JP2017203649A - Prostate cancer determination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a prostate cancer determination method with improved specificity.SOLUTION: A prostate cancer determination method includes using of, as an index, at least one peak selected from peaks in a chromatogram with an m/z value of 80.7-81.7, obtained by GC/MS analysis of a urine sample of a subject, or includes using of, as an index, an amount of at least one biomarker selected from the group consisting of menthol and isomintlactone in urine of a subject (subject biomarker amount).SELECTED DRAWING: None

Description

  The present invention relates to a method for determining prostate cancer, a diagnostic agent for prostate cancer, and the like.

  In Japan, prostate cancer has the highest incidence in men, and the number of cases is about 100,000 per year. Moreover, with the westernization and aging of dietary habits, it is predicted that the incidence of prostate cancer and the number of patients will continue to increase.

  The diagnosis of prostate cancer is usually performed based on the concentration (PSA value) of prostate specific antigen (PSA = Prostate Specific Antigen) in the blood. In general, the PSA value is 4.0 ng / mL or more as an abnormal value although it varies depending on the age of the subject. However, the PSA value may show an abnormal value due to prostatic hypertrophy, inflammation, or the like, and not all subjects with abnormal PSA values have prostate cancer. Therefore, when the PSA value is an abnormal value, a definitive diagnosis is performed by an ultrasound-guided prostate needle biopsy.

  However, even if a prostate needle biopsy is performed on a subject with an abnormal PSA value, the subject is about 20 to 40% in the case of 4.0 to 10 ng / mL, and about 50% in the case of 10 ng / mL or more. Prostate cancer can only be found in these subjects. Prostate needle biopsy is usually performed by hospitalization for about two days a night, and the burden on the subject is large. Furthermore, a prostate needle biopsy is a highly invasive test that can cause complications such as pain, infection, bloody stool, hematuria, urinary retention.

  Thus, since the specificity of the PSA test is low, the current diagnostic method based on the test is effective for many subjects whose PSA values are abnormal although they are not actually prostate cancer. Prostate needle biopsy with physical and financial burden and risk of complications.

  On the other hand, in recent years, it has been reported that a trained dog can discriminate prostate cancer from urine smell (Non-patent Document 1). It has also been reported that various diagnostic markers for prostate cancer are present in urine samples.

THE JOURNAL OF UROLOGY, Vol.193, 1382-1387, April 2015.

  An object of the present invention is to provide a method for determining prostate cancer with higher specificity.

  As a result of intensive research, the inventor surprisingly found that a peak in a chromatogram having an m / z value of 80.7 to 81.7, obtained by GC / MS analysis of a urine sample of a subject. It was found that the above-mentioned problem can be solved by using at least one peak selected from as an index. As a result of further analysis on these peaks, a novel prostate cancer diagnostic marker was found. That is, the present invention includes the following aspects.

Item 1. Including, as an index, at least one peak selected from peaks in a chromatogram having an m / z value of 80.7 to 81.7 obtained by GC / MS analysis of a urine sample of a subject, Prostate cancer determination method.
Item 2. Item 2. The method for determining prostate cancer according to Item 1, comprising using as a parameter the total area (test area) of at least one peak selected from the peaks in the chromatogram.
Item 3. The test area is the total area of the corresponding peaks in the chromatogram having a m / z value of 80.7 to 81.7 obtained by GC / MS analysis of a urine sample of a non-prostate cancer specimen (control) Item 3. The method for determining prostate cancer according to Item 1 or 2, comprising determining that the subject has prostate cancer when the area is greater than (Area).
Item 4. Item 4. The prostate cancer determination method according to any one of Items 1 to 3, comprising steps (a1) to (c1):
(A1) GC / MS analysis of a urine sample of a subject to obtain a chromatogram having an m / z value of 80.7 to 81.7,
(B1) Obtained by GC / MS analysis of the total area (test area) of at least one peak selected from the peaks in the chromatogram obtained in step (a1) and a urine sample of a non-prostate cancer specimen Comparing the total area (control area) of the corresponding peak in the chromatogram having an m / z value of 80.7 to 81.7, and (c1) the test area is larger than the control area And determining that the subject has prostate cancer.
Item 5. Item 5. The prostate cancer determination method according to any one of Items 1 to 4, wherein the blood PSA value of the subject is 4.0 ng / mL or more.
Item 6. Item 6. The prostate cancer determination method according to any one of Items 1 to 5, wherein the urine sample is a sample derived from urine collected from a subject after undergoing prostate palpation.
Item 7. A method for determining prostate cancer, comprising using as an index the amount of at least one biomarker selected from the group consisting of menthol and isomint lactone in the urine of a subject (amount of test biomarker).
Item 8. Determining the subject to be prostate cancer if the amount of the test biomarker is greater than the amount of the biomarker in the urine of a non-prostate cancer sample (control biomarker amount), 8. The prostate cancer determination method according to 7.
Item 9. Item 9. The method for determining prostate cancer according to Item 7 or 8, comprising the steps (a2) to (c2):
(A2) a step of measuring the amount of the biomarker (the amount of the test biomarker) in the urine of the subject,
(B2) a step of comparing the amount of the test biomarker with the amount of the biomarker in the urine of the non-prostate cancer sample (control biomarker amount), and (c2) the amount of the test biomarker is the control biomarker amount Determining that the subject has prostate cancer if more.
Item 10. Item 10. The prostate cancer determination method according to any one of Items 7 to 9, wherein the blood PSA value of the subject is 4.0 ng / mL or more.
Item 11. Item 11. The prostate cancer determination method according to any one of Items 8 to 10, wherein the urine is urine collected from a subject after undergoing prostate palpation.
Item 12. A diagnostic agent for prostate cancer, comprising a detection agent for at least one biomarker selected from the group consisting of menthol and isomint lactone.

  According to the present invention, a method for determining prostate cancer can be provided. According to this method, it is also possible to determine prostate cancer with higher specificity than the conventional determination method using the blood PSA value as an index. Furthermore, since this method is non-invasive, there is no complication risk as in the case of prostate needle biopsy.

The gas chromatogram whose m / z value is 80.70-81.70 is shown (Example 1). In the right end of FIG. 1, “Negative” indicates the result of a subject with a prostate needle biopsy “negative”, and “Cancer” indicates the result of a subject with a prostate needle biopsy “positive”. In each chromatogram, the horizontal axis represents retention time and the vertical axis represents abundance. The circled circle indicates a peak specific to the chromatogram of a positive subject. A representative example of the enlarged view of the chromatogram of FIG. 1 is shown (Example 1). The numbers in the upper right of each chromatogram indicate the subject number (23076 and 51326 are “positive” prostate needle biopsy, “25151 and 16551 are“ negative ”prostate needle biopsy). In each chromatogram, the horizontal axis represents retention time and the vertical axis represents abundance. In FIG. 2, the peaks indicated by the arrows indicate seven peaks (peaks 1 to 7) that are circled in FIG. 1 and are characteristic of the positive subject's chromatogram. The graph which compared the total area of the peaks 1-7 by prostate needle biopsy "positive" and prostate needle biopsy "negative" is shown (Example 1). In the horizontal axis of FIG. 3, “Negative” indicates the result of a subject with a negative prostate needle biopsy, “Cancer” indicates the result of a subject with a positive prostate needle biopsy, and the vertical axis indicates a peak 1 Indicates a total area of ~ 7. Each plot (● or ■) shows data for each subject. The central horizontal line indicates the average value, and the horizontal lines above and below the horizontal line indicate the standard deviation. The figure on the left shows a graph comparing the total area of all peaks between prostate needle biopsy “positive” and prostate needle biopsy “negative” (Example 1). In the horizontal axis on the left side of FIG. 4, “Negative” indicates the result of a subject with a negative prostate needle biopsy, “Cancer” indicates the result of a subject with a positive prostate needle biopsy, and the vertical axis indicates all The total area of the peaks is shown. Each plot (● or ■) on the left side of FIG. 4 shows the data of each subject. The horizontal line at the center on the left side of FIG. 4 indicates the average value, and the horizontal lines above and below the horizontal line indicate the standard deviation. The right figure shows the ROC curve of the left data. The vertical axis represents sensitivity, and the horizontal axis represents specificity. The graph which compares the blood PSA value with prostate needle biopsy "positive" and prostate needle biopsy "negative" is shown (Example 1). In the horizontal axis of FIG. 5, “Negative” indicates the result of a subject with a prostate needle biopsy “negative”, “Cancer” indicates the result of a subject with a prostate needle biopsy “positive”, and the vertical axis indicates in blood. Indicates PSA value. Each plot (● or ■) shows data for each subject. The central horizontal line indicates the average value, and the horizontal lines above and below the horizontal line indicate the standard deviation.

  In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

1. Prostate cancer determination method 1
In one aspect thereof, the present invention provides at least one selected from peaks in a chromatogram having an m / z value of 80.7 to 81.7, which is obtained by GC / MS analysis of a urine sample of a subject. The present invention relates to a prostate cancer determination method including using a peak as an index (in this specification, sometimes referred to as “prostate cancer determination method 1 of the present invention”). This will be described below.

  The subject is not particularly limited as long as it is an animal that is a determination target for prostate cancer. Examples of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

  With current diagnostic methods based on PSA testing, many patients with abnormal PSA levels, even though they are not actually prostate cancer, can be treated with prostatic needles with physical and financial burdens and complication risks. We perform inspection. On the other hand, the determination method 1 of the present invention can determine prostate cancer with high specificity even for a subject whose blood PSA value is an abnormal value. From the viewpoint that the superiority of the present invention can be exhibited, it is desirable that the blood PSA value of the subject is an abnormal value. Whether or not the value is an abnormal value is determined according to or according to a known standard. Typically, an abnormal value is obtained when the blood PSA value is 4.0 ng / mL or more.

  The urine sample is not particularly limited as long as it is a sample derived from urine of a subject in a state suitable for gas chromatography analysis. The urine sample can be prepared from the urine of the subject according to or according to a conventional method.

  The urine of the subject used for urine sample preparation may be urine collected from the subject after undergoing prostate palpation. The prostate palpation may be performed by a method according to or according to a standard method. The time for prostate palpation is not particularly limited, but is, for example, 3 to 30 seconds. The time from prostate palpation to collection of urine is not particularly limited, but is, for example, about 1 hour after prostate palpation.

  The urine sample must be prepared using a volatile organic substance adsorbing material (that is, a volatile organic substance adsorbing material on which urine components are adsorbed) from the viewpoint that sensitivity can be increased. Is preferred. Although it does not restrict | limit especially as a volatile organic substance adsorption material, For example, a silicone resin is mentioned, For example, A silicone elastomer is mentioned preferably. Examples of the silicone resin and silicone elastomer include polydimethylsiloxane (PDMS) or an adsorbent made of a modified silicone compound in which a methyl group of a polymer chain in the silicone compound is substituted with a vinyl group or a phenyl group. The volatile organic substance adsorbing material may be used in combination with a suitable carrier. Typically, a volatile organic substance adsorbing material coated on the surface of a stirrer (star bar) can be used.

  The method for preparing the urine sample using the volatile organic substance adsorbing material is not particularly limited, and a method according to or according to a known method can be adopted. For example, urine components can be adsorbed on the volatile organic substance adsorbing material by bringing the urine of the subject into contact with the volatile organic substance adsorbing material.

  The mode of contact is not particularly limited. For example, it is performed by immersing a volatile organic substance adsorbing material in urine. At this time, it is desirable to stir the liquid. When the volatile organic substance adsorbing material is coated on the surface of a stirrer (star bar), it can be efficiently stirred by rotating the stirrer.

  Although the temperature at the time of a contact is not restrict | limited in particular, For example, it is 5-40 degreeC, Preferably it is 15-25 degreeC.

  The contact time is not particularly limited and can be appropriately adjusted depending on the contact mode and temperature. The time is, for example, about 30 minutes to 8 hours, preferably about 1 to 4 hours.

  After contact between the urine of the subject and the volatile organic substance adsorbing material, it is preferable to remove excess urine by washing the volatile organic substance adsorbing material with water or the like. Moreover, after contact or washing, it is desirable to remove moisture by wiping or the like.

  By the preparation method described above, a volatile organic substance adsorbing material on which urine components are adsorbed can be obtained as a urine sample.

  GC / MS analysis (gas chromatography / mass spectrometry analysis) is performed by separating a urine sample of a subject by gas chromatography and performing mass spectrometry on each separated fraction.

  Gas chromatography can be performed according to or according to a known method.

  For vaporization and introduction of the urine sample in gas chromatography, an appropriate method may be selected from known methods according to the state of the urine sample. For example, if the urine sample is a volatile organic substance adsorbing material on which urine components are adsorbed, it can be performed by heat desorption.

  The carrier gas in gas chromatography is not particularly limited as long as it is an inert gas. Examples of the carrier gas include helium, nitrogen, and argon, and preferably helium.

  The stationary phase for gas chromatography is not particularly limited. Examples of the stationary phase include polyethylene glycol, polymethylcyanoalkylsiloxane, polydimethylsiloxane / diphenylsiloxane, and polydimethylsiloxane. Among these, a more polar stationary phase is preferable, specifically, polyethylene glycol, polymethylcyanoalkylsiloxane, polydimethylsiloxane / diphenylsiloxane and the like are preferable, polyethylene glycol, polymethylcyanoalkylsiloxane and the like are more preferable, More preferred is polyethylene glycol. Other examples of the stationary phase include silica gel, activated carbon, zeolite, activated alumina, and the like.

  The detector for gas chromatography is not particularly limited as long as various components can be widely detected. Examples of the detector include a thermal conductivity detector (TCD), a flame ionization detector (FID), and an electron capture detector (ECD).

  Mass spectrometry can be performed according to or according to a known method.

  The ionization method in mass spectrometry is not particularly limited. Examples of the ionization method include an electron ionization method (EI method), a positive chemical ionization method (PCI method), a negative chemical ionization method (NCI method), and preferably an electron ionization method.

  The method for separating ionized sample molecules in mass spectrometry is not particularly limited. As the separation method, for example, a time-of-flight type, a magnetic field deflection type, a quadrupole type, an ion trap type, a Fourier transform ion cyclotron resonance type, a tandem type, or the like can be adopted.

  A chromatogram having an m / z value of 80.7 to 81.7 is obtained as follows. First, by the gas chromatography, a chromatogram (chromatogram 1) is obtained, in which the horizontal axis represents the retention time and the vertical axis represents the detected component amount. Next, each peak in the chromatogram is subjected to mass spectrometry, and each chromatogram peak is decomposed into peaks for each m / z value. Based on this result, from chromatogram 1, the chromatogram relating to only the m / z value of 80.7 to 81.7, in other words, data other than the chromatogram 1 having the m / z value of 80.7 to 81.7. A chromatogram (chromatogram 2) from which is eliminated is obtained. Therefore, even if there is a sharp peak in retention time X of chromatogram 1, if there is no peak having an m / z value of 80.7 to 81.7 when the peak is subjected to mass spectrometry, retention time X of chromatogram 2 There is no peak in. Moreover, even if the peak at the retention time Y of chromatogram 1 is a relatively weak peak, when the peak was subjected to mass spectrometry, only the peak having an m / z value of 80.7 to 81.7, The peak at the retention time Y of the chromatogram 2 can be a relatively strong peak.

  In the case of a prostate cancer “positive” specimen that has been confirmed by prostate needle biopsy or the like, many peaks are seen in a chromatogram having an m / z value of 80.7 to 81.7, whereas “negative” In the case of the analyte of “”, almost no peak is observed in the chromatogram (see FIG. 1 and the like). Therefore, prostate cancer can be determined by using a peak in a chromatogram having an m / z value of 80.7 to 81.7 as an index.

  A specific aspect of the determination is not particularly limited as long as at least one peak selected from peaks in a chromatogram having an m / z value of 80.7 to 81.7 is used as an index. For example, the presence / absence of at least one peak selected from peaks in a chromatogram having an m / z value of 80.7 to 81.7 and the total area of at least one peak can be used as an index.

  When the presence or absence of a peak is used as an index, the presence or absence of one peak or two or more peaks appearing at a specific holding time can be used as an index.

  When the total area of at least one peak is used as an index, the peak area of one peak appearing at a specific holding time can be used as an index. In addition, the total area of each peak area of two or more peaks appearing at a specific holding time can be used as an index. In addition, the total area of the peak areas of all peaks in the chromatogram can be used as an index.

  When the area of one peak or the total area of two or more or all peaks is used as an index, the area of one peak or two or more or all peaks in a chromatogram having an m / z value of 80.7 to 81.7 Is the area of the corresponding peak in the chromatogram having an m / z value of 80.7 to 81.7, obtained by GC / MS analysis of a urine sample of a non-prostate cancer sample. Alternatively, if the total area (control area) is larger, the subject can be determined to be prostate cancer.

A more specific aspect of the prostate cancer determination method 1 of the present invention includes steps (a1) to (c1):
(A1) GC / MS analysis of a urine sample of a subject to obtain a chromatogram having an m / z value of 80.7 to 81.7,
(B1) Obtained by GC / MS analysis of the total area (test area) of at least one peak selected from the peaks in the chromatogram obtained in step (a1) and a urine sample of a non-prostate cancer specimen Comparing the total area (control area) of the corresponding peak in the chromatogram having an m / z value of 80.7 to 81.7, and (c1) the test area is larger than the control area And determining the subject as having prostate cancer.

  In the step (c1), for example, the test area is, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, further preferably 5 times or more, more preferably 10 times the control area. If it is more than twice, it can be determined that the subject has prostate cancer.

2. Prostate cancer determination method 2
In one aspect of the present invention, the present invention includes using as an index the amount of at least one biomarker selected from the group consisting of menthol and isomint lactone in the urine of a subject (amount of test biomarker). And a prostate cancer determination method (in this specification, sometimes referred to as “prostate cancer determination method 2 of the present invention”). This will be described below.

  The subject and urine are the same as in “1. Prostate cancer determination method 1”.

  Although menthol has a plurality of stereoisomers, the menthol may be any stereoisomer. The biomarker also includes other isomers of menthol. Furthermore, a plurality of menthol isomers may be used as biomarkers.

Although isomint lactone has a plurality of stereoisomers, the isomint lactone may be any stereoisomer. The biomarker also includes other isomers of isomint lactone. Further, a plurality of isomint lactone isomers may be used as biomarkers.
The method for measuring the amount of various biomarkers in urine is not particularly limited as long as it is a method capable of detecting and quantifying the biomarker, and a method according to or according to a known method can be adopted. Examples of the method for measuring the amount of biomarker include gas chromatography, liquid chromatography and other chromatography, mass spectrometry, GC / MS, LC / MS, luminescence method using specific chemical reaction, odor analysis sensor system Etc. The odor analysis sensor system is a system that detects changes in the signal by adsorbing a volatile substance to a special film (sensitive film) or sensor, and an ultra-small sensor element developed by Kyocera in collaboration with the National Institute for Materials Science. This is a system that uses MSS (Membrane-type Surface Stress Sensor).

  Alternatively, the method for measuring the amount of biomarker may be, for example, a measurement method using the physiological activity of the biomarker to be measured. For example, if the biomarker to be measured is a substance (for example, menthol) that binds to a specific receptor (for example, a cold receptor such as TRPM8), the measurement can be performed as follows. That is, whether or not the reaction between the receptor and the antibody that specifically binds to the ligand binding region of the receptor is inhibited by the test sample (sample derived from urine). It can be measured by evaluating. More specifically, for example, a test sample (a sample derived from urine) is brought into contact with a receptor (or an antibody against the receptor) immobilized on a solid phase together with an antibody (or receptor) against the receptor. After that, the substance not immobilized on the solid phase is removed, and the antibody (or receptor) immobilized on the solid phase is detected (after adding a label, color development operation, etc., if necessary). It can be measured. The measurement method can be performed, for example, by ELISA method, Western blot method or the like.

  The receptor may be subjected to modifications such as amino acid mutation, chemical modification, and protein tag addition, as long as its function (binding ability with the biomarker to be measured) is not impaired.

  The receptor includes, for example, chemical synthesis, purification from mammalian cells or tissues (for example, serum, etc.), receptor, etc. according to or according to known methods based on amino acid sequences published on NCBI and other databases. It can be obtained by purification from a transformant containing a polynucleotide encoding. When obtained by purification from a transformant, the transformant is not particularly limited as long as it is a cell capable of expressing the receptor from a polynucleotide encoding the receptor, and bacteria such as E. coli, insect cells, and mammals Various cells such as cells can be used.

As insect cells, for example, Sf cells, MG1 cells, High Five ^TM cells, BmN cells and the like are used. As Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells and the like are used.

  Examples of animal cells that can be used include monkey COS-7 cells, monkey Vero cells, Chinese hamster cells CHO, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.

  The antibody includes a part of the antibody having antigen-binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. The antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, which is at least continuous among the amino acid sequences of the receptor.

  The antibody is preferably an antibody having an antigen binding property to the amino acid sequence of the ligand binding site. The ligand binding sites of various receptors are known, and such antibodies can be produced based on this known information.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12-11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the receptor is synthesized using a receptor expressed and purified in Escherichia coli according to a conventional method, or according to a conventional method. Thus, it is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, a spleen cell obtained by immunizing a non-human animal such as a mouse with a receptor expressed and purified in Escherichia coli according to a conventional method, or an oligopeptide having a partial amino acid sequence of the receptor, and It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocol in Molecular Biology edit. Aubel et al. (1987) Publisher. John Wiley and Sons. 11.1-11. Section 11.1. ).

  Receptors used as immunizing antigens for antibody production are based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of transformants and recovery of proteins from the culture. It can obtain by operation of. These operations are in accordance with methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRES (1985)) and the like. Can be done.

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding the receptor in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. The protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture. The partial peptide of the receptor can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

  The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of a receptor. The oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as the receptor. An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the receptor can be exemplified.

  Production of antibodies against such oligo (poly) peptides can also be carried out by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.

  The receptor or antibody may be in a state to which an appropriate label, for example, a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent substance, biotin, or the like is added.

  The specific mode of determination is not particularly limited as long as the amount of the biomarker is used as an index. For example, when the amount of the biomarker in the urine of the subject (the amount of the test biomarker) is larger than the amount of the biomarker in the urine of the non-prostate cancer sample (the amount of the control biomarker) Can be determined to be prostate cancer.

A more specific aspect of the prostate cancer determination method 2 of the present invention includes steps (a2) to (c2):
(A2) a step of measuring the amount of the biomarker (the amount of the test biomarker) in the urine of the subject,
(B2) a step of comparing the amount of the test biomarker with the amount of the biomarker in the urine of the non-prostate cancer sample (control biomarker amount), and (c2) the amount of the test biomarker is the control biomarker amount Determining that the subject has prostate cancer, if more.

  In the step (c2), for example, the amount of the test biomarker is, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, more preferably 5 times or more, more than the control biomarker amount. More preferably, when the ratio is 10 times or more, the subject can be determined to have prostate cancer.

3. Prostate cancer diagnostic agent The present invention, in one embodiment thereof, contains a detection agent for at least one biomarker selected from the group consisting of menthol or an isomer thereof and isomintlactone or an isomer thereof. The present invention relates to a cancer diagnostic agent (in this specification, sometimes referred to as “prostate cancer diagnostic agent of the present invention”). This will be described below.

  The biomarker detection agent is not particularly limited as long as the biomarker can be detected and quantified in urine of a subject or a sample derived from urine. For example, the biomarker detection agent is based on a known one or a known theory. Can be widely used. Specifically, for example, the receptor and the antibody in “2. Prostate cancer determination method 2” can be mentioned. In addition to these, the diagnostic agent for prostate cancer of the present invention may contain reagents necessary for the ELISA method and the like (for example, a label, a detection agent for the label, a buffer solution, a plate, etc.). The prostate cancer diagnostic agent of the present invention may be in the form of a kit.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Exploration of indicators for prostate cancer determination method Subjects with a blood PSA level of 4 ng / mL, subjects who are relatively young and have a blood PSA level of 3 ng / mL or more, and prostate by rectal examination A subject suspected of having cancer was subjected to prostate palpation according to a standard method, and then urine was collected. On the other hand, the subject was subjected to prostate needle biopsy according to a conventional method to determine whether the prostate cancer was positive or negative.

Information on 18 subjects is as follows:
・ Age: Median 67 years (distribution 59-79 years)
・ PSA level in blood: Median 8.05 (distribution 3.07-15.38)
・ Nine prostate needle biopsy negative ・ Nine prostate needle biopsy positive.

  5 mL of urine was transferred to a vial. A twister (length 10 mm, film thickness 0.5 mm, manufactured by GERSTEL) was added thereto, and the mixture was extracted by stirring at room temperature for 2 hours. The twister was taken out and washed with pure water, and then water was wiped off. Analysis by thermal desorption-GC / MS.

The analyzer and analysis conditions are as follows:
<Analyzer>
・ Pretreatment equipment: GERSTEL MPS
-Gas chromatograph: Agilent Technologies 7890
Mass spectrometer: Agilent Technologies 5975C MSD
<Analysis conditions>
Column: DB-WAX (60 m, diameter 0.25 mm, film thickness 0.25 μm)
Carrier gas: helium, 2.11 mL / min
-Ionization method: EI (electron ionization) method-Ionization voltage: 70 eV.

  Based on the analysis results, gas chromatograms with m / z values of 80.7 to 81.7 were prepared. The results are shown in FIG. In the right end of FIG. 1, “Negative” indicates the result of a subject with a prostate needle biopsy “negative”, and “Cancer” indicates the result of a subject with a prostate needle biopsy “positive”. As shown in FIG. 1, a number of unique peaks were observed in the chromatogram of positive subjects (peaks circled).

  A representative example of an enlarged view of the chromatogram is shown in FIG. In FIG. 2, the numbers in the upper right of each chromatogram indicate the subject number (23076 and 51326 are “positive” prostate needle biopsy, and 25151 and 16551 are “negative” prostate needle biopsy). In FIG. 2, the peaks indicated by the arrows indicate seven peaks (peaks 1 to 7) that are circled in FIG. 1 and are characteristic of the positive subject's chromatogram.

  The result of measuring the total area of peaks 1 to 7 is shown in FIG. In FIG. 3, “Negative” indicates the result of a subject with prostate needle biopsy “negative”, “Cancer” indicates the result of a subject with prostate needle biopsy “positive”, and the vertical axis indicates peaks 1 to 7. Total area is shown. As shown in FIG. 3, the total area of peaks 1-7 was significantly greater for prostate needle biopsy “positive” subjects than for prostate needle biopsy “negative” subjects. Therefore, it was shown that prostate cancer can be determined by using the total area of these seven specific peaks as an index.

  The result of measuring the total area of all peaks is shown in FIG. FIG. 4 shows that prostate cancer can be determined by using the total area of all peaks as an index. For comparison, FIG. 5 shows a comparison result of blood PSA values. In FIG. 5, “Negative” indicates the result of a subject with prostate needle biopsy “negative”, “Cancer” indicates the result of a subject with prostate needle biopsy “positive”, and the vertical axis indicates the blood PSA value. Show. As shown in FIG. 5, the blood PSA value was not significantly different between subjects with a negative prostate needle biopsy “negative” and subjects with a positive prostate needle biopsy “positive”.

  From the above, it was suggested that prostate cancer can be determined by using a peak in a chromatogram having an m / z value of 80.7 to 81.7 obtained by GC / MS analysis of a urine sample as an index. This determination method was also shown to have higher specificity than the conventional determination method using blood PSA value as an index.

Example 2 Analysis of the components indicated by analysis peaks 1 to 7 of the prostate cancer determination marker according to a standard method revealed that peak 2 was menthol or its isomer. It was also revealed that peak 7 is likely to be isomint lactone or its isomer. From these results, it was suggested that prostate cancer can be determined by using the amounts of these components as indices.

Claims (12)

Including, as an index, at least one peak selected from peaks in a chromatogram having an m / z value of 80.7 to 81.7 obtained by GC / MS analysis of a urine sample of a subject, Prostate cancer determination method. The method for determining prostate cancer according to claim 1, comprising using, as an index, a total area (test area) of at least one peak selected from peaks in the chromatogram. The test area is the total area of the corresponding peaks in the chromatogram having a m / z value of 80.7 to 81.7 obtained by GC / MS analysis of a urine sample of a non-prostate cancer specimen (control) 3. The method for determining prostate cancer according to claim 1, comprising determining that the subject has prostate cancer when the area is larger than (Area). 4. The method for determining prostate cancer according to any one of claims 1 to 3, comprising steps (a1) to (c1):
(A1) GC / MS analysis of a urine sample of a subject to obtain a chromatogram having an m / z value of 80.7 to 81.7,
(B1) Obtained by GC / MS analysis of the total area (test area) of at least one peak selected from the peaks in the chromatogram obtained in step (a1) and a urine sample of a non-prostate cancer specimen Comparing the total area (control area) of the corresponding peak in the chromatogram having an m / z value of 80.7 to 81.7, and (c1) the test area is larger than the control area And determining the subject as having prostate cancer. The prostate cancer determination method according to claim 1, wherein the subject has a blood PSA value of 4.0 ng / mL or more. The prostate cancer determination method according to claim 1, wherein the urine sample is a urine-derived sample collected from a subject after undergoing prostate palpation. A method for determining prostate cancer, comprising using, as an index, the amount of at least one biomarker selected from the group consisting of menthol and isomint lactone in the urine of a subject (amount of test biomarker). Determining the subject to be prostate cancer if the amount of the test biomarker is greater than the amount of the biomarker in the urine of a non-prostate cancer sample (control biomarker amount). Item 8. The method for determining prostate cancer according to Item 7. The method for determining prostate cancer according to claim 7 or 8, comprising steps (a2) to (c2):
(A2) a step of measuring the amount of the biomarker (the amount of the test biomarker) in the urine of the subject,
(B2) a step of comparing the amount of the test biomarker with the amount of the biomarker in the urine of the non-prostate cancer sample (control biomarker amount), and (c2) the amount of the test biomarker is the control biomarker amount Determining that the subject has prostate cancer, if more. The prostate cancer determination method according to any one of claims 7 to 9, wherein the blood PSA value of the subject is 4.0 ng / mL or more. The method for determining prostate cancer according to any one of claims 8 to 10, wherein the urine is urine collected from a subject after undergoing prostate palpation. A diagnostic agent for prostate cancer, comprising a detection agent for at least one biomarker selected from the group consisting of menthol and isomint lactone.