WO2019168085A1 - 固定化細胞又はffpe組織切片から抗原性を増強した細胞核を脱離する方法並びにそのための抗原賦活剤及びキット - Google Patents
固定化細胞又はffpe組織切片から抗原性を増強した細胞核を脱離する方法並びにそのための抗原賦活剤及びキット Download PDFInfo
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Definitions
- the present invention relates to a pretreatment method for detecting a cell containing a Ki-67 protein positive cell nucleus using a Ki-67 antibody, a detection method of the cell, a kit used for the pretreatment method or the detection method, and the detection It relates to treatment regime selection using the method.
- Formalin fixation of surgically removed tissue is the world's most common method and standard pathological technique for preserving cancer tissue samples.
- the most common method of storing tissue is to immerse the entire tissue in a formalin aqueous solution for extended periods (8-48 hours), and then place the fixed tissue in paraffin wax for long-term storage at room temperature. To embed.
- the molecular analysis method for analyzing formalin-fixed cancer tissue is the most frequently used method for analyzing cancer patient tissue, for example.
- An immunohistochemical staining method which is one of diagnostic techniques using formalin-fixed paraffin-embedded (FFPE) tissue, is present on the surface of a pathological section tissue or cell after slicing the tissue and pasting it on a slide glass.
- FFPE formalin-fixed paraffin-embedded
- HER2 human epidermal growth factor receptor
- ER estrogen receptor
- PgR progesterone receptor
- Ki-67 a factor in which the presence or absence of the factor correlates with the prognosis.
- IHC method immunohistochemical method of a tumor tissue sample is frequently used. A method that considers both the staining intensity of the tumor cells and the ratio of the stained cells (All red Score, etc.) Either one is used.
- the IHC method is used. If the result is 0 or 1+, the result is negative. If the result is 2+, the result is positive. If it is positive, it is determined as negative. In the case of ER, 3 to 8 is positive in All red Score, while 10% was often used as the cut-off value when judged by the ratio of stained cells. Some say it should be positive. In the case of Ki-67, a method of calculating a positive ratio in the whole tissue, a method of selecting and calculating a region called a hot spot where positive cells gather are mixed. In any case, if a certain cut-off value is set, it can be a guideline for an effective treatment regimen when these effect predictors are judged to be positive.
- Luminal A (like) type ER / PgR positive, HER2 negative, Ki-67 low ( ⁇ 14-20%)
- Luminal B (like) type HER2 negative
- ER ⁇ PgR positive, HER2 negative Ki-67 high ( ⁇ 14-20%)
- Luminal B (like) type HER2 positive
- Non-luminal type ER / PgR negative, HER2 positive
- Triple negative type ER negative, PgR negative, HER2 negative Affects the selection of treatment regimen.
- Non-patent Documents 6, 10 to 12 when it is judged that luminal A (like) type has a low Ki-67 level, endocrine therapy alone is mainly selected, whereas luminal B (like) has a high Ki-67 level.
- the type is determined to be HER2 negative, a combination of endocrine therapy and chemotherapy is mainly selected (Non-patent Documents 6, 10 to 12).
- the above determination is performed by visual determination using a microscope, and the counting is performed manually. Therefore, even an experienced pathologist needs time and effort.
- Ki-67 is a nuclear protein and a commonly used cell proliferation marker. Molecules expressed on the nucleolus and mitotic chromosomes of proliferating cells, expressed in all phases of the cell cycle, changes in expression from the late G1 phase, and increased in the S phase Occurs and is greatest in the M period. Therefore, the cells in which Ki-67 expression is observed only indicate that they are in the cell cycle.
- reagents and methods for diagnosing cancer using Ki-67 as one of the biomarkers are known (Patent Documents 1 to 5), and cells are automatically selected to save time and labor for the measurement.
- Patent Documents 6 to 8 There are known cell image automatic analysis apparatuses and methods for automated tissue analysis.
- Non-Patent Document 4 an antibody targeting the same Ki-67 protein was detectable with clone name: S5 but not with clone name: MIB-1 having a different antigen recognition site. It has been reported that Furthermore, in the case of formalin-fixed tissue or thrombin: (plasma) fibronectin-fixed tissue, the MIB-1 antibody may give false positive or false negative results (Non-patent Document 9).
- kits for extracting nucleic acids have been put into practical use, but remain in research reagents and have not yet been put into practical use.
- the immunohistological staining method is performed by visual judgment using a microscope, and the count is also performed manually. Therefore, even if the same pathological tissue section is used, the judgment is often different depending on the pathologist ( Comparative Example 2). Since the abundance ratio (%) (cutoff value) of Ki-67 positive cells affects the selection of treatment regimen, a more objective, reproducible, and universal quantification method for Ki-67 positive cell abundance ratios is required. It was.
- a tissue sample containing a pathological tissue section is prepared and stored as a standard pathological technique at the time of cancer resection or biopsy, and it should be a quantitative method in which the stored sample can be used. desirable.
- FFPE tissue FFPE tissue
- digestion with these enzymes can cleave the recognition site of the target antigen, limiting the antibodies that can be used. Therefore, cell dispersion and a method for detaching the cell nucleus other than the above existing methods are required.
- the inventors of the present application have studied a pretreatment method using an enzyme that is not suitable for cell dispersion and cell detachment, and a tissue or cell with an enzyme that does not recognize the epitope of MIB-1 (rare cutter enzyme).
- the Ki-67 antigen was successfully activated while enhancing the antigenicity of other antigens including cytokeratin.
- cell nuclei are detached from FFPE sections with enhanced antigenicity, Ki-67 protein present in the nuclei is targeted, and specific antibodies are reacted to make it more objective and reproducible.
- a highly universal method for detecting Ki-67 positive cells was completed.
- the present invention is as follows [1] to [45].
- [1] A method for detecting a cell containing a Ki-67 positive cell nucleus in an immobilized cell group using an anti-Ki-67 antibody, 1) a step of pre-treating the immobilized cell group with a hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2 to activate the antigen; then 2) a step of staining with an anti-Ki-67 antibody; )
- a method comprising detecting stained Ki-67 positive cells or Ki-67 positive cell nuclei; [2] The method according to [1], wherein the antibody is at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047; [3] The method according to [1] or [2], wherein the enzyme is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline endopeptidase and hy
- [7] The method according to any one of [1] to [6], further comprising specifically staining cell nuclei and detecting (eg, counting) the stained cell nuclei; [8] The method according to any one of [1] to [7], further comprising staining cytokeratin with an anti-cytokeratin antibody (for example, fluorescent staining) and detecting stained positive cells. the method of; [9] Further, ER and / or PgR is stained (for example, fluorescent staining) using an anti-estrogen receptor (ER) antibody and / or anti-progesterone receptor (PgR) antibody, and the stained positive cells or positive cell nuclei are stained.
- ER and / or PgR is stained (for example, fluorescent staining) using an anti-estrogen receptor (ER) antibody and / or anti-progesterone receptor (PgR) antibody, and the stained positive cells or positive cell nuclei are stained.
- a method for determining a cancer treatment regimen wherein the ratio of Ki-67 positive cells in a cell group is calculated by the method according to [20] or [21], and the ratio is a cutoff value.
- [25] Calculate the ratio of Ki-67 positive cells in the cell group by the method described in [20] or [21]. If the ratio is equal to or higher than the cut-off value, select a combination of endocrine therapy and chemotherapy. If the percentage is less than the cut-off value, endocrine therapy alone is selected and administered to the patient to treat the cancer.
- [26] A method of activating Ki-67 by pretreating the immobilized tumor cell group or tumor tissue with a hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2;
- An antigen activator for use in a cell or tissue sample that simultaneously detects Ki-67 and cytokeratin by immunostaining comprising a hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2; [31] The antigen activator according to any one of [27] to [30], which is further used for a cell or tissue sample to be detected by immunostaining ER and / or PgR; [32] The antigen activator according to any one of [27] to [31], which is further used for a sample for detecting a cell in which the HER2 gene is amplified;
- a kit for detecting Ki-67 positive cells in immobilized cells A hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2, A kit comprising an anti-Ki-67 antibody;
- the hydrolase is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline endopeptidase and hyaluronidase;
- the hydrolase is thrombin and / or hyaluronidase;
- Any one of [33] to [35], wherein the anti-Ki-67 antibody is at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047.
- a kit according to [37] in combination with Ki-67, includes a ligand for detecting other markers used to aid in the diagnosis of cancer or to help diagnose the prognosis of cancer treatment [33] to [36], a kit according to any one of the above; [38] The kit according to [37], wherein the ligand is at least one selected from the group consisting of an anti-cytokeratin antibody, an anti-ER antibody, an anti-PgR antibody, and a probe that hybridizes to the HER2 gene; [39] The kit according to any one of [33] to [38], further comprising a compound for nuclear staining; [40] The kit according to any one of [33] to [39], further comprising a buffer for dispersing the cells, wherein the buffer contains a surfactant; [41] The kit according to any one of [33] to [40], further comprising a heat treatment antigen activator; [42] The kit according to any one of [33] to [41], which assists diagnosis of cancer.
- a kit for crushing cells fixed by shear stress generated by water flow, ultrasonic waves, etc., and extracting cell nuclei A kit comprising a cell dispersion buffer containing a surfactant; [47] A method of extracting cell nuclei by crushing fixed cells by shear stress generated by water flow, ultrasonic waves, etc. A method in which the immobilized cells are dispersed in a buffer containing a surfactant to disrupt the cells; and [48] a cell in which the target antigen in the immobilized cell group is present in the cell nucleus.
- a method of detecting using an antibody of the antigen 1) a step of pretreating the immobilized cell group with a hydrolase that does not cleave the antigen recognition site of the antibody to activate the antigen; then 2) a step of staining the cell with the antibody; And 3) a method comprising a step of detecting stained antigen-positive cells or antigen-positive cell nuclei.
- a method for detecting (quantifying) Ki-67 positive cells with high objectivity, reproducibility, and universality is established.
- detachment of cell nuclei with enhanced antigenicity of the target antigen is achieved from the FFPE tissue section.
- the collected dispersed cell nuclei can be analyzed as a single cell nucleus. For example, proteins, nucleic acids, etc. on the nuclear membrane or in the nucleus can be detected, and can be detected by staining with dyes (fluorescent substances, chemical issuing substances, enzymes, etc.) or using antibodies modified with these dyes. .
- the ratio of positive nuclei of the protein of interest in the detached nuclei can be rapidly calculated by flow cytometry analysis.
- the cut-off value for Ki-67 required a visual determination and counting process by a pathologist, and thus varied and was different for each facility.
- the protocol (pretreatment reagent and process) provided by the present invention it is possible to provide a global standard of cut-off values for pathological diagnosis with little variation. And it is possible to provide a patient with a high QOL diagnostic regimen using such a cutoff value.
- FIG. 3 is a histogram in which a region surrounded by a frame in FIG. 2 is gated as a cell nucleus region, and the fluorescence intensity of DAPI is shown on the horizontal axis and the number of cells (nuclei) is shown on the vertical axis for the data of the gating fraction.
- Formalin-fixed breast cancer cells are subjected to antigen activation by heat treatment, immunofluorescent staining with anti-cytokeratin antibody and anti-Ki-67 antibody or their isotype control antibody, followed by analysis with a flow cytometer and selection as a cell nucleus region
- the histogram which showed the fluorescence intensity as a horizontal axis
- Formalin-fixed tumor cell lines (MB231, T47D, SKBR) are subjected to antigen activation by heat treatment, and then, with or without antigen activation by thrombin, immunofluorescence staining with anti-Ki-67 antibody or its isotype control antibody , A histogram showing the fluorescence intensity on the horizontal axis and the number of cells (nuclei) on the vertical axis for the data of the gating fraction that was analyzed with a flow cytometer and selected as the cell nucleus region. The graph which shows the change of Ki-67 positive rate in each tumor cell line by the presence or absence of thrombin treatment.
- Formalin-fixed T47D cell line was subjected to antigen activation by heat treatment, and then treated with hyaluronidase reagent, or immunofluorescent staining with anti-Ki-67 antibody or its isotype control antibody without such treatment, Histogram showing the fluorescence intensity on the horizontal axis and the number of cells (nuclei) on the vertical axis for the data of the gating fraction analyzed by the flow cytometer and selected as the cell nucleus region.
- FFPE tissue sections of breast cancer tissue are deparaffinized / hydrophilized, antigen-activated by heat treatment, cell disruption by water flow, and immunofluorescence staining with anti-cytokeratin antibody and anti-Ki-67 antibody or their isotype control antibody Then, a histogram showing the fluorescence intensity on the horizontal axis and the number of cells (nuclei) on the vertical axis for the data of the gating fraction selected as the cell nucleus region analyzed by a flow cytometer.
- FFPE tissue sections of breast cancer tissue were deparaffinized / hydrophilized and antigen-activated by heat treatment, and the cells were disrupted by water flow with or without antigen activation by thrombin, and then anti-cytokeratin antibody and anti-Ki- Immunofluorescent staining with 67 antibodies or their isotype control antibodies, followed by analysis with a flow cytometer, and for the data of the gating fraction selected as the cell nucleus region, the horizontal axis represents the fluorescence intensity and the vertical axis represents the number of cells (nuclei). Histogram shown as. This confirms the enhancement of cytokeratin and Ki-67 signal in the FFPE tissue section specimen by thrombin treatment.
- Each formalin-fixed tumor cell line (MB231, T47D, SKBR) is subjected to antigen activation by heat treatment, and then activated by any of five digestive enzymes (thrombin, trypsin, proteinase K, dispase, proline endopeptidase).
- FFPE tissue section of breast cancer tissue is deparaffinized / hydrophilized and antigen activated by heat treatment, antigen activated by any of five digestive enzymes (thrombin, trypsin, proteinase K, dispase, proline endopeptidase), and water flow
- the immunofluorescent staining with anti-Ki-67 antibody or its isotype control antibody followed by analysis with a flow cytometer, the fluorescence intensity is plotted on the horizontal axis for the data of the gating fraction selected as the cell nucleus region.
- a histogram showing the number of cells (nuclei) as a vertical axis.
- FFPE tissue section of breast cancer tissue is deparaffinized / hydrophilized and antigen activated by heat treatment, antigen activated by any of five digestive enzymes (thrombin, trypsin, proteinase K, dispase, proline endopeptidase), and water flow
- thrombin trypsin, proteinase K, dispase, proline endopeptidase
- water flow After crushing the cells, immunofluorescent staining with an anti-cytokeratin antibody or its isotype control antibody, followed by analysis with a flow cytometer, the data of the gating fraction selected as the cell nucleus region, the fluorescence intensity on the horizontal axis, A histogram showing the number of cells (nuclei) as a vertical axis.
- FFPE tissue sections of breast cancer tissue were deparaffinized / hydrophilized and antigen-activated by heat treatment, antigen-activated by thrombin, and cells were crushed by water flow, followed by two anti-Ki-67 antibodies (MIB-) with different recognition sites (1 clone and S5 clone) and anti-cytokeratin antibodies or their isotype control antibodies, and immunofluorescent staining, followed by analysis with a flow cytometer. Is a histogram showing the horizontal axis and the number of cells (nuclei) as the vertical axis.
- FFPE tissue sections of breast cancer tissue are deparaffinized / hydrophilized and then antigen-stimulated with trypsin, and one of two anti-Ki-67 antibodies (MIB-1 clone and S5 clone) having different recognition sites And immunofluorescent staining with anti-cytokeratin antibodies or their isotype control antibodies, followed by analysis with a flow cytometer, and the data of the gating fraction selected as the cell nucleus region, the fluorescence intensity is plotted on the horizontal axis, the number of cells (nuclei) Histogram showing the vertical axis.
- FFPE tissue sections with known ER and PgR positive or negative were subjected to deparaffinization / hydrophilization, antigen activation by heat treatment, antigen activation with thrombin, and further disruption of cells by water flow, anti-ER antibody and Immunofluorescent staining with anti-PgR antibodies or their isotype control antibodies, followed by analysis with a flow cytometer, and for the data of the gating fraction selected as the nucleus region, the fluorescence intensity is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis. Histogram shown as axis.
- FFPE tissue section of breast cancer tissue is deparaffinized / hydrophilized, antigen activated by heat treatment, antigen activated with thrombin, 4 different crushing methods (combination of masher, mortar, ultrasonic destruction, water flow destruction and ultrasonic destruction) ), followeded by immunofluorescence staining with anti-cytokeratin antibody and anti-Ki-67 antibody or their isotype control antibody, followed by analysis with a flow cytometer, and the gating fraction selected as the cell nucleus region A histogram showing the fluorescence intensity on the horizontal axis and the number of cells (nuclei) on the vertical axis.
- Formalin-immobilized two breast cancer cell lines SKBr3 and MDA-MB-231 and lymphoblastic cell line Jurkat were subjected to antigen activation treatment by heat treatment, antigen activation with thrombin, and disruption of cells by ultrasound Thereafter, cell nuclei were stained, then analyzed with a flow cytometer, and the obtained data was analyzed.
- Predetermined FFPE tissue section of breast cancer tissue treated with thrombin, further disrupted cells by water flow, dispersed cells in a buffer containing various surfactants and disrupted cells by ultrasound, A scattergram in which cell nuclei are stained and then analyzed by a flow cytometer, and the obtained data is analyzed to show forward scatter as the horizontal axis and side scatter as the vertical axis.
- slice of the breast cancer tissue by the IHC method computed by different pathologists.
- Formalin-fixed tumor cell lines (MB231, T47D, SKBR) were subjected to antigen activation by heat treatment, and after antigen activation with any of the three digestive enzymes (thrombin, proteinase K, dispase), anti-Ki-67 antibody Immunofluorescence staining with the S5 clone (Ki-S5) or its isotype control antibody, analysis with a flow cytometer, and the data of the gating fraction selected as the cell nucleus region, the fluorescence intensity on the horizontal axis, the number of cells (nuclei) Histogram showing the vertical axis.
- the three digestive enzymes thrombin, proteinase K, dispase
- Immunofluorescence staining with the S5 clone (Ki-S5) or its isotype control antibody
- analysis with a flow cytometer and the data of the gating fraction selected as the cell nucleus region, the fluorescence intensity on the horizontal axis, the number of cells
- Formalin-fixed tumor cell lines (MB231, T47D, SKBR) were subjected to antigen activation by heat treatment, and after antigen activation with any of the three digestive enzymes (thrombin, proteinase K, dispase), anti-cytokeratin antibodies Alternatively, a histogram showing immunofluorescence staining with an isotype control antibody, analysis with a flow cytometer, and gating fraction data selected as the cell nucleus region, with the fluorescence intensity on the horizontal axis and the number of cells (nuclei) on the vertical axis .
- Anti-cytokeratin after FFPE tissue section of breast cancer tissue is deparaffinized / hydrophilized and antigen activated by heat treatment, antigen activated with thrombin, cell disruption by water flow, and cell disruption by ultrasound Immunofluorescence staining with antibodies and anti-Ki-67 antibodies or their isotype control antibodies, followed by analysis with a flow cytometer, with respect to the data of the gating fraction selected as the cell nucleus region, ) Histogram with number on the vertical axis. This figure shows the positive rate when the threshold for determining positive nuclei is varied.
- FFPE tissue sections with known HER2 positive and negative were subjected to antigen activation by deparaffinization / hydrophilization and heat treatment, followed by antigen activation with thrombin, cell disruption with water flow, and cell disruption with ultrasound.
- Cell immobilization means that a sample is immersed in a fixative to stabilize cell morphology and tissue structure by molecular cross-linking or protein insolubilization.
- a known method may be used for immobilization, for example, a method using formalin solution, paraformaldehyde solution, glutaraldehyde solution, osmium tetroxide solution, alcohol acetate, methanol, ethanol, acetone or the like. Or you may fix by the freezing method.
- Ki-67 antigen (MKI67: Marker Of Proliferation Ki-67)” is a kind of nuclear protein, and in the case of humans, it is represented by the amino acid sequence of SEQ ID NO: 1 (3256 amino acids) (UniProtKB-P46013 (KI67_HUMAN) )). Ki-67 is the name of an antibody originally discovered as an autoantibody in the blood of leukemia patients. In this application, unless otherwise specified, “Ki-67” refers to a “Ki-67 antigen” protein. Thus, “anti-Ki-67 antibody” refers to an “antibody” that recognizes the Ki-67 antigen.
- Antibodies may be full-length (IgG, IgA, IgM, IgD, IgE) immunoglobulins, and include fragments (so-called fragment antibodies (Fab, Fab ′, F (ab ′) 2) containing the antigen-binding recognition region thereof. Etc.))). “Antibodies” may be derived from mammals such as humans, mice, rats, goats, horses and camels, as well as fish (including sharks) and birds (chicken). “MIB-1” or “MIB-1 antibody” is one of clones obtained by monoclonalizing Ki-67 antibody. Although it does not specifically limit, it can purchase from Dako company and Immunotech company.
- Anti-Ki-67 antibody refers to an antibody that recognizes and binds to Ki-67.
- MIB-1, MIB-2, MIB-5, MIB-7, MIB-21 and MIB-24 Antibodies belonging to the MIB (registered trademark) family, such as DAKO-PC, Ki-S5, A0047, etc. can also be used as “anti-Ki-67 antibodies”, and other characteristics same as those used in commercial or clinical tests These antibodies can also be used as “anti-Ki-67 antibodies”.
- the anti-Ki-67 antibody can be prepared by a known method using the Ki-67 antigen or a part thereof as an antigen.
- the “anti-Ki-67 antibody” used in the present invention preferably includes an antibody that recognizes the above-mentioned epitope.
- Thrombin (factor IIa) is an enzyme (serine protease) involved in blood coagulation (EC number: EC 3.4.21.5).
- the hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2 refers to a hydrolase that does not recognize and cleave the epitope of MIB-1 antibody.
- a proteolytic enzyme having such characteristics can be known from a known database (http://web.expasy.org/peptide_cutter/) (Table 1). Also, whether an enzyme has such properties can be confirmed by analyzing whether the enzyme cleaves the epitope using MIB-1 or another antibody that recognizes the same epitope.
- Such an enzyme is preferably capable of recognizing and cleaving collagen, which is an extracellular matrix, in order for the antibody to infiltrate cells and / or to extract cell nuclei.
- thrombin Arg-C (clostripain) peptidase (EC 3.4.22.8), proline endopeptidase (EC 3.4.21.26) and the like can be mentioned.
- an enzyme that hydrolyzes a component in an extracellular matrix other than a protein such as hyaluronidase is also preferred.
- Hyaluronidase (EC 3.2.1.35) is a hydrolase that degrades the ⁇ -1-4 bond of hyaluronic acid and does not cleave the peptide of SEQ ID NO: 2. This property of hyaluronidase promotes infiltration of antibodies into cells.
- hyaluronidase examples include hyaluronidase, glycosidase (EC 3.2.1), N-glycanase (EC 3.5.1.52), and hyaluronidase is preferable.
- the above-mentioned protein enzyme that does not recognize and cleave the peptide of SEQ ID NO: 2 is common to hyaluronidase in that it does not cleave the peptide of SEQ ID NO: 2, while both are enzymes having completely different actions, so that both must be combined. It is expected that the antigen inactivating activity is further increased.
- a combination of thrombin and hyaluronidase is preferable.
- Enzyme activation by enzyme is a process in which antigen or extracellular matrix is cleaved by enzyme treatment to amplify the reactivity of the antibody.
- the sample is immersed in a reagent in which the enzyme is dissolved in a pH-adjusted buffer solution. Can be done. Heat at a temperature suitable for the enzyme reaction, and stop the reaction after a predetermined time. To stop the reaction, reagent removal, temperature change, chelator addition or the like is used.
- “Staining with antibodies” may be performed by directly binding a label such as a fluorescent compound, enzyme, chemiluminescent substance, etc. to each antibody (direct method), and binding the labeled antibody to an antigen.
- a label such as a fluorescent compound, enzyme, chemiluminescent substance, etc.
- An antibody that binds to an antigen is used as an unlabeled primary antibody, and is labeled indirectly by binding a secondary antibody that is specific to the primary antibody and bound to the label (indirect method). Also good.
- the “fluorescent compound” is not particularly limited, and examples thereof include fluorescent substances such as cyanine dyes such as Cy3, fluorescein isothiocyanate (FITC), allophycocyanin, and rhodamine. It is preferable to label each antibody (or its secondary antibody) with fluorescent dyes having different emission wavelengths (for example, Alexa Fluor (registered trademark) series of fluorescent substances).
- fluorescent dyes having different emission wavelengths (for example, Alexa Fluor (registered trademark) series of fluorescent substances).
- Labeleling enzyme is not particularly limited, and examples thereof include alkaline phosphatase and horseradish peroxidase.
- the “chemiluminescent substance” is not particularly limited, and examples thereof include luminol, AMPPD (registered trademark), CSPD (registered trademark), and CDP-Star (registered trademark).
- each ligand for example, anti-cytokeratin antibody, anti-ER antibody, anti-PgR antibody, HER2
- FFPE fluorescence wavelengths with respect to the same
- Nucleic acids that hybridize to genes, or secondary antibodies or nucleic acids that bind to them) may be labeled and detected / quantified, or 2) multiple (FFPE) tissue sections hypothesized to be homogeneous from the same tissue block May be prepared, and each target substance may be stained (for example, immunofluorescent staining, FISH) for detection / quantification.
- FFPE multiple fluorescent staining
- Specific staining of cell nuclei refers to labeling with a compound that specifically stains cell nuclei in immobilized cells, and is not limited to this, but a compound for staining a cell-impermeable nucleic acid is not limited thereto.
- Preferable examples include fluorescent dyes such as DAPI and propidium iodide (PI).
- Cytometry Fluorescently stained cells or cell nuclei can be counted by cytometry.
- Cytometry is a cell measurement method that quantitatively measures a large amount (thousands to millions) of cells one by one in a short time (several seconds to several minutes).
- flow cytometry Flow Cytometry
- Imaging cytometry that extracts fluorescent cell images, scattered light images, transmitted light images, etc. by laser scanning the cell population attached on the slide glass or the slide glass, and extracts information for each cell by cell image processing ( Imaging Cytometry).
- the process of extracting cell nuclei refers to extracting cell nuclei from fixed cells (tissues) while maintaining their structure. It can be performed using means for generating shear stress on the cells and crushing the cells, but is not limited thereto, for example, water flow crushing, masher, mortar, ultrasonic crushing, mesh, French press, homogenizer treatment, glass beads It can carry out using well-known methods, such as a process.
- the present invention is a method for detaching cell nuclei and further enhancing a staining signal without losing antigenicity from the FFPE tissue and without limiting the antibody or detection method used. including: 1) Deparaffinization / hydrophilization of sliced FFPE sections 2) Antigen activation by heat treatment 3) Antigen activation by enzymes 4) Disruption of tissues and cells to extract cell nuclei
- the sliced FFPE tissue section to be used can detect a desired marker as long as the thickness of the sliced tissue is 60 ⁇ m or less in order to improve the efficiency of the pretreatment process. Recommended but not limited to this.
- the number of slices can be increased to a plurality.
- Embedding means that tissue pieces (lumps) are made to have a constant and uniform hardness, fill the inner cavity of the tissue, have a strength that does not peel off when sliced, and increase storage stability.
- Embedding agent examples include, but are not limited to, paraffin, paraffin derivative, celloidin, carbowax, agarose, non-heparin-treated serum, collagen, cellulose derivative, chitin derivative, chitosan derivative, and a mixture thereof.
- de-embedding / hydrophilization refers to removal of an embedding agent (for example, paraffin) used for embedding and replacement of an organic solvent used for the removal with an aqueous solvent. Remove the embedding agent by immersing the slice in an organic solvent typified by xylene, and then change the concentration of ethanol solution with a concentration gradient to replace the organic solvent. Infiltrate sequentially. Examples of the ethanol concentration gradient include, but are not limited to, 100%, 95%, 90%, 70%, and 50%.
- “antigen activation by heat treatment” is a step of removing an antigen recognition site by heat treatment when the antigen recognition site is masked by cross-linking during fixation.
- a heat treatment antigen activator containing a citrate buffer, a surfactant, a chelating agent, a reducing agent and the like is used.
- commercially available Histo VT One Nacalai Tesque
- antigen activation solution pH9 Nacalai Tesque
- ImmunoSaber Nashin EM
- “Crushing by water flow” means that cells and tissues are crushed by a shearing force of water flow.
- RP-10 water flow shearing device manufactured by Sysmex, for example, blades per minute under ice cooling. Cells and tissues can be crushed by the water flow generated by rotating at 10,000 rpm.
- Ultrasonic disruption means that cells and tissues are disrupted by ultrasonic shearing force.
- an ultrasonic disruption device (VCX130PB) manufactured by SONICS & MATERIALS is used, and cells and Tissues can be exposed and crushed.
- the crushing process by ultrasonic waves is useful when crushing lymphocytes preferentially over other blood cells and crushing not only the cell walls of lymphocytes but also the lymphocyte nuclei. Therefore, when it is desired to disrupt lymphocytes preferentially, such as when the presence of lymphocytes hinders analysis of other cells, it is preferable to include an ultrasonic disruption step.
- two or more of the above-described methods may be combined, and a combination of water shearing and ultrasonic disruption is preferred.
- the tissue or cells may be immersed or dispersed in a commonly used buffer, such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, carbonate buffer, glycine buffer, An acetate buffer, a tartrate buffer, a citrate buffer, a triethanolamine buffer, a borate buffer, a Good buffer, or the like may be used.
- a commonly used buffer such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, carbonate buffer, glycine buffer, An acetate buffer, a tartrate buffer, a citrate buffer, a triethanolamine buffer, a borate buffer, a Good buffer, or the like may be used.
- a buffer solution to which a surfactant is added.
- the surfactant is not particularly limited as long as it does not affect the cell nucleus, and is an anionic surfactant (carboxylic acid type, sulfonic acid type, sulfate ester type, phosphate ester type, etc.), cationic surfactant ( Quaternary ammonium salt type, alkylamine salt type, type having pyridine ring, etc.), amphoteric surfactant (betaine type, sulfobetaine type, amine oxide type, alkylimidazole type, amino acid type, etc.), nonionic surfactant
- the surfactant include an ester type, an ether type, an ester ether type, an alkanolamide type, and an alkyl glycoside.
- Preferred examples include nonionic surfactants Triton X-100, Triton X-405, NP-40, Briji-35, Briji-58, Tween-20, Tween-80, BPSH-25, Octyl Glucoside, and Octylthio Glucoside. .
- a malignant tumor is a tumor that infiltrates surrounding tissues or causes metastasis in a cell population (including tumors, benign tumors and malignant tumors) that has grown autonomously and uncontrolled by genetic mutation.
- the term malignant tumor is classified into 1) carcinoma (carcinoma): malignant tumor derived from epithelial tissue 2) sarcoma (malignant tumor derived from non-epithelial tissue 3) and others: leukemia and the like.
- carcinoma carcinoma
- sarcoma malignant tumor derived from non-epithelial tissue 3
- leukemia leukemia and the like.
- cancer means carcinoma.
- head and neck cancer maxillary cancer, (upper, middle, lower) pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer
- breast cancer breast cancer
- lung cancer non-small cell lung cancer, small cell lung cancer
- Gastrointestinal cancer esophageal cancer, stomach cancer, duodenal cancer, colon cancer (colon cancer, rectal cancer)
- liver cancer hepatocellular carcinoma, cholangiocellular carcinoma
- gallbladder cancer gallbladder cancer
- bile duct cancer pancreatic cancer, anal cancer, urinary cancer (renal cancer) , Ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, testicular cancer, genital cancer (uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, vulvar cancer, vaginal cancer), skin cancer (basal)
- cancer cells such as cell carcinoma and squamous cell carcinoma).
- Ki-67 alone or in combination with other markers, can be used to assist in the diagnosis of cancer or to help diagnose the prognosis of cancer treatment.
- Cytokeratin is an example of an epithelial cell marker used to distinguish between carcinoma and sarcoma. Cytokeratin is a major skeletal protein of epithelial cells, and about 20 to 30 subtypes (molecular weight 40 to 68 KDa) have been reported.
- Antibodies that recognize known broad subtypes are used to detect cytokeratin as an epithelial cell marker.
- the estrogen receptor is one of the molecules belonging to the steroid receptor superfamily, and is also called follicular hormone receptor.
- ER ⁇ and ER ⁇ are two isoforms of ER, respectively. These are produced from independent genes (ESR1, ESR2).
- ESR1 is present at 6q25.1 and ESR2 is present at 14q21-22.
- Progesterone receptor PR or PgR is a nuclear protein belonging to subfamily 3 group C of the nuclear receptor superfamily.It is encoded by a single PgR gene present in 11q22 and has a different molecular weight. Two isoforms are known.
- HER2 (Hertwo) is a glycoprotein of about 185 kDa that exists on the cell surface and is a receptor tyrosine kinase. It has a structure similar to the epidermal growth factor receptor (EGFR, also known as ERBB1) and is also called EGFR2, ERBB2, CD340, or NEU.
- EGFR epidermal growth factor receptor
- ERBB1 epidermal growth factor receptor
- ERBB2 also known as ERBB1
- CD340 or NEU.
- the gene encoding the HER2 protein is HER2 / neu, erbB-2, and is present on the long arm of chromosome 17.
- HER2 is a protein belonging to human epidermal growth factor receptor (HER / EGFR / ERBB) family (EGFR family).
- the HER2 protein is involved in the regulation of cell proliferation and differentiation in normal cells, but if for some reason HER2 gene amplification or gene mutation occurs, the cell growth / differentiation cannot be controlled, and the cell becomes malignant. Turn into.
- the HER2 gene is also an oncogene, and gene amplification is observed in many types of cancer.
- the sample obtained in the above-described antigen activation step with an enzyme and further in the step of extracting cell nuclei can be used for analysis of these markers in combination with Ki-67 to assist diagnosis.
- enzyme treatment combining thrombin and hyaluronidase is preferable when these markers are also analyzed.
- the HER2 gene may be detected using a nucleic acid extracted by a known method after detachment of the nucleus.
- a “patient” may be a mammal and may be a “human” or “non-human mammal”.
- tissue section refers to a section of isolated tissue of a human or non-human animal. It may be a “tissue section” taken by biopsy. The “tissue section” may be cryopreserved after isolation.
- Biopsy means that a portion of the pathological tissue is collected with a scalpel or a needle for observation with a microscope during diagnosis.
- "resected biopsy” total lump removal of tissue
- “incision biopsy” partially removed
- “core biopsy” extract part of sputum tissue with thick needle
- FNA fine needle aspiration
- the “antigen activator by degrading enzyme” and “antigen activator by heat treatment” may both exist in the form of a solution in which the components are dissolved, or may be in a dry solid state. When either or both of these are in a solid state, the “kit” may contain a “solvent” for dissolving these solid components.
- Antigen activating agent used in a sample for detecting Ki-67 by immunostaining refers to a sample for detecting a cell or cell nucleus using an anti-Ki-67 antibody, typically a fixed cell group or tissue. The activator by the enzyme used for is pointed out.
- “Prognosis of cancer treatment” means that patients are treated with chemotherapy (anticancer drug treatment), endocrine therapy, surgery (exclusion of tumor), radiation therapy, etc., and the effect of the treatment is confirmed and predicted. .
- the “kit for detecting Ki-67 positive cells in an immobilized cell group” includes a hydrolase that does not recognize and cleave the peptide of SEQ ID NO: 2 and an anti-Ki-67 antibody.
- the hydrolase is preferably at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline endopeptidase and hyaluronidase, more preferably thrombin and / or hyaluronidase.
- the anti-Ki-67 antibody is preferably at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047.
- the anti-Ki-67 antibody may be directly labeled with a fluorescent dye, an enzyme, a chemiluminescent substance, a radioactive element, or the like, or the kit includes a secondary antibody that binds to the anti-Ki-67 antibody. , May be labeled with a fluorescent dye, an enzyme, a chemiluminescent substance, a radioactive element, or the like.
- the kit may be combined with Ki-67 to detect ligands (eg, antibodies or to detect other markers used to aid in the diagnosis of cancer or to help diagnose the prognosis of cancer treatment. Probes, etc.).
- kits may further include a buffer for immersing or dispersing the tissue or cells used in the “step of extracting cell nuclei”, and preferably the buffer includes a surfactant.
- Preferred surfactants include CHAPS, NP-40, and Triton-X100, with NP-40 and Triton-X100 being more preferred.
- kits for extracting cell nuclei from cells usually immobilized cells
- a cell dispersion buffer containing a surfactant comprising the liquid.
- “Cutoff value” refers to a numerical value that distinguishes between positive and negative test results. In the case of Ki-67, a value of 14 to 20% is currently recommended for classifying the luminal A (like) type and the luminal B (like) type.
- Anticancer agent refers to a medicament for treating or preventing cancer. Although not limited to this, it classify
- Endocrine therapy is a treatment that prevents the use of female hormones (estrogens) that promote the growth of hormone-dependent breast cancer, including but not limited to antiestrogens, Lh-RH agonist formulations, aromatase inhibition Administration of hormonal agents such as pharmaceuticals and progesterone preparations.
- “Chemotherapy” refers to treating cancer with a chemical substance that exhibits an anticancer effect.
- anthracycline anticancer agents that are DNA synthesis and replication inhibitors (daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, etc.), and taxane anticancer agents that suppress cell proliferation Including single administration or combination administration of agents (paclitaxel (taxol), docetaxel (taxotere), etc.), platinum (platinum) preparations (cisplatin, oxaliplatin, etc.) that prevent DNA replication, and preferably anthracycline Including co-administration of cancer drugs and taxane anticancer agents.
- a “hormone agent” when a “hormone agent” is administered, it is included in “endocrine therapy”.
- FFPE tissue sections of breast cancer tissue were deparaffinized / hydrophilized and antigen-activated by heat treatment, dispersed using a water flow shearing device, and the recovered product was immunofluorescent stained and observed with a microscope.
- 1-1. FFPE tissue block The FFPE tissue block of the breast cancer tissue purchased from Proteogenex was used.
- the activated tissue was disrupted in 1 mL of TBS at 10,000 rpm for 1 minute using a water flow shearing device (RP-10) manufactured by Water Flow Crushing Sysmex. 1-7.
- RP-10 water flow shearing device
- 4% BSA / TBS supplemented with 10% immunofluorescent-stained normal goat serum (Wako) was added to a microtube containing a crushed product by water flow disruption, and allowed to stand at room temperature for 30 minutes for blocking treatment.
- a pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody: Anti-wide spectrum Cytokeratin antibody (AB9377-500)) was used as the primary antibody, and the secondary antibody was Gobanti Anti-Rabbit Secondary 48 from Abcam. It was used.
- the primary antibody reaction time was 50 minutes
- the secondary antibody reaction time was 30 minutes
- a dye DAPI Solution (Wako) for staining cell nuclei was added when the secondary antibody reaction time passed. All reactions were at room temperature, and were performed in the dark after the addition of the secondary antibody. 0.5% BSA / TBS was used as the antibody diluent, and a washing operation with 0.5% BSA / TBS was performed once between each step. 5 ⁇ L of DAPI embedding agent (Wako) was placed on a slide glass, and the sample after the secondary antibody reaction was placed thereon. Then, after covering with a cover glass, it left still for 5 minutes, pushed vertically from the top, and sealed the circumference
- the light was shielded until observation and stored at 4 ° C. 1-8.
- a fluorescence microscope an EVOS all-in-one microscope and a fluorescence microscope (Thermoscience) were used.
- a DAPI light cube Ex 357 nm, Em 447 nm
- a GFP light cube Ex 470 nm / Em 510 nm
- the eyepiece used 10 times and the objective lens used 40 times.
- FIG. 1 shows cell nuclei observed with a fluorescence microscope. As shown in the left figure, nucleic acids are present in a circular shape, and as shown in the right figure, cytokeratin is present around the nucleic acids. Therefore, it was confirmed that the cell nucleus and the skeleton were kept intact without being damaged, and were recovered as a single cell nucleus without aggregation.
- ⁇ Reference Example 2> Confirmation of cytokeratin and Ki-67-derived signals in formalin-fixed breast cancer cells Materials and Methods Breast cancer cells fixed with formalin were subjected to antigen activation by heat treatment, immunofluorescence staining was performed, and cytokeratin and Ki-67 signals were confirmed with a flow cytometer. 1-1. Cells Three breast cancer cell lines, T47D, MDA-MB231 (denoted MB231 or 231 in FIG.), And SKBr3 a (denoted SKBR in the figure), were obtained from ATCC (American Type Culture Collection), were used . 1-2.
- T47D cell line was cultured in RPMI-1640 medium
- MDA-MB-231 cell line was cultured in Leibovitz's L-15 medium and SKBr3 cells were cultured in McCoy's 5A medium, and 10% fetal bovine serum ( FBS) was supplemented.
- FBS fetal bovine serum
- the culture solution was aspirated and washed with PBS, and then TrypLE Express (Thermofisher) was added. The cells were collected and then centrifuged and washed with PBS. Suspend cells sufficiently with PBS, dispense into 1 ⁇ 10 6 cells, centrifuge, remove PBS, add 10% neutral buffered formalin solution (Wako Pure Chemicals), and fix at 4 ° C.
- the primary antibody is Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody (AB9377-500)), and the secondary antibody is Thermo Fisher Goat. Anti-Mouse Secondary Antibody Alexa 647 and Abcam Goat anti-Rabbit Secondary Antibody Alexa 488 were used.
- the primary antibody reaction time was 50 minutes
- the secondary antibody reaction time was 40 minutes
- a dye DAPI Solution (Wako) for staining cell nuclei was added 20 minutes after the addition of the secondary antibody. All reactions were at room temperature, and were performed in the dark after the secondary antibody was added.
- BSA / TBS 0.5% BSA / TBS was used as the antibody dilution, and a washing operation with 0.5% BSA / TBS was performed once between each step.
- a negative control isotype control
- Ki-67 used a mouse IgG antibody from Dako
- cytokeratin used a rabbit IgG antibody from Cellsignaling technology. 1-8.
- the measurement was performed according to the instrument manual. 1-9. Calculation of cytokeratin and Ki-67 positive rates The analysis of the measurement data obtained was performed using Soft Flow Jo v10 manufactured by FLOWJO LLC. Data analysis was performed in the following order according to the instructions attached to the software. The main regions shown in FIG. 2 were selected on the scattergram with forward scattering as the horizontal axis and side scattering as the vertical axis. Next, gating was performed using the DAPI fluorescence intensity as the horizontal axis, the number of cells (nuclei) as the vertical axis, and the main region as the cell nucleus (FIG. 3).
- the positive rate of cytokeratin and Ki-67 was calculated as the ratio of the number of cytokeratin positive nuclei and the number of Ki-67 positive nuclei in the total cell nuclei.
- the threshold for positive nuclei was the 95th percentile of isotype control.
- FIG. 4 shows a chart in which the histograms of the fluorescence intensities detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (solid black line) and the histograms of the fluorescence intensities detected with their isotype control antibodies (grayed dashed line) are superimposed. . In all cell lines, the black peak showed higher fluorescence intensity than gray. From these, the presence of cytokeratin and Ki-67 in formalin-fixed breast cancer cells was confirmed.
- Example 1 Enhancement of Ki-67 signal in formalin-fixed cells by antigen activation with thrombin
- FIG. 1-1 Three breast cancer cell lines used in Cell Reference Example 2 were used.
- 1-2 Three breast cancer cell lines used in Cell Reference Example 2 were used.
- 1-2 Cell culture and formalin fixation were performed in the same manner as in Reference Example 2.
- 1-4 The same treatment as in Reference Example 1 for antigen activation by heat treatment was performed. 1-5.
- Thrombin reagent 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1000 KU / L thrombin, 10 mM CaCl 2 ) was added to cells after antigen activation by enzyme-induced heat treatment with enzyme, and heated at 37 ° C. for 20 minutes in a heat block. did. 1-7.
- Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) was used as the primary antibody, and Thermofisher Goat anti-Mouse Secondary Antibody Alexa 647 was used as the secondary antibody.
- Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of Ki-67 positive rate The calculation was performed in the same manner as in Reference Example 2.
- FIG. 5 shows a chart in which the fluorescence intensities detected with the anti-Ki-67 antibody (black solid line) and the isotype control antibody (gray broken line) are superimposed, and FIG. 6 and Table 2 show changes in the Ki-67 positive rate. .
- the fluorescence intensity increased, and the Ki-67 positive rate was increased accordingly.
- Example 2 Enhancement of Ki-67 signal in formalin-fixed cells by antigen activation with hyaluronidase Materials and Methods Breast cancer cells immobilized with formalin were used to compare signal intensities with and without hyaluronidase treatment. 1-1. The T47D cell line used in Cell Reference Example 2 was used. 1-2. Cell culture and formalin fixation were performed in the same manner as in Reference Example 2. 1-4. The same treatment as in Reference Example 1 for antigen activation by heat treatment was performed. 1-5.
- Hyaluronidase reagent 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 4000-10000 KU / L hyaluronidase IS, 7500-30000 KU / L hyaluronidase IV-S, 27 mM KCl .
- Hyaluronidase reagent 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 4000-10000 KU / L hyaluronidase IS, 7500-30000 KU / L hyaluronidase IV-S, 27 mM KCl .
- Ki-67 antibody Dako, clone: MIB-1, mouse monoclonal antibody
- Thermofisher Goat anti-Mouse Secondary Antibody Alexa 647 was used as the secondary antibody.
- Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of Ki-67 positive rate The calculation was performed in the same manner as in Reference Example
- FIG. 7 shows an overlay chart of the fluorescence intensities detected with the anti-Ki-67 antibody (black solid line) and the isotype control antibody (grey broken line). The fluorescence intensity increased by the treatment with hyaluronidase, and the Ki-67 positive rate was increased accordingly.
- the positive rate of cytokeratin and Ki-67 was calculated as the ratio of the number of cytokeratin positive nuclei and the number of Ki-67 positive nuclei in the total cell nuclei.
- the threshold for positive nuclei was the 95th percentile of isotype control.
- FIG. 8 shows a chart in which the fluorescence intensity histograms detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (solid black line) and the fluorescence intensity histograms detected with the isotype control antibody (gray broken line) are superimposed. .
- the black peak showed higher fluorescence intensity than gray.
- FFPE tissue blocks Two types of FFPE tissue blocks purchased from IProteogenex and obtained from two breast cancer patients with different Ki-67 positive rates by the HC method were used.
- 1-2 Preparation of FFPE section Using a Thermofisher sliding microtome, the FFPE tissue block was sliced to prepare two sections having a thickness of 20 ⁇ m and used for the examination.
- 1-3 Deparaffinization / hydrophilization The same procedure as in Reference Example 1 was performed. 1-4.
- Antigen activation by heat treatment was performed in the same manner as in Reference Example 1. 1-5. Antigen activation by enzyme The same procedure as in Example 1 was performed. 1-6. It carried out like water flow crushing reference example 1. 1-7. The same procedure as in Reference Example 2 for immunofluorescent staining was performed. 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of cytokeratin and Ki-67 positive rate was performed in the same manner as in Reference Example 3.
- FIG. 9A shows a chart in which a histogram of fluorescence intensity detected with anti-cytokeratin antibody and anti-Ki-67 antibody (black solid line) and a histogram of fluorescence intensity detected with these isotype control antibodies (grey broken line) are superimposed.
- treatment with thrombin increased the fluorescence intensity of cytokeratin and Ki-67, and the positive rate increased accordingly (FIG. 9B).
- FFPE tissue block used in FFPE tissue block reference example 1 was used.
- FFPE tissue block reference example 1 was used.
- 1-2. Preparation of FFPE sections Thin slice samples of 20 ⁇ m 3, 30 ⁇ m 2 and 60 ⁇ m 1 were prepared from the same FFPE tissue block using a Thermofisher sliding microtome.
- 1-3. Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5.
- Example 2 Antigen activation by enzyme The same treatment as in Example 1 was performed. 1-6. It carried out like water flow crushing reference example 1. 1-7. The same procedure as in Reference Example 2 for immunofluorescent staining was performed. 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of cytokeratin and Ki-67 positive rate was performed in the same manner as in Reference Example 3.
- FIG. 10 shows changes in the thickness of sliced sections and the positive rates of cytokeratin and Ki-67.
- the detection efficiency was the best with 3 sheets of 20 ⁇ m, but cytokeratin (CK) and Ki-67 (Ki67) could be detected at any thickness of 20 to 60 ⁇ m.
- FFPE tissue block was used to compare the Ki-67 positive rate by the flow cytometer and the positive rate by the IHC method.
- 1-1 FFPE tissue blocks
- FFPE tissue blocks Nineteen FFPE tissue blocks purchased from Proteogenex and obtained from 19 patients with different Ki-67 positive rates by the IHC method were used.
- 1-2 Preparation of FFPE section The same procedure as in Reference Example 3 was performed.
- 1-3 Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5.
- Ki-67 positive rate calculation by IHC method Ki-67 positive rate by IHC method was calculated by hot spot method using Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) as the primary antibody. In addition, the average value of two pathologists was used in consideration of the variation by the examiner.
- Example 6> Detection of Ki-67 and cytokeratin upon antigen activation with various digestive enzymes Materials and Methods for breast cancer cells immobilized with formalin, the signal intensities of Ki-67 and cytokeratin were compared for antigen activation with five different digestive enzymes.
- Antigen activation by enzyme 250 ⁇ L of trypsin reagent (25 mM TBS pH 7.4, 1M CaCl2, 1 mg / mL trypsin) was added to the cells that had been antigen-stimulated by heat treatment, and heated at 37 ° C. for 20 minutes. After the enzyme treatment, the enzyme was removed by washing with TBS. 1-5C.
- Antigen activation by protein 600 mA Son U / mL proteinase K (Takara Bio) was diluted 40 times (v / v) with 25 mM TBS pH 7.4 to obtain a proteinase K solution.
- Proline endopeptidase Proline endopeptidase (Toyobo) was dissolved in 25 mM TBS pH 7.4 to obtain a proline endopeptidase solution (10 U / mL). 250 ⁇ L of proline endopeptidase solution was added to the cells activated by antigen by heat treatment, and heated at 37 ° C. for 20 minutes. After completion of the reaction, the enzyme was removed by washing with TBS. 1-7. The same procedure as in Reference Example 2 for immunofluorescent staining was performed.
- Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the primary antibody, and Thermofisher Goat anti- A Mouse Secondary Antibody Alexa 647, and Abcam Goat anti-Rabbit Secondary Antibody Alexa 488 were used. 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of cytokeratin and Ki-67 positive rate was performed in the same manner as in Reference Example 2.
- thrombin and proline endopeptidase that did not recognize and cleave the amino acid sequence of SEQ ID NO: 2 increased the positive rate of Ki-67, indicating that the antigenicity of Ki-67 antigen was enhanced (FIG. 12A).
- FFPE sections of breast cancer tissue were antigen-stimulated with 5 different digestive enzymes and the signal intensities of Ki-67 and cytokeratin were compared.
- 1-1 The FFPE tissue block used in FFPE tissue block reference example 1 was used.
- 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed.
- 1-3. Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5A.
- Antigen activation by enzyme thrombin
- Antigen activation by enzyme trypsin
- the same operation as in Example 6 was performed.
- 1-5C Antigen activation by protein (proteinase K)
- 1-5D Antigen activation by enzyme (dispase)
- 1-5E Antigen activation by enzymes (proline endopeptidase)
- the same operation as in Example 6 was performed. 1-6. It carried out like water flow crushing reference example 1. 1-7. The same procedure as in Reference Example 2 for immunofluorescent staining was performed.
- Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the primary antibody, and Thermofisher Goat anti- A Mouse Secondary Antibody Alexa 647, and Abcam Goat anti-Rabbit Secondary Antibody Alexa 488 were used. 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of cytokeratin and Ki-67 positive rate was performed in the same manner as in Reference Example 3.
- FIGS. 13A and 13B are charts in which a histogram of fluorescence intensity detected with anti-Ki-67 antibody or anti-cytokeratin antibody (solid black line) and a histogram of fluorescence intensity detected with their isotype control (grey dashed line) are superimposed.
- Cytokeratin was detectable with any proteolytic enzyme (FIG. 13B).
- trypsin, dispase, or proteinase K enzyme capable of recognizing and cleaving the peptide of SEQ ID NO: 2 as in Example 6, the fluorescence intensity of the Ki-67 antibody (black solid line) is greatly reduced. (FIG. 13A).
- FFPE tissue sections of breast cancer tissue were confirmed with a flow cytometer.
- the Ki-67 antibody the MIB-1 clone and the S5 clone as an antibody having a different recognition site were used.
- 1-1 The FFPE tissue block used in FFPE tissue block reference example 1 was used.
- 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed.
- 1-3. Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5.
- Antigen activation treatment with enzyme The same treatment as in Example 1 was performed. 1-6. It carried out like water flow crushing reference example 1. 1-7. The same procedure as in Reference Example 2 for immunofluorescent staining was performed. Here, Ki67 MIB-1 antibody (Dako, mouse monoclonal antibody) or Ki67 S5 antibody (Millipore, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) are used as the primary antibody, and the secondary antibody. Thermo Fisher Goat anti-Mouse Secondary Antibody Alexa 647 and Abcam Goat anti-Rabbit Secondary Alexa 488 were used. 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of cytokeratin and Ki-67 positive rate was performed in the same manner as in Reference Example 3.
- FIG. 14 shows a chart in which a histogram of the fluorescence intensity detected with the anti-Ki-67 antibody or the anti-cytokeratin antibody (black solid line) and a histogram of the fluorescence intensity detected with the isotype control (gray) are overlaid ( Upper: Ki-67; Lower: cytokeratin).
- the peak of the Ki-67 antibody showed higher fluorescence intensity than the isotype control. This confirmed the detection of cytokeratin and Ki-67 in FFPE tissue sections, suggesting that Ki-67 can be detected not only in the Ki-67 MIB-1 clone but also in the S5 clone. It was.
- FFPE tissue block reference example 1 The FFPE tissue block used in FFPE tissue block reference example 1 was used. 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed. 1-3. Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-5.
- Antigen activation by enzyme 250 ⁇ L of trypsin reagent (25 mM PBS pH 7.4, 1 mg / ml CaCl 2 , 1 mg / mL trypsin) was added to a tissue section hydrophilized by an existing method and heated at 37 ° C. for 70 minutes. . After the enzyme treatment, the enzyme was removed by washing with PBS. 1-7. Immunofluorescence staining 4% BSA / TBS supplemented with 10% normal goat serum (Wako) was added to the microtube containing the sample after antigen activation with the enzyme, and allowed to stand at room temperature for 30 minutes to perform blocking treatment.
- Immunofluorescence staining uses Ki67 MIB-1 antibody (Dako, mouse monoclonal antibody) or Ki67 S5 antibody (Millipore, mouse monoclonal antibody) and cytokeratin antibody (Abcam, rabbit polyclonal antibody) as the primary antibody.
- Ki67 MIB-1 antibody Dako, mouse monoclonal antibody
- Ki67 S5 antibody Millipore, mouse monoclonal antibody
- cytokeratin antibody Abcam, rabbit polyclonal antibody
- Thermofisher Goat anti-Mouse Secondary Antibody Alexa 647 and Abcam Goat anti-Rabbit Secondary Alexa 488 were used.
- the primary antibody reaction time was 4 ° C. overnight.
- the secondary antibody was allowed to react for 40 minutes, and a dye DAPI Solution (Wako) that stains cell nuclei was added 20 minutes after the addition of the secondary antibody. All the steps after the addition of the secondary antibody were performed at room temperature under light shielding.
- ER positive specimens All red Score ER7
- PgR positive specimens All red Score PgR8
- ER and PgR were negative samples of ER and PgR.
- FFPE tissue sections from negative specimens All red Score ER0 and PgR0
- FFPE section It was prepared in the same manner as Reference Example 3. 1-3. Deparaffinization / hydrophilization The same treatment as in Reference Example 1 was performed. 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5. Antigen activation by enzyme After antigen activation by heat treatment, ER positive specimens were antigen-activated by thrombin. In addition, PgR positive specimens and ER and PgR negative specimens were antigen-stimulated with thrombin or proline endopeptidase. Antigen activation with thrombin was performed in the same manner as in Example 1, and antigen activation with proline endopeptidase was performed in the same manner as in Example 6. 1-6.
- the secondary antibody was reacted at room temperature for 40 minutes, and a dye DAPI Solution (Wako) for staining cell nuclei was added 20 minutes after the addition of the secondary antibody.
- 1-8. Flow cytometer measurement was performed in the same manner as in Reference Example 2.
- 1-9. Calculation of ER and PgR positive rate
- software FlowJo v10 manufactured by FLOWJO LLC was used. Gating was performed with the DAPI fluorescence amount on the horizontal axis, the number of cells (nuclei) on the vertical axis, and the region on the horizontal axis with a fluorescence amount of 5-150 as the cell nucleus.
- the positive rate of ER and PgR was calculated as the ratio of the number of ER positive nuclei and the number of positive nuclei of PgR in all cell nuclei.
- the threshold for positive nuclei was the 90th percentile of isotype control.
- FIG. 16 shows a chart in which the histogram of the fluorescence intensity detected with the anti-ER antibody or the anti-PgR antibody (solid black line) and the histogram of the fluorescence intensity detected with these isotype control antibodies (gray broken line) are superimposed.
- Example 10 Comparison of Ki-67 and cytokeratin signals in immobilized breast cancer cells using different heat-treated antigen activators 1.
- Materials and Methods Breast cancer cell line MDA-MB-231 immobilized with formalin was antigen-activated by heat treatment using three types of antigen activators, and after thrombin enzyme treatment, immunofluorescent staining was performed, and cytokeratin and Ki -67 was detected with a flow cytometer. 1-1. The breast cancer cell line MDA-MB-231 obtained from the cell ATCC (American Type Culture Collection) was used. 1-2. Cell culture and formalin fixation were performed in the same manner as in Reference Example 2. 1-4A.
- Antigen activation by heat treatment Histo VT ONE
- Antigen activation by heat treatment antigen activation solution pH 9
- Antigen activation solution pH 9 (Nichirei Bioscience)
- the processing procedure and conditions were the same as in Reference Example 1.
- 1-4C Antigen activation by heat treatment (immunosaver)
- Antigen activation liquid immunosaver (Nisshin EM) was used.
- the processing procedure and conditions were the same as in Reference Example 1. 1-5.
- Antigen activation by enzyme The same treatment as in Example 1 was performed. 1-7.
- the same procedure as in Reference Example 2 for immunofluorescent staining was performed.
- Flow cytometer measurement was performed in the same manner as in Reference Example 2. 1-9. Calculation of positive rate of cytokeratin and Ki-67 The same procedure as in Reference Example 3 was performed.
- FIG. 17 shows a chart in which a fluorescence intensity histogram (black solid line) detected with anti-Ki-67 antibody or anti-cytokeratin antibody and a fluorescence intensity histogram (grey dashed line) detected with these isotype control antibodies are superimposed.
- a fluorescence intensity histogram black solid line
- a fluorescence intensity histogram grey dashed line
- the black peak showed higher fluorescence intensity than gray. From these, the detection of cytokeratin and Ki-67 in formalin-fixed cells was confirmed, and it was confirmed that it was useful even if the activation solution was changed.
- Example 11 Effects of various disruption methods in the process of extracting cell nuclei on the detection of cytokeratin and Ki-67 in FFPE tissue sections
- the FFPE tissue block used in FFPE tissue block reference example 1 was used.
- 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed.
- 1-3. Deparaffinization / hydrophilization The same procedure as in Reference Example 1 was performed.
- Antigen activation by heat treatment was performed in the same manner as in Reference Example 1.
- cytokeratin and Ki-67 positive rate The analysis of the obtained measurement data used software FlowJo v10 manufactured by FLOWJO LLC. Gating was performed with the DAPI fluorescence amount on the horizontal axis, the number of cells (nuclei) on the vertical axis, and the region on the horizontal axis with a fluorescence amount of 5-150 as the cell nucleus.
- the positive rate of cytokeratin and Ki-67 was calculated as the ratio of the number of cytokeratin positive nuclei and the number of positive nuclei of Ki-67 in all cell nuclei.
- the threshold for positive nuclei was the 90th percentile of isotype control.
- FIG. 18 shows a chart in which a histogram of the fluorescence intensity detected with the anti-cytokeratin antibody or anti-Ki-67 antibody (black solid line) and a histogram of the fluorescence intensity detected with these isotype control antibodies (grey broken line) are superimposed. .
- black solid line a histogram of the fluorescence intensity detected with these isotype control antibodies
- grey broken line a histogram of the fluorescence intensity detected with these isotype control antibodies
- Example 12 Preferential disruption of lymphocytes by ultrasonic disruption Materials and Methods Breast cancer cell lines and lymphoblastic cell lines immobilized with formalin were used to confirm whether ultrasonic disruption affects the acquisition of cell nuclei. 1-1. Breast cancer cell lines SKBr3 and MDA-MB-231 and lymphoblastic cell line Jurkat were obtained and used from cells ATCC (American Type Culture Collection). 1-2. Cell culture and formalin fixation 1-4. Treatment was performed in the same manner as in Reference Example 1 for antigen activation by heat treatment . 1-5. Antigen activation treatment with enzyme The same treatment as in Example 1 was performed. 1-6.
- FIG. 19 shows a scattergram of each cell line with forward scatter as the horizontal axis and side scatter as the vertical axis.
- breast cancer cells SKBr3 and MDA-MB-231 it was confirmed that the position of the main cell nucleus region did not change depending on the presence or absence of ultrasonic disruption.
- lymphocyte Jurkat the main cell nucleus region disappeared by ultrasonic disruption. Therefore, it was suggested that epithelial cells are not destroyed by ultrasonic disruption, and that lymphoblastic cells are preferentially ultrasonically disrupted.
- FFPE tissue blocks Two types of FFPE tissue blocks purchased from Proteogenex and obtained from two breast cancer patients with different Ki-67 positive rates by the IHC method were used. 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed. 1-3. Deparaffinization / hydrophilization The same procedure as in Reference Example 1 was performed. 1-4. Antigen activation by heat treatment was performed in the same manner as in Reference Example 1. 1-5.
- FIG. 20 shows a case where the forward scattering is plotted on the horizontal axis and the side in the case where the surfactant is ultrasonically crushed in a buffer solution to which no surfactant is added and in the case where the surfactant is ultrasonically crushed in a buffer solution to which the various surfactants are added.
- a scattergram with the ordinate as the vertical axis is shown, and Table 3 shows the total number of analyzed nuclei at the time of flow cytometer measurement. It was confirmed that the total number of analyzed cell nuclei was increased by the addition of the surfactant.
- FFPE tissue blocks 38 FFPE tissue blocks obtained from Proteogenex and obtained from 38 patients with different Ki-67 positive rates by the IHC method were used. 1-10. Calculation of Ki-67 positive rate by IHC method The same procedure as described in Example 5 was performed.
- ⁇ Reference Example 4> Detection with anti-Ki-67 antibodies with different recognition sites after antigen activation with various digestive enzymes
- Three breast cancer cell lines used in Cell Reference Example 2 were used.
- 1-2. Cell culture and formalin fixation were performed in the same manner as in Reference Example 2.
- the same treatment as in Reference Example 1 for antigen activation by heat treatment was performed.
- 1-5A Antigen activation by enzyme (thrombin) The same treatment as in Example 1 was performed.
- Results Overlaid on FIGS. 22A and B are histograms of fluorescence intensities detected with anti-Ki-67 antibody (Ki-S5) or anti-cytokeratin antibody (solid black line) and histograms of fluorescence intensities detected with their isotype control (dashed gray line). The combined chart is shown. Even when the antigen was activated with any enzyme, the peak of the black solid line showed higher fluorescence intensity than the gray broken line. From these results, the antigen recognition site of anti-Ki-67 antibody (Ki-S5) and anti-cytokeratin antibody is retained and Ki-67 and cytokeratin can be detected regardless of which enzyme is used to activate the antigen. It was confirmed.
- Example 14 Examination of threshold value in cytokeratin and Ki-67 positive nucleus determination in FFPE tissue section Materials and Methods Signals derived from cytokeratin and Ki-67 in FFPE tissue sections were detected with a flow cytometer, and the positive rate when the threshold for positive nuclei was changed was calculated. 1-1. The FFPE tissue block used in FFPE tissue block reference example 1 was used. 1-2. Preparation of FFPE section The same procedure as in Reference Example 3 was performed. 1-3. Deparaffinization / hydrophilization The same procedure as in Reference Example 1 was performed. 1-4. Antigen activation by heat treatment was performed in the same manner as in Reference Example 1. 1-5. Antigen activation by enzyme The same procedure as in Example 1 was performed.
- cytokeratin and Ki-67 positive rate The analysis of the obtained measurement data used software FlowJo v10 manufactured by FLOWJO LLC. Gating was performed with the DAPI fluorescence amount on the horizontal axis, the number of cells (nuclei) on the vertical axis, and the region on the horizontal axis with a fluorescence amount of 5-150 as the cell nucleus.
- the positive rate of cytokeratin and Ki-67 was calculated as the ratio of the number of cytokeratin positive nuclei and the number of positive nuclei of Ki-67 in all cell nuclei.
- the threshold for positive nuclei was 60, 70, 80, 90 and 95 percentile values of isotype control. The positive rate was calculated by the value detected by a flow cytometer ⁇ (100 ⁇ threshold).
- FIG. 23 shows a histogram of fluorescence intensities detected with anti-cytokeratin antibody and anti-Ki-67 antibody (black solid line) and a histogram of fluorescence intensities detected with these isotype control antibodies (gray broken line) with different threshold values. The combined chart is shown. The positive rate was calculated regardless of which threshold was used. Therefore, cytokeratin and Ki-67 positive rates can be calculated regardless of the threshold.
- the FFPE tissue block used in FFPE tissue block reference example 1 was used.
- Preparation of FFPE section The same procedure as in Reference Example 3 was performed.
- Deparaffinization / hydrophilization The same procedure as in Reference Example 1 was performed.
- Antigen activation by heat treatment was performed in the same manner as in Reference Example 1. 1-5.
- Antigen activation by enzyme The same procedure as in Example 1 was performed. 1-6. Using a water shearing device (RP-10) manufactured by Sysmex Corporation in combination with water disruption and ultrasonic disruption, the tissue after enzyme treatment was disrupted for 1 minute at 10,000 rpm in 1 mL of TBS under ice cooling. Furthermore, using an ultrasonic crusher (VCX130PB) manufactured by SONICS & MATERIALS, the tissue after water flow crushing in 1 mL of TBS was cooled for 30 seconds at an output intensity of 20% under ice cooling. 1-7. Detection of HER2 by FISH The sample after tissue disruption was placed on a slide glass, dried, and then immersed in 10% neutral buffered formalin at room temperature for 10 minutes.
- RP-10 water shearing device
- VCX130PB ultrasonic crusher
- the slide glass was washed with TBS, air-dried, and then FISH-stained using a Pathvision® HER-2 DNA probe kit (Abbott) (operation was in accordance with the kit attachment). 1-8.
- An all-in-one fluorescence microscope BZ-X710 Keyence was used for fluorescence microscope observation. During observation, a DAPI filter (Ex 360 nm, Em 460 nm) and a TRITC filter (Ex 545 nm, Em 605 nm) were used. The objective lens used 20 times.
- FIG. 24 shows cell nuclei observed with a fluorescence microscope. It was confirmed that a fluorescent signal derived from HER2 could be detected in a HER2-positive FFPE tissue section, but not detected in a HER2-negative FFPE tissue section.
- a method for detecting (quantifying) Ki-67 positive cells with high objectivity, reproducibility, and universality is established.
- ER positive cells PgR positive cells, Her2 positive cells
- the protocol provided by the present invention including a pretreatment process including antigen activation by a predetermined enzyme
- detachment of cell nuclei with enhanced antigenicity of the target antigen is achieved from the FFPE tissue section.
- the collected dispersed cell nuclei can be analyzed as a single cell nucleus.
- proteins and nucleic acids on the nuclear membrane and in the nucleus are analyzed, and these objects can be analyzed by observing the morphology by dye (fluorescence) staining or using ligands such as antibodies or nucleic acids labeled with enzymes or fluorescent dyes. Can be detected.
- the method, antigen activator and kit of the present invention can be used for morphological observation with a microscope, analysis with IHC, EIA, CLEIA and digital PCR, and cytometry analysis with an imaging cytometer or flow cytometer.
- it can be used for calculation of the ratio of positive nuclei (especially Ki-67 positive rate) of the target protein in the detached nuclei by flow cytometry analysis.
- the protocol provided by the present invention including a pretreatment process including antigen activation by a predetermined enzyme
- an optimal diagnostic regimen for a patient by using an index with little variation.
- Anticancer drug treatment itself has a heavy burden on the patient, and it is important for the patient to maintain a QOL (Quality of Life) to decide a treatment regimen suitable for the patient before the start of treatment.
- QOL Quality of Life
- it can be applied not only to a chemotherapy regimen (preoperative anticancer drug treatment) before surgery but also to a treatment regimen (prognosis) after tumor removal by surgery.
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Abstract
Description
一方、その因子の有無と予後が相関する因子のことを予後因子という。Ki-67は現在予後因子とも考えられている。効果予測因子の検出には、主として腫瘍組織サンプルの免疫組織化学的方法(IHC法)が多用されている。腫瘍細胞の染色強度と染色された細胞の比率の両方を加味する方法(All red Score等)や染色強度を評価せず染色された腫瘍細胞の比率のみで判定する方法(J-Score等)のいずれかが用いられる。
HER2の場合、一般にIHC法で検査し、その結果が0あるいは1+の場合は陰性、3+の場合は陽性、2+の場合はFISH法(Fluorescence in situ hybridization)によって増幅の有無を調べ、増幅があれば陽性、なければ陰性と判定する。
ERの場合、All red Scoreでは3~8が陽性とされる一方、染色細胞の比率で判定する場合は10%をカットオフ値とすることが多かったが、1%でも存在する場合は、ER陽性と判定すべきとの意見もある。
Ki-67の場合、組織全体で陽性割合を算出する方法、陽性細胞の集まるホットスポットと呼ばれる領域を選択して算出する方法等が混在している。
いずれにせよ、一定のカットオフ値を設定すれば、これらの効果予測因子が陽性と判断された場合、効果的な治療レジメンの指針となりうる。
1)ルミナルA(様)型:ER・PgR陽性、HER2陰性、Ki-67低値(<14~20%)
2)ルミナルB(様)型(HER2陰性):ER・PgR陽性、HER2陰性、Ki-67高値(≧14~20%)
3)ルミナルB(様)型(HER2陽性):ER・PgR陽性、HER2陽性
4)非ルミナル型:ER・PgR陰性、HER2陽性
5)トリプルネガティブ型:ER陰性、PgR陰性、HER2陰性
これらの分類は治療レジメンの選定にも影響し、例えばKi-67低値であるルミナルA(様)型と判断された場合、主として内分泌療法単独が選択され、一方Ki-67高値であるルミナルB(様)型(HER2陰性)と判断された場合は、主として内分泌療法と化学療法の併用が選択される(非特許文献6、10~12)。
さらに、ホルマリン固定組織やトロンビン:(血漿)ファイブロネクチン固定組織の場合、MIB-1抗体は偽陽性や偽陰性の結果を生じる可能性があった(非特許文献9)
通常、FFPE組織から単一の細胞レベルで解析を行う場合、細胞間の接着を担う細胞外基質を切断して抗原を賦活化する必要がある。しかしながら、これらの酵素を用いた消化は、標的とする抗原の認識部位を切断する可能性があり、使用可能な抗体を制限する。そのため、上記の既存方法以外で細胞分散や、細胞核を脱離する方法が必要となる。
[1] 固定化された細胞群中のKi-67陽性細胞核を含む細胞を、抗Ki-67抗体を用いて検出する方法であって、
1)固定化された細胞群を配列番号2のペプチドを認識切断しない加水分解酵素で前処理して抗原を賦活化する工程、その後
2)抗Ki-67抗体を用いて染色する工程、及び
3)染色されたKi-67陽性細胞又はKi-67陽性細胞核を検出する工程を含む方法;
[2] 前記抗体が、MIB-1、DAKO-PC、Ki-S5及びA-0047からなる群から選択される少なくとも一種である[1]に記載の方法;
[3] 前記酵素が、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である[1]又は[2]に記載の方法;
[4] 前記酵素が、トロンビン及び/又はヒアルロニダーゼであり、前記抗体が、MIB-1である[1]に記載の方法;
[5] 前記固定化された細胞群が、ホルマリン、グルタルアルデヒド、アルコール、アセトン及びそれらの組み合わせからなる群から選択される固定化剤で固定されている、[1]~[4]のいずれか一項に記載の方法;
[6] 工程1)の前に熱処理により賦活化することを含む、[1]~[5]のいずれか一項に記載の方法;
[8] さらに、抗サイトケラチン抗体を用いてサイトケラチンを染色(例えば、蛍光染色)し、染色された陽性細胞を検出することを含む、[1]~[7]のいずれか一項に記載の方法;
[9] さらに抗エストロジェン受容体(ER)抗体及び/又は抗プロゲステロン受容体(PgR)抗体を用いてER及び/又はPgRを染色(例えば、蛍光染色)し、染色された陽性細胞又は陽性細胞核を検出(例えば、カウント)することを含む、[1]~[8]のいずれか一項に記載の方法;
[10] さらにHER2遺伝子にハイブリダイズするプローブを用いて、HER2遺伝子が増幅されている細胞又は細胞核を検出することを含む、[1]~[9]のいずれか一項に記載の方法。
[11] 前記固定化された細胞群が組織切片に含まれている、[1]~[10]のいずれか一項に記載の方法;
[12] 前記組織切片が、包理剤で包理されており、
前記加水分解酵素で抗原を賦活化する工程の前に該包理剤を除去し、該組織切片を親水化する工程を含む、[11]に記載の方法;
[14] 水流、超音波などで生じるせん断応力により前記細胞を破砕して、細胞核を抽出する、[13]に記載の方法;
[15] 界面活性剤を含む緩衝液中で、前記細胞核を抽出する[13]又は[14]に記載の方法;
[16] 前記界面活性剤が、CHAPS、NP-40、およびTriton-X100から選択される少なくとも一種である、[15]に記載の方法;
[17] 前記染色されたKi-67陽性細胞又はKi-67陽性細胞核の数をカウントする、[1]~[16]のいずれか一項に記載の方法;
[18]前記Ki-67陽性細胞又は前記Ki-67陽性細胞核を、蛍光染色し、フローサイトメトリーを用いてカウントする、[17]に記載の方法;
[19] 前記固定化された細胞又は前記組織切片が患者由来である、[1]~[18]のいずれか一項に記載の方法;
[20] 前記固定化された細胞又は前記組織切片が患者の腫瘍組織由来である、[1]~[18]のいずれか一項に記載の方法;
[21] 前記固定化された細胞又は前記腫瘍組織が、乳癌細胞又は乳癌組織である、[20]に記載の方法;
[22] がんの診断を補助するための、[20]又は[21]に記載の方法;
[23] がん治療の予後を診断するのを補助するための、[20]又は[21]に記載の方法;
[25] [20]又は[21]に記載の方法により、細胞群内のKi-67陽性細胞の割合を算定し、割合がカットオフ値以上の場合は内分泌療法と化学療法の併用を選択し、割合がカットオフ値未満の場合は、内分泌療法単独を選択して、患者に施し、癌を治療する方法。
[27] 配列番号2のペプチドを認識切断しない加水分解酵素を含む、Ki-67を免疫染色で検出する細胞又は組織試料に用いられる抗原賦活化剤;
[28] 前記酵素が、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である、[27]に記載の抗原賦活化剤;
[29] 前記酵素がトロンビン及び/又はヒアルロニダーゼである、[28]に記載の抗原賦活化剤。
[31] 更に、ER及び/又はPgRを免疫染色して検出する細胞又は組織試料に用いられる[27]~[30]のいずれか一項に記載の抗原賦活化剤;
[32] 更に、HER2遺伝子を増幅している細胞を検出する試料に用いられる[27]~[31]のいずれか一項に記載の抗原賦活化剤;
配列番号2のペプチドを認識切断しない加水分解酵素と、
抗Ki-67抗体と
を含む、キット;
[34] 前記加水分解酵素は、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である、[32]に記載のキット;
[35] 前記加水分解酵素は、トロンビン及び/又はヒアルロニダーゼである、[34]に記載のキット;
[36] 前記抗Ki-67抗体は、MIB-1、DAKO-PC、Ki-S5及びA-0047からなる群から選択される少なくとも一種である、[33]~[35]のいずれか一項に記載のキット;
[37] 更に、Ki-67と組み合わせて、がんの診断を補助するため、又はがんの治療の予後を診断するのを補助するために用いられる他のマーカーを検出するためのリガンドを含む、[33]~[36]のいずれか一項に記載のキット;
[38] 前記リガンドは、抗サイトケラチン抗体、抗ER抗体、抗PgR抗体、及びHER2遺伝子にハイブリダイズするプローブからなる群から選択される少なくとも一種である、[37]に記載のキット;
[39] 更に、核染色用の化合物を含む、[33]~[38]のいずれか一項に記載のキット;
[40] 更に、細胞を分散させるための緩衝液を含み、該緩衝液は、界面活性剤を含有する、[33]~[39]のいずれか一項に記載のキット;
[41] 更に、熱処理抗原賦活化剤を含む、[33]~[40]のいずれか一項に記載のキット;
[42] がんの診断を補助するための、[33]~[41]のいずれか一項に記載のキット。
[43] がん治療の予後を診断するのを補助するための、[33]~[41]のいずれか一項に記載のキット;
[44] 癌の治療レジメンを決定するための、[33]~[41]のいずれか一項に記載のキット;
[45] 前記治療レジメンの決定は、細胞群内のKi-67陽性細胞の割合を算定し、割合がカットオフ値以上の場合は内分泌療法と化学療法の併用を選択し、割合がカットオフ値未満の場合は、内分泌療法単独を選択することによって行なわれる、キット;
界面活性剤を含有する細胞分散用緩衝液を含む、キット;
[47] 水流、超音波などで生じるせん断応力により固定化された細胞を破砕して、細胞核を抽出する方法であって、
該固定化された細胞を界面活性剤を含有する緩衝液に分散して該細胞の破砕を行う、方法;及び
[48] 固定化された細胞群中の目的の抗原が細胞核に存在する細胞を、該抗原の抗体を用いて検出する方法であって、
1)該固定化された細胞群を、該抗体の抗原認識部位を切断しない加水分解酵素で前処理して抗原を賦活化する工程、その後
2)該抗体を用いて該細胞を染色する工程、及び
3)染色された抗原陽性細胞又は抗原陽性細胞核を検出する工程を含む方法。
本発明より提供されるプロトコール(前処理試薬とプロセス)を用いることで、FFPE組織切片から標的抗原の抗原性を増強した細胞核の脱離を達成する。回収した分散細胞核は、単一の細胞核として解析することが可能である。例えば、核膜上や核内のタンパク質、核酸等が検出対象となり、色素(蛍光物質、化学発行物質、酵素など)による染色や、これらの色素により修飾された抗体を用いて検出することができる。特に、フローサイトメトリー解析により脱離核中の対象タンパク質の陽性核の割合(Ki-67陽性率等)を迅速に算出することが可能である。
Ki-67のカットオフ値は、病理医による、目視判定とカウントの工程を必要としていたため、ばらつきがあり、従来施設ごとに異なっていた。しかしながら本発明より提供されるプロトコール(前処理試薬とプロセス)を用いることで、ばらつきの少ない病理診断のカットオフ値の世界基準を提供することが可能である。
そして、かかるカットオフ値を用いて、患者にQOLの高い診断レジメンを提供することが可能である。
「抗体」は、完全長(IgG、IgA、IgM、IgD、IgE)イムノグロブリンであってもよく、その抗原結合認識領域を含む断片(いわゆる断片抗体(Fab、Fab’、F(ab’)2など))であってもよい。また「抗体」は、ヒト、マウス、ラット、ヤギ、ウマ、ラクダなど哺乳動物のほか、魚類(サメを含む)や鳥類(ニワトリ)に由来するものであってよい。
「MIB-1」又は「MIB-1抗体」は、Ki-67抗体をモノクローナル化したクローンの1つである。特に限定しないが、Dako社やImmunotech社から購入可能である。Ki-67中、PKEKAQALEDLAGFKELFQT(配列番号2)からなるエピトープを認識して結合する。
「抗Ki-67抗体」とは、Ki-67を認識し、これに結合する抗体を指し、MIB-1の他、MIB-2、MIB-5、MIB-7、MIB-21およびMIB-24などのMIB(登録商標)ファミリーに属する抗体、DAKO-PC、Ki-S5、A0047なども「抗Ki-67抗体」として用いることができ、それ以外の市販或いは臨床検査上使用されている同じ特性の抗体も「抗Ki-67抗体」として用いることができる。抗Ki-67抗体は、Ki-67抗原またはその一部を抗原として用い、公知の方法によって調製することができる。本発明で用いられる「抗Ki-67抗体」は、上記エピトープを認識する抗体を含むことが好ましい。
これに限定しないが、公知のデータベースにより(http://web.expasy.org/peptide_cutter/)、このような特性を有する蛋白分解酵素を知る事ができる(表1)。また、ある酵素がこのような特性を有するかは、MIB-1、又は同じエピトープを認識する他の抗体を用いて当該酵素が当該エピトープを切断するかを分析して確認することができる。
抗体が細胞内に浸潤するため或いは/及び細胞核の抽出のため、かかる酵素は細胞外マトリックスである、コラーゲンを認識切断できることが好ましい。これに限定しないが、トロンビン、Arg-C(クロストリパイン)ペプチターゼ(EC 3.4.22.8)、プロリンエンドペプチターゼ(EC 3.4.21.26)などが挙げられる。
本発明の目的には、ヒアルロニダーゼなどの蛋白以外の細胞外基質中の成分を加水分解する酵素も好ましい。ヒアルロニダーゼ(EC 3.2.1.35)は、ヒアルロン酸のβ‐1‐4結合を分解する加水分解酵素であり、配列番号2のペプチドを切断しない。ヒアルロニダーゼのこの特性により、抗体の細胞内への浸潤が促進される。このような酵素としてヒアルロニダーゼ、グリコシダーゼ(EC 3.2.1)、N-グリカナーゼ(EC 3.5.1.52)等を挙げることができ、ヒアルロニダーゼが好ましい。
また、上述した配列番号2のペプチドを認識切断しない蛋白酵素は、配列番号2のペプチドを切断し無い点でヒアルロニダーゼと共通する一方、両者は、全く異なる作用の酵素であるため、両者を組み合わせることで抗原不活化活性がより高まることが期待される。特に、トロンビンとヒアルロニダーゼの組み合わせは好ましい。
「標識酵素」は、特に限定しないが、アルカリフォスファターゼ、ホースラディッシュペルオキシダーゼが挙げられる。
「化学発光物質」は、特に限定しないが、ルミノール、AMPPD(登録商標)、CSPD(登録商標)、CDP-Star(登録商標)が挙げられる。
1)同一(FFPE)組織切片に対し、異なる蛍光波長の蛍光色素などの他のリガンドとの識別が可能になる標識で各リガンド(例えば、抗サイトケラチン抗体、抗ER抗体、抗PgR抗体、HER2遺伝子にハイブリダイズする核酸、あるいはそれらに結合する二次抗体又は核酸)を標識し、検出/定量してもよいし、或いは
2)同一組織ブロックから均質と仮定される複数の(FFPE)組織切片を作製し、各標的物質について染色(例えば、免疫蛍光染色、FISH)を行い検出/定量してもよい。
1) 薄切FFPE切片の脱パラフィン/親水化
2) 熱処理による抗原賦活化
3) 酵素による抗原の賦活化
4) 組織及び細胞を破砕して細胞核を抽出
超音波による破砕工程は、他の血液細胞よりリンパ球を優先的に破砕し、リンパ球の細胞壁のみならずリンパ球の核を破砕する際に有益である。従って、リンパ球の存在が他の細胞の分析の妨げになる場合など、リンパ球を優先的に破砕したい場合には、超音波による破砕工程を含むことが好ましい。
1)癌腫(Carcinoma):上皮組織由来の悪性腫瘍
2)肉腫(Sarcoma):非上皮組織由来の悪性腫瘍
3)その他:白血病など
に分類され、本願において「がん」とは癌腫を意味する。特に限定しないが、頭頸部癌(上顎癌、(上、中、下)咽頭癌、喉頭癌、舌癌、甲状腺癌)、胸部癌(乳癌、肺癌(非小細胞肺癌、小細胞肺癌))、消化器癌(食道癌、胃癌、十二指腸癌、大腸癌(結腸癌、直腸癌)、肝癌(肝細胞癌、胆管細胞癌)、胆嚢癌、胆管癌、膵癌、肛門癌、泌尿器の癌(腎癌、尿管癌、膀胱癌、前立腺癌、陰茎癌、精巣(睾丸)癌)、生殖器癌(子宮癌(子宮頸癌、子宮体癌)、卵巣癌、外陰癌、膣癌)、皮膚癌(基底細胞癌、有棘細胞癌)などのがん細胞がこれにあたる。
癌腫と肉腫を区別するために用いられる上皮性細胞のマーカーとしては、サイトケラチンが挙げられる。サイトケラチンは上皮細胞の主要な骨格タンパク質であり、約20~30種類のサブタイプ(分子量40~68KDa)が報告されている。上皮性細胞マーカーとしてサイトケラチンを検出するのに、公知の広いサブタイプを認識する抗体(ポリクローナル又はモノクローナル抗体)が用いられるが、各サブタイプに特異的に結合する公知の抗体を複数含むカクテルを用いてもよい。
プロゲステロン受容体(Progesterone receptor (PRまたはPgR)は、核内受容体スーパーファミリーのサブファミリー3グループCに属する核内タンパクである。11q22に存在する単一のPgR遺伝子によってコードされ、分子量の異なる2つのアイソフォームが知られている。
HER2タンパクは、正常細胞において細胞の増殖、分化などの調節に関与しているが、何らかの理由でHER2遺伝子の増幅や遺伝子変異が起こると、細胞の増殖・分化の制御ができなくなり、細胞は悪性化する。HER2遺伝子はがん遺伝子でもあり、多くの種類のがんで遺伝子増幅がみられる。
「組織切片」とはヒト又は非ヒト動物の単離された組織の切片をいう。生検で採取された「組織切片」であってよい。「組織切片」は単離後、凍結保存などされていてもよい。
キットは、更に、「細胞核を抽出する工程」で用いられる組織又は細胞を浸漬又は分散させるための緩衝液を含むことができ、好ましくはこの緩衝液は界面活性剤を含む。界面活性剤としては、好ましくはCHAPS、NP-40、Triton-X100が挙げられ、NP-40、Triton-X100がより好ましい。従って、本願発明によれば、水流又は超音波などで生じるせん断応力により細胞(通常、固定化された細胞)から細胞核を抽出するためのキットであって、界面活性剤を含有する細胞分散用緩衝液を含む、キットが提供される。
「内分泌療法」とは、ホルモン依存性の乳がんの増殖を促す女性ホルモン(エストロゲン)が働かないようにする治療法であり、これに限定しないが、抗エストロゲン剤、Lh-RHアゴニスト製剤、アロマターゼ阻害剤、プロゲステロン製剤などのホルモン剤を投与することを含む。
「化学療法」とは、抗がん作用を奏する化学物質を用いてがんを治療することをいう。これに限定しないが、DNA合成及び複製阻害剤であるアントラサイクリン系抗がん剤(ダウノルビシン(ダウノマイシン)、ドキソルビシン(アドリアマイシン)、エピルビシン、イダルビシンなど)、細胞増殖の抑制剤であるタキサン系抗がん剤(パクリタキセル(タキソール)、ドセタキセル(タキソテール)など)、DNAの複製を妨げるプラチナ(白金)製剤(シスプラチン、オキサリプラチン等)などを単投与または併用投与することを含み、好ましくはアントラサイクリン系抗がん剤とタキサン系抗がん剤を併用投与することを含む。本願明細書においては、「ホルモン剤」を投与する場合は、「内分泌療法」に含まれるものとする。
水流せん断による細胞核脱離観測
1.材料及び方法
乳癌組織のFFPE組織切片に脱パラフィン/親水化、熱処理による抗原賦活化を施し、水流せん断装置を用いて分散処理後、回収産物に免疫蛍光染色を行い顕微鏡観察した。
1-1.FFPE組織ブロック
Proteogenex社より購入した乳癌組織のFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
Thermofisher社製滑走式ミクロトームを用いてFFPE組織ブロックを薄切して厚さ20μmの切片を作成した。
1-3.脱パラフィン/親水化
薄切したFFPE切片に十分量のキシレンを添加し、10分間静置後、キシレンを除去した。この工程を再度行い、完全にパラフィンを除去した。次いで、100%エタノール、95%エタノール、70%エタノール、50%エタノール、脱イオン水に順に3分間ずつ浸漬して親水化した。
1-4.熱処理による抗原賦活化
純水で10倍に希釈したナカライテスク社製Histo VT Oneを添加し、ヒートブロックで98℃下、20分間加熱した。加熱後、室温にて20分間静置して後、抗原賦活化液を除去した。
1-6.水流破砕
Sysmex社の水流せん断装置(RP-10)を用い、氷冷下、賦活化済組織を、TBS1mL中で10,000rpm、1分間破砕した。
1-7.免疫蛍光染色
10%正常ヤギ血清(和光)を添加した4%BSA/TBSを水流破砕による破砕物が入ったマイクロチューブに添加し、室温下30分間静置し、ブロッキング処理した。免疫蛍光染色は、1次抗体としてpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体:Anti-wide spectrum Cytokeratin antibody(AB9377-500))を用い、2次抗体はAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。1次抗体反応時間は50分、2次抗体反応時間は30分であり、2次抗体反応時間経過時に細胞核を染色する色素DAPI Solution(和光)を添加した。反応は全て室温であり、2次抗体添加以降は遮光下にて行った。抗体希釈液には0.5%BSA/TBSを使用し、各工程の間には1回ずつ0.5%BSA/TBSによる洗浄操作を行った。スライドガラスにDAPI包埋剤(和光)を5μLのせ、その上に2次抗体反応後のサンプルを乗せた。その後、カバーガラスを被せ、5分間静置後、上から垂直に押し、カバーガラスの周囲をマニキュアでシールした。観察時まで遮光し、4℃下で保存した。
1-8.蛍光顕微鏡
顕微鏡観察には、EVOS オールインワン顕微鏡、蛍光顕微鏡(Thermoscientific)を使用した。観察時にはDAPIライトキューブ(Ex 357nm、Em 447nm)、GFPライトキューブ(Ex 470nm/Em 510nm)を使用した。接眼レンズは10倍、対物レンズは40倍を使用した。
図1に蛍光顕微鏡で観測した細胞核を示す。左の図に示す通り、円形状に核酸が存在し、右図に示す通り、サイトケラチンが核酸の周囲に存在している。よって、細胞核、骨格が破損せず円形を保ち、凝集することなく単一細胞核として回収されたことが確認された。
ホルマリン固定化乳癌細胞中のサイトケラチン及びKi-67由来シグナルの確認
1.材料及び方法
ホルマリンで固定化した乳癌細胞に熱処理による抗原賦活化を施し、免疫蛍光染色を行い、サイトケラチンとKi-67のシグナルをフローサイトメーターで確認した。
1-1.細胞
3種の乳癌細胞株、T47D、MDA-MB-231(図においてMB231又は231と表記)、及びSKBr3(図においてはSKBRと表記)を、ATCC(American Type Culture Collection)より入手し、使用した。
1-2.細胞培養とホルマリン固定
T47D細胞株はRPMI-1640培地、MDA-MB-231細胞株はLeibovitz’s L-15培地およびSKBr3細胞は、McCoy’s 5A培地で培養し、10%のウシ胎児血清(FBS)を補った。細胞が十分に増殖した後に培養液を吸引、PBSで洗浄したのちに、TrypLE Express(Thermofisher)を添加した。細胞を回収した後に遠心分離を行いPBSで洗浄した。さらにPBSで細胞を十分に懸濁後1×106個に分注し、遠心分離、PBS除去を行い、10%中性緩衝ホルマリン液(和光純薬)を添加後、4℃で24時間固定した。細胞は使用する前に、ホルマリン液を除去し、PBSで洗浄した。
1-4.熱処理による抗原賦活化
参考例1と同様の処理を行った。
1-7.免疫蛍光染色
10%正常ヤギ血清(和光)を添加した4%BSA/TBSを熱処理による抗原賦活化後の細胞が入ったマイクロチューブに添加し、室温下30分間静置し、ブロッキング処理した。免疫蛍光染色は、マウス抗体とラビット抗体の混合液を用いて2重染色した。1次抗体はKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体(AB9377-500))を用い、2次抗体はThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。1次抗体反応時間は50分、2次抗体反応時間は40分であり、2次抗体添加後20分後に細胞核を染色する色素DAPI Solution(和光)を添加した。反応は全て室温であり、2次抗体添加以降は遮光下行った。抗体希釈液には0.5%BSA/TBSを使用し、各工程の間には1回ずつ0.5%BSA/TBSによる洗浄操作を行った。また、ネガティブコントロール(アイソタイプコントロール)として1次抗体の代わりにそれぞれ対応する1次抗体と同種、同濃度の抗体を用いた。Ki-67はDako社のマウスIgG抗体を使用し、サイトケラチンはCellsignaling technology社のラビットIgG抗体を用いた。
1-8.フローサイトメーター測定
Falcon(登録商標)セルストレーナー 5mLチューブ用35μm(380メッシュ)(フローサイトメーター用)のフィルターを通過させた後に、指定容器に移し替えてフローサイトメーター(Sysmex:Space)で測定した。測定は、機器マニュアルに従って行った。
1-9.サイトケラチン及びKi-67陽性率の算出
得られた測定データの解析にはFLOWJO LLC社製のソフトFlowJo v10を用いた。データ解析はソフト添付の説明書に従い、以下の順で行った。前方散乱を横軸、側方散乱を縦軸としたスキャッタグラム上で、図2に示す主要な領域を選択した。次に、DAPIの蛍光強度を横軸、細胞(核)個数を縦軸として、主要な領域を細胞核としてゲーティングした(図3)。サイトケラチン及びKi-67の陽性率は全細胞核中のサイトケラチン陽性核数、及びKi-67陽性核数の割合として算出した。陽性核の閾値はアイソタイプコントロールの95パーセンタイル値とした。
図4に抗サイトケラチン抗体及び抗Ki-67抗体で検出した蛍光強度のヒストグラム(黒色実線)、及びそれらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。いずれの細胞株でも、黒色のピークが灰色と比較し高い蛍光強度を示した。これらより、ホルマリン固定化乳癌細胞中のサイトケラチン及びKi-67の存在を確認した。
トロンビンでの抗原賦活化によるホルマリン固定化細胞中のKi-67シグナルの増強
1.材料及び方法
ホルマリンで固定化した乳癌細胞を用い、トロンビン処理の有無でのシグナル強度を比較した。
1-1.細胞
参考例2で用いた3種の乳癌細胞株を用いた。
1-2.細胞培養とホルマリン固定
参考例2と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様の処理を行った。
1-5.酵素による抗原賦活化
熱処理による抗原賦活化後の細胞にトロンビン試薬(25mM Tris-HCl pH7.4、150mM NaCl、1000KU/L トロンビン、10mM CaCl2)を添加しヒートブロックで37℃下、20分間加熱した。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体としてKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.Ki-67陽性率の算出
参考例2と同様に行った。
図5に抗Ki-67抗体(黒色実線)及びアイソタイプコントロール抗体(灰色破線)で検出した蛍光強度を重ね合わせたチャートを示し、図6及び表2にKi-67陽性率の変化を示した。いずれの細胞株でも、トロンビンによる抗原賦活化を行うと蛍光強度が上昇し、それに伴いKi-67陽性率の増加が見られた。
ヒアルロニダーゼでの抗原賦活化によるホルマリン固定化細胞中のKi-67シグナルの増強
1.材料及び方法
ホルマリンで固定化した乳癌細胞を用い、ヒアルロニダーゼ処理の有無でのシグナル強度を比較した。
1-1.細胞
参考例2で用いたT47D細胞株を用いた。
1-2.細胞培養とホルマリン固定
参考例2と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様の処理を行った。
1-5.酵素による抗原賦活化
熱処理による抗原賦活化後の細胞にヒアルロニダーゼ試薬(25mM Tris-HCl pH7.4、150mM NaCl、4000-10000KU/L ヒアルロニダーゼI―S、7500-30000KU/L ヒアルロニダーゼIV―S、27mM KCl)を添加しヒートブロックで37℃下、20分間加熱した。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体としてKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa647を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.Ki-67陽性率の算出
参考例2と同様に行った。
図7に抗Ki-67抗体(黒色実線)及びアイソタイプコントロール抗体(灰色破線)で検出した蛍光強度の重ね合わせチャートを示した。ヒアルロニダーゼ処理により蛍光強度が上昇し、それに伴いKi-67陽性率の増加が見られた。
FFPE組織切片中のサイトケラチン及びKi-67由来シグナルの確認
1.材料及び方法
乳癌組織のFFPE組織切片中のサイトケラチン及びKi-67のシグナルをフローサイトメーターにて確認した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
Thermofisher社製滑走式ミクロトームを用いてFFPE組織ブロックを薄切して厚さ20μmの切片2枚を作成し、検討に用いた。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
得られた測定データの解析にはFLOWJO LLC社製のソフトFlowJo v10を用いた。データ解析は以下の順で行った。まず、DAPIの蛍光強度を横軸、細胞(核)個数を縦軸として、横軸の蛍光量が5~150の領域を細胞核としてゲーティングした。サイトケラチン及びKi-67の陽性率は全細胞核中のサイトケラチン陽性核数、及びKi-67陽性核数の割合として算出した。陽性核の閾値はアイソタイプコントロールの95パーセンタイル値とした。
図8に抗サイトケラチン抗体及び抗Ki-67抗体で検出した蛍光強度のヒストグラム(黒色実線)、及びそれらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。黒色のピークが灰色と比較し高い蛍光強度を示した。これらより、FFPE組織切片中のサイトケラチン及びKi-67の検出を確認した。
トロンビンでの抗原賦活化によるFFPE組織切片中のサイトケラチン及びKi-67シグナルの増強
1.材料及び方法
乳癌組織のFFPE組織切片を用い、トロンビン処理の有無でのシグナル強度を比較した。
1-1.FFPE組織ブロック
IProteogenex社より購入した、HC法によってKi-67陽性率が異なった2人の乳癌患者から得られた2種のFFPE組織ブロックを使用した。
1-2.FFPE切片の作成
Thermofisher社製滑走式ミクロトームを用いてFFPE組織ブロックを薄切して厚さ20μmの切片2枚を作成し、検討に用いた。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-5.酵素による抗原賦活化
実施例1と同様に行った。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
参考例3と同様に行った。
図9Aに抗サイトケラチン抗体及び抗Ki-67抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。いずれのFFPE切片でも、トロンビンによる処理を行うとサイトケラチン、Ki-67の蛍光強度がいずれも上昇し、それに伴い陽性率の増加が見られた(図9B)。
薄切切片の厚さによるサイトケラチン及びKi-67シグナル強度への影響
1.材料及び方法
FFPE切片の厚さによってサイトケラチン及びKi-67のシグナル強度に影響を及ぼすか確認した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
Thermofisher社製滑走式ミクロトームを用いて同一FFPE組織ブロックより20μm 3枚、30μm 2枚、60μm 1枚の薄切サンプルを作成した。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化
実施例1と同様に処理した。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
参考例3と同様に行った。
薄切切片の厚さとサイトケラチン及びKi-67の陽性率の変化を図10に示す。
20μm 3枚で検出効率が最も良好であったが、20~60μmいずれの厚さにおいてもサイトケラチン(CK)及びKi-67(Ki67)の検出が可能であった。
フローサイトメーターによるKi-67陽性率とIHC法による陽性率の比較検討
1.材料及び方法
FFPE組織ブロックを用いてフローサイトメーターによるKi-67陽性率とIHC法による陽性率を比較した。
1-1.FFPE組織ブロック
Proteogenex社より購入した、IHC法によってKi-67陽性率が異なった19人の患者より得られた19種のFFPE組織ブロックを使用した。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化
実施例1と同様に処理した。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体はKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体)を用い、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.フローサイトメーターによるKi-67陽性率の算出
参考例3と同様に行った。
1-10.IHC法によるKi-67陽性率算出
IHC法によるKi-67陽性率は1次抗体はKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)を用い、ホットスポット法により算出された。なお、鏡検者によるバラツキを考慮して病理医2名の平均値とした。
Ki-67陽性率におけるIHC法との相関性を図11に示す。
IHC法をX、本法をYとして相関性を確認した。また、陽性核のカットオフをアイソタイプコントロールの95パーセンタイル値としているため、切片は10と設定した。図11に示したようにY=0.5632X+10、相関係数0.7697と良好な結果が得られた。これより本法はFFPE組織切片中のKi-67を検出出来ているといえる。
各種消化酵素での抗原賦活化をした際のKi-67及びサイトケラチンの検出
1.材料及び方法
ホルマリンで固定化した乳癌細胞を用い、5種の異なる消化酵素での抗原賦活化について、Ki-67及びサイトケラチンのシグナル強度を比較した。
1-1.細胞
参考例2で用いた3種の乳癌細胞株を用いた。
1-2.細胞培養とホルマリン固定
参考例2と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様の処理を行った。
1-5A.酵素による抗原賦活化(トロンビン)
実施例1と同様に処理した。
1-5B.酵素による抗原賦活化(トリプシン)
トリプシン試薬(25mM TBS pH7.4、 1M CaCl2、1mg/mLトリプシン)250μLを、熱処理による抗原賦活化をした細胞に添加し、37℃下、20分間加熱した。酵素処理後はTBSで洗浄して酵素を取り除いた。
1-5C.酵素による抗原賦活化(プロテイナーゼK)
600 mAnson U/mLプロテイナーゼK(タカラバイオ)を25mM TBS pH7.4で40倍(v/v)に希釈し、プロテイナーゼK溶液とした。プロテイナーゼK溶液250μLを、熱処理による抗原賦活化をした細胞へ添加し、37℃下、20分間加熱した。酵素処理後はTBSで洗浄して酵素を取り除いた。
1-5D.酵素による抗原賦活化(ディスパーゼ)
ディスパーゼ(和光)を25mM TBS pH7.4に溶解し、ディスパーゼ溶液(3,000PU/mL)とした。ディスパーゼ溶液250μLを抗原賦活化した細胞へ添加し37℃下、20分間加熱した。反応終了後は、1M EDTA溶液(和光)を2μL添加して転倒混和した後、TBSで洗浄して酵素を取り除いた。
1-5E.酵素による抗原賦活化(プロリンエンドペプチダーゼ)
プロリンエンドペプチダーゼ(東洋紡)を25mM TBS pH7.4に溶解し、プロリンエンドペプチダーゼ溶液(10U/mL)とした。プロリンエンドペプチダーゼ溶液250μLを、熱処理による抗原賦活化をした細胞へ添加し、37℃下、20分間加熱した。反応終了後は、TBSで洗浄して酵素を取り除いた。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体はKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体)を用い、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
参考例2と同様に行った。
図12A及びBに、抗Ki-67抗体又は抗サイトケラチン抗体で検出した蛍光強度のヒストグラム(黒色実線)とそれらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示した。サイトケラチンはいずれのタンパク質分解酵素を用いた場合でも、検出可能であった(図12B)。それに対して、配列番号2のアミノ酸配列を認識切断しうるトリプシン、ディスパーゼ、及びプロテイナーゼKの3種のタンパク質分解酵素を用いた場合は、シグナルが減少し、Ki-67の陽性率が著しく低下した。一方、配列番号2のアミノ酸配列を認識切断しないトロンビン及びプロリンエンドペプチダーゼはKi-67の陽性率が上昇し、Ki-67抗原の抗原性を増強していることが示された(図12A)。
各種消化酵素を使用した際の乳癌組織のFFPE切片中のKi-67及びサイトケラチンの検出
1.材料及び方法
乳癌組織のFFPE切片を5種の異なる消化酵素にて抗原賦活化し、Ki-67及びサイトケラチンのシグナル強度を比較した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5A.酵素による抗原賦活化(トロンビン)
実施例1と同様に行った。
1-5B.酵素による抗原賦活化(トリプシン)
実施例6と同様に行った。
1-5C.酵素による抗原賦活化(プロテイナーゼK)
実施例6と同様に行った。
1-5D.酵素による抗原賦活化(ディスパーゼ)
実施例6と同様に行った。
1-5E.酵素による抗原賦活化(プロリンエンドペプチダーゼ)
実施例6と同様に行った。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体はKi-67抗体(Dako、クローン:MIB-1、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体)を用い、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
参考例3と同様に行った。
図13A及びBに抗Ki-67抗体又は抗サイトケラチン抗体で検出した蛍光強度のヒストグラム(黒色実線)とそれらのアイソタイプコントロールで検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。サイトケラチンはいずれのタンパク質分解酵素を用いた場合でも、検出可能であった(図13B)。それに対して、実施例6と同様に配列番号2のペプチドを認識切断しうるトリプシン、ディスパーゼ、プロテイナーゼKの酵素を使用した際にはKi-67抗体(黒色実線)の蛍光強度が大幅に減少していた(図13A)。
認識部位の異なる抗体を用いたKi-67の検出
1.材料及び方法
乳癌組織のFFPE組織切片中のサイトケラチン及びKi-67のシグナルをフローサイトメーターにて確認した。Ki-67抗体は、MIB-1クローンとこれと認識部位の異なる抗体としてS5クローンとを使用した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化処理
実施例1と同様に処理した。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体としてKi67 MIB-1抗体(Dako、マウスモノクローナル抗体)もしくはKi67 S5抗体(Millipore、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体)を用い、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
参考例3と同様に行った。
図14に、抗Ki-67抗体又は抗サイトケラチン抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロールで検出した蛍光強度のヒストグラム(灰色)を重ね合わせたチャートを示す(上段:Ki-67;下段:サイトケラチン)。Ki-67抗体のピークがアイソタイプコントロールと比較し高い蛍光強度を示した。これにより、FFPE組織切片中のサイトケラチン及びKi-67の検出を確認し、Ki-67のMIB-1クローンに限らず、S5クローンでも検体中のKi-67を検出可能であることが示唆された。
公知の方法によるFFPE組織切片中のKi-67シグナルの検出
1.材料及び方法
公知の先行文献法(非特許文献4:Cytometry 27:283-289)により乳癌組織のFFPR組織切片中のサイトケラチン及びKi―67シグナルの検出を行った。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化(トリプシン):既存法
親水化処理した組織切片にトリプシン試薬(25mM PBS pH7.4、1mg/ml CaCl2、1mg/mLトリプシン)250μLを添加し37℃下、70分間加熱した。酵素処理後はPBSで洗浄して酵素を取り除いた。
1-7.免疫蛍光染色
10%正常ヤギ血清(和光)を添加した4%BSA/TBSを酵素による抗原賦活化後の試料が入ったマイクロチューブに添加し、室温下30分間静置し、ブロッキング処理した。免疫蛍光染色は1次抗体としてKi67 MIB-1抗体(Dako、マウスモノクローナル抗体)もしくはKi67 S5抗体(Millipore、マウスモノクローナル抗体)、及びサイトケラチン抗体(Abcam、ラビットポリクローナル抗体)を用い、2次抗体はThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。1次抗体反応時間は4℃オーバーナイトで反応させた。
2次抗体は40分間反応させ、2次抗体添加後20分後に細胞核を染色する色素DAPI Solution(和光)を添加した。2次抗体添加以降は全て室温で遮光下にて行った。抗体希釈液には0.5%BSA/TBSを使用し、各工程の間には1回ずつ0.5%BSA/TBSによる洗浄操作を行った。また、ネガティブコントロールとして1次抗体の代わりにそれぞれ対応する1次抗体と同種、同濃度の抗体を用いた。マウス抗体はDako社のマウスIgG抗体を使用し、ラビット抗体はCellsignaling technology社のラビットIgG抗体を用いた。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.フローサイトメーターによるサイトケラチン及びKi-67陽性率の算出
参考例3と同様に行った。
図15(上段:Ki-67;下段:サイトケラチン)に示すとおり、非特許文献4に記載された既存法では、Ki-67のS5クローンを使用した場合、検出することができたが、MIB-1クローン抗体を用いたサンプルではKi-67由来の蛍光ピークが大幅に減少しており、Ki-67を検出することができなかった。
FFPE切片中のER及びPgRの検出
1.材料及び方法
ER及びPgRの陽性及び陰性が既知となっているFFPE組織切片を用い、トロンビンで抗原賦活化し、水流破砕により細胞核を抽出し、フローサイトメーターを用いてER及びPgRのシグナル確認を行った。
1-1.FFPE組織ブロック
ER及びPgRの陽性試料として、Proteogenex社より購入したER陽性検体(All red Score ER7)、及びPgR陽性検体(All red Score PgR8)を用い、ER及びPgRの陰性試料として、ER及びPgR陰性検体(All red Score ER0及びPgR0)のFFPE組織切片を用いた。
1-2.FFPE切片の作成
参考例3と同様にして作成した。
1-3.脱パラフィン/親水化
参考例1と同様に処理した。
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化
熱処理による抗原賦活化後、ER陽性検体は、トロンビンで抗原賦活化した。また、PgR陽性検体とER及びPgR陰性検体は、トロンビンまたはプロリンエンドペプチダーゼで抗原賦活化した。トロンビンでの抗原賦活化は実施例1と同様に行い、プロリンエンドペプチダーゼでの抗原賦活化は実施例6と同様に行った。
1-6.水流破砕
参考例1と同様に行った。
1-7.免疫蛍光染色
10%正常ヤギ血清(和光)を添加した4%BSA/TBSを水流破砕後の試料が入ったマイクロチューブに添加し、室温下30分間静置し、ブロッキング処理した。免疫蛍光染色は1次抗体としてER抗体(Abcam、クローン:SP1、ウサギモノクローナル抗体)もしくはPgR抗体(Thermofisher、クローン:SP2、ウサギモノクローナル抗体)を用い、2次抗体はAbcam社Goat anti-Rabbit Mouse Secondary Antibody Alexa 488を使用した。1次抗体は室温で45分間反応させた。2次抗体は室温で40分間反応させ、2次抗体添加後20分後に細胞核を染色する色素DAPI Solution(和光)を添加した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.ER及びPgR陽性率の算出
得られた測定データの解析にはFLOWJO LLC社製ソフトFlowJo v10を用いた。DAPIの蛍光量を横軸、細胞(核)個数を縦軸として、横軸の蛍光量が5~150の領域を細胞核としてゲーティングした。ER及びPgRの陽性率は、全細胞核中のER陽性核数及びPgRの陽性核数の割合として算出した。陽性核の閾値はアイソタイプコントロールの90パーセンタイル値とした。
図16に抗ER抗体又は抗PgR抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。ER陽性及びPgR陽性FFPE組織切片を配列番号2のペプチドを認識切断しない酵素で処理しても、黒色のピークが灰色と比較し高い蛍光強度を示し、ER及びPgRは共に検出された。一方で、ER及びPgR陰性FFPE組織切片ではER及びPgRは検出されなかった。
異なる熱処理抗原賦活化剤を用いた場合の、固定化乳癌細胞中のKi-67及びサイトケラチンのシグナルの比較
1.材料及び方法
ホルマリンで固定化した乳癌細胞株MDA-MB-231を3種類の抗原賦活化剤を用いて熱処理による抗原賦活化をし、トロンビン酵素処理後、免疫蛍光染色を行い、サイトケラチンとKi-67をフローサイトメーターで検出した。
1-1.細胞
ATCC(American Type Culture Collection)より入手した乳癌細胞株MDA-MB-231を用いた。
1-2.細胞培養とホルマリン固定
参考例2と同様に行った。
1-4A.熱処理による抗原賦活化(Histo VT ONE)
参考例1と同様の処理を行った。
1-4B.熱処理による抗原賦活化(抗原賦活化液pH9)
抗原賦活化液pH9(ニチレイバイオサイエンス)を使用した。処理の手順及び条件は参考例1と同様にして行った。
1-4C.熱処理による抗原賦活化(イムノセイバー)
抗原賦活化液イムノセイバー(日新EM)を使用した。処理の手順及び条件は参考例1と同様にして行った。
1-5.酵素による抗原賦活化
実施例1と同様に処理した。
1-7.免疫蛍光染色
参考例2と同様に行った。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67の陽性率の算出
参考例3と同様に行った。
図17に抗Ki-67抗体又は抗サイトケラチン抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す(上段:Ki-67;下段:サイトケラチン)。いずれの賦活化液でも、黒色のピークが灰色と比較し高い蛍光強度を示した。これらより、ホルマリン固定化細胞中のサイトケラチン及びKi-67の検出を確認し、賦活化液を変更しても有用であることを確認した。
細胞核を抽出する工程における各種破砕方法の、FFPE組織切片中のサイトケラチン及びKi-67の検出への影響
1.材料及び方法
乳癌組織のFFPE組織切片を用い、3種の破砕方法、及び破砕方法の組み合わせを用いて細胞核を抽出し、フローサイトメーターを用いてサイトケラチン及びKi-67のシグナル強度を比較した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-5.酵素による抗原賦活化
実施例1と同様に処理した。
1-6A.マッシャー
ニッピ社のバイオマッシャー(登録商標)IIを用い、氷冷下、TBS1mL中で酵素処理後の組織を20回すり潰した。
1-6B.乳鉢
乳鉢と乳棒を用い、TBS1mL中で酵素処理後の組織をすり潰した。
1-6C.超音波破砕
SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS1mL中で酵素処理後の組織を出力強度40%で20秒間破砕した。
1-6D.水流破砕と超音波破砕の組み合わせ
Sysmex社の水流せん断装置(RP-10)を用い、氷冷下、TBS1mL中で酵素処理後の組織を10,000rpm、1分間破砕した。さらに、SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS1mL中で水流破砕後の組織を出力強度20%で30秒間破砕した。
1-7.免疫蛍光染色
参考例2と同様に行った。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
得られた測定データの解析にはFLOWJO LLC社製ソフトFlowJo v10を用いた。DAPIの蛍光量を横軸、細胞(核)個数を縦軸として、横軸の蛍光量が5~150の領域を細胞核としてゲーティングした。サイトケラチン及びKi-67の陽性率は、全細胞核中のサイトケラチン陽性核数及びKi-67の陽性核数の割合として算出した。陽性核の閾値はアイソタイプコントロールの90パーセンタイル値とした。
図18に抗サイトケラチン抗体又は抗Ki-67抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。いずれの破砕方法でも、黒色実線のピークが灰色破線と比較して高い蛍光強度を示した。これらより、各種の破砕方法を用いてFFPE組織切片中のサイトケラチン及びKi-67の検出が可能であることを確認した。
超音波破砕によるリンパ球の優先的破砕
1.材料及び方法
ホルマリンで固定化した乳癌細胞株及びリンパ芽球性細胞株を用い、超音波破砕が細胞核の取得に影響を及ぼすかを確認した。
1-1.細胞
ATCC(American Type Culture Collection)より乳癌細胞株SKBr3及びMDA-MB-231、並びにリンパ芽球性細胞株Jurkatを入手して、使用した。
1-2.細胞培養とホルマリン固定
1-4.熱処理による抗原賦活化
参考例1と同様に処理した。
1-5.酵素による抗原賦活化処理
実施例1と同様に処理した。
1-6.超音波破砕
SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS1mL中で酵素処理後の細胞を出力強度20%で30秒間破砕した。
1-7.細胞核染色
細胞核を染色する色素DAPI Solution(和光)を添加し、遮光20分間反応した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
図19に、前方散乱を横軸、側方散乱を縦軸とした各細胞株のスキャッタグラムを示す。乳癌細胞SKBr3及びMDA-MB-231において、超音波破砕の有無により、主要な細胞核領域の位置が変化しないことを確認した。一方で、リンパ球Jurkatにおいて、超音波破砕により主要な細胞核領域が消失した。したがって、上皮細胞は、超音波破砕により破壊されず、リンパ球芽球性細胞が優先的に超音波破砕されることが示唆された。
界面活性剤の添加による解析細胞核数の増加
1.材料及び方法
乳癌組織のFFPE組織切片を用い、超音波破砕時に界面活性剤を添加し、細胞核の取得に変化を及ぼすかを確認した。
1-1.FFPE組織ブロック
Proteogenex社より購入した、IHC法によってKi-67陽性率が異なった2人の乳癌患者から得られた2種のFFPE組織ブロックを使用した。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-5.酵素による抗原賦活化処理
実施例1と同様に処理した。
1-6.超音波破砕
SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS、1%CHAPS(和光)を含むTBS、1%NP-40(和光)を含むTBS、または1%Triton-X100(和光)を含むTBS1mL中で酵素による抗原賦活化後の組織及び細胞を出力強度20%で30秒間破砕した。
1-7.細胞核染色
細胞核を染色する色素DAPI Solution(和光)を添加し、遮光20分間反応した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
図20に、界面活性剤を無添加の緩衝液中で超音波破砕した場合と、上記各種界面活性剤を添加した緩衝液中で超音波破砕した場合とで、前方散乱を横軸、側方散乱を縦軸としたスキャッタグラムを示し、表3にフローサイトメーター測定時の総解析細胞核数を示した。界面活性剤の添加により、総解析細胞核数が増加することが確認された。
異なる病理医によるKi-67陽性細胞の測定
1.材料及び方法
2名の病理医によって算出されたIHC法によるFFPE組織切片中のKi-67陽性率の相関性を確認した。
1-1.FFPE組織ブロック
Proteogenex社より購入した、IHC法によってKi-67陽性率が異なった38人の患者から得られた38種のFFPE組織ブロックを使用した。
1-10.IHC法によるKi-67陽性率算出
実施例5で言及したのと同様に実施した。
図21に結果を示す。同一サンプルであっても病理医によって、Ki-67陽性率が異なる値となり、ばらつきが比較的大きいことがわかった。
各種消化酵素での抗原賦活化後の認識部位の異なる抗Ki-67抗体での検出
1.材料及び方法
ホルマリンで固定化した乳癌細胞を用い、3種の消化酵素(トロンビン、ディスパーゼ、プロテイナーゼK)の何れかで抗原賦活化後、蛍光免疫染色し、フローサイトメーターを用いてKi-67及びサイトケラチンを検出した。
1-1.細胞
参考例2で用いた3種の乳癌細胞株を用いた。
1-2.細胞培養とホルマリン固定
参考例2と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様の処理を行った。
1-5A.酵素による抗原賦活化(トロンビン)
実施例1と同様に処理した。
1-5B.酵素による抗原賦活化(ディスパーゼ)
実施例6と同様に処理した。
1-5C.酵素による抗原賦活化(プロテイナーゼK)
実施例6と同様に処理した。
1-7.免疫蛍光染色
参考例2と同様に行った。ここでは、1次抗体はKi-67抗体(Millipore、クローン:S5(Ki-S5)、マウスモノクローナル抗体)、及びpan-サイトケラチン抗体(Abcam、ウサギポリクローナル抗体)を用い、2次抗体としてThermofisher社Goat anti-Mouse Secondary Antibody Alexa 647、及びAbcam社Goat anti-Rabbit Secondary Antibody Alexa 488を使用した。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.Ki-67陽性率の算出
参考例3と同様に行った。
図22A及びBに抗Ki-67抗体(Ki-S5)又は抗サイトケラチン抗体で検出した蛍光強度のヒストグラム(黒色実線)とそれらのアイソタイプコントロールで検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。いずれの酵素で抗原賦活化した場合でも、黒色実線のピークが灰色破線と比較して高い蛍光強度を示した。これらより、いずれの酵素で抗原賦活化した場合でも、抗Ki-67抗体(Ki-S5)及び抗サイトケラチン抗体の抗原認識部位が保持され、Ki-67及びサイトケラチンの検出が可能であることを確認した。
FFPE組織切片中のサイトケラチン及びKi-67陽性核判定における閾値の検討
1.材料及び方法
FFPE組織切片中のサイトケラチン及びKi-67由来のシグナルをフローサイトメーターにて検出し、陽性核の閾値を変化させた際の陽性率を算出した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-5.酵素による抗原賦活化
実施例1と同様に行った。
1-6.水流破砕と超音波破砕の組み合わせ
Sysmex社の水流せん断装置(RP-10)を用い、氷冷下、TBS1mL中で酵素処理後の組織を10,000rpm、1分間破砕した。さらに、SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS1mL中で水流破砕後の組織を出力強度20%で30秒間破砕した。
1-7.免疫蛍光染色
参考例2と同様に行った。
1-8.フローサイトメーター測定
参考例2と同様に行った。
1-9.サイトケラチン及びKi-67陽性率の算出
得られた測定データの解析にはFLOWJO LLC社製ソフトFlowJo v10を用いた。DAPIの蛍光量を横軸、細胞(核)個数を縦軸として、横軸の蛍光量が5~150の領域を細胞核としてゲーティングした。サイトケラチン及びKi-67の陽性率は、全細胞核中のサイトケラチン陽性核数及びKi-67の陽性核数の割合として算出した。陽性核の閾値はアイソタイプコントロールの60、70、80、90及び95パーセンタイル値とした。陽性率はフローサイトメーターによる検出値-(100-閾値)で算出した。
図23に閾値を変化させ、抗サイトケラチン抗体及び抗Ki-67抗体で検出した蛍光強度のヒストグラム(黒色実線)と、それらのアイソタイプコントロール抗体で検出した蛍光強度のヒストグラム(灰色破線)を重ね合わせたチャートを示す。いずれの閾値を用いた場合でも陽性率が算出された。従って、サイトケラチン及びKi-67陽性率は閾値に依らず算出される事が可能である。
FFPE組織切片中のHER2の検出
1.材料及び方法
HER2陽性(検体A:Score 3+)及び陰性(検体B:Score 0)が既知となっているFFPE組織切片をそれぞれ用い、水流破砕及び超音波破砕により細胞核を抽出し、FISH法によりHER2の増幅を確認した。
1-1.FFPE組織ブロック
参考例1で用いたFFPE組織ブロックを用いた。
1-2.FFPE切片の作成
参考例3と同様に行った。
1-3.脱パラフィン/親水化
参考例1と同様に行った。
1-4.熱処理による抗原賦活化
参考例1と同様に行った。
1-5.酵素による抗原賦活化
実施例1と同様に行った。
1-6.水流破砕と超音波破砕の組み合わせ
Sysmex社の水流せん断装置(RP-10)を用い、氷冷下、TBS1mL中で酵素処理後の組織を10,000rpm、1分間破砕した。さらに、SONICS&MATERIALS社の超音波破砕装置(VCX130PB)を用い、氷冷下、TBS1mL中で水流破砕後の組織を出力強度20%で30秒間破砕した。
1-7.FISHによるHER2の検出
組織破砕後のサンプルをスライドガラスに乗せ、乾燥後、10%中性緩衝ホルマリンに室温で10分間浸漬した。スライドガラスをTBSで洗浄し、風乾後、パスビジョン(R)HER-2 DNAプローブキット(アボット)を用いてFISH染色を行った(操作は、キット添付書類に従った)。
1-8.蛍光顕微鏡
顕微鏡観察には、オールインワン蛍光顕微鏡BZ-X710(キーエンス)を使用した。観察時にはDAPIフィルタ(Ex 360nm、Em 460nm)、TRITCフィルタ(Ex 545nm、Em 605nm)使用した。対物レンズは20倍を使用した。
図24に蛍光顕微鏡で観測した細胞核を示す。HER2陽性FFPE組織切片中においてHER2由来の蛍光シグナルが検出でき、一方でHER2陰性FFPE組織切片中において、検出されないことを確認した。
本発明より提供されるプロトコール(所定の酵素による抗原賦活化を含む前処理プロセス含む)を用いることで、FFPE組織切片から標的抗原の抗原性を増強した細胞核の脱離を達成する。回収した分散細胞核は、単一の細胞核として解析することが可能である。例えば、核膜上や核内のタンパク質、核酸等が解析対象となり、色素(蛍光)染色による形態の観察や、酵素もしくは蛍光色素で標識した抗体、核酸等のリガンドを用いることで、これらの対象を検出できる。本願発明の方法、抗原賦活剤及びキットは、顕微鏡での形態観測、IHC、EIA、CLEIA、デジタルPCRでの解析や、イメージングサイトメーター又はフローサイトメーターによるサイトメトリー解析に利用することができる。特に、本願発明の好ましい実施形態によれば、フローサイトメトリー解析による脱離核中の対象タンパク質の陽性核の割合(特にKi-67陽性率)の算出に利用することが可能である。
そして、このばらつきの少ない指標を用いて、患者に最適な診断レジメンを提供することが可能である。抗がん剤治療はそれ自体患者に対する負担も大きく、患者にあった治療レジメンを治療開始前に決めることは患者のQOL(QualityofLife)を保つ上でも重要である。本願発明の場合、外科手術前の化学治療(術前抗がん剤治療)の治療レジメンに応用できるだけなく、外科手術で腫瘍摘出後の(予後)治療レジメンにも応用できる。
Claims (47)
- 固定化された細胞群中のKi-67陽性細胞核を含む細胞を、抗Ki-67抗体を用いて検出する方法であって、
1)固定化された細胞群を配列番号2のペプチドを認識切断しない加水分解酵素で前処理して抗原を賦活化する工程、その後
2)抗Ki-67抗体を用いて免疫染色する工程、及び
3)染色されたKi-67陽性細胞又はKi-67陽性細胞核を検出する工程を含む、方法。 - 前記抗体が、MIB-1、DAKO-PC、Ki-S5及びA-0047からなる群から選択される少なくとも一種である、請求項1に記載の方法。
- 前記酵素が、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である、請求項1又は2に記載の方法。
- 前記酵素が、トロンビン及び/又はヒアルロニダーゼであり、前記抗体が、MIB-1である、請求項1に記載の方法。
- 前記固定化された細胞群が、ホルマリン、グルタルアルデヒド、アルコール、アセトン及びそれらの組み合わせからなる群から選択される固定化剤で固定されている、請求項1~4のいずれか一項に記載の方法。
- 工程1)の前に熱処理により賦活化することを含む、請求項1~5のいずれか一項に記載の方法。
- さらに細胞核を特異的に染色し、染色された細胞核を検出することを含む、請求項1~6のいずれか一項に記載の方法。
- さらに、抗サイトケラチン抗体を用いてサイトケラチンを免疫染色し、染色された陽性細胞を検出することを含む、請求項1~7のいずれか一項に記載の方法。
- さらに抗エストロジェン受容体(ER)抗体及び/又は抗プロゲステロン受容体(PgR)抗体を用いてER及び/又はPgRを免疫染色し、染色された陽性細胞又は陽性細胞核を検出することを含む、請求項1~8のいずれか一項に記載の方法。
- さらにHER2遺伝子にハイブリダイズするプローブを用いて、HER2遺伝子が増幅されている細胞又は細胞核を検出することを含む、請求項1~9のいずれか一項に記載の方法。
- 前記固定化された細胞群が組織切片に含まれている、請求項1~10のいずれか一項に記載の方法。
- 前記組織切片が、包理剤で包理されており、
前記加水分解酵素で抗原を賦活化する工程の前に、該包理剤を除去し、該組織切片を親水化する工程を含む、請求項11に記載の方法。 - 工程1)と工程2)の間に、前記細胞を破砕して細胞核を抽出する工程を含む、請求項11又は請求項12に記載の方法。
- せん断応力により前記細胞を破砕して、細胞核を抽出する、請求項13に記載の方法。
- 界面活性剤を含む緩衝液中で、前記細胞核を抽出する請求項13又は14に記載の方法。
- 前記界面活性剤が、CHAPS、NP-40、およびTriton-X100から選択される少なくとも一種である、請求項15に記載の方法。
- 前記染色されたKi-67陽性細胞又はKi-67陽性細胞核の数をカウントする、請求項1~16のいずれか一項に記載の方法。
- 前記Ki-67陽性細胞又は前記Ki-67陽性細胞核を、蛍光染色し、フローサイトメトリーを用いてカウントする、請求項1~17のいずれかに記載の方法。
- 前記固定化された細胞又は前記組織切片が患者由来である、請求項1~18のいずれか一項に記載の方法。
- 前記固定化された細胞又は前記組織切片が患者の腫瘍組織由来である、請求項1~18のいずれか一項に記載の方法。
- 前記固定化された細胞又は前記腫瘍組織が、乳癌細胞又は乳癌組織である、請求項20に記載の方法。
- がんの診断を補助するための、請求項20又は21に記載の方法。
- がん治療の予後を診断するのを補助するための、請求項20又は21に記載の方法。
- 癌の治療レジメンを決定するための方法であって、請求項20又は21に記載の方法により、細胞群内のKi-67陽性細胞の割合を算定し、割合がカットオフ値以上の場合は内分泌療法と化学療法の併用を選択し、割合がカットオフ値未満の場合は、内分泌療法単独を選択する方法。
- 請求項20又は21に記載の方法により、細胞群内のKi-67陽性細胞の割合を算定し、割合がカットオフ値以上の場合は内分泌療法と化学療法の併用を選択し、割合がカットオフ値未満の場合は、内分泌療法単独を選択して、患者に施し、癌を治療する方法。
- 固定化された腫瘍細胞群又は腫瘍組織を配列番号2のペプチドを認識切断しない加水分解酵素で前処理してKi-67を賦活化する方法。
- 配列番号2のペプチドを認識切断しない加水分解酵素を含む、Ki-67を免疫染色で検出する細胞又は組織試料に用いられる抗原賦活化剤。
- 前記酵素が、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である、請求項27に記載の抗原賦活化剤。
- 前記酵素がトロンビン及び/又はヒアルロニダーゼである、請求項28に記載の抗原賦活化剤。
- 配列番号2のペプチドを認識切断しない加水分解酵素を含む、Ki-67及びサイトケラチンを免疫染色で同時検出する細胞又は組織試料に用いられる抗原賦活化剤。
- さらにER及び/又はPgRを免疫染色して検出する細胞又は組織試料に用いられる、請求項27~30のいずれか一項に記載の抗原賦活化剤。
- さらに、HER2遺伝子を増幅している細胞を検出する試料に用いられる、請求項27~31のいずれか一項に記載の抗原賦活化剤。
- 固定化された細胞中のKi-67陽性細胞を検出するためのキットであって、
配列番号2のペプチドを認識切断しない加水分解酵素と、
抗Ki-67抗体と
を含む、キット。 - 前記加水分解酵素は、トロンビン、Arg-C(クロストリパイン)ペプチターゼ、プロリンエンドペプチターゼ及びヒアルロニダーゼからなる群から選択される少なくとも一種である、請求項33に記載のキット。
- 前記加水分解酵素は、トロンビン及び/又はヒアルロニダーゼである、請求項34に記載のキット。
- 前記抗Ki-67抗体は、MIB-1、DAKO-PC、Ki-S5及びA-0047からなる群から選択される少なくとも一種である、請求項33~35のいずれか一項に記載のキット。
- 更に、Ki-67と組み合わせて、がんの診断を補助するため、又はがんの治療の予後を診断するのを補助するために用いられる他のマーカーを検出するためのリガンドを含む、請求項33~36のいずれか一項に記載のキット。
- 前記リガンドは、抗サイトケラチン抗体、抗ER抗体、抗PgR抗体、及びHER2遺伝子にハイブリダイズするプローブからなる群から選択される少なくとも一種である、請求項37に記載のキット。
- 更に、核染色用の化合物を含む、請求項33~38のいずれか一項に記載のキット。
- 更に、細胞を分散させるための緩衝液を含み、該緩衝液は、界面活性剤を含有する、請求項33~39のいずれか一項に記載のキット。
- 更に、熱処理抗原賦活化剤を含む、請求項33~40のいずれか一項に記載のキット。
- がんの診断を補助するための、請求項33~41のいずれか一項に記載のキット。
- がん治療の予後を診断するのを補助するための、請求項33~41のいずれか一項に記載のキット。
- 癌の治療レジメンを決定するための、請求項33~41のいずれか一項に記載のキット。
- 前記治療レジメンの決定は、細胞群内のKi-67陽性細胞の割合を算定し、割合がカットオフ値以上の場合は内分泌療法と化学療法の併用を選択し、割合がカットオフ値未満の場合は、内分泌療法単独を選択することによって行なわれる、キット。
- せん断応力により固定化された細胞を破砕して細胞核を抽出するためのキットであって、
界面活性剤を含有する細胞分散用緩衝液を含む、キット。 - 水流、超音波などで生じるせん断応力により固定化された細胞を破砕して、細胞核を抽出する方法であって、
該固定化された細胞を界面活性剤を含有する緩衝液に分散して該細胞の破砕を行う、方法。
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JP2020503605A JP7025730B2 (ja) | 2018-02-28 | 2019-02-28 | 固定化細胞又はffpe組織切片から抗原性を増強した細胞核を脱離する方法並びにそのための抗原賦活剤及びキット |
EP19761170.0A EP3761031A4 (en) | 2018-02-28 | 2019-02-28 | METHOD FOR ISOLATING CELL NUCLEI HAVING ENHANCED ANTIGENICITY FROM IMMOBILIZED CELLS OR FFPE TISSUE SECTION, ANTIGEN ACTIVATOR AND ASSOCIATED KIT |
KR1020207028077A KR102655467B1 (ko) | 2018-02-28 | 2019-02-28 | 고정화 세포 또는 ffpe 조직 절편으로부터 항원성을 증강한 세포핵을 탈리하는 방법 그리고 그것을 위한 항원 부활제 및 키트 |
US16/975,930 US20200408770A1 (en) | 2018-02-28 | 2019-02-28 | Method for isolating cell nuclei having enhanced antigenicity from fixed cells or ffpe tissue section, and antigen activator and kit therefor |
CN201980015834.0A CN111819443A (zh) | 2018-02-28 | 2019-02-28 | 从固定化细胞或ffpe组织切片脱离增强抗原性的细胞核的方法以及用于该方法的抗原活化剂及试剂盒 |
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WO2022091805A1 (ja) * | 2020-10-29 | 2022-05-05 | シスメックス株式会社 | 試料調製方法、細胞分析方法、試料調製装置および細胞分析装置 |
CN115433764A (zh) * | 2022-08-19 | 2022-12-06 | 杭州联川生物技术股份有限公司 | 一种植物细胞核提取的试剂盒及其应用 |
WO2023053574A1 (ja) | 2021-09-29 | 2023-04-06 | 日東紡績株式会社 | 細胞または細胞核の豊富化方法 |
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EP3761031A1 (en) | 2021-01-06 |
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