Breast cancer diagnostic aid method and breast cancer test kit

Abstract

Provided is a breast cancer diagnosis assistance method comprising: a measurement step for measuring the quantity of laminin 5 or laminin β3 in a sample; and an information provision step for providing information for diagnosing breast cancer, on the basis of the measured amount of laminin 5 or laminin β3. Also provided is a breast cancer test kit including an anti-laminin 5 antibody or an anti-laminin β3 antibody.

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in the diagnosis of breast cancer and a breast cancer test kit. [Background technology]

[0002] The main methods used for breast cancer screening are visual and palpation, mammography, and ultrasound. For those who are suspected of having cancer, a portion of the lesion is taken and pathological examination is performed using cytology or histology (biopsy) (Non-Patent Document 1: Breast Cancer, Examination and Diagnosis, Subtype Classification [searched October 5, 2020], available online).<URL:https: / / ganjoho.jp / public / cancer / breast / diagnosis.html> and Non-Patent Document 2: Nover AB, Jagtap S, Anjum W, Yegingil H, Shih WY, Shih WH, Brooks AD. Int J Biomed Imaging. 2009,902326).

[0003] A method for classifying breast cancer subtypes using genetic testing has been proposed (Non-Patent Document 3: Garrido-Castro AC, Lin NU, Polyak K. Cancer Discov. 2019, 9(2):176-198). However, genetic testing is expensive and difficult to put into practical use. Currently, instead of genetic testing, cancer cells collected by biopsy or surgery are immunostained, and clinical pathology is used to classify cancers based on genetic analysis. Immunostaining targets hormone receptors (estrogen receptor (ER) and progesterone receptor (PgR)), HER2, and Ki67. These are proteins involved in cancer cell proliferation. Breast cancer subtypes are classified when a patient is diagnosed with breast cancer. Because triple-negative breast cancer progresses rapidly and has a poorer prognosis than other subtypes, it is useful to be able to identify triple-negative breast cancer early using a blood test. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Breast cancer, testing, diagnosis, subtype classification [Searched October 5, 2020] Internet<URL:https: / / ganjoho.jp / public / cancer / breast / diagnosis.html> [Non-patent document 2] Nover AB, Jagtap S, Anjum W, Yegingil H, Shih WY, Shih WH, Brooks AD. Int J Biomed Imaging. 2009,902326 [Non-patent document 3] Garrido-Castro AC, Lin NU, Polyak K. Cancer Discov. 2019, 9(2):176-198 Summary of the Invention [Problem to be solved by the invention]

[0005] In order to detect breast cancer early and provide appropriate treatment, it is desirable to have a simple and easy method for testing for breast cancer. An object of the present invention is to provide a new diagnostic auxiliary method for diagnosing breast cancer and a breast cancer testing kit. [Means for solving the problem]

[0006] The present invention relates to a method for assisting in the diagnosis of breast cancer, which includes a measurement step of measuring the amount of laminin 5 or laminin β3 contained in a sample, and an information provision step of providing information for diagnosing breast cancer based on the measured amount of laminin 5 or laminin β3.

[0007] The present invention relates to a breast cancer test kit comprising an anti-laminin 5 antibody or an anti-laminin β3 antibody. [Effects of the Invention]

[0008] According to the present invention, a new diagnostic aid method for diagnosing breast cancer and a breast cancer test kit are provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram illustrating the structure of laminin. [Figure 2] FIG. 1 is a diagram showing an example of a breast cancer test kit according to the present invention. [Figure 3] FIG. 1 is a diagram showing an example of use of the breast cancer test kit according to the present invention. [Figure 4] 1 is a graph showing the expression level of laminin 5 in breast cancer-derived cell lines in Experiment 2 of the Example. [Figure 5] The left figure shows the results of immunochromatographic detection of the amounts of laminin 5 and CD9 (exosome marker) in the plasma of breast cancer patients and healthy individuals in Experiment 3. The right figure is a graph showing the value (laminin 5 / CD9) obtained by normalizing the expression level of laminin 5 with the expression level of CD9 from the immunochromatographic results shown in the left figure. [Figure 6] 1 is a graph showing the amount of laminin 5 in the plasma of HER2 breast cancer patients and triple-negative breast cancer patients normalized by the amount of CD9 (laminin 5 / CD9) in Experiment 4 of the Example. [Figure 7A] FIG. 1 shows the values obtained by standardizing the amount of laminin 5 by the amount of CD9 (laminin 5 / CD9) in breast cancer patients and healthy subjects in Experiment 5 of the Example. [Figure 7B] 1 is an ROC curve showing the ability of laminin 5 to discriminate breast cancer patients in Experiment 5 of the Example. [Figure 8A] FIG. 10 shows the values obtained by standardizing the amount of laminin 5 with the amount of CD9 (laminin 5 / CD9) in early breast cancer patients and healthy subjects in Experiment 5 of the Example. [Figure 8B] 1 is an ROC curve showing the ability of laminin 5 to identify early stage breast cancer patients in Experiment 5 of the Example. [Figure 9A]FIG. 1 shows the values obtained by standardizing the amount of laminin 5 with the amount of CD9 (laminin 5 / CD9) in breast cancer patients and healthy subjects in Experiment 6 of the Example. [Figure 9B] 1 is an ROC curve showing the ability of laminin 5 to discriminate breast cancer patients in Experiment 6 of the Example. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Breast cancer diagnostic aid method> A method for assisting diagnosis of breast cancer according to one aspect of the present invention includes: a measuring step of measuring the amount of laminin 5 or laminin β3 contained in the sample; and an information providing step of providing information for diagnosing breast cancer based on the measured amount of laminin 5 or laminin β3.

[0011] According to the diagnostic support method for breast cancer of the present invention, it is possible to easily provide information for diagnosing breast cancer. The diagnostic support method of the present invention does not include a judgment step by a doctor and does not fall under the category of a method for diagnosing humans.

[0012] Each step will be described in detail below. The diagnostic aid method for breast cancer according to the present invention first measures the amount of laminin 5 or laminin β3 contained in a sample. Hereinafter, this step is also referred to as the "measurement step."

[0013] Laminin is a large protein that constitutes the basement membrane, an extracellular matrix. It is involved in the construction and maintenance of multicellular structures and tissues, as well as cell adhesion, cell migration, and cell proliferation. As shown in Figure 1, laminin forms a heterotrimeric structure consisting of three polypeptide chains—α, β, and γ—linked by disulfide bonds. In humans, there are five α chains (α1, α2, α3, α4, and α5), three β chains (β1, β2, and β3), and γ chains (γ1, γ2, and γ3). At least 17 laminins with different combinations of these have been identified. Laminin-5, also known as laminin-332, is composed of α3, β3, and γ2 chains. Laminin-β3 is the only laminin reported to date. The amount of laminin-5 can be correlated with the amount of laminin-β3. The laminin β3 contained in the sample is thought to include laminin β3 that has formed a trimeric complex and laminin β3 that has not formed a complex.

[0014] The method for measuring the amounts of laminin 5 and laminin β3 is not particularly limited, and any known method capable of accurately and quantitatively measuring proteins can be used. Measurement methods include immunoassays and mass spectrometry. Measurement methods can also be used in combination as appropriate.

[0015] Immunological assays are assay methods that utilize antigen-antibody reactions, and examples include immunochromatography, enzyme immunoassays (EIA, Enzyme-Linked Immunosorbent Assays (ELISA)), chemiluminescent enzyme immunoassays, fluorescent immunoassays, radioimmunoassays, immunoturbidimetry, immunonephelometry, latex agglutination, immunoprecipitation, Western blotting, affinity chromatography, flow cytometry, immunoassays using metal-labeled antibodies and mass spectrometry, etc. From the viewpoints of rapidity and simplicity of measurement, immunochromatography is preferred.

[0016] Antibodies used in immunoassays include anti-laminin 5 antibodies and anti-laminin β3 antibodies. Anti-laminin 5 antibodies and anti-laminin β3 antibodies are used to detect laminin 5 and laminin β3, respectively. From the viewpoint of excellent detection sensitivity and specificity, it is preferable to use anti-laminin 5 antibodies in immunoassays.

[0017] Antibodies used in immunoassays may be labeled with a substance suitable for detection. Examples of labels include metal particles, enzymes, fluorescent substances, radioactive substances, affinity substances, etc. Examples of metal particles include gold nanoparticles, silver nanoparticles, gold nanorods, silver nanorods, gold nanoplates, silver nanoplates, etc., as well as metal nanoparticles of various shapes coated with heterometals. Examples of enzymes include peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc. Examples of fluorescent substances include fluorescein isothiocyanate (FITC), Cy dyes, Alexa Fluor dyes, rhodamine, phycoerythrin (PE), allophycocyanin (APC), other fluorescent proteins, etc. Examples of radioactive substances include: 3 H, 14 C. 32 P, 35 S, 125Examples of affinity substances include biotin and streptavidin. Labels may be attached to anti-laminin 5 or anti-laminin β3 antibodies, or to secondary antibodies that specifically recognize these primary antibodies. From the perspective of enabling highly sensitive detection, antibodies are preferably labeled with metal particles or enzymes. When the substrate is catalyzed by the enzyme to emit chemiluminescence, the signal is amplified, improving detection sensitivity. Antibodies labeled with metal particles change their absorption wavelength upon aggregation, allowing the amount of laminin 5 or laminin β3 to be confirmed as a color change detectable with the naked eye. The color change may be quantified using a known image processing device. Antibodies labeled with metal particles can also be detected by inductively coupled plasma mass spectrometry (ICP-MS), allowing highly sensitive measurement of even small amounts of laminin 5 or laminin β3. Antibodies can be detected using devices commonly used to detect each label.

[0018] As an example of a method for measuring laminin 5 or laminin β3 by immunoassay, immunochromatography may be performed to develop and capture the sample, and then labeled anti-laminin 5 antibody or anti-laminin β3 antibody may be detected using visible light (absorbance), ICP-MS, chemiluminescence, or fluorescence.

[0019] Mass spectrometry methods for measuring laminin 5 and laminin β3 include ionizing proteins using ionization methods such as matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI), and then quantitatively analyzing the signal intensity of each peak resolved by the mass / charge ratio (m / z) of the protein ions. Alternatively, samples can be enzymatically digested with proteases such as trypsin, and the resulting peptide fragments can be separated and concentrated by liquid chromatography (LC), followed by mass analysis using liquid chromatography-mass spectrometry (LC-MS). Suitable mass spectrometers include common single-type mass spectrometers, as well as tandem mass spectrometers such as triple quadrupole (QqQ) mass spectrometers, quadrupole time-of-flight (Q-TOF) mass spectrometers, tandem time-of-flight (TOF-TOF) mass spectrometers, quadrupole ion trap (QIT) mass spectrometers, and quadrupole ion trap time-of-flight (QIT / TOF) mass spectrometers. In LC-MS / MS, multiple reaction monitoring (MRM) may be performed using a triple quadrupole mass spectrometer. Triple quadrupole mass spectrometers offer superior quantitative performance. In MRM, separation from contaminating ions is performed within the instrument, further reducing noise levels and enabling quantitative analysis of even minute amounts of peptides.

[0020] When measuring the amount of laminin 5 or laminin β3 by mass spectrometry, dissociation of the protein complex and fragmentation of the protein may occur, so it is preferable to measure the amount of laminin β3.

[0021] The sample may be cultured cells, cultured tissues, or their culture supernatants, but is preferably a biological sample collected from a subject. The biological sample may be blood obtained from an animal, or may be biological tissue, urine, feces, sweat, saliva, lymph, amniotic fluid, breast milk, pleural effusion, ascites, cerebrospinal fluid, tears, etc. The blood may be whole blood, but is preferably plasma or serum. The sample may be diluted with physiological saline, buffer, etc. Biological samples may be collected by any known method appropriate for the type of biological sample. The subject is preferably a human individual, such as a breast cancer patient, a person suspected of having breast cancer, or a healthy individual. Using a biological sample collected from a subject as a sample can provide information for diagnosing breast cancer in the subject. The sample is preferably blood collected from a human individual. Using blood collected from a human individual as a sample makes it possible to provide information for diagnosing human breast cancer accurately and easily.

[0022] To improve the accuracy of measuring the amounts of laminin-5 and laminin-β3, samples may be purified or pretreated as appropriate, using procedures such as filtration, centrifugation, affinity separation, dialysis, and precipitation. The sample may also be a purified exosome fraction. When the sample is a purified exosome fraction, contaminants in the sample can be reduced, improving measurement accuracy. Exosomes are small vesicles with a diameter of approximately 100 nm secreted from cells and are present in large quantities in body fluids such as blood, saliva, and urine. Exosomes contain lipids and proteins derived from cell membranes on their surface and are known to contain nucleic acids and proteins, such as mRNA and miRNA, inside. Exosome purification can be performed using commercially available purification kits, centrifugation, density gradient centrifugation, size exclusion chromatography, filtration, and other known methods.

[0023] The diagnostic support method for breast cancer according to the present invention includes a step of providing information for diagnosing breast cancer based on the measured amount of laminin 5 or laminin β3. Hereinafter, this step is also referred to as the "information providing step."

[0024] In the information providing step, the amount of laminin 5 or laminin β3 contained in the sample is compared with a predetermined value to obtain information for diagnosing breast cancer. The information for diagnosing breast cancer may include, for example, information for diagnosing (1) whether or not it is breast cancer, (2) breast cancer subtype, and (3) breast cancer stage. One or more of these pieces of information may be provided simultaneously.

[0025] (1) Whether it is breast cancer or not Since the 5-year survival rate of breast cancer patients decreases as the stage progresses, it is preferable to diagnose the disease as early as possible. The diagnostic assistance method according to this embodiment can provide information for diagnosing breast cancer non-invasively and simply, even in the early stages of breast cancer that are difficult to detect using conventional tests.

[0026] Comparison of the amount of laminin 5 or laminin β3 with a predetermined value can provide useful information for diagnosing breast cancer. The predetermined value can be, for example, the amount of laminin 5 or laminin β3 in the culture supernatant of cultured cells not derived from breast cancer, or the amount of laminin 5 or laminin β3 in the body fluids (plasma, serum, etc.) of healthy individuals. Higher amounts of laminin 5 or laminin β3 tend to be detected in breast cancer patients compared to healthy individuals. When the amount of laminin 5 or laminin β3 contained in a sample is higher than the predetermined value, this provides useful information for determining whether the sample is derived from breast cancer.

[0027] (2) Breast cancer subtype Breast cancer subtypes are known to be luminal A, luminal B, HER2, and triple-negative. Triple-negative breast cancer is negative for estrogen receptors (ER), progesterone receptors (PgR), and human epidermal growth factor receptor 2 (HER2). One approach to breast cancer treatment is to classify breast cancers into subtypes and select treatments tailored to the characteristics of the cancer cells, such as endocrine (hormone) therapy, chemotherapy, and molecular targeted therapy. Therefore, in order to provide appropriate treatment for breast cancer, it is necessary to diagnose the breast cancer subtype.

[0028] Breast cancer subtype classification is performed when breast cancer testing suggests the presence of the disease, and is therefore difficult to apply to patients with cancer at a relatively early stage. A method of classifying breast cancer subtypes using genetic testing has been proposed. However, genetic testing is expensive and considered difficult to put into practical use. On the other hand, triple-negative breast cancer progresses quickly, and the 5-year recurrence-free survival rate of patients with triple-negative breast cancer in the later stages (stages III and IV) is low. Therefore, it is necessary to diagnose breast cancer subtypes as early as possible. The diagnostic assistance method of this embodiment can easily provide information for diagnosing breast cancer subtypes.

[0029] Comparison of the amount of laminin 5 or laminin β3 with a predetermined value can provide useful information for diagnosing breast cancer subtypes, particularly triple-negative breast cancer. The predetermined value can be, for example, the amount of laminin 5 or laminin β3 in the culture supernatant of cultured cells not derived from breast cancer or cultured cells not derived from triple-negative breast cancer, or the amount of laminin 5 or laminin β3 in the body fluids (plasma, serum, etc.) of healthy individuals or patients with breast cancer other than triple-negative breast cancer. Patients with triple-negative breast cancer tend to have higher levels of laminin 5 or laminin β3 detected than healthy individuals and patients with breast cancer other than triple-negative breast cancer. When the amount of laminin 5 or laminin β3 in a sample is higher than the predetermined value, it provides useful information for determining that the sample is derived from triple-negative breast cancer.

[0030] (3) Stage of breast cancer The degree of progression of breast cancer is classified into stages 0, I, II (IIA, IIB), III (IIIA, IIIB, IIIC), or IV depending on the size of the lump and the state of metastasis to lymph nodes or other organs. Since treatment is carried out according to the degree of cancer progression, it is necessary to diagnose the stage of breast cancer. The diagnostic assistance method according to this embodiment can easily provide information for diagnosing the stage of breast cancer.

[0031] Comparison of the amount of laminin 5 or laminin β3 with a predetermined value can provide useful information for diagnosing the stage of breast cancer, particularly early stage I and II breast cancer. In this case, the predetermined value can be, for example, the amount of laminin 5 or laminin β3 in the culture supernatant of cultured cells not derived from breast cancer or cultured cells derived from late stage III or stage IV breast cancer, or the amount of laminin 5 or laminin β3 in the body fluids (plasma, serum, etc.) of healthy individuals or patients with late stage III or IV breast cancer. Higher amounts of laminin 5 or laminin β3 tend to be detected in early stage breast cancer patients compared to healthy individuals or patients with late stage breast cancer. A higher amount of laminin 5 or laminin β3 in a sample than the predetermined value provides useful information for determining that the sample is derived from early stage breast cancer.

[0032] In the information providing step, the amount of laminin 5 or laminin β3 may be the measured value itself or the concentration in the sample, but it is preferable to measure the amount of laminin 5 or laminin β3 relative to the amount of a control contained in the sample. The control may be, for example, exosomes. When exosomes are used as a control, the amount of laminin 5 or laminin β3 is standardized by the amount of exosomes in the information providing step. Because the amount of exosomes contained in a sample can vary from sample to sample, standardizing the measured amount of laminin 5 or laminin β3 using a control can improve the accuracy of the information used to diagnose breast cancer. The amount of exosomes can be measured by the amount of exosome markers. Examples of exosome markers include surface antigens such as CD9, CD63, and CD81.

[0033] For example, when the ratio of laminin 5 or laminin β3 to the amount of CD9 contained in a sample is greater than the ratio of laminin 5 or laminin β3 to the amount of CD9 in the plasma of a healthy person, preferably more than twice as much, this provides useful information for determining that the sample is derived from breast cancer, including triple-negative breast cancer, and / or from early-stage breast cancer.

[0034] The amount of exosome markers can be measured by known methods, preferably the same methods as those for measuring the amount of laminin 5 or laminin β3 described above. When measuring the amount of exosome markers by immunoassay, the amount of exosome markers can be measured by the same method as for measuring the amount of laminin 5 or laminin β3 by using an antibody against the exosome marker. When the antibody against the exosome marker is labeled with a substance different from the anti-laminin 5 antibody or anti-laminin β3 antibody (e.g., a different metal particle or a different fluorescent substance), laminin 5 or laminin β3 and the exosome marker can also be detected in the same experimental system. When mass spectrometry is used, any protein can be detected, so the amount of exosome markers and the amount of laminin 5 or laminin β3 can be measured in a single analysis.

[0035] <Breast cancer test kit> A breast cancer test kit according to one embodiment of the present invention comprises an anti-laminin 5 antibody or an anti-laminin β3 antibody. The breast cancer test kit according to the present invention can easily provide information for diagnosing breast cancer by measuring the amount of laminin 5 or laminin β3 contained in a sample. The information for diagnosing breast cancer may include, for example, information for diagnosing (1) whether or not the patient has breast cancer, (2) the breast cancer subtype, and (3) the stage of breast cancer. One or more of these pieces of information may be provided simultaneously.

[0036] The anti-laminin 5 antibody or anti-laminin β3 antibody may be the same as the antibody used in the above-mentioned method for measuring the amount of laminin 5 or laminin β3. The anti-laminin 5 antibody or anti-laminin β3 antibody is used to detect laminin 5 or laminin β3 and is preferably labeled with metal particles or an enzyme. The secondary antibody used to detect the anti-laminin 5 antibody or anti-laminin β3 antibody may also be labeled. When the antibody is labeled with an enzyme, a signal derived from the reaction product of the substrate is detected, enabling highly sensitive detection by extending the reaction and detection times. Furthermore, because the absorption wavelength of antibodies labeled with metal particles changes upon aggregation, the amount of laminin 5 or laminin β3 can be confirmed using visible light (absorbance). Even small amounts of antibodies labeled with metal particles can be detected by inductively coupled plasma mass spectrometry (ICP-MS), allowing highly sensitive measurement of the amount of laminin 5 or laminin β3.

[0037] The test kit is preferably capable of measuring the amount of laminin 5 or laminin β3 contained in a sample by immunoassay or mass spectrometry. From the viewpoint of rapidity and ease of measurement, the test kit preferably utilizes immunochromatography.

[0038] The test kit may further include an immunochromatographic test paper for performing immunochromatography, an antibody for detecting the control, a factor or antibody that captures laminin 5 or laminin β3, a standard for laminin 5 or laminin β3, and an instruction manual. An example of an immunochromatographic test paper included in the test kit of the present invention is shown in Figure 2. The immunochromatographic test paper 10 generally comprises a membrane 11 and an absorbent pad 12 connected together (Figure 2A). A capture antibody 13 is immobilized on the membrane 11 to form a detection line. The capture antibody 13 may be a factor or antibody that captures laminin 5 or laminin β3, or a factor or antibody that captures an exosome marker. The membrane 11 is, for example, a nitrocellulose membrane. The absorbent pad 12 is a portion for quickly absorbing the sample. The immunochromatographic test paper 10 may further include a sample pad 14 (FIG. 2B) or may include both the sample pad 14 and a conjugate pad 15 (FIG. 2C). The sample pad 14 serves as a sample supply section and absorbs a liquid sample, allowing the liquid and the substance to be detected to pass through. The conjugate pad 15 contains a detection antibody and can release the detection antibody to the membrane 11. The detection antibody may be a factor or antibody that captures laminin 5 or laminin β3, or a factor or antibody for capturing an exosome marker. Exosomes can be used as a control. Exosomes can be detected by CD9, a marker protein. The factor or antibody that captures laminin 5 or laminin β3 may be an anti-laminin 5 antibody or anti-laminin β3 antibody immobilized on the membrane. When detecting laminin 5 or laminin β3 contained in exosomes, the factor or antibody that captures laminin 5 or laminin β3 may be an antibody that captures an exosome marker immobilized on a membrane, such as an anti-CD9 antibody.

[0039] The capture antibody 13 and detection antibody used in immunochromatographic testing can be combined, for example, as shown in Table 1. As shown in Pattern 1, using an antibody that captures an exosome marker as the capture antibody and an antibody that captures an exosome marker as the detection antibody allows for the detection of exosomes present in a sample and the quantification of exosomes as a control. As shown in Pattern 2, using an anti-laminin 5 antibody or anti-laminin β3 antibody as the capture antibody and an anti-laminin β3 antibody or anti-laminin β3 antibody as the detection antibody allows for the detection of laminin 5 or laminin β3 present in a sample and the quantification of laminin 5 or laminin β3. As shown in Pattern 3, using an antibody that captures an exosome marker as the capture antibody and an anti-laminin 5 antibody or anti-laminin β3 antibody as the detection antibody allows for the detection of exosomes containing laminin 5 or laminin β3 present in a sample and the quantification of laminin 5 or laminin β3 expressed on exosomes. As shown in Pattern 4, by using an anti-laminin 5 antibody or anti-laminin β3 antibody as the capture antibody and an antibody that captures an exosome marker as the detection antibody, it is possible to detect exosomes containing laminin 5 or laminin β3 present in the sample and quantify the laminin 5 or laminin β3 expressed in exosomes.

[0040] [Table 1]

[0041] The sample used for the test may be the same as the sample used in the above-mentioned method for measuring the amount of laminin 5 or laminin β3. The sample is preferably blood, plasma, serum, or an exosome-purified fraction thereof. The sample is preferably blood collected from a human individual.

[0042] An example of how to use the test kit according to the present invention is shown in FIG. 3. The test kit shown in FIG. 3 includes an immunochromatographic test paper 10. A capture antibody 13 is immobilized on the detection line of the membrane 11 of the immunochromatographic test paper 10. A sample absorbed from the end of the immunochromatographic test paper 10 opposite the absorbent pad 12 is spread across the membrane 11 by capillary action and absorbed into the absorbent pad 12 (Step 1). At this time, a detection target 16 (laminin 5, laminin β3, or exosomes containing these) contained in the sample is captured. After or simultaneously with the sample spreading, a detection antibody 17 labeled with metal particles is spread (Step 2). When the detection antibody 17 is labeled with metal particles, the detection antibody 17 gathers on the detection line, allowing visual confirmation of coloration of the detection line. The detection antibody 17 labeled with metal particles may be detected by ICP-MS. When the detection antibody 17 is labeled with an enzyme or a fluorophore, the detection antibody 17 can be detected by the chemiluminescence or fluorescence generated upon addition of a substrate.

[0043] To more quantitatively evaluate laminin 5 or laminin β3, the amount of CD9 may be measured as a control and the amount of laminin 5 or laminin β3 may be normalized by the amount of CD9 contained in the sample. The amount of CD9 can be measured by immunochromatography using anti-CD9 antibodies as capture and detection antibodies. The amount of CD9 may be measured using the same kit as the test kit of the present invention or a different kit. CD9 and laminin 5 or laminin β3 may be simultaneously detected by using differently labeled anti-CD9 antibodies and anti-laminin 5 or anti-laminin β3 antibodies on exosomes captured by capture antibodies on the same membrane.

[0044] The amount of laminin 5 or laminin β3 measured by the test kit is compared with a predetermined value. The predetermined value may be any of those listed in the "Breast Cancer Diagnostic Support Method," such as the amount of laminin 5 or laminin β3 in the culture supernatant of cultured cells not derived from breast cancer, cultured cells not derived from triple-negative breast cancer, or cultured cells derived from late-stage breast cancer, or the amount of laminin 5 or laminin β3 in the body fluids (plasma, serum, etc.) of healthy individuals, breast cancer patients other than triple-negative breast cancer, or late-stage breast cancer patients. When the test kit detects an amount of laminin 5 or laminin β3 in a sample that is higher than the predetermined value, this provides useful information for determining that the sample is derived from breast cancer, including triple-negative breast cancer, and / or early-stage breast cancer.

[0045] When the ratio of laminin 5 or laminin β3 to the control amount measured by the test kit is greater than the ratio of laminin 5 or laminin β3 to the control amount (optional) in the plasma of a healthy person, preferably more than twice as much, this provides useful information for determining that the sample is derived from breast cancer, including triple-negative type, and / or early-stage breast cancer.

[0046] <Breast cancer diagnostic marker> A breast cancer diagnostic marker according to one embodiment of the present invention comprises laminin 5 or laminin β3 contained in a sample. The breast cancer diagnostic marker according to the present invention can provide information for diagnosing breast cancer, and can easily provide useful information for diagnosing whether a patient has breast cancer, as well as the subtype and stage of breast cancer.

[0047] The sample and laminin 5 or laminin β3 may be as described above. The method for detecting the diagnostic marker of the present invention is not particularly limited, and the amount of laminin 5 or laminin β3 can be measured, for example, by the method described above. Whether a sample is derived from breast cancer, triple-negative breast cancer, or early-stage breast cancer can be determined based on the measured amount of laminin 5 or laminin β3 and the criteria described above.

[0048] <Measurement method> A measurement method according to one embodiment of the present invention measures the amount of laminin 5 or laminin β3 contained in a sample. According to the measurement method of the present invention, the amount of laminin 5 or laminin β3 contained in a sample can be determined. The amount of laminin 5 or laminin β3 can provide information for diagnosing breast cancer, and can be used as information for diagnosing whether or not a patient has breast cancer, as well as the subtype and stage of breast cancer. When the amount of laminin 5 or laminin β3 contained in a sample is equal to or greater than a predetermined value, this information is useful for determining whether the sample is derived from breast cancer, including triple-negative breast cancer, and / or from early-stage breast cancer.

[0049] The measurement method is not particularly limited as long as it can measure the amount of laminin 5 or laminin β3 contained in the sample, and may be the same as the method described in the measurement step section above. The sample and the predetermined value may be as described above.

[0050] <Breast cancer diagnosis methods> A method for diagnosing breast cancer according to one embodiment of the present invention includes determining that a sample is derived from breast cancer when the amount of laminin 5 or laminin β3 contained in the sample is equal to or greater than a predetermined value. The method can also diagnose the breast cancer subtype and stage of the sample. The method for diagnosing breast cancer according to the present invention can easily determine whether a sample is derived from breast cancer, triple-negative breast cancer, or early-stage breast cancer.

[0051] The sample and the predetermined value may be as described above. When the sample is a biological sample collected from a subject, the method for diagnosing breast cancer according to the present invention can easily determine whether the subject is a breast cancer patient, and the breast cancer subtype and stage. The method for diagnosing breast cancer according to the present invention may include the measuring step and the information providing step described above. [Example]

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0053] <Experiment 1> We performed a comprehensive protein analysis to investigate differences in protein expression between triple-negative and non-triple-negative breast cancers. First, exosome fractions were purified by ultracentrifugation from the culture supernatants of a human triple-negative breast cancer cell line (MM231) and a luminal A breast cancer cell line (MCF7). The purified exosome fractions were enzymatically digested and subjected to shotgun proteomics analysis by mass spectrometry (Orbitrap mass spectrometer) to detect proteins contained in the exosome fraction. Laminin β3 was detected at high concentrations in triple-negative breast cancer cell lines but at low concentrations in luminal A breast cancer cell lines. Because laminin β3 is only found in laminin 5, our results suggest that laminin 5 is highly expressed in triple-negative breast cancer cell lines and is abundant in exosomes.

[0054] <Experiment 2> The expression of laminin 5 was confirmed using other human triple-negative breast cancer cell lines. Exosome fractions were purified by ultracentrifugation from the culture supernatants of 11 human triple-negative breast cancer cell lines and the luminal A breast cancer cell line (MCF7), and the expression levels of laminin 5 and CD9 were detected by immunochromatography.

[0055] (Detection of CD9 expression level) A mouse anti-CD9 antibody (manufacturer: Hansa Bio Med LifeSciences, product number: HBM-CD9-100) was immobilized in the center of a half-strip immunochromatographic test paper with a nitrocellulose membrane. The membrane was then blocked with a water-soluble polymer and BSA. The end of the immunochromatographic test paper that did not have an absorbent pad attached was filled with 1 x 10 9The exosomes were immersed in a solution containing 100 exosomes and developed on immunochromatographic test paper. The exosome solution was prepared by diluting the exosome fraction purified in Experiment 1 above with PBS containing 1% BSA and 0.05% Tween 20. Next, the exosomes were developed with a developing solution containing an HRP-labeled mouse anti-CD9 antibody using the Ab-10 Rapid Peroxidase Labeling Kit (manufacturer: Dojindo Laboratories, product number: LK33). The resulting exosomes were then exposed to light using an ImmunoStar® LD (manufacturer: Fujifilm Wako Pure Chemical Corporation, product number: 290-69904), and the light was detected using an Image Quant LAS4000.

[0056] (Detection of laminin 5 expression level) The same procedure was repeated, substituting rabbit anti-LAM5 antibody (manufacturer: Abcam, product number: ab14509) for the above antibody. 10 A solution containing exosomes was developed. Next, a developing solution containing HRP-labeled rabbit anti-LAM5 antibody was developed using the Ab-10 Rapid Peroxidase Labeling Kit (manufacturer: Dojindo Laboratories, product number: LK33). The resulting solution was then irradiated with ImmunoStar® LD and detected using an Image Quant LAS4000.

[0057] (Measurement of detection intensity (brightness difference)) The total brightness of the area around the antibody-immobilized area on the immunochromatography test paper and the total brightness of the same area that did not emit light were measured using ImageJ, and the expression levels of CD9 and laminin 5 were calculated from the difference between them (brightness difference). The normalized value (laminin 5 / CD9) obtained by dividing the expression level of laminin 5 by the expression level of CD9 is shown in Figure 4. Compared to a cell line (MCF7) that was not derived from triple-negative breast cancer, laminin 5 / CD9 was upregulated in all 11 triple-negative breast cancer cell lines examined.

[0058] <Experiment 3> We investigated whether laminin 5 expression was elevated in biological samples from triple-negative breast cancer patients. Plasma samples from three breast cancer patients (HER2, triple-negative, and luminal B) and three healthy controls were used as samples, and laminin 5 and CD9 were detected by immunochromatography.

[0059] (Detection of CD9 expression level) The immunochromatographic test paper used was prepared by immobilizing mouse anti-CD9 antibody using the same method as in Experiment 2 above, and blocking the membrane with a water-soluble polymer and BSA. Plasma was diluted 3-fold with PBS containing 1% BSA and 0.05% Tween 20, and 9 μL of the solution was filtered through a 0.22 μm pore syringe filter. Next, a developing solution containing HRP-labeled mouse anti-CD9 antibody was developed using the same method as in Experiment 2. The sample was then irradiated using ImmunoStar® LD (product number: 290-69904, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the luminescence was detected using an Image Quant LAS4000.

[0060] (Detection of laminin 5 expression level) The immunochromatographic test paper used was prepared by immobilizing rabbit anti-LAM5 antibody using the same method as in Experiment 2 above, and blocking the membrane with a water-soluble polymer and BSA. Plasma was diluted 3-fold with PBS containing 1% BSA and 0.05% Tween 20, and 90 μL of the solution filtered through a 0.22 μm pore syringe filter was applied to the immunochromatographic test paper. Next, a developing solution containing HRP-labeled rabbit anti-LAM5 antibody was applied using the same method as in Experiment 2. The sample was then irradiated using ImmunoStar® LD (product number: 290-69904, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the luminescence was detected using an Image Quant LAS4000.

[0061] As a result, laminin 5 and CD9 were detected, as shown in the left panel of Figure 5. The total brightness of the area around the antibody-immobilized area on the immunochromatography test paper and the total brightness of the same area that did not emit light were measured using ImageJ, and the expression levels of laminin 5 and CD9 were calculated from the difference between these values (brightness difference). The value obtained by dividing the brightness of laminin 5 by the brightness of CD9 is shown in the right panel of Figure 5. The laminin 5 / CD9 ratio in the plasma of breast cancer patients was higher than that of healthy subjects, and the laminin 5 / CD9 ratio in triple-negative breast cancer patients was higher than that in patients with other subtypes of breast cancer.

[0062] <Experiment 4> Furthermore, we measured the expression levels of CD9 and laminin-5 in five samples from HER2 breast cancer patients and 10 samples from triple-negative breast cancer patients. The experimental method was the same as in Experiment 3. Figure 6 shows the laminin-5 intensity divided by the CD9 intensity. Compared to the laminin-5 / CD9 ratio in the plasma of HER2 breast cancer patients, the laminin-5 / CD9 ratio in the plasma of triple-negative breast cancer patients was more than two-fold higher in six of the ten samples. Furthermore, based on the amount of laminin-5, we were able to diagnose the subtype (triple-negative) of breast cancer patients with 100% specificity and 60% sensitivity. These results suggest that laminin-5 can be used as a diagnostic marker for breast cancer subtypes.

[0063] <Experiment 5> We increased the number of samples from breast cancer patients and healthy controls to examine whether laminin 5 expression was elevated in breast cancer patients. We measured the expression levels of CD9 and laminin 5 in the plasma of 20 healthy controls and 45 breast cancer patients. The measurement method was the same as in Experiment 3. The subtypes and stages of breast cancer patients are shown in Tables 2 and 3.

[0064] [Table 2]

[0065] [Table 3]

[0066] The normalized value (laminin 5 / CD9) obtained by dividing the laminin 5 intensity by the CD9 intensity is shown in Figure 7A. The laminin 5 / CD9 ratio in breast cancer patients was statistically significantly (p = 0.0011) higher than that in healthy controls. Figure 7B shows the receiver operating characteristic (ROC) curve demonstrating the discrimination ability of laminin 5 / CD9 in breast cancer patients. The area under the curve (AUC) was 0.80. These results suggest that laminin 5 expression is elevated in breast cancer patients and has a high ability to discriminate between breast cancers.

[0067] We compared the expression levels of laminin 5 in 32 specimens from early-stage breast cancer patients (stages I and II) with those from 20 healthy controls. As shown in Figure 8A, the laminin 5 / CD9 ratio in early-stage breast cancer patients was statistically significantly higher (p = 0.0007) than that in healthy controls. Figure 8B shows the ROC curve demonstrating the discriminatory ability of laminin 5 / CD9 in early-stage breast cancer patients. The AUC was 0.82. Laminin 5 expression is elevated in early-stage breast cancer patients, suggesting its high discriminatory ability.

[0068] <Experiment 6> The performance of laminin-5 as a diagnostic marker for early-stage breast cancer was verified using samples from a different population (20 samples from breast cancer patients and 20 samples from healthy controls) than in Experiment 5. The breast cancer patient stages are shown in Table 4. The expression levels of laminin-5 and CD9 in the plasma of breast cancer patients and healthy controls were measured using the same method as in Experiment 5. The calculated laminin-5 / CD9 ratio is shown in Figure 9A. Laminin-5 expression was statistically significantly elevated in breast cancer patients compared to healthy controls (p = 0.0003). The AUC of the ROC curve for breast cancer patients was 0.89 (Figure 9B), confirming the superiority of laminin-5 in identifying early-stage breast cancer. These results demonstrate that laminin-5 also has high performance as a marker for identifying early-stage breast cancer.

[0069] [Table 4]

[0070] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0071] (Section 1) One embodiment of a method for assisting in the diagnosis of breast cancer includes a measurement step of measuring the amount of laminin 5 or laminin β3 contained in a sample, and an information provision step of providing information for diagnosing breast cancer based on the measured amount of laminin 5 or laminin β3.

[0072] According to the diagnostic assistance method described in item 1, it is possible to easily provide information for diagnosing breast cancer.

[0073] (Section 2) In the diagnostic assistance method described in item 1, the information for diagnosing breast cancer includes information for diagnosing whether or not it is breast cancer.

[0074] According to the diagnostic assistance method described in item 2, information for diagnosing whether or not a patient has breast cancer can be easily provided.

[0075] (Section 3) In the diagnostic assistance method according to item 1 or 2, the information for diagnosing breast cancer includes information for diagnosing breast cancer subtypes.

[0076] According to the diagnostic assistance method described in item 3, information for diagnosing breast cancer subtypes can be easily provided.

[0077] (Section 4) In the diagnostic support methods described in items 1 to 3, the information for diagnosing breast cancer includes information for diagnosing the stage of breast cancer.

[0078] According to the diagnostic assistance method described in item 4, information for diagnosing the stage of breast cancer can be easily provided.

[0079] (Section 5) In the diagnostic auxiliary method according to any one of items 1 to 4, the amount of laminin 5 or laminin β3 is measured by immunoassay or mass spectrometry in the measuring step.

[0080] According to the diagnostic aid method described in item 5, the amounts of laminin 5 and laminin β3 can be measured with high accuracy.

[0081] (Section 6) In the diagnostic aid method described in item 5, the immunological assay is an immunochromatography method.

[0082] According to the diagnostic aid method described in item 6, the amounts of laminin 5 and laminin β3 can be measured quickly and easily.

[0083] (Section 7) In the diagnostic aid method according to item 5 or 6, the antibody used in the immunoassay is labeled with a metal particle or an enzyme.

[0084] When the antibody is labeled with an enzyme, the amount of laminin 5 or laminin β3 can be measured with high sensitivity. When the antibody is labeled with metal particles, the amount of laminin 5 or laminin β3 can be detected using visible light (absorbance).

[0085] (Section 8) In the diagnostic aid method described in item 7, the metal particles are detected by inductively coupled plasma mass spectrometry (ICP-MS).

[0086] According to the diagnostic aid method described in item 8, even small amounts of metal particles can be detected, and therefore the amount of laminin 5 or laminin β3 can be measured with high sensitivity.

[0087] (Section 9) In the diagnostic support method according to any one of items 1 to 8, the amount of laminin 5 or laminin β3 is standardized with the amount of exosomes in the information providing step.

[0088] According to the diagnostic assistance method described in item 9, the accuracy of information for diagnosing breast cancer can be improved.

[0089] (Section 10) In the diagnostic aid method described in item 9, the amount of exosomes is measured by CD9 and normalized to the amount of laminin 5 or laminin β3.

[0090] According to the diagnostic assistance method described in item 10, the accuracy of information for diagnosing breast cancer can be further improved.

[0091] (Section 11) In the diagnostic support methods described in items 1 to 10, the sample is a biological sample collected from a subject.

[0092] According to the diagnostic assistance method described in item 11, it is possible to provide information for diagnosing breast cancer in a subject.

[0093] (Section 12) In the diagnostic support methods described in items 1 to 11, the sample is blood collected from a human individual.

[0094] According to the diagnostic assistance method described in item 12, it is possible to provide information for diagnosing human breast cancer.

[0095] (Section 13) In the diagnostic auxiliary method according to any one of items 1 to 12, the sample is a purified fraction of exosomes.

[0096] According to the diagnostic support method described in item 13, it is possible to reduce impurities in a sample, thereby improving the accuracy of measurement.

[0097] (Section 14) A breast cancer test kit according to one embodiment comprises an anti-laminin 5 antibody or an anti-laminin β3 antibody.

[0098] According to the breast cancer test kit described in item 14, by measuring the amount of laminin 5 or laminin β3 contained in a sample, it is possible to easily provide information for diagnosing breast cancer.

[0099] (Section 15) In the test kit according to item 14, the anti-laminin 5 antibody or anti-laminin β3 antibody is labeled with a metal particle or an enzyme.

[0100] Enzyme-labeled antibodies can measure the amount of laminin 5 or laminin β3 with high sensitivity, while metal particle-labeled antibodies can detect laminin 5 or laminin β3 using visible light (absorbance).

[0101] (Section 16) 16. The test kit according to claim 14 or 15, further comprising an immunochromatographic test paper.

[0102] The test kit described in item 16 allows the amount of laminin 5 or laminin β3 to be measured quickly and easily.

[0103] (Section 17) In one embodiment, the breast cancer diagnostic marker comprises laminin 5 or laminin β3 contained in a sample.

[0104] The diagnostic marker described in item 17 provides useful information for diagnosing breast cancer in a simple and convenient manner.

[0105] (Section 18) A measurement method according to one embodiment measures the amount of laminin 5 or laminin β3 contained in a sample.

[0106] The amount of laminin 5 or laminin β3 contained in a sample can be determined by the measurement method described in item 18. The amount of laminin 5 or laminin β3 can be used as information for determining whether the sample is derived from breast cancer.

[0107] (Section 19) A method for diagnosing breast cancer according to one embodiment includes determining that a sample is derived from breast cancer when the amount of laminin 5 or laminin β3 contained in the sample is equal to or greater than a predetermined value.

[0108] According to the method for diagnosing breast cancer according to item 19, it is possible to easily diagnose whether a sample is derived from breast cancer.

[0109] (Section 20) In one embodiment, an anti-laminin 5 antibody or an anti-laminin β3 antibody is used to diagnose breast cancer.

[0110] The use described in paragraph 20 makes it possible to diagnose breast cancer or provide information for diagnosing it.

[0111] (Section 21) In one embodiment, an anti-laminin 5 antibody or an anti-laminin β3 antibody is used to produce a breast cancer testing kit.

[0112] According to the use described in item 21, a breast cancer test kit can be produced.

[0113] The embodiments and examples disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0114] 10 immunochromatographic test paper, 11 membrane, 12 absorbent pad, 13 capture antibody, 14 sample pad, 15 conjugate pad, 16 detection target, 17 detection antibody.

Claims

1. measuring the amount of laminin 5 or laminin β3 contained in blood collected from a subject to be diagnosed with breast cancer; and providing information for diagnosing breast cancer based on the amount of laminin 5 or laminin β3 obtained by standardizing the measured amount of laminin 5 or laminin β3 with the amount of exosomes contained in the blood, A diagnostic aid method for breast cancer, wherein the step of measuring the amount of laminin 5 or laminin β3 is performed by immunochromatography.

2. The method of claim 1 , wherein the information for diagnosing breast cancer includes information for diagnosing whether or not the patient has breast cancer.

3. The method of claim 1 or 2, wherein the information for diagnosing breast cancer includes information for diagnosing breast cancer subtypes.

4. The method according to any one of claims 1 to 3, wherein the information for diagnosing breast cancer includes information for diagnosing a stage of breast cancer.

5. The method of claim 1 , wherein the antibody used in the immunochromatography method is labeled with a metal particle or an enzyme.

6. The method of claim 5, wherein the metal particles are detected by inductively coupled plasma mass spectrometry (ICP-MS).

7. The method of claim 1, wherein the amount of exosomes is measured by CD9 and normalized to the amount of laminin 5 or laminin β3.

8. an anti-laminin 5 antibody or an anti-laminin β3 antibody; A breast cancer testing kit comprising an antibody that captures an exosome marker.

9. The test kit according to claim 8 , wherein the anti-laminin 5 antibody or anti-laminin β3 antibody is labeled with a metal particle or an enzyme.

10. The test kit according to claim 8 or 9, comprising an immunochromatographic test paper.

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