Method for measuring index used for cancer diagnosis and therapeutic effect evaluation, and reagent composition

Abstract

Provided is a method for measuring a highly accurate and quantitative index that can be used for cancer diagnosis and therapeutic effect evaluation. The measurement method includes: a detection step for detecting, from blood collected from a subject, aggregates in which a central region exhibiting characteristics of enucleated cancer cells is surrounded by a plurality of cells that are white blood cells; and a counting step for counting the number of the aggregates detected in the detection step. The central region may exhibit at least one among: a first feature in which a detection reaction by a cancer epithelial marker is positive and a nucleus is not recognized; a second feature in which a detection reaction by a cancer epithelial marker is negative, a nucleus is not recognized, and a detection reaction by a leukocyte-lineage cell marker is positive; and a third feature in which a detection reaction by a cancer epithelial marker is negative, a nucleus is not recognized, and a detection reaction by a leukocyte-lineage cell marker is negative.

Description

Method for Measuring Index and Reagent Composition Used for Diagnosis of Cancer and Evaluation of Treatment Effect

[0001] The present invention relates to a method for measuring an index and a reagent composition used for diagnosis of cancer and evaluation of treatment effect.

[0002] Conventionally, a method for observing circulating tumor cells (CTC) in blood has been known for diagnosis of cancer and evaluation of treatment effect (see, for example, Patent Documents 1 and 2). In this method, living single cancer cells are separated from other components in blood, and observation is performed by microscopic imaging using a fluorescent dye or a flow cytometer.

[0003] Japanese Patent Application Laid-Open No. 2007-52564, Japanese Patent Application Laid-Open No. 2011-515109

[0004] The number of CTCs present per unit volume of blood is extremely small, and the cell size of CTCs is very small. Therefore, it has been difficult to measure CTCs with high throughput conventionally. Also, although CTCs can be an index for cancer invasion and metastasis, since they are living cells, the mechanism for using only their presence as an index for evaluating treatment effect is not yet clear.

[0005] The present invention has been made in view of the above, and an object thereof is to provide a method for measuring an index and a reagent composition with high precision and quantification, which can be used for diagnosis of cancer and evaluation of treatment effect.

[0006] In order to solve the above problems, a measurement method according to one aspect of the present invention includes a detection step of detecting an aggregate in which a central region exhibiting characteristics of enucleated cancer cells is surrounded by a plurality of cells that are white blood cells, from blood collected from a subject, and a counting step of counting the number of the aggregates detected in the detection step.

[0007] In the above measurement method, the central region may exhibit at least one of the following characteristics: a first characteristic in which the detection reaction to cancer epithelial marker is positive and no nucleus is observed; a second characteristic in which the detection reaction to cancer epithelial marker is negative, no nucleus is observed and the detection reaction to leukocyte marker is positive; and a third characteristic in which the detection reaction to cancer epithelial marker is negative, no nucleus is observed and the detection reaction to leukocyte marker is negative.

[0008] In the above measurement method, the central region may exhibit at least one of the following characteristics: a first characteristic in which the detection reaction to cancer epithelial marker is positive and the detection reaction to nuclear DNA staining reagent is negative; a second characteristic in which the detection reaction to cancer epithelial marker is negative and the detection reaction to nuclear DNA staining reagent is negative and the detection reaction to leukocyte marker is positive; and a third characteristic in which the detection reaction to cancer epithelial marker is negative and the detection reaction to nuclear DNA staining reagent is negative and the detection reaction to leukocyte marker is negative.

[0009] In the above measurement method, the first characteristic is that the detection reaction by a marker for leukocytes may also show a positive result.

[0010] In the above measurement method, the detection reaction for cancer epithelial markers may be a detection reaction using an antibody molecularly labeled with at least one of the following: an antibody against the cytokeratin family, an anti-EpCAM antibody, an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-HER2 antibody, an anti-p53 antibody, an anti-Ki-67 antibody, an anti-BCL-2 antibody, an anti-E-cadherin antibody, an anti-Vimentin antibody, an anti-CEA antibody, an anti-MUC1 antibody, an anti-Napsin A antibody, an anti-SCC antigen antibody, an anti-p40 antibody, an anti-p63 antibody, an anti-Thyroid Translation Factor-1 antibody, an anti-CA125 antibody, an anti-Mesothelin antibody, an anti-HMB-45 antibody, and an anti-Melan-A antibody.

[0011] In the above measurement method, the antibody against the cytokeratin family may include any of the following: anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody.

[0012] In the above measurement method, the central region may show a negative detection reaction with at least one of the following: DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, Propidium Iodide (PI), SYBR Green I, SYBR Green II, Ethidium bromide, Acridine Orange, SYTO 9, SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, and SYBR Safe DNA Gel Stain.

[0013] In the above measurement method, cells surrounding the central region may show a positive detection reaction with an antibody molecularly labeled with an anti-leukocyte marker antibody, which is at least one of the following: anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody.

[0014] In the above measurement method, the detection step may include an extraction step of extracting solid components from blood collected from a subject; a reagent mixing step of mixing the extracted solid components with a molecularly labeled anti-leukocyte marker antibody, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a nuclear DNA staining reagent; and an observation step of spectroscopically observing the sample containing the mixed reagents.

[0015] In the above measurement method, the detection step may include: an extraction step of extracting solid components from blood collected from a subject; a first reagent mixing step of mixing an anti-leukocyte marker antibody and an antibody molecularly labeled with an antibody for specifically detecting proteins or molecules expressed in epithelial cells with the extracted solid components; a second reagent mixing step of further mixing an antibody molecularly labeled with a secondary antibody that binds to the anti-leukocyte marker antibody with the sample mixed with the reagents in the first reagent mixing step; a third reagent mixing step of further mixing a nuclear DNA staining reagent with the sample mixed with the reagents in the second reagent mixing step; and an observation step of spectroscopically observing the sample mixed with the reagents in the third reagent mixing step.

[0016] The above measurement method may further include an estimation step of estimating the likelihood that the subject has cancer by comparing the number of aggregates with a predetermined reference value.

[0017] The above measurement method may further include an estimation step of estimating the effect of cancer treatment on the subject by comparing the number of aggregates with a predetermined reference value.

[0018] Another embodiment of the present invention is a reagent composition comprising a hemolytic reagent, a molecularly labeled anti-leukocyte marker antibody as a staining antibody reagent, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a nuclear DNA staining reagent, and is a reagent composition for detecting aggregates in blood in which leukocytes surround a central region exhibiting the characteristics of enucleated cancer cells.

[0019] Another embodiment of the present invention is a reagent composition comprising a hemolytic reagent, a staining antibody reagent including an anti-leukocyte marker antibody, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a molecularly labeled secondary antibody that binds to the anti-leukocyte marker antibody, and a nuclear DNA staining reagent, and is a reagent composition for detecting aggregates in blood in which leukocytes surround a central region exhibiting the characteristics of enucleated cancer cells.

[0020] In the reagent composition described above, the molecularly labeled antibody for specifically detecting proteins or molecules expressed in the epithelial cells may be at least one of the following: an antibody against the cytokeratin family, an anti-EpCAM antibody, an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-HER2 antibody, an anti-p53 antibody, an anti-Ki-67 antibody, an anti-BCL-2 antibody, an anti-E-cadherin antibody, an anti-Vimentin antibody, an anti-CEA antibody, an anti-MUC1 antibody, an anti-Napsin A antibody, an anti-SCC antigen antibody, an anti-p40 antibody, an anti-p63 antibody, an anti-Thyroid Translation Factor-1 antibody, an anti-CA125 antibody, an anti-Mesothelin antibody, an anti-HMB-45 antibody, and an anti-Melan-A antibody.

[0021] In the reagent composition described above, the antibody against the cytokeratin family may include any of the following: anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody.

[0022] In the reagent composition described above, the anti-leukocyte marker antibody may be at least one of the following: anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody.

[0023] In the above reagent composition, the nuclear DNA staining reagent may contain any of the following: DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, propidium iodide (PI), SYBR Green I, SYBR Green II, ethidium bromide, Acridine Orange, SYTO 9, SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, and SYBR Safe DNA Gel Stain.

[0024] According to the present invention, it is possible to realize a highly accurate and quantitative method for measuring indicators and a reagent composition that can be used in the diagnosis of cancer and evaluation of treatment effectiveness.

[0025] This is a flowchart showing a measurement method according to an embodiment of the present invention. This graph shows the results of nonparametric analysis in Example 4. This graph shows the results of ROC analysis in Example 4. This graph shows the flow cytometry analysis of EpCAM and CK18-positive cells for an MCF-7 spike sample in Example 5. This is a fluorescence microscope image of an MCF-7 spike sample in Example 5. This is a fluorescence microscope image of an MCF-7 spike sample in Example 5. This is a fluorescence microscope image showing an example of detection of EpCAM-positive CTCs. This is a fluorescence microscope image showing an example of detection of EpCAM-positive CESs. This is a fluorescence microscope image showing an example of detection of EpCAM-negative CESs. This is a fluorescence microscope image showing an example of detection of EpCAM-negative and CD45-negative CESs. This graph shows the ROC curves for each tumor marker.

[0026] The measurement method and reagent composition according to embodiments of the present invention will be described below with reference to the drawings. However, the present invention is not limited to these embodiments.

[0027] (Summary of Measurement Method) The measurement method according to the embodiment of the present invention is a method for measuring an index used in the diagnosis of cancer and evaluation of the effectiveness of treatment, and specifically targets a specific cell sphere present in the blood collected from a subject.

[0028] The inventors of this application focused on the possibility that cancer cells circulating in the bloodstream may leave specific traces as a result of immune responses, the degeneration of cancer cell tissue, or chemical and physical treatments that induce these processes. Specifically, they hypothesized that fragments of cells or tissues derived from cancer cells or cancer tissue, or similar waste products, circulate in the bloodstream, and attempted to search for these using morphological and immunochemical methods.

[0029] Through immunochemical observation, the inventors specifically observed a phenomenon in blood samples taken from cancer patients where numerous spherical, cell-like structures, approximately 10 μm in size, covered the surface of irregularly shaped clumps, approximately 5 μm to 100 μm in size. To further clarify this phenomenon, the inventors conducted molecular biological analyses to determine the identity of these clumps.

[0030] As a result, it became clear that the objects observed by the inventors of the present invention were groups of cells (aggregates) in which leukocytes surrounded a central region exhibiting the characteristics of enucleated cancer cells. Therefore, the inventors defined these groups of cells (aggregates) containing the region exhibiting the characteristics of enucleated cancer cells as "Cancer Enucleation Spheres (CES)" and established a method using the number of CES as a diagnostic indicator for cancer, and a method using the fluctuation in the number of CES as an indicator for evaluating the effectiveness of cancer treatment. Furthermore, the inventors of the present invention found that by observing CES, it is possible to evaluate the effectiveness of treatment with high accuracy and quantitatively compared to conventional methods of observing circulating tumor cells (CTCs).

[0031] Figure 1 is a flowchart showing a measurement method according to an embodiment of the present invention. As shown in Figure 1, in the measurement method according to this embodiment, first, CES is detected from blood collected from a subject (step S1: detection step).

[0032] CES is an aggregate in which leukocytes surround a central region exhibiting the characteristics of enucleated cancer cells. Three main types of characteristics of the central region are observed: (i) a central region in which the detection reaction by cancer epithelial marker is positive and no nucleus is observed (first characteristic); (ii) a central region in which the detection reaction by cancer epithelial marker is negative, no nucleus is observed, and the detection reaction by leukocyte marker is positive (second characteristic); and (iii) a central region in which the detection reaction by cancer epithelial marker is negative, no nucleus is observed, and the detection reaction by leukocyte marker is negative (third characteristic).

[0033] Alternatively, the characteristics of the central region described above can also be defined as follows: (i') a central region in which the detection reaction by cancer epithelial marker is positive and the detection reaction by nuclear DNA staining reagent is negative (first characteristic); (ii') a central region in which the detection reaction by cancer epithelial marker is negative and the detection reaction by nuclear DNA staining reagent is negative and the detection reaction by leukocyte marker is positive (second characteristic); (iii') a central region in which the detection reaction by cancer epithelial marker is negative and the detection reaction by nuclear DNA staining reagent is negative and the detection reaction by leukocyte marker is negative (third characteristic).

[0034] Here, (i) a central region in which the detection reaction by cancer epithelial marker is positive and no nucleus is observed, or (i') a central region in which the detection reaction by cancer epithelial marker is positive and the detection reaction by nuclear DNA staining reagent is negative, may also show a positive detection reaction by a marker for leukocytes.

[0035] For example, a central region that is positive for cytokeratin (CK) 18 or EpCAM (Epithelial Cell Adhesion Molecule) and negative for DAPI (4',6-Diamidino-2-phenylindole) can be considered to be an enucleated cancer cell. Furthermore, the leukocytes surrounding it are negative for epithelial cancer markers.

[0036] Table 1 shows the characteristics of three types of CES. Note that Table 1 shows the case where the cancer epithelial marker is CK18 or EpCAM, the nuclear DNA staining reagent is DAPI, and the leukocyte marker is CD45.

[0037]

[0038] Step S1 includes the following steps: First, solid components are extracted from the blood collected from the subject (extraction step). The solid components can be extracted, for example, by the following procedure: First, the cells are fixed by fixing the blood with paraformaldehyde and incubating it for a predetermined time, and then a hemolytic agent is added to lyse the blood. This allows for the removal of most of the red blood cells from the blood. After that, the sample is centrifuged to form a pellet of cells in the blood. This allows for the removal of most of the plasma and platelets from the sample.

[0039] Alternatively, another step in the extraction process involves first adding a hemolytic agent to the blood to lyse it. This largely removes the red blood cells from the blood. The lysed sample is then centrifuged to pelletize the cells in the blood. This removes the plasma and platelets.

[0040] Next, the sample containing the extracted solid components is mixed with a molecularly labeled anti-leukocyte marker antibody, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a nuclear DNA staining reagent (reagent mixing step). Reagent mixing can be carried out, for example, by the following procedure: the pelletized sample is suspended in phosphate-buffered saline (PBS), and then the reagents are added and mixed. The sample may be blocked before adding the reagents.

[0041] The reagents may be added all at once or sequentially. In the latter case, for example, PBS or similar material may be added to a sample containing solid components to suspend it, then molecularly labeled antibodies (e.g., the two types mentioned above) may be added and mixed, and the sample mixed with these antibodies may be centrifuged to extract the solid components. After that, the extracted solid components may be suspended and mixed with the nuclear DNA staining reagent.

[0042] As another example of the reagent mixing step, the sample containing the solid components extracted in the extraction step is mixed with an anti-leukocyte marker antibody and an antibody molecularly labeled with an antibody for specifically detecting proteins or molecules expressed in epithelial cells (first reagent mixing step). The sample mixed with the reagents in the first reagent mixing step is further mixed with an antibody molecularly labeled with a secondary antibody that binds to the anti-leukocyte marker antibody (second reagent mixing step). The sample mixed with the reagents in the second reagent mixing step may further be mixed with a nuclear DNA staining reagent (third reagent mixing step).

[0043] Examples of anti-leukocyte marker antibodies include anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody. Furthermore, examples of conjugate dyes used for molecular labeling of such antibodies include FITC, PE (Phycoerythrin), PerCP (Peridinin Chlorophyll Protein), APC (Allophycocyanin), TRITC (Tetramethylrhodamine Isothiocyanate), Texas Red, Alexa Fluor series, GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), YFP (Yellow Fluorescent Protein), CFP (Cyan Fluorescent Protein), HiLyte Fluor series, Quantum Dots, and PE-Cy5.Examples of the molecule-labeled anti-leukocyte marker antibody include, as a specific example of the molecule-labeled anti-CD45 antibody, FITC-labeled anti-CD45 antibody, PerCP (Peridinin Chlorophyll Protein)-labeled anti-CD45 antibody, APC (Allophycocyanin)-labeled anti-CD45 antibody, TRITC (Tetramethylrhodamine Isothiocyanate)-labeled anti-CD45 antibody, Texas Red-labeled anti-CD45 antibody, Alexa Fluor-labeled anti-CD45 antibody, GFP (Green Fluorescent Protein)-labeled anti-CD45 antibody, RFP (Red Fluorescent Protein)-labeled anti-CD45 antibody, YFP (Yellow Fluorescent Protein)-labeled anti-CD45 antibody, CFP (Cyan Fluorescent Protein)-labeled anti-CD45 antibody, HiLyte Fluor-labeled anti-CD45 antibody, Quantum Dots (quantum dot)-labeled anti-CD45 antibody, conjugate dye-labeled anti-CD45 antibody such as PE-Cy5, and the like can be used.

[0044] Antibodies for specifically detecting proteins or molecules expressed in epithelial cells include, specifically, antibodies against the cytokeratin family, anti-EpCAM antibodies, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-HER2 antibodies, anti-p53 antibodies, anti-Ki-67 antibodies, anti-BCL-2 antibodies, anti-E-cadherin antibodies, anti-Vimentin antibodies, anti-CEA antibodies, anti-MUC1 antibodies, anti-Napsin A antibodies, anti-SCC antigen antibodies, anti-p40 antibodies, anti-p63 antibodies, anti-Thyroid Translation Factor-1 antibodies, anti-CA125 antibodies, anti-Mesothelin antibodies, anti-HMB-45 antibodies, anti-Melan-A antibodies, and other epithelial cancer markers. Examples of antibodies against the cytokeratin family include anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody. Examples of molecular labels for such antibodies include conjugate dyes such as FITC, PE (Phycoerythrin), PerCP (Peridinin Chlorophyll Protein), APC (Allophycocyanin), TRITC (Tetramethylrhodamine Isothiocyanate), Texas Red, Alexa Fluor series, GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), YFP (Yellow Fluorescent Protein), CFP (Cyan Fluorescent Protein), HiLyte Fluor series, Quantum Dots, and PE-Cy5. Examples of molecularly labeled antibodies for specifically detecting proteins or molecules expressed in epithelial cells include HiLyte Fluor555-labeled anti-CK18 antibody and PE (Phycoerythrin)-labeled anti-EpCAM antibody.

[0045] As nuclear DNA staining reagents, DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, Propidium Iodide (PI), SYBR Green I, SYBR Green II, Ethidium Bromide, Acridine Orange, SYTO series such as SYTO 9 and SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, SYBR Safe DNA Gel Stain, etc. can be used.

[0046] Next, the sample mixed with the reagent is spectroscopically observed (observation step). As methods for spectroscopic observation, absorption spectroscopy, fluorescence spectroscopy, emission spectroscopy, scattering spectroscopy, time-resolved spectroscopy, etc. can be mentioned, and any of them may be used. In this embodiment, any of them may be used. As a specific example, it can be observed using a cell observation device such as a fluorescence microscope, fluorescence observation imaging, flow cytometer, or cell sorter.

[0047] In this observation step, if an aggregate surrounded by a plurality of cells showing positive for a leukocyte marker and positive for the detection reaction by the nuclear DNA staining reagent is observed, with a central region showing positive for the detection reaction by an antibody for specifically detecting a protein or molecule expressed in epithelial cells and negative for the detection reaction by the nuclear DNA staining reagent, then it is a CES.

[0048] Next, the number of CESs detected in step S1 is counted (step S2: counting step). The method of counting is not particularly limited. For example, an operator may count visually, or clusters may be automatically counted by image processing, or classification and counting may be performed using a flow cytometer.

[0049] Next, the number of CES counted in step S2 is compared with a predetermined reference value (step S3). This allows for the estimation of the possibility that the subject has cancer (estimation step). That is, if the number of CES is equal to or greater than the reference value (for example, 10 per 1 mL of blood), it can be estimated that the subject may have cancer. Alternatively, the effectiveness of cancer treatment in the subject can be evaluated by observing the trend in the number of CES during regular examinations.

[0050] (Reagent Composition) In the measurement method according to this embodiment, a reagent composition (reagent composition kit) having the following configuration can be used.

[0051] That is, the reagent composition according to this embodiment can be configured as a reagent composition kit comprising, for example, a first reagent composition (hemolytic reagent) for hemolyzing a blood sample, a second reagent composition (staining antibody reagent) for detecting antigens in the blood, and a third reagent composition (nuclear DNA staining reagent) for detecting living cells in the blood. The staining antibody reagent includes a molecularly labeled anti-leukocyte marker antibody and a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells. In addition to these reagents, the reagent composition kit may also include a cell fixative and a blocking reagent.

[0052] Hemolysis reagents are used to ensure that red blood cells are not included in the solid components extracted from blood. Hemolysis reagents include, for example, ammonium chloride, EDTA-4Na (tetrasodium ethylenediaminetetraacetate), and carbonate buffer.

[0053] Among the staining antibody reagents, molecularly labeled anti-leukocyte marker antibodies can be used, such as at least one of the following: anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody. Specifically, FITC-labeled anti-CD45 antibody, PerCP (Peridinin Chlorophyll Protein)-labeled anti-CD45 antibody, APC (Allophycocyanin)-labeled anti-CD45 antibody, TRITC (Tetramethylrhodamine Isothiocyanate)-labeled anti-CD45 antibody, Texas Red-labeled anti-CD45 antibody, Alexa Fluor-labeled anti-CD45 antibody, GFP (Green Fluorescent Protein)-labeled anti-CD45 antibody, RFP (Red Fluorescent Protein)-labeled anti-CD45 antibody, YFP (Yellow Fluorescent Protein)-labeled anti-CD45 antibody, CFP (Cyan Fluorescent Protein)-labeled anti-CD45 antibody, HiLyte Fluor-labeled anti-CD45 antibody, Quantum Examples include Dots (quantum dot) labeled anti-CD45 antibodies and conjugate dye-labeled anti-CD45 antibodies such as PE-Cy5.

[0054] Among the staining antibody reagents, as antibodies molecularly labeled to specifically detect proteins or molecules expressed in epithelial cells, for example, antibodies molecularly labeled with at least one of the following can be used: antibodies against the cytokeratin family, anti-EpCAM antibodies, anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-HER2 antibodies, anti-p53 antibodies, anti-Ki-67 antibodies, anti-BCL-2 antibodies, anti-E-cadherin antibodies, anti-Vimentin antibodies, anti-CEA antibodies, anti-MUC1 antibodies, anti-Napsin A antibodies, anti-SCC antigen antibodies, anti-p40 antibodies, anti-p63 antibodies, anti-Thyroid Translation Factor-1 antibodies, anti-CA125 antibodies, anti-Mesothelin antibodies, anti-HMB-45 antibodies, and anti-Melan-A antibodies. Specifically, examples include HiLyte Fluor555-labeled anti-CK18 antibody and PE (Phycoerythrin)-labeled anti-EpCAM antibody. Antibodies against the cytokeratin family include anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody.

[0055] Nuclear DNA staining reagents that can be used include DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, propidium iodide (PI), SYBR Green I, SYBR Green II, ethidium bromide, Acridine Orange, SYTO series such as SYTO 9 and SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, and SYBR Safe DNA Gel Stain.

[0056] For example, paraformaldehyde can be used as a cell fixative. For example, normal goat serum can be used as a blocking reagent.

[0057] Alternatively, in the reagent composition kit according to this embodiment, the second reagent composition (staining antibody reagent) can be replaced with an anti-leukocyte marker antibody, an antibody molecularly labeled for the specific detection of proteins or molecules expressed in epithelial cells, and a secondary antibody molecularly labeled for binding to the anti-leukocyte marker antibody. In this case, as the secondary antibody molecularly labeled for binding to the anti-leukocyte marker antibody, you can use an antibody molecularly labeled with anti-mouse IgG antibody, anti-rabbit IgG antibody, anti-rat IgG antibody, anti-goat IgG antibody, anti-donkey IgG antibody, anti-chicken IgG antibody, anti-horse IgG antibody, anti-sheep IgG antibody, or anti-guinea pig IgG antibody.

[0058] (Example 1: Detection of CES by CK18) CES contained in blood samples was detected by the following procedure. 1. Detection procedure (1) Blood was collected from the veins of healthy individuals and cancer patients using blood collection tubes manufactured by Streck, Inc. (Cell-Free DNA BCT CE, Streck, Inc.) and stored at room temperature until processing began.

[0059] (2) The blood was fixed with 4% paraformaldehyde in an amount equal to one-tenth of the whole blood and incubated at room temperature for 20 minutes. After cell fixation, a hemolytic agent containing 150 mM ammonium chloride was added in an amount equal to 14 times the whole blood, and incubated at room temperature for more than 4 minutes to completely lyse the blood.

[0060] (3) The hemolyzed sample was centrifuged at 500 G at room temperature to form a cell pellet, and the supernatant was discarded. This procedure was repeated to wash the cells. The resulting pellet was suspended, normal goat serum was added, and the cells were blocked by incubation at room temperature for 5 minutes. PBS was added to this sample to suspend the cells.

[0061] (4) HiLyte Fluor555-labeled anti-CK18 antibody and FITC-labeled anti-CD45 antibody were added to the sample and incubated overnight at 4°C.

[0062] (5) After incubation at 4°C, each sample was washed with PBS, centrifuged at 200G at room temperature for 5 minutes, and the supernatant was removed. The resulting pellet was suspended, DAPI was added, and it was allowed to stand at room temperature for 2 minutes. Then, PBS was added to the sample to suspend the cells, which were then transferred to a plate for cell observation and observed under a fluorescence microscope.

[0063] 2. Observation Results In the blood of cancer patients, aggregates were observed in which numerous spherical, cell-like structures of about 10 μm covered the surface of irregularly shaped clumps of about 5 μm to 100 μm in size. In these aggregates, the central clump showed positivity for CK18, an antigen derived from epithelial cancer cells, and was negative for DAPI (no fluorescence signal detected by DAPI staining). Furthermore, the spherical, cell-like structures surrounding this clump showed positivity for CD45 and was positive for DAPI (fluorescence signal detected by DAPI staining). From this, it was determined that the central clump was enucleated cancer cells, and the surrounding spherical cells were leukocytes or similar lymphocytes, or inflammatory cells. The inventors of this application defined such aggregates as "enucleated cancer cell spheres (CES)".

[0064] On the other hand, CES was not observed at all in the blood of most healthy individuals. Although CES was observed in the blood of some healthy individuals, the number was small. Based on these observations, it was confirmed that the observation of CES may be usable as an indicator for cancer diagnosis and evaluation of the effectiveness of cancer treatment.

[0065] (Example 2: Detection of CES by EpCAM) Molecular biological observations were also performed on EpCAM (CD326), a target of epithelial cancer cells, using the following procedure.

[0066] (1) Blood samples were collected from the veins of healthy individuals and cancer patients using blood collection tubes manufactured by Streck, Inc. (Cell-Free DNA BCT CE, Streck, Inc.) and stored at room temperature until processing began.

[0067] (2) Cells in the blood were fixed and blocked in the same manner as in the procedure (2) to (3) of Example 1.

[0068] (3) PE-labeled anti-EpCAM antibody and FITC-labeled anti-CD45 antibody were added to the sample and incubated overnight at 4°C.

[0069] (4) The sample was processed in the same manner as in (5) of Example 1 and observed with a fluorescence microscope.

[0070] 2. Observation Results Similar aggregates to those in Example 1 were observed in samples from cancer patients. The central mass of this aggregate showed positive results for EpCAM and negative results for DAPI. On the other hand, the surrounding spherical cells showed positive results for CD45 and DAPI. This confirmed that the central mass of the aggregate consisted of enucleated cancer cells, the surrounding spherical cells were immune cells, and that this aggregate was a CES similar to that in Example 1. This indicates that CES originates from cancer cells and that CES is formed when immune cells surround cancer cells that have lost their nucleus and living cell function.

[0071] On the other hand, CES was not observed at all in the blood of most healthy individuals. Although CES was observed in the blood of a small number of healthy individuals, the number was small.

[0072] (Example 3: Monitoring of therapeutic effects by CES) CES was detected in blood samples from cancer patients before and after cancer treatment using the following method.

[0073] 1. Detection Procedure (1) Blood samples were collected from one patient each with colorectal cancer, breast cancer, pancreatic cancer, and gastric cancer, both before and after hyperthermia therapy, using blood collection tubes manufactured by Streck (Cell-Free DNA BCT CE, Streck, Inc.), and stored at room temperature until processing began.

[0074] (2) The number of CES cells in the blood was measured in the same manner as in the procedure (2) to (4) of Example 2.

[0075] 2. As shown in Measurement Results Table 2, the number of CESs after treatment was significantly higher than the number before treatment. In other words, it can be considered that the primary tumor was shrinking as a result of the treatment. This suggests that treatments such as hyperthermia tend to be immunologically eliminated by cancer cells, and that changes in the number of CESs may be a quantitative indicator of the treatment effect.

[0076] (Example 4: Reagent composition and protocol) A ready-to-use reagent composition (reagent composition kit) was prepared to facilitate the observation of CES, and CES was detected using this kit.

[0077] 1. Components of the Reagent Composition Kit The reagent composition kit was configured as follows: First Reagent Composition (Hemolytic Reagent) Ammonium chloride 150 mM EDTA-4Na (Ethylenediaminetetraacetate tetrasodium salt) 1 mM Carbonate buffer (pH 7.4) 70 mM Second Reagent Composition (Staining Antibody Reagent) FITC-labeled anti-CD45 antibody PE-labeled anti-EpCAM antibody HiLyte Fluor555-labeled anti-CK18 antibody Third Reagent Composition (Nuclear DNA Staining Reagent) DAPI 50 ng / mL Fourth Reagent Composition (Cell Fixative) Paraformaldehyde 4% Fifth Reagent Composition (Blocking Reagent) Normal goat serum

[0078] 2. Protocol (1) Blood was collected from the veins of healthy individuals and cancer patients using blood collection tubes manufactured by Streck, Inc. (Cell-Free DNA BCT CE, Streck, Inc.) and stored at room temperature until processing began.

[0079] (2) Using the reagent composition described above, cells in the blood were fixed and blocked in the same manner as in steps (2) to (3) of Example 1.

[0080] (3) The sample was divided into two vials. HiLyte Fluor555-labeled anti-CK18 antibody and FITC-labeled anti-CD45 antibody were added to one of the samples, and PE-labeled anti-EpCAM antibody and FITC-labeled anti-CD45 antibody were added to the other sample. Both samples were incubated overnight at 4°C.

[0081] The sample was processed in the same manner as in procedure (5) of Example 1 and observed with a fluorescence microscope.

[0082] 3. Observation Results Using the reagent composition kit and protocol described above, CES in the blood of healthy individuals (N=24) and cancer patients (N=14) were observed. The average number of observed CES was 27.14 per 1 mL of blood in the cancer patient group and 0.58 per 1 mL in the healthy control group.

[0083] A nonparametric analysis (Mann-Whitney U test) was performed on the mean number of CESs in the healthy control group and the cancer patient group. The results are shown in Figure 2. As shown in Figure 2, the p-value was P < 0.01, indicating that the number of detected CESs was significantly higher in the cancer patient group compared to the healthy control group.

[0084] Furthermore, Figure 3 shows the ROC analysis results when the number of CES cells is used as a threshold, and the presence of cancer is considered positive. The largest area under the curve was obtained when the COI (Cut Off Index) was 10. The largest area under the curve was 0.83, and the 95% confidence interval was 0.69–0.97.

[0085] Based on these analysis results, the sensitivity, specificity, positive predictive value, and negative predictive value for diagnosing cancer when the number of CES cells is 10 or more per 1 mL of blood were calculated. As a result, when the number of CES cells is 10 or more, the sensitivity was 71.4%, the specificity was 95.8%, the positive predictive value was 90.9%, and the negative predictive value was 85.1%, demonstrating that the possibility of cancer can be estimated with high accuracy, with an overall agreement rate of 86.8%.

[0086] (Example 5) CES and CTC were detected in patients undergoing cancer treatment (disease group) and healthy individuals with no history of the disease (healthy control group), and statistical analyses including ROC analysis were performed.

[0087] 1. Subjects and Sample Collection: The diseased group consisted of 46 patients receiving standard treatment. These included 11 cases of pancreatic cancer, 9 cases of colorectal cancer, 10 cases of breast cancer, 4 cases of uterine cancer, 3 cases of gastric cancer, 3 cases of renal cell carcinoma, 2 cases of prostate cancer, 1 case of esophageal cancer, 1 case of ovarian cancer, 1 case of renal pelvis cancer, and 1 case of cervical cancer. The healthy control group consisted of 60 individuals with no history of cancer.

[0088] Furthermore, as cases where comparisons before and after hyperthermia therapy were possible, six cases were selected from the diseased group, and the dynamics of CES and CTC before and after treatment were examined.

[0089] Whole blood was collected venously from all subjects using Streck blood collection tubes. The collected samples were pre-treated by hemolysis, promptly subjected to immunofluorescence staining for target markers, and then analyzed using a fluorescence microscope and flow cytometer.

[0090] 2. Detection and Measurement of CES and CTCs: CES and CTCs were detected by analyzing marker expression and cell morphology according to the following procedure.

[0091] 2-1. Flow Cytometry (FCM) Two mL of blood samples were stained and fixed using DURAClone RE CTC and PerFix CTC kits (Beckman Coulter), and measured using a CytoFLEX flow cytometer (same as above). The evaluation items were EpCAM, CK18 (Cytokeratin 18), and CD45, and multi-parameter analysis was performed in accordance with Beckman Coulter's recommended protocol.

[0092] In setting the criteria for evaluation, samples were prepared by spiking 100 cells of the breast cancer cell line MCF-7 in 1 mL of whole blood from healthy individuals. The distribution of each biomarker was obtained from the flow cytometry analysis results of EpCAM and CK18-positive cells. In this analysis, nuclear-positive cells were first identified by DAPI staining (DAPI ＋ Doublets and cell aggregates were excluded by gating the cells and further setting up singlet gates based on FSC-A and SSC-A.

[0093] Figure 4 is a graph showing the flow cytometry analysis of EpCAM and CK18-positive cells in an MCF-7 spiked sample from Example 5. The spiked sample yielded the two-dimensional plot shown in Figure 4. The left side of Figure 4 shows CK18 versus CD45, the center shows EpCAM versus CD45, and the right side shows EpCAM versus CK18. In all plots, a population positive for epithelial tumor markers (CK18 or EpCAM) and negative for CD45 was clearly detected within the rectangular frame. This population is thought to correspond to the spiked MCF-7 cells. This demonstrates that this method can detect cancer-derived cells in the blood.

[0094] 2-2. Fluorescence Microscopy (Manual Method) Cells were fixed by adding 4% PFA (paraformaldehyde) to 3 mL of blood sample, and then hemolyzed with ammonium chloride hemolytic agent. After centrifuging the hemolyzed sample, the supernatant was discarded, and the cell components were obtained by permeabilization with 60% methanol and washing with PBS.

[0095] Cellular components washed with PBS were fluorescently stained with the following antibodies: Anti-EpCAM Mouse IgG Monoclonal Antibody (PE labeled, clone: ​​9c4, BioLegend), Anti-Cytokeratin 18 Mouse IgG Monoclonal Antibody (PE labeled, clone: ​​1G11C4, Proteintech), and Anti-CD45 Mouse IgG Monoclonal Antibody (FITC labeled, clone: ​​J33, Beckman Coulter). Nuclear staining was then performed using DAPI (Nacalai Tesque). The stained cells were then observed using a fluorescence microscope (Eclipse 2, Nikon), and subjects exhibiting CES and CTC characteristics were extracted.

[0096] Figures 5 and 6 are fluorescence microscope images of MCF-7 spike samples from Example 5, showing cells that were DAPI-positive and EpCAM-positive, and cells that were DAPI-positive and CK18-positive.

[0097] Figure 5 shows immunofluorescence staining images of cells that are EpCAM-positive and CD45-negative. In Figure 5, the upper right is the EpCAM image, the lower right is the CD45 image, the lower left is the DAPI image, and the upper left is a merged image of these two images. In the EpCAM-positive region of the EpCAM image in Figure 5, CD45 is negative while DAPI is positive (see merged image), which is consistent with the phenotype of CTCs. In other words, Figure 5 shows nucleated cancer cells, i.e., CTCs.

[0098] Figure 6 shows immunofluorescence staining images of cells that are CK18-positive and CD45-negative. In Figure 6, the upper right is the CK18 image, the lower right is the CD45 image, the lower left is the DAPI image, and the upper left is a merged image of these two images. In the CK18-positive region of the CK18 image in Figure 6, CD45 is negative while DAPI is positive (see merged image), which is consistent with the phenotype of CTCs. In other words, Figure 6 shows nucleated cancer cells, i.e., CTCs.

[0099] Figures 5 and 6 clearly show cells that are positive for EpCAM or CK18. This demonstrates that this method can detect cells expressing EpCAM or CK18.

[0100] 2-3. Comparison with the FDA-Approved Method (CellSearch® System) The CellSearch® System defines CTCs as being EpCAM-positive, positive for one of CK8 / CK18 / CK19, CD45-negative, and DAPI-positive (Reference: Andree KC, et al., “Challenges in circulating tumor cell detection by the CellSearch system”, Molecular Oncology 2016 Mar; 10(3): 395-407). However, observations by the inventors of this application revealed that a certain number of CD45-positive cells were also observed in MCF-7 spike samples. Therefore, even if cells were CD45-positive, they were counted as CES or CTC candidates, taking into account cell morphology and other marker expression patterns.

[0101] 2-4. Statistical Analysis Statistical analysis was performed on the number of CES and CTC detections in the non-cancer group and the cancer group using SPSS ver.24 (IBM, USA). Significance testing was performed using the Mann-Whitney U test, and diagnostic performance was evaluated using ROC analysis.

[0102] 3. Results 3-1. Fluorescence Observation of CES and CTC The fluorescence observation results of CES and CTC are summarized below. CES was classified into three types according to the antigen expressed in the central region.

[0103] CTCs are cells that are positive for tumor antigens (EpCAM or CK18), negative for CD45, and positive for DAPI. They exist as single cells and are not surrounded by cell clusters (see Figures 5 and 6).

[0104] Figure 7 shows fluorescence microscope images illustrating an example of EpCAM-positive CTC detection. In Figure 7, the upper right is the EpCAM image, the lower right is the CD45 image, the lower left is the DAPI image, and the upper left is a merged image of these two. The positive areas in the EpCAM image, i.e., the areas considered to be cancer cells, exist as single cell regions without surrounding cell clusters, which is a typical morphological pattern for CTCs.

[0105] CES is a cell aggregate in which a central region exhibiting characteristics of enucleated cancer cells is surrounded by multiple leukocytes, and has the following characteristics: (i) The central region is DAPI-negative, i.e., has a cell-like structure lacking a nucleus. (ii) The multiple cells surrounding the central region are DAPI-positive and CD45-positive, and are EpCAM-negative and CK18-negative.

[0106] CES is classified into three types based on the antigen expressed in its central region. Figures 8 to 10 show fluorescence images of each type of CES. In Figures 8 to 10, the top row is the EpCAM image, the middle row is the CD45 image, and the bottom row is the DAPI image.

[0107] Figure 8 is a fluorescence microscope image showing an example of the detection of an EpCAM-positive CES. This CES was detected in the peripheral blood of a patient with advanced cancer. Note that if CK18 is detected as an epithelial marker, it will be classified as CK18-positive. The detected CES is a roughly spherical structure with a diameter of approximately 50–100 μm. The central region shows the structure of a cell-like object that lacks a DAPI-positive nucleus (denucleate) and colocalizes EpCAM (or CK18) and CD45. The surrounding cells are numerous leukocytes that are both CD45-positive and DAPI-positive.

[0108] Figure 9 shows a fluorescence microscope image illustrating an example of the detection of EpCAM-negative CES. This CES was detected from a blood sample from a patient with advanced cancer. Note that if CK18 is detected as an epithelial marker, it will be classified as CK18-negative. The DAPI image (bottom panel) in Figure 9 shows a cluster of numerous DAPI-positive globules (leukocytes) forming a structure approximately 50–100 μm in diameter. However, a DAPI-negative region (anucleated structure) exists in its center. In the EpCAM image (top panel) corresponding to this center, no fluorescence signal is detected, indicating a negative epithelial marker. On the other hand, a strong fluorescence signal is observed in the outer layer of the CD45 image (middle panel) corresponding to this center, suggesting the involvement of leukocyte-derived components.

[0109] Figure 10 is a fluorescence microscope image showing an example of the detection of EpCAM-negative and CD45-negative CES. This CES was detected in the peripheral blood of a patient with advanced cancer. Note that when CK18 is detected as an epithelial marker, the CES will be CK18-negative and CD45-negative. The DAPI image (bottom panel) in Figure 10 shows an aggregation of numerous DAPI-positive globules (leukocytes), forming a structure with a diameter of approximately 50–100 μm. On the other hand, no fluorescence signal was observed in the central region of the EpCAM image (top panel) and CD45 image (middle panel).

[0110] 3-2. Detection Rate in Each Group The detection status of CES and CTC in the advanced cancer patient group (n=46) and the healthy control group (n=60) was compared and evaluated using FCM and imaging methods. Table 3 shows the number of positive cases for CTC-related markers in the advanced cancer and healthy control groups (comparison of FCM and imaging methods).

[0111]

[0112] First, in the detection of CTC markers by FCM, in the advanced cancer group, there were 3 cases (6.5%) in which EpCAM-positive cells were detected, 8 cases (17.4%) in which CK18-positive cells were detected, and 8 cases (17.4%) in which both EpCAM-positive and CK-positive cells were detected (excluding cases where only one of them was detected). On the other hand, in the healthy control group, the detection rates were low, with 0 cases (0%), 1 case (1.7%), and 1 case (1.7%), respectively.

[0113] On the other hand, detection of CES was difficult with FCM. This is likely because, compared to the size of white blood cells, CES are large cell aggregates with diameters exceeding 100 μm, and due to the flow cytometer's channel diameter and hydrodynamic characteristics, they cannot pass through the measuring device or are excluded during the gating process in the analysis.

[0114] In CTC marker evaluation using imaging methods, EpCAM-positive cells were detected in 6 cases (13.0%) and CK18-positive cells in 14 cases (30.4%) in the advanced cancer group.

[0115] In the imaging-based evaluation of CES, EpCAM-positive CES was detected in 25 cases (54.3%) and CK18-positive CES in 21 cases (45.6%) in the advanced cancer group, both of which were high frequencies. On the other hand, the detection rates in the healthy control group were low, with only 1 case (1.7%) and 0 cases (0%), respectively.

[0116] 3-3. Changes in the number of detected cells before and after hyperthermia The measurement results of CES and CTC before and after hyperthermia in six patients with advanced cancer are as follows. Specifically, for CTC, cases were observed where the number of detected cells remained the same, increased, or decreased before and after treatment. Specifically, EpCAM-positive CTCs were detected in two cases, of which one decreased after treatment (FCM in Case 3 of Table 4) and one increased after treatment (Imaging in Case 5 of Table 4). Furthermore, CK-positive CTCs were detected in three cases, of which two decreased after treatment (FCM in Case 2 of Table 4 and Imaging in Case 4 of Table 4), and one case showed both an increase after treatment (Imaging in Case 5) and remaining the same (FCM in Case 5). In contrast, EpCAM-positive CES and CK-positive CES increased after treatment in some cases (see Case 2 Colon Cancer and Case 3 Breast Cancer in Table 4).

[0117] Table 4 shows a comparison of the positive rates for each biomarker and the positive rate for CES before and after hyperthermia therapy (evaluated by FCM and imaging methods).

[0118]

[0119] 3-4. Statistical Analysis Results ROC (Receiver Operating Characteristic) analysis was performed for each tumor-related marker. Figure 11 is a graph showing the ROC curve for each tumor marker.

[0120] As shown in Figure 11, the Area Under the Curve (AUC) values ​​for EpCAM-positive CES and CK18-positive CES detected by imaging were 0.748 and 0.730, respectively, demonstrating high diagnostic accuracy.

[0121] On the other hand, the AUC for EpCAM positivity alone using FCM was 0.533, and the AUC for CK18 positivity alone was 0.579. Furthermore, when the Mann-Whitney U test was performed on the cancer group and the non-cancer group, the p-values ​​for each method were as follows: EpCAM-positive CTC (FCM): 0.046 CK-positive CTC (FCM): 0.004 EpCAM / CK-positive CTC (FCM): 0.009 EpCAM-positive CTC (imaging method): 0.120 CK-positive CTC (imaging method): 0.005 EpCAM-positive CES: <0.001 CK-positive CES: <0.001

[0122] 4. Discussion 4-1. Pathological Characteristics of CES and CTCs CTCs exhibiting a pattern of EpCAM positivity, CK18 positivity, CD45 negativity, and DAPI positivity were consistent with the characteristics widely reported as typical cancer cells. These CTCs are thought to have the ability to survive in the bloodstream for extended periods by utilizing immune evasion mechanisms. Pathologically, reports suggest that this immune evasion increases the risk of cancer metastasis. Rarely, cells showing CD45 negativity to weak positivity (mild to moderate luminescence intensity) were observed, which were confirmed by both FCM and imaging methods.

[0123] Comparing FCM and imaging methods, imaging methods detected a higher number of CTCs. This is likely because imaging methods allow for simultaneous observation of cell morphology and nuclear structure, enabling more accurate identification of cells possessing the morphological characteristics of CTCs.

[0124] CES exhibits a characteristic morphology, lacking a nucleus in the center (DAPI negative), with EpCAM and CK18 localized at the periphery, and surrounded by numerous leukocytes. This structure is highly likely to be a tumor-derived structure formed during the immune response or cell death process, and could serve as a novel pathological marker distinct from conventional single-cell type CTCs.

[0125] While there are multiple morphological patterns for CES, they all share the common structure of aggregates in which leukocytes surround the central region (see Table 1). Therefore, differences in CES morphology may reflect the gradual changes that cancer cells undergo during immune responses and treatment stress (Reference 1: Galluzzi L, et al., “Immunogenic cell death in cancer and infectious disease”, Nature Reviews Immunology vol. 17, p. 97-111 (2017), Reference 2: Ahmed A, et al., “Targeting immunogenic cell death in cancer”, Molecular Oncology Vol. 14(12), p. 2994-3006 (2020)).

[0126] In other words, in the initial stage, cancer cells begin to partially lose their nuclei, making them more susceptible to immune responses. In the intermediate stage, enucleation progresses, and tumor antigens are exposed on the surface, enhancing recognition by immune cells. In the final stage, complete enucleation occurs, and the cells are recognized as spherical structures surrounded by white blood cells, and it is thought that the immune clearance process progresses.

[0127] 4-2. Changes in CES due to thermotherapy: In some cases, an increase in the number of detected CES cells was observed after thermotherapy. Hyperthermia has been reported to induce cancer cell death and stimulate the immune response by releasing tumor antigens and damage-associated molecular patterns (DAMPs) (References: 3: Krysko D V., et al., “Immunogenic cell death and DAMPs in cancer therapy”, Nature Reviews Cancer vol. 12, p. 860-875 (2012); 4: Overgaard J, “The current and potential role of hyperthermia in radiotherapy”, Int J Radiation Oncology Biol Phys., vol. 16(3):535-49 (1989); 5: Datta NR, et al., “Local hyperthermia combined with radiotherapy and- / or chemotherapy: Recent advances and promises for the future”, Cancer Treat Rev., vol. 41(9), p. 742-53 (2015); 6: Toraya-Brown) S, et al., “Local tumor hyperthermia as immunotherapy for metastatic cancer”, International Journal of Hyperthermia vol. 30(8), p. 531-9 (2014)). Therefore, the appearance of CES may reflect tumor changes and immune activation associated with treatment.

[0128] 4-3. Statistical Analysis In Example 5, CES and CTC were detected in patients with advanced cancer using FCM and imaging methods. As a result, it was found that the detection frequency of CES and CTC was significantly higher in the group of patients with advanced cancer compared to the healthy control group. In particular, EpCAM-positive CES and CK18-positive CES were found in 52.2% and 47.8% of the group with advanced cancer, respectively, compared to only 5.0% and 3.3% of the healthy control group, suggesting that they can be used as candidate biomarkers specific to advanced cancer.

[0129] ROC analysis revealed that the AUC for EpCAM-positive CES was 0.748, and the AUC for CK18-positive CES was 0.730, both of which were high. In other words, CES shows superior diagnostic ability compared to EpCAM-positive CTC (AUC = 0.533) and CK18-positive CTC (AUC = 0.579) as determined by FCM. The results of the significance test also show that the p-value for CES is lower than that for CTC, indicating that CES is a useful diagnostic marker for cancer.

[0130] As described above, according to the embodiments of the present invention, the number of CES cells can be used as a highly accurate and quantitative indicator in the diagnosis of cancer and evaluation of treatment effectiveness. Therefore, by observing CES cells in the blood and counting their number, it becomes possible to provide useful information in the implementation of cancer treatment and evaluation of its effectiveness.

[0131] Furthermore, when processing samples using the above reagent composition kit, it is possible to apply the findings to fluorescence observation using a fluorescence microscope, fluorescence imaging, and observation using cell observation devices such as flow cytometers and cell sorters.

[0132] (Modification) In the above reagent composition kit, the second reagent composition (staining antibody reagent) can be changed as appropriate. In other words, the target marker can be arbitrarily selected according to the number of wavelengths to be simultaneously observed, depending on the purpose. For example, epithelial markers and leukocyte markers can be selected for the detection of CES. Also, when identifying cancer type, cancer-specific tumor markers and leukocyte markers can be selected. When observing organ specificity or subtype, organ-specific tumor markers and leukocyte markers can be selected. For evaluating therapeutic effect, target markers and leukocyte markers arbitrarily selected from cancer-specific tumor markers and organ-specific tumor markers can be used.

[0133] The present invention described above is not limited to the embodiments and modifications, and various inventions can be formed by appropriately combining the multiple components disclosed in the above embodiments and modifications. For example, the invention may be formed by excluding some components from all the components shown in the above embodiments and modifications, or by appropriately combining the components shown in the above embodiments and modifications.

Claims

1. A measurement method comprising: a detection step of detecting aggregates in which multiple cells, which are leukocytes, surround a central region exhibiting characteristics of enucleated cancer cells from blood collected from a subject; and a counting step of counting the number of aggregates detected in the detection step.

2. The measurement method according to claim 1, wherein the central region exhibits at least one of the following characteristics: a first characteristic in which a detection reaction to a cancer epithelial marker is positive and no nucleus is observed; a second characteristic in which a detection reaction to a cancer epithelial marker is negative and no nucleus is observed and a detection reaction to a leukocyte marker is positive; and a third characteristic in which a detection reaction to a cancer epithelial marker is negative and no nucleus is observed and a detection reaction to a leukocyte marker is negative.

3. The measurement method according to claim 1, wherein the central region exhibits at least one of the following characteristics: a first characteristic in which the detection reaction to cancer epithelial marker is positive and the detection reaction to nuclear DNA staining reagent is negative; a second characteristic in which the detection reaction to cancer epithelial marker is negative and the detection reaction to nuclear DNA staining reagent is negative and the detection reaction to leukocyte marker is positive; and a third characteristic in which the detection reaction to cancer epithelial marker is negative and the detection reaction to nuclear DNA staining reagent is negative and the detection reaction to leukocyte marker is negative.

4. The measurement method according to claim 2 or 3, wherein the first feature is further that the detection reaction by a marker for leukocytes shows a positive result.

5. The measurement method according to any one of claims 1 to 4, wherein the detection reaction for cancer epithelial markers is a detection reaction using an antibody molecularly labeled with at least one of the following: an antibody against the cytokeratin family, an anti-EpCAM antibody, an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-HER2 antibody, an anti-p53 antibody, an anti-Ki-67 antibody, an anti-BCL-2 antibody, an anti-E-cadherin antibody, an anti-Vimentin antibody, an anti-CEA antibody, an anti-MUC1 antibody, an anti-Napsin A antibody, an anti-SCC antigen antibody, an anti-p40 antibody, an anti-p63 antibody, an anti-Thyroid Translation Factor-1 antibody, an anti-CA125 antibody, an anti-Mesothelin antibody, an anti-HMB-45 antibody, and an anti-Melan-A antibody.

6. The measurement method according to claim 5, wherein the antibody against the cytokeratin family includes any one of anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody.

7. The measurement method according to any one of claims 1 to 6, wherein the central region shows a negative detection reaction with at least one of the following: DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, Propidium Iodide (PI), SYBR Green I, SYBR Green II, Ethidium bromide, Acridine Orange, SYTO 9, SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, and SYBR Safe DNA Gel Stain.

8. The measurement method according to any one of claims 1 to 7, wherein the cells surrounding the central region show a positive detection reaction with an antibody molecularly labeled with an anti-leukocyte marker antibody, which is at least one of the following: anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody.

9. The measurement method according to any one of claims 1 to 8, wherein the detection step comprises: an extraction step of extracting solid components from blood collected from a subject; a reagent mixing step of mixing a molecularly labeled anti-leukocyte marker antibody, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a nuclear DNA staining reagent with the extracted solid components; and an observation step of spectroscopically observing the sample with the reagents mixed.

10. The measurement method according to any one of claims 1 to 8, wherein the detection step comprises: an extraction step of extracting solid components from blood collected from a subject; a first reagent mixing step of mixing an anti-leukocyte marker antibody and an antibody molecularly labeled with an antibody for specifically detecting proteins or molecules expressed in epithelial cells with the extracted solid components; a second reagent mixing step of further mixing an antibody molecularly labeled with a secondary antibody that binds to the anti-leukocyte marker antibody with the sample mixed with the reagents in the first reagent mixing step; a third reagent mixing step of further mixing a nuclear DNA staining reagent with the sample mixed with the reagents in the second reagent mixing step; and an observation step of spectroscopically observing the sample mixed with the reagents in the third reagent mixing step.

11. The measurement method according to any one of claims 1 to 10, further comprising an estimation step of estimating the likelihood that a subject has cancer by comparing the number of aggregates with a predetermined reference value.

12. The measurement method according to any one of claims 1 to 10, further comprising an estimation step of estimating the cancer treatment effect in a subject by comparing the number of aggregates with a predetermined reference value.

13. A reagent composition for detecting aggregates in blood in which leukocytes surround a central region exhibiting characteristics of enucleated cancer cells, comprising a hemolytic reagent, a molecularly labeled anti-leukocyte marker antibody as a staining antibody reagent, and a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a nuclear DNA staining reagent.

14. A reagent composition for detecting aggregates in blood in which leukocytes surround a central region exhibiting characteristics of enucleated cancer cells. This aggregate comprises a hemolytic reagent, a staining antibody reagent including an anti-leukocyte marker antibody, a molecularly labeled antibody for specifically detecting proteins or molecules expressed in epithelial cells, and a molecularly labeled secondary antibody that binds to the anti-leukocyte marker antibody, and a nuclear DNA staining reagent.

15. The reagent composition according to claim 13 or 14, wherein the molecularly labeled antibody for specifically detecting a protein or molecule expressed in the epithelial cells is at least one of the following: an antibody against the cytokeratin family, an anti-EpCAM antibody, an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-HER2 antibody, an anti-p53 antibody, an anti-Ki-67 antibody, an anti-BCL-2 antibody, an anti-E-cadherin antibody, an anti-Vimentin antibody, an anti-CEA antibody, an anti-MUC1 antibody, an anti-Napsin A antibody, an anti-SCC antigen antibody, an anti-p40 antibody, an anti-p63 antibody, an anti-Thyroid Translation Factor-1 antibody, an anti-CA125 antibody, an anti-Mesothelin antibody, an anti-HMB-45 antibody, and an anti-Melan-A antibody.

16. The reagent composition according to claim 15, wherein the antibody against the cytokeratin family comprises any one of anti-CK18 antibody, anti-CK7 antibody, anti-CK20 antibody, anti-CK5 antibody, anti-CK6 antibody, anti-CK17 antibody, anti-CK8 antibody, and anti-CK19 antibody.

17. The reagent composition according to any one of claims 13 to 16, wherein the anti-leukocyte marker antibody is at least one of anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD5 antibody, anti-CD28 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD38 antibody, anti-CD16 antibody, anti-CD56 antibody, anti-CD15 antibody, anti-CD125 antibody, anti-CD123 antibody, anti-CD203c antibody, anti-CD14 antibody, anti-CD11b antibody, anti-CD68 antibody, anti-CD11c antibody, anti-HLA-DR antibody, and anti-CD34 antibody.

18. The reagent composition according to any one of claims 13 to 17, wherein the nuclear DNA staining reagent comprises any of DAPI (4',6-Diamidino-2-phenylindole), Hoechst 33342, Hoechst 33258, propidium iodide (PI), SYBR Green I, SYBR Green II, ethidium bromide, Acridine Orange, SYTO 9, SYTO 13, YO-PRO-1, YOYO-1, TOTO-1, TO-PRO-3, and SYBR Safe DNA Gel Stain.

Images