biomarkers may include one or two types of urinary tumor markers. This can also be combined with other biomarkers to be used as a pan-cancer test (analyzing various cancers at once).

[0064] Next, the testing center will hand over the test kit required for urine collection to the subject. If necessary, it will be sent by mail. After receiving the test kit, the subject will hand over or send the sample to the testing center. At the testing center, the sample will be frozen at approximately -80°C as needed for subsequent testing. However, if it is known that the target urinary metabolites are stable over time and temperature, the sample may be stored frozen at approximately -5°C, refrigerated at approximately 5°C, or at room temperature, rather than at -80°C. The testing center will then perform the initial test and send the test results to the subject.

[0065] After receiving the results of the primary test, the patient can apply for a secondary test or undergo a more detailed diagnosis depending on the results, which can confirm the suspicion of pancreatic cancer from the primary test and even identify the stage of pancreatic cancer.

[0066] Furthermore, the urinary tumor markers used in the present invention can be used to evaluate the effectiveness of a treatment (therapeutic agent or therapy) for pancreatic cancer or to screen candidate therapeutic agents for pancreatic cancer. Specifically, the method for evaluating the effectiveness of a treatment for pancreatic cancer or the method for screening candidate therapeutic agents for pancreatic cancer include: (a) measuring urinary tumor markers in urine samples from animals with pancreatic cancer treated with a test therapeutic agent or therapy; (b) Evaluating the efficacy of the test therapeutic drug or treatment for pancreatic cancer based on the measurement results of (a). Includes:

[0067] In the method of the present invention, urine samples are collected from patients with pancreatic cancer or humans without pancreatic cancer, and urinary tumor markers in the urine samples are measured. Preferably, urine samples are collected from humans with pancreatic cancer before treatment with a test therapeutic drug or therapy, and urinary tumor markers in the urine samples are measured. After treatment of an animal with pancreatic cancer with a test therapeutic drug or therapy, urine samples are collected at appropriate time points, and urinary tumor markers in the urine samples are measured. For example, urine samples are collected immediately after treatment, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours (1 day), 2 to 10 days, 10 to 20 days, 20 to 30 days, and 1 month to 6 months later. Measurement of urinary tumor markers in urine samples and assessment of pancreatic cancer can be performed as described above.

[0068] The type of test therapeutic agent or treatment to be evaluated or screened is not particularly limited. For example, the test therapeutic agent or treatment may be any material factor, specifically, naturally occurring molecules such as amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, carbohydrates (such as sugars), steroids, glycopeptides, glycoproteins, and proteoglycans; synthetic analogs or derivatives of naturally occurring molecules such as peptidomimetics, nucleic acid molecules (such as aptamers, antisense nucleic acids, and double-stranded RNA (RNAi)); non-naturally occurring molecules such as small organic compounds (such as inorganic and organic compound libraries or combinatorial libraries); and mixtures thereof. The test therapeutic agent or treatment may be a single substance, a complex composed of multiple substances, or a food or diet. Furthermore, the test therapeutic agent or treatment may be radiation, ultraviolet light, or the like in addition to the material factors described above.

[0069] The efficacy of a test therapeutic agent or treatment can also be examined under several conditions. Such conditions include the time or duration of treatment with the test therapeutic agent or treatment, the amount (large or small), and the number of times. For example, multiple doses can be established by preparing a dilution series of the test therapeutic agent. Furthermore, when examining the additive or synergistic effects of multiple test therapeutic agents or treatments, a combination of therapeutic agents or treatments may be used.

[0070] By measuring the urinary tumor marker in a urine sample collected after treatment with the test therapeutic drug or therapy and comparing the amount or concentration with that before treatment, it is possible to evaluate whether the test therapeutic drug or therapy is effective in eliminating pancreatic cancer, shrinking pancreatic cancer, improving symptoms caused by pancreatic cancer, or stopping or slowing the progression of pancreatic cancer.

[0071] For example, in the case of glycochenodeoxycholic acid 3-sulfate, a lower post-treatment measurement value compared to a pre-treatment measurement value in a pancreatic cancer patient, and / or a higher post-treatment measurement value compared to a pre-treatment measurement value in the case of δ-valerobetaine, indicates that the test therapeutic agent or therapy is effective in eliminating, shrinking, ameliorating symptoms of, or halting the progression of pancreatic cancer. On the other hand, a post-treatment measurement value for glycochenodeoxycholic acid 3-sulfate that is equal to or greater than the pre-treatment measurement value or is not significantly different from the pre-treatment measurement value, and / or a post-treatment measurement value for δ-valerobetaine that is equal to or less than the pre-treatment measurement value or is not significantly different from the pre-treatment measurement value, indicates that the test therapeutic agent or therapy is not effective in treating pancreatic cancer.

[0072] As described above, the method for evaluating the effectiveness of a treatment for pancreatic cancer according to the present invention makes it possible to find a therapeutic agent or a treatment method for treating or preventing pancreatic cancer, and further to confirm the effectiveness of the therapeutic agent or treatment method.

[0073] The present invention will be specifically described below by way of examples, but these examples are provided merely to illustrate the present invention and are not intended to limit or restrict the scope of the invention disclosed in this application. [Example]

[0074] [Example 1] Comprehensive analysis of urinary metabolites associated with pancreatic cancer Urine samples were collected with permission from 14 cases of malignant pancreatic cancer (with jaundice), 23 cases of malignant pancreatic cancer (without jaundice), 23 cases of benign tumors, and 25 healthy individuals at Nagoya University Hospital. Urine samples from these four case groups were comprehensively analyzed using liquid chromatography-high-resolution mass spectrometry (LC / HRMS). The m / z and retention times of peaks detected in the mass spectra were compared with a metabolite database to identify detected metabolites. The ion intensities of each sample were compared between the sample groups to identify metabolites whose ion intensities varied significantly depending on the presence or absence of pancreatic cancer.

[0075] As a result, glycochenodeoxycholic acid 3-sulfate and delta-valerobetaine were identified as potential markers associated with pancreatic cancer.

[0076] [Example 2] Quantitative analysis of marker candidates A quantitative mass spectrometry protocol was developed for glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine, two candidate markers discovered by the LC / HRMS analysis described in Example 1. A liquid chromatography triple quadrupole mass spectrometer was used as the analytical instrument. A reversed-phase column was used as the LC column. When measuring glycochenodeoxycholic acid 3-sulfate, mobile phase A was 0.1% acetic acid in water, and mobile phase B was 0.1% acetic acid in acetonitrile. When measuring δ-valerobetaine, mobile phase A was 0.1% nonafluorovaleric acid (NFVA) in water, and mobile phase B was 0.1% NFVA in acetonitrile. An ion-pair reagent was used to improve retention of δ-valerobetaine on the LC column. Thus, the concentrations of the candidate markers were absolutely quantified using quantitative MS, and their applicability for pancreatic cancer testing was analyzed.

[0077] Urine samples were collected from patients diagnosed with pancreatic cancer ("pancreatic cancer," n = 52), patients suspected of pancreatic cancer who visited a doctor but tested negative in detailed examinations ("negative," n = 20), healthy individuals who collected urine samples at a health checkup center ("health checkup," n = 49–62), and patients diagnosed with colorectal cancer ("colorectal cancer," n = 14). An isotopic internal standard (internal standard) was added to the urine sample, and the sample was diluted with organic solvent to precipitate proteins. The supernatant was then centrifuged and collected. The supernatant was further diluted with water and analyzed. The resulting ion intensity was normalized to the internal standard and then quantified. The quantitative values ​​were further normalized by the amount of urinary creatinine to analyze differences between sample groups. A Wilcoxon rank-sum test was used for statistical testing between groups, with a significance level of p < 0.05.

[0078] The results are shown in Figure 1 and Table 1. Figure 1 is a graph showing the marker quantitative values / creatinine quantitative values ​​in each group: pancreatic cancer, negative, health check, and colon cancer. Table 1 also shows the p-values ​​from the Wilcoxon rank sum test. These results show that the pancreatic cancer patient group, negative group, and health check group have different profiles, and that there are significant differences in the Wilcoxon rank sum test. Furthermore, since the quantitative values ​​in colon cancer patients do not change from those in healthy individuals, even though they are in the same digestive system, these markers are thought to be markers that specifically recognize pancreatic cancer.

[0079] [Table 1]

[0080] [Example 3] Discriminant analysis of marker candidates Logistic discriminant analysis was performed on the two candidate markers identified in Example 1 when used alone and in combination. Discriminant analysis was performed on the pancreatic cancer patient group vs. the negative + health check group, and ROC curves and AUCs were calculated. The results are shown in Figure 2 and Table 2. In Figure 2 and Table 2, A indicates the results for glycochenodeoxycholic acid 3-sulfate, B indicates δ-valerobetaine, and C indicates the results for the combination of glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine. The AUC value represents the discriminant ability for pancreatic cancer when the indicated marker is used. The closer the AUC value is to 1, the higher the discriminant ability; generally, an AUC value of 0.7 or higher is considered to be a good model or to have good discriminant ability.

[0081] [Table 2]

[0082] As a result, it was found that glycochenodeoxycholic acid 3-sulfate and delta-valerobetaine, either alone or in combination, could distinguish between the urine of pancreatic cancer patients and non-cancer patients with good discrimination ability. Therefore, since the abundance of these markers changes depending on the presence or absence of pancreatic cancer, measuring these markers may enable early detection and screening of pancreatic cancer.

[0083] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for determining pancreatic cancer in a subject, comprising: measuring a urinary tumor marker in a urine sample from a subject, the urinary tumor marker comprising at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; determining whether the subject has pancreatic cancer based on the measurement results; A method comprising:

2. 2. The method of claim 1, wherein the urinary tumor markers include glycochenodeoxycholic acid 3-sulfate and delta-valerobetaine.

3. The method of claim 1, wherein a higher level of glycochenodeoxycholic acid 3-sulfate than the reference value indicates that the subject is likely to have pancreatic cancer.

4. 2. The method of claim 1, wherein a lower level of delta-valerobetaine than the reference value indicates that the subject may have pancreatic cancer.

5. The method of claim 1, wherein the assessment of pancreatic cancer is detection of pancreatic cancer in a subject, risk prediction of pancreatic cancer in a subject, stage assessment of pancreatic cancer in a subject, prognosis assessment of pancreatic cancer in a subject, monitoring of pancreatic cancer in a subject, or monitoring the effect of treatment for pancreatic cancer present in a subject.

6. The method of claim 1, wherein the measurement of the urinary tumor marker is performed by liquid chromatography mass spectrometry (LC / MS).

7. The method according to claim 1, wherein the measured value of the urinary tumor marker is normalized by the amount of urinary creatinine or the osmotic pressure of urine.

8. The method according to claim 1, wherein an isotope of the urinary tumor marker is measured simultaneously, and the measurement value of the urinary tumor marker is corrected by the measurement value of the isotope.

9. 2. The method according to claim 1, wherein an isotope of a urinary metabolite other than the urinary tumor marker is measured simultaneously, and the measurement value of the urinary tumor marker is corrected by the measurement value of the isotope.

10. A pancreatic cancer diagnosis device, comprising: a measurement unit for measuring a urinary tumor marker in a urine sample, the urinary tumor marker comprising at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; a comparison unit that compares the measured value of the urinary tumor marker measured by the measurement unit with a reference value or a previous measured value; a determination unit for determining pancreatic cancer based on the comparison results obtained by the comparison unit; An apparatus comprising:

11. The device of claim 10 , wherein the measurement unit comprises a liquid chromatography mass spectrometry (LC / MS) device.

12. A method for evaluating the efficacy of a treatment for pancreatic cancer, comprising: measuring urinary tumor markers in urine samples from animals with pancreatic cancer treated with a test therapeutic agent or therapy, wherein the urinary tumor marker comprises at least one urinary tumor marker selected from glycochenodeoxycholic acid 3-sulfate and δ-valerobetaine; A step of evaluating the efficacy of the test therapeutic drug or treatment method for pancreatic cancer based on the measurement results. A method comprising:

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