JP2010071953A - Breast cancer marker, and diagnosis using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new diagnosis marker for breast cancer capable of determining breast cancer quickly and precisely, and enabling early discovery of a disease and early treatment, and to provide an effective diagnosis method of breast cancer utilizing the marker. <P>SOLUTION: In an examination method for diagnosing breast cancer in a subject, the amount of one or more kinds of peptides selected from a group consisting of ten kinds of peptides having each molecular weight of about 2,030, 2,143, 2,553, 2,860, 3,570, 3,836, 3,947, 4,829, 6,739, and 9,990 in a biosample (preferably body fluid, more preferably serum, plasma or the like) collected from the subject is measured (preferably, measured by using mass spectrometry or an antibody for recognizing the peptide specifically). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel diagnostic marker for breast cancer, a method for diagnosing breast cancer using the same, and the like.

  Breast cancer is the second most common cause of death in women in the United States, after skin cancer, and the most common cause of death in women's cancer, followed by lung cancer. During the year 2001, about 200,000 women were diagnosed with breast cancer in the United States, of which it is estimated that about 1 in 5 people die from breast cancer. That is, breast cancer is a disease that can be fully cured if diagnosed and treated early. Therefore, in the case of breast cancer, it is much more important to always keep early detection rather than improvement of risk factors for onset.

Breast cancer screening includes breast palpation, mammary ultrasonography, and mammary x-ray examination (mammography), but in many cases, it is often completed only by breast palpation. However, some breast cancers do not make lumps, and small lumps may be overlooked by palpation alone, so be sure to have a highly accurate screening with breast ultrasonography or mammograms as much as possible. Is desirable. In fact, of breast cancers found by visual inspection, 50% are early cancers, while 90% are early cancers in mammography screening.
However, in order to conduct screening effectively with mammography, equipment and film that meet certain standards, the ability of radiographers and doctors to interpret images are indispensable. Has been. Therefore, a new technique that enables simpler and more accurate breast cancer diagnosis is desired.

With the progress of proteomics research that comprehensively analyzes protein expression in vivo, search for new biomarkers using proteomics has been energetically performed. For example, Honda et al. (Non-Patent Document 1) analyzed plasma samples of patients with pancreatic cancer using SELDI-TOFMS technology, and found four mass peaks (m / z 8,766) with significantly different peak intensities compared to healthy individuals. , 17,272, 28,080, 14,779) have been identified and reported as pancreatic cancer markers. In addition, proteolytic product patterns in the serum of several cancer patients were examined, and fibrinopeptide A (FPA) and its fragments, fibrinogen alpha chain fragments and ITIH4 fragments varied in prostate cancer, bladder cancer and breast cancer It has been reported (Non-Patent Document 2).
Honda et al., “Cancer Res.”, Volume 65 (No. 22), pp. 10613-10622 (2005) Villanueva et al., "The Journal of Clinical Investigation", Volume 116 (No. 1), pp. 271-284 (2006)

  An object of the present invention is to provide a novel diagnostic marker for breast cancer and an effective diagnostic method for breast cancer using the same.

The inventors first examined sera collected from breast cancer patients and breast benign tumor patients by mass spectrometry, conducted differential profiling analysis between both patient sera, and selected a peptide group that gave a P value of 0.05 or less by T test. Of these, 52 markers were selected by excluding those that seemed inappropriate by visual inspection. Using these 52 markers, clustering of all patient samples was performed by the random forest method, and the five markers most effective for the clustering result were selected. Next, a random forest was performed again on the training set using the selected five markers, and the results were verified with the test set. As a result, the sensitivity (prevalence of prevalence) was about 98%, and the specificity ( It was found that malignant / benign can be judged with extremely high reliability (no disease correct diagnosis rate) of about 89%.
The inventors have also found six peptides that vary significantly between both groups, including one of the five markers described above, from a two-group comparison between malignant / benign tumors.
Based on these findings, the present inventors have newly identified these 10 peptides as breast cancer diagnostic markers, and have completed the present invention.

That is, the present invention
[1] A group consisting of 10 peptides having molecular weights of about 2030, about 2143, about 2553, about 2860, about 3570, about 3836, about 3947, about 4829, about 6739 and about 9990 in biological samples collected from subjects. A test method for diagnosing breast cancer in the subject, characterized by measuring the amount of one or more peptides selected from;
[2] The method according to [1] above, wherein the peptide to be measured is selected from the group consisting of five peptides having a molecular weight of about 2030, about 2143, about 3570, about 3836 and about 4829;
[3] The method according to [1] or [2] above, wherein 2 to 5 peptides are measured;
[4] The method according to [2] above, wherein the peptides to be measured are five peptides having molecular weights of about 2030, about 2143, about 3570, about 3836 and about 4829;
[5] The method according to any one of [1] to [4] above, wherein the biological sample is a body fluid;
[6] The above [5], wherein the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid ] The method according to
[7] The method according to any one of [1] to [6] above, comprising subjecting the biological sample to mass spectrometry;
[8] The method according to any one of [1] to [6] above, wherein an antibody that specifically recognizes each peptide is used;
[9] The method according to any one of [1] to [8] above, wherein a biological sample is collected from a patient in time series, and the time course of the amount of each peptide in the sample is examined; ] A test method for evaluating therapeutic effects in breast cancer patients, wherein the molecular weights of biological samples collected from the patient before and after treatment are about 2030, about 2143, about 2553, about 2860, about 3570, about The present invention provides a method characterized by examining changes in the amount of one or more peptides selected from the group consisting of 10 peptides of 3836, about 3947, about 4829, about 6739 and about 9990.

  According to the present invention, breast cancer can be determined quickly and accurately, so that early detection and early treatment of the disease are possible.

  The present invention provides 10 new and useful breast cancer diagnostic marker peptides (hereinafter, the 10 peptides may be collectively referred to as “the peptides of the present invention”).

  Ten peptides of the present invention have an apparent molecular weight of about 2030, about 2143, about 2553, about 2860, about 3570, about 3836, about 3947, about 4829, about 6739 and about 9990 (may be referred to as peptide 1, peptide 2,... Peptide 10 in this order). The actual measurement value of the molecular weight may slightly vary depending on the measurement method / measurement equipment used. Therefore, “about” in these molecular weights is used, for example, in the sense of including an error of ± 0.5%, preferably ± 0.3%, more preferably ± 0.1% in the case of a method using a mass spectrometer.

  Among these peptides, the serum levels of peptide 1 (about 2030), peptide 2 (about 2143), peptide 3 (about 2553) and peptide 6 (about 3836) are compared in non-breast cancer patients or healthy individuals in breast cancer patients. (The numerical value in parentheses indicates the molecular weight. The same applies hereinafter). On the other hand, the serum levels of peptide 4 (about 2860), peptide 5 (about 3570), peptide 7 (about 3947), peptide 8 (about 4829), peptide 9 (about 6739) and peptide 10 (about 9990) There is a marked increase in patients compared to non-breast cancer patients or healthy individuals.

The present invention also provides a test method for diagnosing breast cancer in a patient by measuring the amount of one or more peptides of the present invention in a biological sample from a patient suspected of having breast cancer. provide. Here, “test for diagnosis” means measurement of the amount of the peptide and, if necessary, comparison with the measured value in a control sample. The “patient suspected of having breast cancer” may be subjectively suspected by the patient himself or based on some objective basis. Patients who have been judged by a doctor to have a reasonable possibility of having breast cancer as a result of clinical examination and / or examination, or who have an equivalent pathological condition. In particular, the present invention is useful for determining whether a tumor is malignant or benign in a patient whose tumor has been confirmed in the breast.
In addition, in the treatment of breast cancer, early detection and early treatment are the major principles. However, the peptide of the present invention can also be used in non-breast cancer patients or in early cases of all six stages of disease classification (1997, UICC). Since the living body level fluctuates with a significant difference from healthy individuals, it may be possible to contribute to early detection and early treatment of breast cancer by adopting periodic diagnosis using the peptide of the present invention as an index.

  The peptide to be measured is not particularly limited as long as it is selected from the above 10 peptides of the present invention. Preferably, peptide 1 (about 2030), peptide 2 (about 2143), peptide 5 (about 3570), The peptide is selected from five peptides, peptide 6 (about 3836) and peptide 8 (about 4829). Further, as long as the peptide of the present invention, preferably at least one peptide selected from the above five peptides, is included, the amount of one or more peptides that can be a breast cancer marker other than the peptide of the present invention can be further measured. Good. Examples of such peptides include various protein fragments disclosed in Non-Patent Document 2.

The patient-derived biological sample to be the test sample is not particularly limited, but is preferably one that is less invasive to the patient, for example, blood, plasma, serum, saliva, urine, tears that can be easily collected from the living body And those collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor.
When serum or plasma is used, it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component. The peptide of the present invention as a detection target can be separated and removed in advance using a spin column or the like, if necessary, such as a high molecular weight protein fraction.

  The detection of the peptide of the present invention in a biological sample can be performed, for example, by measuring various molecular weights of the biological sample, for example, gel electrophoresis or various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity). Chromatography, reverse phase chromatography, etc.), ionization methods (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption ionization (MALDI), electrospray ionization Etc.), mass spectrometers (eg double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.) , Detect bands, spots, or peaks that match the molecular weight of the peptide. It can be performed by, but not limited to. Since the amino acid sequence of the peptide of the present invention is known, a method of preparing an antibody that recognizes the amino acid sequence and detecting the peptide by Western blotting or various immunoassays can be used more preferably. Furthermore, the hybrid detection method of the above method is also effective.

  Each of the peptides of the present invention has a molecular weight as described above, but it goes without saying that the actual measurement values may vary slightly depending on the measurement method and instrument used. For example, in the case of a method using a mass spectrometer, it is preferable to measure the peak intensity appearing at the position of the calculated value ± 0.5% (preferably ± 0.3%, more preferably ± 0.1%).

One of the particularly preferable measurement methods in the inspection method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of a component captured on the plate surface is measured using a time-of-flight mass spectrometer. The method of measuring by is mentioned.
The plate that can be adapted to the time-of-flight mass spectrometer may be any plate as long as it has a surface structure that can efficiently adsorb the peptide of the present invention to be detected. Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicon, a wide range of gels or polymers (eg (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof). Examples of surface structures having a plurality of monomer or polymer sequences include linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having α-, β- or ω-amino acids, gel surfaces used in chromatography Carriers (anionic / cationic compounds, hydrophobic compounds comprising carbon chains 1-18, hydrophilic compounds (eg, carriers cross-linked with silica, nitrocellulose, cellulose acetate, agarose, etc.), artificial homopolymers) (For example, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, etc.) Any known drug or natural compound is bound to any of the above compounds (covalent and non-covalent bonds). ) Coated with heteropolymer etc. Packaging and the like.

  In a preferred embodiment, the support used as a plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF [usually a plate for mass spectrometry For example, insulators (glass, ceramics, plastics, resins, etc.), metals (aluminum, stainless steel, etc.), conductive polymers, composites thereof, etc. Although an aluminum plate is preferably used, see WO 2004/031759). The shape of the support can be appropriately devised into a shape suitable for the sample analyzer, in particular, the sample introduction port, but is not limited thereto. As such a plate for mass spectrometry coated with a thin layer with PVDF, a blot chip (registered trademark) manufactured by Protocera is preferably used.

Preferably, the coating refers to a thin layer formed by depositing on a support in a state where coating molecules are dispersed, instead of overlaying a pre-formed structure like a membrane on the support. The manner in which the coating molecules are deposited is not particularly limited, but means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
The thickness of the thin layer can be appropriately selected within a range that does not adversely affect the transfer efficiency of the molecules contained in the tissue or cells and the measurement sensitivity of mass spectrometry, etc., for example, about 0.001 to about 100 μm, Preferably, it is about 0.01 to about 30 μm.

Although the plate for mass spectrometry (support) can be prepared by a method known per se, for example, the above preferred mass spectrometry plate is prepared by thinly coating the surface of the support with a coating molecule such as PVDF. The Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
In the case of “application”, a coating molecule is dissolved in an appropriate solvent, for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (for example, about 1 to about 100 mg / mL) ( The coating molecule-containing solution) can be applied to the substrate using a suitable tool such as a brush.
In the case of “spraying”, the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.
In the case of “evaporation”, the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the base material using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer of molecules can be formed.
In the case of “immersing”, the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.
In the case of “printing”, various printing techniques that can be normally used depending on the material of the substrate can be appropriately selected and used. For example, screen printing or the like is preferably used.
In the case of “sputtering”, for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas collides with the molecule. The repelled coating molecules can be deposited on the substrate to form a thin layer.
The coating may be applied to the entire surface of the substrate, or may be applied only to the surface (fraction) subjected to mass spectrometry.

The coating molecule can be used in a preferred form as appropriate depending on the coating means. For example, the coating molecule can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form. “Apply” refers to bringing the coating molecule into contact with the support so that the coating molecules remain and deposit on the support after contact. The amount of application is not particularly limited, and examples of the coating molecular weight include about 10 to about 100,000 μg / cm ² , preferably about 50 to about 5,000 μg / cm ² . After the application, the solvent is removed by natural drying, vacuum drying or the like.

  The substrate of the mass spectrometric plate may be modified (processed) in advance by an appropriate physical or chemical technique before coating with the coating molecule. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.

  Transfer of the test sample to the mass spectrometric plate (support) can be performed by leaving the patient-derived biological sample as the test sample untreated or after removing and concentrating the high molecular weight protein using an antibody column or other method. -It is performed by subjecting to polyacrylamide gel electrophoresis or isoelectric focusing, and transferring (blotting) the gel after contact with the plate. A known transfer device can be used. The transfer method itself is known. Preferably electrotransfer is used. The sample developed on the gel after the electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.). As a buffer used for electrotransfer, it is preferable to use a buffer having a pH of 7 to 9 and a low salt concentration. Specific examples include Tris buffer, phosphate buffer, borate buffer, and acetate buffer. Tris / Glycine / Methanol buffer, SDS-Tris-Tricine buffer, etc. as Tris buffer, ACN / NaCl / Isotonic phosphate buffer, Sodium phosphate / ACN, Boric acid, etc. as phosphate buffer Examples of the buffer include sodium borate-hydrochloric acid buffer, Tris-borate / EDTA, and borate / ACN. Examples of the acetate buffer include Tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate-hydrochloric acid buffer. Examples of the composition of the tris / glycine / methanol buffer include Tris 10-15 mM, glycine 70-120 mM, and methanol 7-13%. Examples of the composition of the sodium borate-hydrochloric acid buffer include about 5-20 mM sodium borate.

  Thereby, the molecules present in the test sample including the target molecules are efficiently captured on the support surface. After the plate is dried, a reagent called matrix is added to absorb the laser light and promote ionization of analyte molecules through energy transfer, which is advantageous for later mass spectrometry (when using MALDI method) You can also. As the matrix, those known in mass spectrometry can be used. For example, sinapinic acid (SPA (= 3,5-dimethoxy-4-hydoroxycinammic acid)), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic acid (DHB) ), Α-cyano-4-hydroxycinnamic acid (CHCA), and the like, but is not limited thereto. Preferably, it is DHB or CHCA.

The presence and amount of the peptide of the present invention, which is a target molecule, can be identified from the information on the molecular weight by mass spectrometry of molecules in the test sample captured on the support surface by the above method.
The mass spectrometer measures the molecular weight of a substance by ionizing a gaseous sample and then putting the molecule or molecular fragment into an electromagnetic field, separating it by mass number / charge number from its movement, and obtaining the spectrum of the substance. -It is a device to detect. Matrix-assisted laser deionization (MALDI), in which a sample and a matrix that absorbs laser light are mixed, dried and crystallized, and ionized analytes are brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation. And the MALDI-TOFMS method using time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration. Principles of ionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solutions are electrically sprayed into the atmosphere and individual analyte multivalent ions are unfolded into the gas phase A mass spectrometer based on can be used.
A method for mass spectrometry of molecules on a plate for mass spectrometry is known per se. For example, the method described in WO 2004/031759 can be appropriately modified and used as necessary.

  From the result of mass spectrometry, the presence or absence and the amount of the target molecule in the test sample can be identified based on the molecular weight information of the target molecule. In this step, the information from the mass spectrometer may be output as differential information by comparing with mass spectrometry data in a biological sample derived from a non-breast cancer patient or a healthy person using an arbitrary program. Is possible. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.

  In a particularly preferred embodiment, each of the above steps is performed using a blot chip manufactured by Protocera as a plate for mass spectrometry, and the peptides of the present invention are quantitatively compared (differential analysis) using a MALDI mass spectrometer. Furthermore, if necessary, the peptides remaining on the same chip can be identified. Alternatively, a quantitative comparison (differential analysis) of the test sample is performed using a blot chip system manufactured by Protocera, and the peptide is identified by a combination device (LC-MS / LC / MS / MS / MS). MS).

  Measurement of the peptide of the present invention in the test method of the present invention can also be performed using an antibody against it. In such a method, if an optimized immunoassay system is constructed and a kit is made, the peptide can be detected with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.

  An antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a biological sample derived from a patient expressing the peptide and immunizing an animal using the peptide as an antigen. Alternatively, when the amount of the obtained peptide is small, the peptide is partially digested with peptidase or the like, the amino acid sequence of the obtained fragment is determined by the Edman method or the like, and the nucleic acid encoding the peptide based on the sequence A hybridizable oligonucleotide is synthesized and a cDNA library derived from the patient is used as a template to obtain a cDNA encoding a protein containing the peptide, or a cDNA derived from the patient is used as a primer. By performing RT-PCR using RNA as a template, a cDNA fragment encoding the peptide is obtained, the cDNA fragment is incorporated into an appropriate expression vector, introduced into an appropriate host cell, and the resulting transformant is cultured. Thus, the peptide of the present invention can be prepared in large quantities by collecting the recombinant peptide. You can. Alternatively, the peptide of the present invention can be obtained by using a cell-free transcription / translation system using the cDNA obtained as described above as a template. Further, it can be prepared in a large amount by an organic synthesis method.

  Alternatively, in the detection by mass spectrometry, the amino acid sequence of the peptide of the present invention is directly identified by using a tandem mass spectrometry (MS / MS) method, and all or part of the peptide is obtained based on the sequence information. It can be synthesized and used as an antigen (hapten). Peptide identification method using MS / MS method includes de novo sequencing method to determine amino acid sequence by analyzing MS / MS spectrum and partial sequence information (mass tag) contained in MS / MS spectrum A database search using the method for identifying peptides and the like can be mentioned.

  The antibody against the peptide of the present invention (hereinafter sometimes referred to as “the antibody of the present invention”) may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

  For example, the polyclonal antibody is cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin, if necessary) prepared by any of the above methods or other methods. Can also be administered as an antigen, together with a commercially available adjuvant (eg, complete or incomplete Freund's adjuvant) subcutaneously or intraperitoneally in animals every 2-4 weeks (partial) The antibody titer of the collected blood is measured by a known antigen-antibody reaction and the increase is confirmed), and it can be obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum. . Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the peptide of the present invention or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. Cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. The culture of the hybridoma producing the monoclonal antibody can be performed in vitro or in vivo such as mouse or rat, or preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively. .

  The test method of the present invention using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is determined by chemical or physical means. Any measurement method may be used as long as it is a measurement method that is detected and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

  For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, a test sample is reacted with an insolubilized antibody of the present invention (primary reaction), and another labeled antibody of the present invention is reacted (secondary reaction), followed by a labeling agent on an insolubilized carrier. By measuring the amount (activity), the amount of the peptide of the present invention in the test sample can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

The monoclonal antibody against the peptide of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competition method, the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( B / F separation), measure the amount of labeled B or F, and quantify the amount of antigen in the test sample. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or The first antibody is soluble, and the second antibody is a solid phase method using a solid phase antibody.
In the immunometric method, the antigen of the test sample and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen and excess in the test sample are separated. Then, the solid phase antigen is added, then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the peptide of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)), ibid Vol 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (Above, published by Academic Press) It is possible to irradiation.

  Since the peptide of the present invention is composed of proteolytic products, various molecules such as undegraded proteins and similar peptides having a common cleavage site may affect the measurement value in the usual “sandwich ELISA system”. Therefore, in the first step, a biological sample is immunoaffinity purified with an antibody, the fraction bound to the antibody is subjected to mass spectrometry in the second step, and so-called immunomass spectrometry, in which identification and quantification are performed based on a precise molecular weight. (See, for example, Rapid Commun. Mass Spectrom. 2007, 21: 352-358). For example, when a blood sample is used as a biological sample, a biomarker peak is not observed even if the sample is directly measured with a MALDI mass spectrometer. However, according to immunomass spectrometry, undegraded protein is also a similar peptide. Is completely separated by a mass spectrometer, and can be quantified with high specificity and sensitivity based on the exact molecular weight of the biomarker.

  Alternatively, as another test method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, and a test sample is applied to the antibody on the probe. Examples include a method of detecting a peak corresponding to the molecular weight of a marker peptide recognized by the antibody by subjecting the biological sample component captured by the antibody to mass spectrometry.

  When the level of the peptide of the present invention in a sample derived from a subject measured by any of the above methods is significantly varied compared to the level of the peptide in a control sample derived from a non-breast cancer patient or a healthy person, The subject can be diagnosed as having a high probability of having breast cancer.

  More specifically, when the peptide of the present invention is peptide 1 (about 2030), peptide 2 (about 2143), peptide 3 (about 2553) or peptide 6 (about 3836), the level of the peptide in the sample is If the level of the peptide in the control sample is significantly reduced, the subject can be diagnosed as likely to have breast cancer.

  On the other hand, the peptide of the present invention is peptide 4 (about 2860), peptide 5 (about 3570), peptide 7 (about 3947), peptide 8 (about 4829), peptide 9 (about 6739) or peptide 10 (about 9990). In some cases, if the level of the peptide is significantly elevated compared to the level of the peptide in a control sample, the subject can be diagnosed as likely to have breast cancer.

  Each of the 10 peptides of the present invention can be used alone as a diagnostic marker for breast cancer, but by combining two or more of them, sensitivity (prevalence of correct diagnosis) and specificity (prevalence of non-pathological diagnosis) Can be increased. The number of peptides used as markers and combinations thereof are not particularly limited, but preferably 2 to 5 types, more preferably peptide 1 (about 2030), peptide 2 (about 2143), peptide 5 (about 3570), peptide 6 2 to 5 peptides selected from 5 peptides (approximately 3836) and peptide 8 (approximately 4829).

  As a diagnostic method when two or more peptides are used as markers, for example, (1) when the level of all peptides to be measured varies significantly, it is determined that the cancer is breast cancer, and the level of any peptide (2) If the level of all peptides to be measured does not vary significantly, it is determined that the cancer is not breast cancer, and the level of any of the peptides varies significantly. (3) Of the n peptides to be measured, for example, 2 to (n-1) or more peptides are breast cancer when the level varies significantly And a method of giving a weight between peptides. However, according to the above method (1), the frequency of false positives can be reduced (that is, the specificity can be increased), but the frequency of false negatives increases, and the sensitivity may decrease. On the other hand, according to the method (2), the frequency of false negatives can be reduced (that is, the sensitivity can be increased), but the frequency of false positives increases and the specificity may be lowered. Preferably, a discriminant (multiple equation) is created based on the measured values of each peptide in a sample derived from a known number of known breast cancer patients and non-breast cancer controls, and the actual value of each peptide in the subject-derived sample is used as the discriminant. To find a solution, and according to the value, determine whether the subject has breast cancer.

  However, when a method with a large coefficient of variation (cv) such as mass spectrometry is used as the peptide measuring means of the present invention, it is difficult to create the discriminant, so in this embodiment, a group learning method is used. It is preferable to determine whether the subject has breast cancer. The group learning method is a method of improving accuracy by combining a plurality of results that are not necessarily highly accurate. Typical examples include a bagging method, a boosting method, a random forest method, and the like, and any method can be applied in the inspection method of the present invention, but the random forest method is preferable.

  The diagnosis of breast cancer using the random forest method will be described using Example 1 as a specific example. First, 30 known breast cancer patients and 23 breast benign tumor patients are divided into a training set (22 breast cancers, 16 benign tumors) and a test set (8 breast cancers, 7 benign tumors). For 10 training sets Bi (i = 1-10) randomly generated, peptide 1 (about 2030), peptide 2 (about 2143), peptide 5 (about 3570), peptide 6 (about 3836) and peptide 8 ( The maximum undecidated decision tree Ti is generated using five types of peptides of about 4829) as variables. Since each branch node when constructing Ti generates many different trees, m variables are sampled at random, and the variable with the best branch is used. Fit each test set's data to each decision tree and calculate sensitivity and specificity. In Example 1, the average sensitivity of 10 times is about 98% and the average specificity is about 89%, which indicates that the diagnosis by this method is extremely reliable. Next, peptides 1, 2, 5, 6, and 8 in the sample derived from the subject are measured, and the obtained measurement data is applied to each of the decision trees Ti to determine whether the subject's tumor is malignant or benign. A majority decision is made on the 10 judgment results Ri, and finally malignant / benign are judged.

  In another embodiment, when 2 or 3 peptides of the invention are used as markers, the measured values of each peptide in a sample from a known number of breast cancer patients and non-breast cancer controls in 2D or 3D coordinates. By plotting and creating a scatter plot, it is visualized in which region breast cancer and non-breast cancer are distributed, and then by plotting the measured values of each peptide in the subject-derived sample on the scatter plot, It can be easily determined whether or not the subject suffers from breast cancer. The determination can also be made by determining the cut-off value of each peptide based on the scatter diagram and comparing the measured value of each peptide in the subject-derived sample.

  The test method of the present invention is preferably carried out by collecting biological samples from patients in time series and examining changes with time in the expression of the peptide of the present invention in each sample. The collection interval of the biological sample is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL. For example, when plasma or serum is used as a sample, blood is collected at intervals of about 1 minute to about 12 hours. It is preferable to carry out. Among the peptides of the present invention, peptide 1 (about 2030), peptide 2 (about 2143), peptide 3 (about 2553) and peptide 6 (about 3836) tend to decrease in serum and plasma levels as breast cancer progresses. is there. Therefore, if the level of these peptides increases over time, it can be determined that there is a high probability that breast cancer in the patient has improved. On the other hand, peptide 4 (about 2860), peptide 5 (about 3570), peptide 7 (about 3947), peptide 8 (about 4829), peptide 9 (about 6739) and peptide 10 (about 9990) progress in breast cancer. Serum and plasma levels tend to increase according to Therefore, when the level of these markers decreases with time, it can be determined that there is a high possibility that breast cancer in the patient is improved.

  Further, the breast cancer inspection method based on the above time-series sampling has a therapeutic effect when the treatment for the disease is performed on the patient who is the subject between the previous sampling and the current sampling. Can be used to evaluate That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.

  Furthermore, the peptide of the present invention can provide an aggressive target for breast cancer drug discovery in addition to diagnosis. That is, when the peptide itself has a physiological function in the direction of treatment (remission) of the disease (referred to as “therapeutic peptide”), by administering to the patient a substance that increases the amount or activity of the peptide, When the peptide itself has physiological functions in the direction of exacerbation of the disease (referred to as “exacerbation peptide”), the disease can be treated by administering a substance that reduces the amount or activity of the peptide.

  The invention also increases the amount or activity of the peptide when the peptide of the invention acts as a therapeutic peptide, and / or the amount or amount of the peptide when the peptide of the invention acts as an exacerbation peptide. A method of treating breast cancer by reducing activity is provided. The therapeutic method specifically comprises an effective amount of a substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide, Administration to breast cancer patients. Accordingly, the present invention also comprises a substance for increasing the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance for reducing the amount or activity of the peptide of the present invention as an exacerbation peptide. Provide the agent.

Specifically, examples of the substance that increases the activity of the peptide of the present invention as a therapeutic peptide include the peptide itself or a molecule having an agonistic action similar thereto. Alternatively, as a substance that increases the activity of the peptide of the present invention as a therapeutic peptide, a non-neutralizing antibody, preferably an agonist antibody, of the peptide can also be mentioned. On the other hand, examples of the substance that reduces the activity of the peptide of the present invention as an exacerbation peptide include a molecule having an antagonistic action of the peptide, or a neutralizing antibody against the peptide.
The substance that increases the production of the peptide of the present invention as a therapeutic peptide includes a degrading enzyme that releases the peptide from a parent protein present in the living body, and the degradation on the N-terminal side and / or C-terminal side of the peptide. A substrate or substrate analog molecule of the decomposing enzyme further comprising an amino acid sequence recognized and cleaved by an enzyme, a molecule (including an analogous compound) that promotes the production of the decomposing enzyme, a molecule that promotes the activity of the decomposing enzyme, Examples include molecules that suppress the production of inhibitors of degradation enzymes. The presence of a degrading enzyme that liberates the peptide is suggested from the amino acid sequence at the N-terminal side and / or C-terminal side of the peptide, and degradation using the amino acid sequence at the N-terminal side and / or C-terminal side of the peptide as a probe Enzyme search and identification becomes possible. A substrate or substrate analog molecule of a decomposing enzyme thus identified, that is, a peptide molecule further comprising an amino acid sequence recognized and cleaved by the decomposing enzyme on the N-terminal side and / or C-terminal side of the peptide, In this way, the peptide of the present invention or its analog molecule as a therapeutic peptide is released by being cleaved by the degrading enzyme, so that the same therapeutic effect can be obtained. On the other hand, substances that promote the production and / or activity of the identified degrading enzymes can also indirectly increase the production of the peptides of the invention as therapeutic peptides. If the target degrading enzyme is identified, these substances can be screened or molecularly designed by a method known per se.

  On the other hand, the substance that reduces the production of the peptide of the present invention as an exacerbation peptide includes a molecule that suppresses the production of a degrading enzyme that liberates the peptide from a protein present in the living body, an inhibitor of the decomposing enzyme, and the production of the inhibitor And molecules that promote The degrading enzyme that liberates the peptide of the present invention as an exacerbation peptide can be searched and identified by the same method as the peptide of the present invention as the therapeutic peptide. By using a degrading enzyme thus identified, a substance that suppresses (inhibits) the production or activity of the degrading enzyme directly or indirectly can be screened or molecularly designed by a method known per se.

Substances that increase the amount or activity of the peptides of the invention as therapeutic peptides and substances that reduce the amount or activity of the peptides of the invention as exacerbation peptides can be formulated according to conventional means.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-mentioned compound or a salt thereof with an ordinary suppository base.

The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of dosage forms of such dosage units include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for injections, for each dosage unit dosage form. Other dosage forms preferably contain 10 to 250 mg of the above compound.
Each of the above-described compositions may be mixed with a substance that increases the amount or activity of the peptide of the present invention as the therapeutic peptide or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. Other active ingredients may be contained as long as the action is not caused.

Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, orally or parenterally to humans.
A substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide are determined by its action, administration route, patient severity, Although there are differences depending on age, weight, drug acceptability, etc., for example, the amount of active ingredient per day for an adult is about 0.0008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg, This can be administered once or in several divided doses.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Reference Example 1 Profiling analysis using BlotChip Serum from 30 breast cancer patients (early 25 cases, late 3 cases, 2 unknown cases) and 1.5 μL serum from 23 patients with benign mammary tumor patients (NuPAGE) (Trademark) LDS Sample Buffer 4x; Invitrogen) was mixed with 4.5 μL, heat-treated at 70 ° C. for 10 minutes, applied to 4-12% gradient polyacrylamide gel (Invitrogen), and electrophoresed. After the completion of electrophoresis, the gel was cut out, laminated on BLOTCHIP (registered trademark) (Protosera, Inc.), and transferred in an electric transfer buffer (BLOTBuffer ^™ ; Protosera, Inc.) at 90 mA for 120 minutes. After transfer, rinse the surface of the chip with ultrapure water, apply matrix (α-Cyano-4-hydroxy cinamic acid) to the entire chip, and then apply matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) Mass spectrometry was performed with a mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics). The measurement parameters were Detector voltage 1685V, Supression 1000, Laser Intensity 28-35 Fuzzy mode, 415 points per chip, 500 times laser irradiation per chip, total 207,500 times laser irradiation. Each peak intensity in the obtained spectrum was integrated for each M / z and converted into one integrated spectrum. Using ClinProTools (Bruker Daltonik GmbH) for the integrated spectrum, differential profiling analysis is performed between breast cancer patient serum and benign tumor patient serum, giving a P value of 0.05 or less regardless of peak intensity or malignant / benign peak ratio As a result of selecting a peptide group and excluding those which seemed inappropriate by visual observation, 52 marker candidates were extracted. Using these 52 markers, cluster (heat map) analysis was performed on the 53 cases. As a result, as shown in FIG. 1, clusters were formed with malignant tumors and benign tumors.

Reference Example 2 Random Forest and Reduction of Variables Random forest was performed using 43 biomarkers obtained by removing 1,000 biomolecules from the 52 biomarkers obtained in Reference Example 1. The 53 cases were divided into a test set (8 cases of malignancy, 7 cases of benign) and a training set (22 cases of malignancy, 16 cases of benign), and clustering was performed on 15 randomly selected training sets. Each set was verified using test set data. As a result, it was shown that malignancy / benignity of breast tumors can be determined with extremely high accuracy, with an average sensitivity of 97.5% and an average specificity of 93.3% (Table 1).

Example 1 Extraction of 5 Biomarkers by Random Forest Using the 52 biomarkers obtained in Reference Example 1, the above 53 cases were clustered by random forest, and the importance ranking of variables was constructed to rank the top 5 Individual biomarkers (molecular weights of about 2030, about 2143, about 3570, about 3836, about 4829) were selected. Using these 5 biomarkers, a random forest was performed again on the training set (22 malignant cases, 16 benign cases) and the results were randomly assigned to 10 test sets (8 malignant cases, 7 benign cases). Example), and sensitivity and specificity were calculated. As a result, it was revealed that malignancy / benignity of breast tumors can be determined with extremely high accuracy using only 5 biomarkers with an average sensitivity of 97.5% and an average specificity of 88.6% (Table 2). In addition, the serum levels of the five selected biomarkers were displayed as boxplots for each malignant tumor (30 cases) and benign tumors (23 cases). It was shown that a large marker was selected (Figure 2).

Example 2 Selection of Biomarkers by Differential Profiling Analysis From 52 biomarkers obtained in Reference Example 1, P value of 0.01 or less, peak intensity (patient or control, whichever is greater) 20 or more, patient / control peak ratio The markers were narrowed down based on the criteria that is greater than 2 or less than 0.5. As a result, six biomarkers (molecular weights of about 2143, about 2553, about 2860, about 3947, about 6739, and about 9990, respectively) were extracted (FIGS. 3A to F). The biomarker having a molecular weight of about 2143 was the same peptide as one of the markers selected by the random forest method in Example 1.
Next, when plotting the peak intensity of the biomarker with a molecular weight of 2143 and the peak intensity of each of the other five biomarkers to create a two-dimensional scatter plot, the cluster area of the malignant tumor and the benign tumor It was clearly distinguished from the cluster region (FIGS. 4A-E).

  The clinical examination method using the novel breast cancer diagnostic marker of the present invention is useful in that it can quickly and accurately determine breast cancer, and thus enables early detection and early treatment of the disease. In addition, since the peptide of the present invention, which is a measurement target in the present invention, can itself be a drug discovery target in these diseases, it can be used for screening for new therapeutic agents for breast cancer and for treating the diseases using them. It is extremely useful.

It is a figure which shows the result of a cluster analysis of 30 malignant tumors (breast cancer) and 23 benign tumors using 52 biomarkers selected in Reference Example 1. Of the five biomarkers selected in Example 1, 1. 30 malignant tumors (breast cancer) and It is a box-and-whisker plot of serum levels in 23 benign tumors. Integrated spectrum of peaks (left figure) and plot of peak intensity in each case (right figure) of 30 malignant tumors (breast cancer) and 23 benign tumors of the biomarker selected in Example 2 (molecular weight about 2143) FIG. Integrated spectrum of peaks (left figure) and plots of peak intensities in each case (right figure) of the biomarkers selected in Example 2 (molecular weight about 2553) in 30 cases of malignant tumor (breast cancer) and 23 cases of benign tumor FIG. Integrated spectrum of peaks (left figure) and plots of peak intensities in each case of the biomarkers (molecular weight of about 2860) selected in Example 2 in mass analysis of 30 cases of malignant tumors (breast cancer) and 23 cases of benign tumors (right) FIG. Integrated spectrum of peaks (left figure) and plot of peak intensity in each case (right figure) of 30 malignant tumors (breast cancer) and 23 benign tumors of the biomarker selected in Example 2 (molecular weight about 3947) FIG. Integrated spectrum of peaks (left figure) and plots of peak intensities in each case (right figure) of 30 malignant tumors (breast cancer) and 23 benign tumors of the biomarker selected in Example 2 (molecular weight about 6739) FIG. Integrated spectrum of peaks (left figure) and plot of peak intensities in each case of the biomarkers (molecular weight about 9990) selected in Example 2 in mass analysis of 30 cases of malignant tumor (breast cancer) and 23 cases of benign tumor (right) FIG. It is a two-dimensional scatter diagram of peak intensities in mass spectrometry of 30 cases of malignant tumors (breast cancer) and 23 cases of benign tumors of the biomarker having a molecular weight of about 2143 and the biomarker having a molecular weight of about 2553 selected in Example 2. It is a two-dimensional scatter diagram of peak intensities in mass spectrometry of 30 malignant tumors (breast cancer) and 23 benign tumors of the biomarker having a molecular weight of about 2143 and the biomarker having a molecular weight of about 2860 selected in Example 2. It is a two-dimensional scatter diagram of peak intensities in mass spectrometry of 30 malignant tumors (breast cancer) and 23 benign tumors of the biomarker having a molecular weight of about 2143 and the biomarker having a molecular weight of about 3947 selected in Example 2. It is a two-dimensional scatter diagram of peak intensities in mass spectrometry of 30 cases of malignant tumor (breast cancer) and 23 cases of benign tumor of the biomarker having a molecular weight of about 2143 and the biomarker having a molecular weight of about 6739 selected in Example 2. It is a two-dimensional scatter diagram of peak intensities in mass spectrometry of 30 cases of malignant tumor (breast cancer) and 23 cases of benign tumor of the biomarker having a molecular weight of about 2143 and the biomarker having a molecular weight of about 9990 selected in Example 2.

Claims (10)

  Selected from the group consisting of 10 peptides having a molecular weight of about 2030, about 2143, about 2553, about 2860, about 3570, about 3836, about 3947, about 4829, about 6739 and about 9990 in a biological sample collected from a subject. A test method for diagnosing breast cancer in the subject, comprising measuring the amount of one or more peptides.   The method of claim 1, wherein the peptide to be measured is selected from the group consisting of five peptides having molecular weights of about 2030, about 2143, about 3570, about 3836 and about 4829.   The method according to claim 1 or 2, wherein the number of peptides to be measured is 2 to 5.   The method of claim 2, wherein the peptides to be measured are five peptides having a molecular weight of about 2030, about 2143, about 3570, about 3836 and about 4829.   The method according to claim 1, wherein the biological sample is a body fluid.   The method according to claim 5, wherein the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid. .   The method according to claim 1, comprising subjecting the biological sample to mass spectrometry.   The method according to any one of claims 1 to 6, wherein an antibody that specifically recognizes each peptide is used.   The method according to any one of claims 1 to 8, wherein a biological sample is collected from a patient in time series, and a change with time of the amount of each peptide in the sample is examined.   A test method for evaluating therapeutic effects in breast cancer patients, wherein the molecular weights of biological samples collected from the patient before and after treatment are about 2030, about 2143, about 2553, about 2860, about 3570, about 3836 A change in the amount of one or more peptides selected from the group consisting of 10 peptides of about 3947, about 4829, about 6739 and about 9990.