JP7336097B2 - Peptide markers for breast cancer - Google Patents

Description

The present invention relates to a method for detecting breast cancer using peptide markers, and more specifically, determination of breast cancer using peptide markers, determination of breast cancer patients (responders) who respond to therapeutic drugs (companion diagnostic method), and detection of preventive effects. The present invention relates to determination, determination of therapeutic effect, test method for early diagnosis, test method for early treatment, and screening method for substances.

Breast cancer is one of the most common cancers in Japan, and the estimated number of breast cancer cases in 2018 is 86,500, and the number of deaths is 14,800 women, accounting for about 9% of all cancer deaths among women. (National Cancer Center, September 15, 2018).

There are the following types of means (breast cancer screening) for examining whether a patient has breast cancer: (1) interview/physical examination, (2) mammography screening, and (3) ultrasound screening (echo).

However, (1) it is difficult to detect breast cancer early by interview/physical examination, and it is difficult to distinguish unless the tumor is larger than a certain size. (2) Mammography screening is not suitable for young women because it is difficult to distinguish between mammary glands and cancer in young women with well-developed mammary glands. In addition, there are problems such as pain and radiation exposure. (3) Ultrasound examination (echo) requires a high level of skill of the doctor who makes the diagnosis. Microcalcifications are also difficult to detect.

Under these circumstances, if a simple and high-performance method for detecting breast cancer from a single drop of blood with minimal invasiveness is provided, the problems of existing detection methods will be resolved, contributing to preventive medicine and early treatment of breast cancer. Furthermore, it is possible to select responders for treatment effect (prognosis) and treatment method selection.

Peptidome analysis is a systematic and comprehensive analysis of endogenous small proteins and peptides less than 20 kDa. Peptidome analysis includes all the information gleaned from the genome and transcriptome, including post-translational modifications (phosphorylation, glycosylation, etc.), and provides insight into biological systems, including structures, physicochemical properties and their functions. A large-scale analysis of all proteins in , which integrates genetic and epigenetic features to detect dynamic changes in protein production and degradation. Also, both the concentrations of proteins and their post-translational modifications can change during disease progression, both demonstrating the risk of contracting the disease and assisting in monitoring therapeutic efficacy.

The applicant, Protocera, has simplified the process from one-dimensional electrophoresis to mass spectrometry (MALDI-TOF-MS) and has developed a new measurement plate (BLOTCHIP) for MALDI-TOF-MS that enables high-speed processing of large volumes of samples. (registered trademark)) was developed (Non-Patent Document 1). The BLOTCHIP®-MS method is the only method that can measure the total amount of peptides adsorbed to proteins and the total amount of free peptides. trademark). The adsorbed and free peptides on the BLOTCHIP® are then measured by MALDI-TOF-MS. In other words, there is no need for pretreatment such as protein removal or enzymatic digestion, and at low concentrations (femtomoles to picomoles) that could not be detected in the past, MALDI-TOF (/TOF) can be used to collectively search for peptides in the molecular weight range of 1-20 kDa. Quantitative analysis and sequence analysis can be handled.

Tanaka K. et al., Biochem. Biophys. Res. Commun. 379, 110-114. (2009)

An object of the present invention is to detect breast cancer by measuring biomarker peptides in biological samples of subjects. A further object is to detect patients who respond well to the therapy (responders) before selecting the therapy. A further object is to detect the effect (prognosis) of a therapeutic method by measuring a biomarker peptide in a biological sample of a patient administered a therapeutic agent.

In order to achieve the above object, the present inventors investigated serum samples of breast cancer patients, and discovered 7 types of biomarker candidate peptides, thereby completing the present invention.

That is, according to the present invention, the following aspects are provided.

Item 1, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 3, and a sequence in a biological sample of a subject A peptide consisting of the amino acid sequence represented by number 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 A method for detecting breast cancer in a subject, comprising measuring one or more peptides selected from the group consisting of peptides.

Item 2, the biological sample is selected from the group consisting of blood, plasma, serum, saliva, urine, cerebrospinal fluid, bone marrow fluid, pleural fluid, ascitic fluid, synovial fluid, sweat, tears, aqueous humor, vitreous humor and lymphatic fluid Item 1. The method of Item 1, comprising a bodily fluid.

Item 3, the method of Item 1 or 2, comprising subjecting the biological sample to mass spectrometry.

Item 4, peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, amino acid represented by SEQ ID NO: 4 selected from the group consisting of a peptide consisting of the sequence, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 Item 3. The method according to Item 1 or 2, wherein an antibody that specifically recognizes the peptide is used.

Item 5, the detection of breast cancer includes determination of breast cancer, determination of breast cancer preventive effect, determination of breast cancer therapeutic effect, determination of breast cancer patients who respond to therapeutic drugs, determination of therapeutic drugs that respond to individual breast cancer patients, breast cancer Item 5. The method according to any one of Items 1 to 4, which is a testing method for diagnosing or treating breast cancer.

Item 6, peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, amino acid represented by SEQ ID NO: 4 selected from the group consisting of a peptide consisting of the sequence, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 A breast cancer detection kit comprising an internal standard in which one or more peptides or fragment ions thereof are labeled with stable isotopes.

Item 7, stable isotope-labeled peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, A peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 breast cancer detection agent containing one or more peptides selected from the group consisting of peptides or fragment ions thereof.

Item 8, A computer-implemented method for determining the susceptibility of breast cancer in a subject, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, in a biological sample of the subject a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5; Acquiring quantitative data on one or more peptides selected from the group consisting of a peptide consisting of the amino acid sequence represented by number 6 and a peptide consisting of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 and
applying the obtained data to a multivariate logistic regression model that is a function of the one or more peptides to determine a predictive value of breast cancer susceptibility in a subject.

Item 9, peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, amino acid represented by SEQ ID NO: 4 selected from the group consisting of a peptide consisting of the sequence, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 as a marker for detecting breast cancer.

Item 10, peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, amino acid represented by SEQ ID NO: 4 selected from the group consisting of a peptide consisting of the sequence, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 as a marker for detecting the responsiveness of breast cancer patients to therapeutic agents.

Item 11, peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, amino acid represented by SEQ ID NO: 4 A peptide consisting of a peptide consisting of the sequence, a peptide consisting of the amino acid sequence represented by SEQ ID NO:5, a peptide consisting of the amino acid sequence represented by SEQ ID NO:6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO:7.

According to the present invention, since breast cancer can be determined rapidly and with extremely high reliability from an early stage, it is possible to determine the disease, determine breast cancer patients (responders) who respond to therapeutic agents (companion diagnostic method), and determine preventive effects. , determination of therapeutic effects, early diagnosis, testing methods for early treatment, and screening methods for substances.

A preferred embodiment of the present invention enables diagnosis of stage 0, which has been difficult with conventional examination methods. Therefore, since breast cancer can be determined quickly and with extremely high reliability from an early stage, it is possible to determine the disease, determine breast cancer patients (responders) who respond to therapeutic agents (companion diagnostic method), determine preventive effects, and determine therapeutic effects. Test methods for determination, early diagnosis, early treatment, and screening methods for substances become possible.

The 10-year survival rate for Stage 0 is almost 100%, and we can expect the advent of an era in which breast cancer is no longer a disease for women.

Because it is a simple test, early detection can be achieved by conducting tests in a short time span for women who are considered to have a high risk of developing breast cancer due to familial or hereditary factors.

In addition, similar to the already marketed Protkey colorectal cancer risk test (Protocella), it is expected that a method will be established for use in breast cancer screening to determine the effectiveness of treatment. In other words, a flow is assumed in which early-stage breast cancer (stage 0) is discovered when the risk value increases through chronological examinations, and the risk value decreases after removal. Furthermore, it is expected that it will be possible to diagnose recurrence after breast cancer surgery and to determine the effect of recurrence treatment.

In addition, responders when selecting treatment methods for breast cancer (e.g., surgery, breast reconstruction, radiotherapy, drug therapy, hormone therapy (endocrine therapy), chemotherapy (cytotoxic anticancer agents), recurrence, metastasis, etc.) It is also expected that companion diagnosis will become possible as an examination method of choice.

Boxplots showing predicted probabilities for each stage of breast cancer (stages 0-III) by regression model.

The present invention provides novel and useful breast cancer detection marker peptides (hereinafter sometimes collectively referred to as "peptides of the present invention").

In the present specification, "detection" of breast cancer includes determination of breast cancer, determination of breast cancer patients (responders) who respond to therapeutic drugs (companion diagnostic method), determination of preventive effect of breast cancer, determination of therapeutic effect of breast cancer. , an examination method for breast cancer diagnosis (particularly early diagnosis), and an examination method for breast cancer treatment (particularly early treatment). "Determination" of breast cancer includes not only determining the presence or absence of breast cancer, but also prophylactically determining the possibility of developing breast cancer, predicting the prognosis of breast cancer after treatment, and determining the availability of therapeutic agents for breast cancer. Determining therapeutic efficacy is included. Screening methods for substances include screening methods for substances useful in "detection", "determination" and "treatment" of breast cancer.

As used herein, "morbidity" includes "development."

As used herein, "treatment" means curing or ameliorating a disease or symptom, or suppressing symptoms, and includes "prevention." "Prevention" means to forestall the onset of a disease or condition.

As used herein, the term "peptide" refers to a molecule formed by binding 2 to 50 amino acids.

The present invention provides a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 3, in a biological sample of a subject. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 A method for detecting breast cancer in the subject, comprising measuring one or more peptides selected from the group consisting of peptides.

The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a partial sequence of the fibrinogen α-chain (isoform 1, hereinafter the same unless otherwise specified). A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a partial sequence of the fibrinogen α-chain. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 is a partial sequence of α-2-HS-glycoprotein. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is a partial sequence of the fibrinogen α-chain. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 5 is a partial sequence of fibrinogen α-chain. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is a partial sequence of fibrinogen α-chain isoform 2. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is a partial sequence of α-2-HS-glycoprotein. Peptides encompassed by the present invention and methods of testing, determination, diagnosis, diagnosis, and substance screening using them are applicable to the detection of breast cancer.

The peptides used in the method for detecting breast cancer in a subject of the present invention include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a peptide found in a biological sample of a subject. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 and a peptide consisting of the amino acid sequence represented by SEQ ID NO:7. Measurements of these peptides correlate with breast cancer incidence. The amino acid sequences of SEQ ID NOs: 1-7 are shown in Table 1 below.

As used herein, when referring to "a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1", unless otherwise specified, such peptides include those represented by SEQ ID NO: 1 in which each amino acid is unmodified In addition to peptides consisting of the amino acid sequence represented by SEQ ID NO: 1, peptides consisting of the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids are modified while maintaining the type of amino acid are also included. Such modifications include oxidation by binding of an oxygen atom, phosphorylation, N-acetylation, S-cysteinylation, and the like. "Peptide consisting of the amino acid sequence represented by SEQ ID NO: 2", "Peptide consisting of the amino acid sequence represented by SEQ ID NO: 3", "Peptide consisting of the amino acid sequence represented by SEQ ID NO: 4", "Peptide consisting of the amino acid sequence represented by SEQ ID NO: 5" Similarly, unless otherwise specified, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 6, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of an amino acid sequence represented by SEQ ID NO: 7 Peptides in which each amino acid is unmodified or modified are intended to be included.

In this specification, an amino acid residue contained in an amino acid sequence may be simply referred to as "amino acid".

Unmodified amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. It refers to 20 kinds of amino acids that constitute proteins encoded by genetic information. A modified amino acid refers to an amino acid that has been chemically modified after protein synthesis.

The number of modified amino acids is not limited. For example, it can be about 1 to 5 pieces, preferably about 1 to 3 pieces, more preferably about 1 or 2 pieces, and still more preferably 1 piece.

Examples of specific amino acid modifications of the peptide of the present invention include oxygen oxidation of the 9th (536th from the N-terminal of the fibrinogen α-chain) methionine in the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (methionine sulfoxide (methionine sulfoxide)), oxidation of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (358th from the N-terminus of α-2-HS-glycoprotein) (cysteic acid), SEQ ID NO: Oxidation of methionine (methionine sulfoxide) at position 9 (536th from the N-terminus of the fibrinogen α-chain) of the peptide consisting of the amino acid sequence represented by 4, consisting of the amino acid sequence represented by SEQ ID NO: 6 Oxidation of methionine with oxygen (methionine sulfoxide) at the 28th peptide (603rd from the N-terminus of fibrinogen α-chain isoform 2), 18th peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (α 358th from the N-terminus of 2-HS-glycoprotein) S-cysteinylation (cystine). Such cases are also included in the scope of the present invention as long as they are peptides consisting of the amino acid sequences represented by SEQ ID NOS: 1-7. Of course, SEQ ID NOS: 1 to 7 (for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: A peptide consisting of the amino acid sequence represented by 2) in which all amino acids are unmodified is also included in the present invention. The modified peptides consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 7 can be distinguished from unmodified peptides by mass spectrometry, and can be used for detection, determination and treatment of diseases. It is within the scope of the invention to use any of the modified peptides in appropriate circumstances.

The present invention includes a method for detecting or determining breast cancer in a subject, comprising measuring one or more of the seven peptides of the present invention in a biological sample of the subject.

In a further preferred embodiment, the method for detecting or determining breast cancer in a subject includes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 in a biological sample of the subject. , a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 and 2, 3, 4, 5, 6, or 7, preferably 3, 4, selected from the group consisting of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, It comprises measuring 5, 6 or 7, more preferably 4, 5 or 6 peptides.

A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 a plurality of amino acids from the group consisting of a peptide, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 When selecting peptides with the same amino acid sequence, a combination of one or more modified and unmodified peptides or a combination of multiple modified peptides with different modifications can be selected. For example, a peptide having an amino acid sequence in which the 18th Cys (cysteine) in the amino acid sequence represented by SEQ ID NO: 3 is an oxidized amino acid (cysteic acid), and an unmodified amino acid sequence represented by SEQ ID NO: 3 Peptides having the same can be used in combination.

Subjects include patients suspected of having breast cancer. not only those who wish to undergo preventive examinations), but also those who have been determined or diagnosed with breast cancer based on some objective evidence (e.g., conventionally known clinical tests (e.g., , breast interview, visual inspection and palpation, mammography examination, ultrasound (echo) examination, blood test, and "clinical and pathological breast cancer treatment regulations 18th edition" (edited by the Japanese Breast Cancer Society) and/or examination results, rationality of breast cancer (Persons who have been determined by a doctor to be potentially susceptible to disease.) "Measuring a peptide" refers to measuring the concentration, amount, or signal intensity of the peptide.

Although the subject-derived biological sample to be the test sample is not particularly limited, it is preferably one that is less invasive to the subject. and those that are relatively easily collected such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, synovial fluid, aqueous humor, vitreous humor, and lymph. In one embodiment, the biological sample is selected from the group consisting of blood, plasma, serum, saliva, urine, cerebrospinal fluid, bone marrow fluid, pleural fluid, ascitic fluid, synovial fluid, tear fluid, sweaty aqueous humor, vitreous humor and lymphatic fluid. Consists of body fluids. When serum or plasma is used, a test sample for analysis can be prepared by collecting blood from a subject according to a conventional method and directly without pretreatment, or by separating liquid components. If necessary, the peptide of the present invention to be detected can be separated and removed in advance from high-molecular-weight protein fractions using an antibody column, other adsorbent column, spin column, or the like.

The peptide of the present invention can be detected in a biological sample, for example, by subjecting the biological sample to various molecular weight measurement methods, such as gel electrophoresis, and various separation and purification methods (e.g., ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reversed-phase chromatography, etc.), surface plasmon resonance method, ionization method (e.g. electron impact ionization method, field desorption method, secondary ionization method, fast atom bombardment method, matrix-assisted laser desorption ionization (MALDI) method, electrospray) ionization method, etc.), and a mass spectrometer (e.g., double focusing mass spectrometer, quadrupole spectrometer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, immunomass spectrometer, A mass spectrometer using a stable isotope peptide as an internal standard, an MS-MS mass spectrometer using a stable isotope-labeled fragment ion as an internal standard, an immunomicroscope, a mass microscope, etc.) or fragment ions, spots, or peaks of the peptide, but are not limited to these.

A method of purifying the peptides of the present invention to prepare antibodies that recognize them, and detecting the peptides by ELISA, RIA, immunochromatography, surface plasmon resonance, western blotting, immunomass spectrometry, various immunoassays, and immunomicroscopy. can also be preferably used. Furthermore, a hybrid detection method of the above method is also effective.

One of the particularly preferred measurement methods in the detection or determination method of the present invention is to bring the test sample into contact with the surface of a plate used for time-of-flight mass spectrometry, and measure the mass of the component captured on the plate surface. A method of measuring with an analyzer can be mentioned. A plate compatible with a time-of-flight mass spectrometer has a surface structure (e.g., functional group-added glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicones, various gels or polymer coatings).

In a preferred embodiment, the support used as a mass spectrometry plate is a substrate thinly coated with polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF (WO 2004/031759 ). Such substrates are not particularly limited as long as they are used in mass spectrometry plates, and examples thereof include insulators, metals, conductive polymers, composites thereof, and the like. As such a plate for mass spectrometry coated with a thin layer of PVDF, BLOTCHIP (registered trademark) manufactured by Protocera Co., Ltd. is preferably used. Alternatively, the mass spectrometry plate can be prepared by known methods by coating the support surface by known means such as painting, spraying, vapor deposition, dipping, printing, sputtering and the like. Also, the method itself for mass spectrometry of molecules on a mass spectrometry plate is known (for example, WO 2004/031759). The method described in WO 2004/031759 can be used with appropriate modifications as necessary.

The transfer of the test sample to the mass spectrometry plate (support) can be carried out either by leaving the subject-derived biological sample as the test sample untreated, or after removing and concentrating high-molecular-weight proteins using an antibody column or other method. , SDS-polyacrylamide gel electrophoresis or isoelectric focusing, and after electrophoresis, the gel is brought into contact with a plate for transfer (blotting). The transfer method itself is known, and electric transfer is preferably used. As a buffer for electrotransfer, it is preferable to use a known buffer having a pH of 7 to 9 and a low salt concentration (eg, Tris buffer, phosphate buffer, borate buffer, acetate buffer, etc.).

By performing mass spectrometry on the molecules in the test sample captured on the support surface by the above method, the presence and amount of the peptide of the present invention, which is the target molecule, can be identified from the information on the molecular weight. Information from the mass spectrometer can be compared, using any program, to mass spectrometry data in biological samples from unaffected cancer patients, post-treatment patients (follow-up), or healthy individuals to determine the differential ( differential) information. 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 order to obtain highly accurate results of mass spectrometry, analysis is performed using a triple quadrupole mass spectrometer or the like connected to high-performance liquid chromatography. A stable isotope-labeled peptide of the target molecule is synthesized, mixed with a test sample as an internal standard in a known amount, and the peptide fraction is crudely purified using a reversed-phase solid-phase carrier or the like. After introduction into high-performance liquid chromatography, each separated peptide is ionized in a mass spectrometer, then fragmented in a collision cell, and the resulting peptide fragments are quantified by the multiple reaction monitoring method. In this case, by using a stable isotope-labeled peptide as an internal standard, measured data with a CV value of 5% or less can be obtained. Stable isotope ^- labeled peptides ^were purchased from Cambridge Isotope Laboratory (MA, USA _{) .} An existing synthetic method (e.g., by F-moc) is substituted at the sequence position of the original amino acid by replacing the amino acid a with 2 isotopic nitrogen (atomic weight 15) substitutions with an 8 atomic weight increase from the original amino acid. solid phase reaction). Mass spectrometry can also be performed with the BLOTCHIP (registered trademark) system from Protothera, Inc., and these methods can be clinically used as detection devices that do not use antibodies.

In the detection by mass spectrometry described above, peptides can be identified using a tandem mass spectrometry (MS/MS) method. and a method of identifying peptides by performing a database search using partial sequence information (mass tag) contained in the MS/MS spectrum. Also, by using the MS/MS method, the amino acid sequence of the peptide of the present invention is directly identified, all or part of the peptide is synthesized based on the sequence information, and used as an antigen for the following antibodies. can also

Measurement of the peptide of the present invention can also be performed using an antibody against it. Therefore, the present invention includes breast cancer detection or determination methods using antibodies that specifically recognize peptides, breast cancer detection or determination agents containing such antibodies, and breast cancer detection or determination kits containing such antibodies. Such a method can detect the peptide with high sensitivity and accuracy without using a special device such as the above mass spectrometer by constructing an optimized immunoassay system and making it into a kit. 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 patient-derived biological sample expressing the peptide, and immunizing an animal with the peptide as an antigen. Alternatively, when the amount of the peptide obtained is small, a large amount of the peptide can be prepared by well-known genetic engineering techniques such as amplification of a cDNA fragment encoding the peptide by RT-PCR, or such cDNA can be prepared. The peptide of the present invention can also be obtained using a cell-free transcription/translation system as a template. Furthermore, it is also possible to prepare a large amount by an organic synthesis method.

Antibodies against the peptides of the present invention (hereinafter sometimes referred to as "antibodies of the present invention") may be either polyclonal antibodies or monoclonal antibodies, and can be produced by well-known immunological techniques. The antibodies also include not only complete antibody molecules but also fragments thereof, such as Fab, F(ab')2, ScFv, minibodies, and the like.

For example, the polyclonal antibody is administered subcutaneously or intraperitoneally to an animal with a commercially available adjuvant (e.g., complete or incomplete Freund's adjuvant) using the peptide of the present invention as an antigen, about 2 to 4 times every 2 to 3 weeks, It can be obtained by collecting whole blood after the final immunization and purifying the antiserum. Animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, sheep, horses, guinea pigs, and hamsters from which the desired antibody can be obtained.

Monoclonal antibodies can also be produced by the cell fusion method. A description of techniques for preparing monoclonal antibodies can be found in Stites et al, Basic and Clinical Immunology. (Lang Medical Publications Los Altos. CA. ^4th Edition) and references therein, in particular Koehler, G. & Milstein, C. Nature 256, 495-497 (1975). For example, the peptide of the present invention is subcutaneously or intraperitoneally administered to mice 2 to 4 times together with a commercially available adjuvant, and after the final administration, the spleen or lymph nodes are collected, and leukocytes are collected. This leukocyte and myeloma cell (eg, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. Hybridomas that produce the desired monoclonal antibody can be selected by detecting antibodies that specifically bind to the antigen from the culture supernatant using well-known EIA or RIA methods. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo in mice or rats, preferably mouse ascites, etc. Antibodies can be obtained from hybridoma culture supernatants and animal ascites, respectively. .

The detection or determination method of the present invention using an antibody 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 detected by chemical or physical means. However, any measurement method may be used as long as it is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method, sandwich method and the like are preferably used. For measurement, antibodies or antigens can be conjugated with labeling agents such as radioisotopes, enzymes, fluorescent or luminescent substances. Furthermore, a biotin-avidin system can also be used for binding the antibody or antigen to the labeling agent. These individual immunoassay methods can be applied to the quantification method of the present invention according to the ordinary skill of those skilled in the art.

Since the peptide of the present invention is composed of proteolytic products, various molecules such as undegraded proteins and similar peptides having common cleavage sites may affect the measured values. Therefore, in the first step, the biological sample is immunoaffinity purified with an antibody, and the antibody-bound fraction is subjected to mass spectrometry in the second step to identify and quantify based on precise molecular weight, so-called immunomass spectrometry. can be utilized (see, for example, Rapid Commun. Mass Spectrom. 2007, 21: 352-358). According to immunomass spectrometry, both intact proteins and similar peptides are completely separated by mass spectrometry, enabling quantification with high specificity and sensitivity based on the exact molecular weight of biomarkers.

Alternatively, as another detection or determination method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a chip compatible with a mass spectrometer as described above, and the antibody on the chip is tested. Examples include a method of contacting a sample, subjecting biological sample components captured by the antibody to mass spectrometry, and detecting a peak corresponding to the molecular weight of the marker peptide recognized by the antibody.

The level of the peptide of the present invention in a subject-derived sample measured by any of the above methods is significantly higher than the peptide level in a control sample from a non-breast cancer patient, a post-treatment patient, or a healthy subject. If it varies, it can be determined that the subject is likely to have breast cancer.

Each of the peptides of the present invention can be used as a breast cancer detection marker alone, but by combining two or more of them, the sensitivity (prevalence accuracy) and specificity (no disease accuracy) can be further increased. can be done.

As a detection method when using two or more peptides as markers, for example, (1) when the levels of all the peptides to be measured vary significantly, it is determined that the patient has breast cancer; (2) determining that the patient does not have breast cancer when the levels of all the peptides to be measured do not significantly change; A method for determining that the patient has breast cancer when the level of any of the peptides varies significantly, (3) among the n peptides to be measured, for example, 2 to (n-1) or more For peptides, a method to determine that one has breast cancer if the level fluctuates significantly, a method to assign weights among peptides, and (4) mechanical methods such as bagging, boosting, and random forest methods. learning method, etc., but it is particularly preferable to use multivariate logistic regression analysis, which is an analysis method capable of handling a plurality of marker peptides as one marker set. In this case, the number of peptides used as markers is not particularly limited, but preferably two or more.

In one embodiment, the peptide used for the above analysis is at least one, preferably two or more, of the seven peptides consisting of the amino acid sequences represented by SEQ ID NOs: 1-7.

In the present invention, multivariate logistic regression models for candidate peptides identified by mass spectrometry were constructed by maximum likelihood and showed high ROC area under the curve (AUC) (e.g., greater than 0.85 for multiple marker peptides). ) It was found that detection or determination of breast cancer with extremely high reliability is possible.

The number of peptides to be detected or measured is preferably such that the AUC in the test method of the present invention exceeds a certain threshold. A typical threshold is 0.9. For example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and an amino acid represented by SEQ ID NO: 4 According to the logistic regression model, which is a function of the blood concentration of the peptide consisting of the sequence, the area under the curve (AUC) of ROC plotting sensitivity against specificity exceeds 0.85 (especially exceeds 0.9). ), which can detect breast cancer with extremely high accuracy.

Therefore, in a preferred embodiment of the detection or determination method of the present invention, the method for detecting or determining breast cancer in a subject includes: A peptide consisting of the amino acid sequence represented by , a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, and four peptides of SEQ ID NO: 4 are measured.

The detection method of the present invention can also be carried out by collecting biological samples from a patient in chronological order and examining changes over time in the expression of the peptide of the present invention in each sample. The sampling 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 QOL of the patient. It is preferable to The peptide of the present invention tends to change (increase or decrease) in serum or plasma levels as breast cancer improves. Therefore, when the levels of these markers change over time (increase or decrease, changes approaching the serum or plasma levels of healthy subjects), it is determined that there is a high possibility that breast cancer in the patient is improving. be able to.

Furthermore, the method for detecting breast cancer by time-series sampling is, between the previous sampling and the current sampling, if a therapeutic measure for the disease is taken for the patient who is the subject, by the measure It can be used to assess therapeutic efficacy. That is, if the sample sampled before and after the treatment is judged to show a decrease in peptide (improvement of pathology) compared to the condition before treatment, the treatment is evaluated as having an effect. can do. On the other hand, if there is no decrease in peptide (improvement of pathology) in the condition after treatment compared to the condition before treatment, or if it is judged to be worse, the treatment is evaluated as ineffective. can do.

Furthermore, the test method for detecting breast cancer by time-series sampling is to evaluate the preventive effect after taking measures to reduce the risk of developing breast cancer, such as intake of health foods, smoking cessation, exercise therapy, and isolation from harmful environments. can be used for That is, for samples sampled before and after the implementation of measures to reduce the risk of morbidity, when it is determined that the state after implementation does not increase peptides (onset or progression of pathology) compared to the state before implementation , it can be evaluated that the enforcement of the measure was effective. On the other hand, if it is judged that there is no decrease in peptide (improvement of pathology) in the condition after treatment compared to the condition before treatment, or the condition is further worsened, the effect of the implementation of the measure It can be evaluated that there was no

Therefore, the peptide and method of the present invention can serve not only as a marker for diagnosing or detecting breast cancer, but also as a marker for predicting the prognosis of breast cancer and a marker for judging therapeutic effects. That is, the peptide and method of the present invention can be used as a companion diagnostic for screening drug discovery target molecules for the treatment of breast cancer and/or for selecting patients (responders) or adjusting the dose (dosage) of therapeutic agents. can do.

Examples of breast cancer treatment methods for determining therapeutic efficacy include surgery, breast reconstruction, radiotherapy, drug therapy, hormone therapy (endocrine therapy), and chemotherapy. Drug therapy includes therapy using a molecular target drug such as trastuzumab.

In addition, the peptides and methods of the present invention can be used in substance screening methods. Substances in this case include health foods that prevent breast cancer at the pre-disease stage, foods for specified health uses (FOSHU products), foods with function claims, markers for diagnosing or detecting breast cancer, and breast cancer after diagnosis. Includes pharmaceuticals such as therapeutic agents for treating

In one embodiment, the present invention provides a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 breast cancer detection kits containing antibodies to 1, 2, 3, 4, 5, 6, or 7 peptides selected from the group consisting of As a preferred embodiment of the breast cancer detection kit of the present invention, a peptide having an amino acid sequence in which the 9th methionine in the amino acid sequence represented by SEQ ID NO: 1 is an oxidized amino acid (methionine sulfoxide), unmodified SEQ ID NO: 2 A peptide having an amino acid sequence represented by, a peptide having an amino acid sequence in which the 18th Cys (cysteine) in the amino acid sequence represented by SEQ ID NO: 3 is an oxidized amino acid (cysteic acid), and an unmodified SEQ ID NO: A breast cancer detection kit containing antibodies against four types of peptides having the amino acid sequence represented by 3 is exemplified.

In another embodiment, the present invention provides a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a sequence A peptide consisting of the amino acid sequence represented by number 4, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 A breast cancer detection agent containing, as a detection reagent, an antibody against 1, 2, 3, 4, 5, 6, or 7 peptides selected from the group consisting of peptides is included. As a preferred embodiment of the agent for detecting breast cancer of the present invention, a peptide having an amino acid sequence in which the 9th methionine in the amino acid sequence represented by SEQ ID NO: 1 is an oxidized amino acid (methionine sulfoxide), unmodified SEQ ID NO: 2 A peptide having an amino acid sequence represented by, a peptide having an amino acid sequence in which the 18th Cys (cysteine) in the amino acid sequence represented by SEQ ID NO: 3 is an oxidized amino acid (cysteic acid), and an unmodified SEQ ID NO: A breast cancer detection agent containing antibodies against four peptides of the peptide having the amino acid sequence represented by 3 can be mentioned.

In yet another embodiment, the invention provides a computer-implemented method for determining the susceptibility of breast cancer in a subject, wherein the amino acid sequence represented by SEQ ID NO: 1 a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4; 1, 2, 3, or 4 selected from the group consisting of a peptide consisting of an amino acid sequence, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 6, and a peptide consisting of an amino acid sequence represented by SEQ ID NO: 7. , obtaining quantitative data for 5, 6, or 7 peptides; and applying said obtained data to a multivariate logistic regression model that is a function of said plurality of peptides to determine breast cancer in a subject. and determining the predictive value of the likelihood of the disease. Here, the quantitative data of the peptide refers to quantitative measurement values such as the expression level and blood concentration of the peptide measured using, for example, mass spectrometry or an antibody against the peptide. As a preferred embodiment of the computer-implemented method for determining the morbidity of breast cancer in a subject of the present invention, the 9th methionine in the amino acid sequence represented by SEQ ID NO: 1 in the subject's biological sample is A peptide having an amino acid sequence that is an oxidized amino acid (methionine sulfoxide), a peptide having an unmodified amino acid sequence represented by SEQ ID NO: 2, and a peptide having an amino acid sequence represented by SEQ ID NO: 3 in which the 18th Cys (cysteine) is Obtaining quantitative data for a peptide having an amino acid sequence that is an oxidized amino acid (cysteinic acid) and a peptide having an unmodified amino acid sequence represented by SEQ ID NO: 3; applying a multivariate logistic regression model that is a function of the four peptides to obtain a predictive value for the likelihood of susceptibility to breast cancer in the subject.

The computer-implemented method may further comprise, after determining the predictive value, determining the likelihood of breast cancer in the subject based on the predictive value. For example, when the calculated predicted value exceeds a certain threshold, the subject is determined to have breast cancer. Usually the threshold is 0.5, preferably 0.1 or less.

EXAMPLES The present invention will be described in more detail with reference to examples below, but it goes without saying that the present invention is not limited to these.

The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entireties.

1. Patients and Blood Sampling At Kyoto Prefectural University of Medicine, blood was collected from breast cancer patients (n=75) and healthy subjects (n=68) to obtain serum. Specifically, venous blood was collected from the patient's radial vein using a 22G needle and placed in a blood collection tube. The samples were allowed to stand at room temperature for 1 hour and centrifuged at 3,000 rpm for 10 minutes at room temperature to obtain serum. The supernatant was stored in aliquots at -80°C until use.

The breakdown of breast cancer patients is 17 for stage 0 (St.0), 32 for stage I (St. I), 20 for stage II (St. II), and 6 for stage III (St. III). there were. The average age (mean ± standard deviation (S.D.)) of the patient group was 59.6 ± 13.4. For stage classification, "clinical and pathological breast cancer treatment regulations 18th edition" (edited by the Japanese Breast Cancer Society) was used as diagnostic criteria.

The average age (average ± standard deviation) of the healthy group was 61.1 ± 13.5.

2. Mass spectrometry by BLOTCHIP® Mass spectrometry analysis of peptides in serum was performed by BLOTCHIP® mass spectrometry, a one-step direct transcription technology, a rapid quantitative method for peptidome profiling (Biochem. Biophys. Res. Commun. 2009;379(1):110-4).
First, serum samples were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) to separate peptides from proteins. Next, the peptides in the gel were electrotransferred to BLOTCHIP (registered trademark) (Protocera Co., Ltd., Amagasaki City). After the transfer, the surface of the chip was rinsed with ultrapure water, and a matrix (α-cyano-4-hydroxycinnamic acid, Sigma-Aldrich Co., Missouri, USA) was applied directly to BLOTCHIP®, followed by ultraflextreme TOF/ Mass spectrometric analysis was performed on a TOF mass spectrometer (Bruker Daltonics, MA, USA) in linear mode as described in Proteomics 11:2727-2737. Peptide profiles were obtained.

3. Statistical Analysis Samples were analyzed in quadruplicate by BLOTCHIP® mass spectrometry. In order to find more statistically significant peaks, the four data were used as independent data, and the analysis software ClinProTools 2.2 (Bruker Daltonics) was used to calculate the p-value of the Mann-Whitney U test, p A value of 0.05 or less was considered significant.

Wilcoxon test using statistical analysis software R (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.) To calculate the p-value of , we evaluated the diagnostic performance of the peptides using the average value of four data per sample, and found useful biomarker peptides with high diagnostic performance. Statistical analysis software R (R Core Team (2013) was used to construct the model.

ROC analysis was performed to evaluate the diagnostic ability of the constructed model. The R package “Epi package” (A package for statistical analysis in epidemiology, Version 1.149, http://cran.r-project.org/web/packages/Epi/index.html) was used. AUC was calculated from the ROC curve. The optimal cut-off value for diagnosis was determined according to Youden's index in Cancer 1950;3:32-5.

4. Peptide Identification Serum containing each target peptide was collected for its identification. Peptides were extracted with 80% v/v acetonitrile (ACN) in water containing 0.1% trifluoroacetic acid using Sep-Pak C18 solid-phase extraction cartridges (Waters Corporation, Milford, MA, USA). The eluate was concentrated to 100 μL or less using a CC-105 centrifugal concentrator (Tomy Seiko Co., Ltd., Tokyo). Next, the solution was diluted with 400 μL of 2% v/v ACN aqueous solution containing 0.065% TFA (referred to as eluent A), and applied to a C18 silica column (COSMOSIL (registered trademark) 5C18-AR-II) (Nacalai Tesque Co., Ltd., Kyoto). ) equipped with an AEKTA purifier (GE Healthcare UK Ltd, Buckinghamshire, UK). The eluate was divided into 22 fractions (1 mL each) with a linear gradient of 0-100% at a flow rate of 1.0 mL/min in an aqueous solution of 80% v/v ACN containing 0.05% TFA for eluent A. . Each fraction was concentrated to 10 μL or less with a CC-105 centrifugal concentrator, and peptide sequences were analyzed by MALDI-TOF/TOF (ultraflextreme TOF/TOF) and LC-MS/MS (Q-Exactive; Thermo Fisher Scientific Inc, Massachusetts, USA). State Waltham) was used for analysis.

(result)
1. Peptide Analysis of Breast Cancer Patient Serum Peptide analysis of serum was performed by BLOTCHIP® mass spectrometry. Mass spectral data obtained from each peptidome profile were stored in a database. After all MS measurements are completed, using the analysis software ClinProTools 3.0, differential analysis is performed between the two groups of breast cancer patients and healthy subjects, and there is a statistically significant difference between the two groups for each peak. I considered whether.

2. Identified Peptides For the above significant peaks, MALDI-TOF/TOF and LC-MS/MS peptide sequencing analyzes were performed with partially purified serum peptides by reversed-phase chromatography, and 8 peptides were identified. (hereinafter sometimes referred to as peptides #1 to #8) (Table 3).

The first peptide (#1) was oxidized (methionine sulfoxide) at the 9th Met (methionine) position in the amino acid sequence represented by SEQ ID NO:1. The third peptide (#3) was oxidized (cysteic acid) at the 18th Cys (cysteine) position in the amino acid sequence represented by SEQ ID NO:3. The 5th peptide (#5) was oxidized (methionine sulfoxide) at the 9th Met (methionine) position in the amino acid sequence represented by SEQ ID NO:4. The seventh peptide (#7) was oxidized (methionine sulfoxide) at the 28th Met (methionine) position in the amino acid sequence represented by SEQ ID NO:6. The eighth peptide (#8) was S-cysteinylated at the 18th Cys (cysteine) position in the amino acid sequence of SEQ ID NO:7.

The second peptide (#2) was the unmodified amino acid sequence represented by SEQ ID NO:2. The fourth peptide (#4) was the unmodified amino acid sequence represented by SEQ ID NO:3. The sixth peptide (#6) was the unmodified amino acid sequence represented by SEQ ID NO:5.

The mass spectrometry apparent molecular weights [M+H] ⁺ of peptides #1-#8 were about 5078.36, about 1465.66, about 2787.51, about 2739.52, about 5206.44, about 2659.26, about 5917.69, and about 2858.53, respectively.

3. Rate of Change and Diagnostic Performance of Each Peptide For the identified eight peptides, it was examined whether there was a statistically significant difference between the breast cancer group and the healthy subject group. The specific host is the calculation of the area under the curve (AUC), sensitivity, specificity and cutoff for each peptide, and the rate of change in content in samples (content in breast cancer patient samples/ content in samples from healthy subjects) was calculated.

Table 4 shows the results. The p-values were confirmed to be significant for all peptides. Each peptide had various sensitivities and specificities as breast cancer markers, and the AUC, which indicates diagnostic performance, ranged from 0.60 to 0.79.

The marker list includes peptides whose content increases due to disease (peptide #1, peptide #6, peptide #7) and peptides whose content decreases (peptide #2, peptide #3, peptide #4, peptide #5, There was peptide #8). The peptide with the largest change in content (peptide #1) nearly doubled.

4. Multivariate logistic regression analysis Among the 8 peptides identified, a combination of peptide #1, peptide #2, peptide #3, and peptide #4 (multimarker set [#1-4]) was selected to predict breast cancer incidence A regression model equation for calculating the risk index was obtained by multivariate logistic regression analysis.

The resulting equation is shown below.

[#1] to [#4] represent the blood concentration of each peptide.

Each coefficient is shown in the table below.

Table 6 shows each stage and diagnostic performance of the above regression model. In the table, "CL" is the 68 healthy subjects, "St0" is the 17 stage 0 breast cancer patients, "St1" is the 32 stage I breast cancer patients, and "St2" is the stage II breast cancer patients. 20, "St3" refers to 6 patients with stage III breast cancer, and "all BC" refers to 75 patients with all breast cancer.

Fig. 1 shows the predicted probability of each stage of breast cancer (stages 0 to III) using a multivariate logistic regression model. As is clear from FIG. 1, sensitivity is high from relatively early stages, and high AUC is exhibited in all stages 0-III. This indicates that the above regression model is superior in terms of early detection of risks.

Based on the above results, the predictive values obtained from the blood concentrations of these peptide markers using a multivariate logistic regression model are useful for the determination of breast cancer, the determination of breast cancer patients (responsors) who respond to therapeutic drugs, and the individual breast cancer patients. It is suggested that it is useful for determination of effective therapeutic drugs (companion diagnostic method), determination of preventive effect, determination of therapeutic effect, test method for early diagnosis, test method for early treatment, and screening method for substances. was done.

Claims (11)

A method for detecting breast cancer in a subject, comprising measuring a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in a biological sample of the subject. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, and SEQ ID NO: 6 in the subject's biological sample In claim 1, further comprising measuring one or more peptides selected from the group consisting of a peptide consisting of an amino acid sequence represented by and a peptide consisting of an amino acid sequence represented by SEQ ID NO: 7 Detection method as described . The biological sample consists of a body fluid selected from the group consisting of blood, plasma, serum, saliva, urine, cerebrospinal fluid, bone marrow fluid, pleural effusion, ascitic fluid, synovial fluid, sweat, tears, aqueous humor, vitreous humor and lymphatic fluid. , a method according to claim 1 or 2 . A method according to any one of claims 1 to 3 , comprising subjecting the biological sample to mass spectrometry. 4. The method according to any one of claims 1 to 3, wherein an antibody that specifically recognizes the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is used. the method of. The detection of breast cancer includes determination of breast cancer, determination of breast cancer preventive effect, determination of breast cancer therapeutic effect, determination of breast cancer patients who respond to therapeutic drugs, determination of therapeutic drugs that respond to individual breast cancer patients, and determination of breast cancer diagnosis. The method according to any one of claims 1 to 5 , which is an examination method for breast cancer or an examination method for treatment of breast cancer. Detection of breast cancer comprising an internal standard in which the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 or fragment ions thereof are labeled with stable isotopes kit. A breast cancer detection agent comprising a stable isotope-labeled peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4, or fragment ions thereof. A computer-implemented method for determining the susceptibility of breast cancer in a subject, comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or represented by SEQ ID NO: 4 in a biological sample of the subject. obtaining quantitative data for a peptide consisting of an amino acid sequence obtained from
applying the obtained data to a multivariate logistic regression model that is a function of the one or two peptides to obtain a predictive value of the likelihood of susceptibility to breast cancer in a subject. A method of using a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 as a marker for detecting breast cancer. A method of using the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and/or the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 as a marker for detecting the responsiveness of breast cancer patients to therapeutic agents.

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