JP2018057359A - Method of predicting effect of anticancer drug administration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that can more accurately detect genetic mutations over time in tumor cells and predict the effect of anticancer drug administration in cancer patients with high accuracy.SOLUTION: The method of predicting the effect of anticancer drug administration comprises: (1) a step of collecting samples other than cancer primary tissue and cancer metastatic tissue from a cancer patient; (2) a step of recovering tumor cells from the sample collected in (1); and (3) a step of identifying mutations that cannot be detected in cancer primary tissue or tumor cells derived from cancer metastatic tissue or circulating free DNA or mutations that are not predominant by analysing genetic mutations in the tumor cells recovered in (2). The mutations identified in (3) are used as markers for the effect of administering the anticancer drug.SELECTED DRAWING: None

Description

  The present invention relates to a method for predicting the administration effect of an anticancer agent. In particular, the present invention relates to a method for predicting the administration effect of an anticancer agent using tumor cells derived from a sample other than cancer primary tissue and cancer metastatic tissue (eg, blood sample).

  In recent years, companion diagnostics based on genetic mutations in tumor cells derived from patient specimens have been used to determine the administration of anticancer agents to cancer patients. Based on the companion diagnosis, the doctor determines the administration of the anticancer agent, thereby reducing the risk of performing unnecessary treatment (administration of unnecessary anticancer agent) on the cancer patient. Therefore, it contributes to the reduction of medical costs and also improves the prognosis of patients through optimal treatment.

  In companion diagnosis based on gene mutations in tumor cells derived from patient specimens, tumor cells derived from primary cancer tissues or cancer metastasis tissues obtained by biopsy or tissue excision are usually subjected to genetic analysis. For example, when examining the administration of vemurafenib, an anticancer agent for melanoma, the gene mutation of tumor cells derived from primary cancer tissue or metastasis tissue of a melanoma patient was analyzed, and as a result, the gene mutation was found at the position of V600 in the BRAF gene. If there is, it is judged that vemurafenib shows a high response.

  On the other hand, it is also known that tumor cells with different gene mutations occur over time in cancer primary tissues or cancer metastasis tissues due to changes in the external environment or cell copy errors caused by X-ray irradiation or drug administration. Yes. However, tumor cells having such different gene mutations often have a small proportion in the cancer tissue or are ubiquitous in the cancer tissue, and the tumor cell collected from the cancer tissue is used as a sample. It was generally difficult to detect tumor cells with mutations. In addition, when a primary cancer tissue or the like is completely excised by surgery or the like, it is impossible to follow changes over time in gene mutations in the tissue. For these reasons, it has not been possible to accurately reflect changes over time in gene mutations in tumor cells collected from primary cancer tissue or cancer metastasis tissue in the decision to administer anticancer drugs to cancer patients. .

  In recent years, it has been known that trace amounts of DNA released together with cell contents when cells die in the body exist in the blood (such DNA is referred to as “circulating free DNA (blood free DNA, cell Attempts have been made to use the target gene in the DNA for genetic diagnosis. For example, when a tumor cell derived from a cancer primary tissue or a cancer metastatic tissue dies, genomic DNA derived from the cell is released. The released genomic DNA is digested by phagocytic cells such as macrophages, but undigested genomic DNA flows through blood vessels as circulating free DNA together with undigested genomic DNA derived from normal cells. By using the circulating free DNA derived from the tumor cells, gene mutation analysis can be performed less invasively than the collection of cancer tissue.

  However, as described above, tumor cells having different gene mutations often occupy a small proportion of the cancer tissue or are ubiquitous in the cancer tissue, and occupy the entire circulating free DNA released from the cancer tissue. Since the percentage of the DNA having the different gene mutation is extremely small, it is still difficult to detect the different gene mutation using circulating free DNA.

  On the other hand, even when tumor cells having different gene mutations as described above are not detected, by monitoring the number of circulating tumor cells (hereinafter referred to as “CTC”), the patient's prognosis or Evaluating multiple patient pathologies including therapeutic effects. In recent years, CTCs have also been used as target cells for gene analysis in place of cancer primary tissues or cancer metastatic tissues. For example, Non-Patent Document 1 discloses genetic analysis of CTCs collected from blood derived from cancer patients. In this document, genetic analysis of CTC in a single cell is also performed. Further, in Patent Document 1, by analyzing the RNA expression level of AR-V7 (Androgen Receptor Splice Variant-7) in CTCs collected from prostate cancer patients, it is possible to determine the administration of enzalutamide and abiraterone, which are drugs for prostate cancer. It is disclosed that.

  Furthermore, in Patent Document 2, a first profile for one or more markers of a disease or symptom is determined from a cell-free body fluid sample, and from a phagocytic cell population (2n phagocytic cell population) or a non-phagocytic cell population having a DNA amount of 2n. Determining a second profile for at least one of the one or more markers, identifying a difference between the first profile and the second profile for at least one of the markers, Based on this, it is disclosed that the presence of the disease or symptom in the sample donating patient, the risk of developing it or its evaluation, prognosis prediction or its assistance, or diagnosis or its assistance.

  However, in CTC, it is possible to detect the presence or absence of genetic mutations that are not detected in cancer primary tissues, cancer metastasis tissues or circulating free DNA, and their changes over time, and to evaluate the pathology of multiple cancer patients using them as indicators. Not reported until.

WO2016 / 033114 Japanese Unexamined Patent Publication No. 2017-60479

K. Sakaizawa et al. , British Journal of Cancer, 106, 939-946 (2012).

  The present invention has been made in view of such a situation, and an object of the present invention is to accurately detect temporal changes in gene mutations in tumor cells and predict the effects of anticancer drug administration in cancer patients with high accuracy. It is to provide a method.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and as a result, by using tumor cells derived from samples other than cancer primary tissues and cancer metastasis tissues, in particular, CTC, gene mutations in tumor cells over time. As a result, the present inventors have found that it is possible to detect such a change sensitively, and that the anticancer agent administration effect can be predicted with high accuracy.

That is, the first aspect of the present invention is a method for predicting the effect of anticancer drug administration,
(1) a step of collecting a sample other than a cancer primary tissue and a cancer metastatic tissue from a cancer patient;
(2) a step of recovering tumor cells from the sample collected in (1), and (3) analyzing a genetic mutation in the tumor cells recovered in (2) to obtain tumor cells derived from cancer primary tissue or cancer metastatic tissue or Identifying mutations that are not detected or are not major in circulating free DNA;
And using the mutation identified in (3) as an indicator of the effect of administering an anticancer agent.

Furthermore, the second aspect of the present invention is a method for predicting the effect of anticancer drug administration,
(1) a step of collecting a sample other than a cancer primary tissue and a cancer metastatic tissue from a cancer patient;
(2) a step of collecting tumor cells from the sample collected in (1), and (3) measuring the number of tumor cells collected in (2), and gene mutation in the tumor cells collected in (2) To identify mutations that are not detected or are not major in tumor cells or circulating free DNA from primary or metastatic tissue of cancer,
The number of tumor cells measured in (3) and the mutation identified in (3) are used as an index of the effect of administering an anticancer drug.

  In a third aspect of the present invention, the step (2) includes a cell holding means provided with a plurality of holding portions capable of holding tumor cells contained in a sample and a means for generating a dielectrophoretic force. The method according to any one of the first and second aspects, which is performed using a cell recovery apparatus.

  Further, a fourth aspect of the present invention is the method according to any one of the first to third aspects, wherein the sample other than the primary cancer tissue and the cancer metastatic tissue is a blood sample, and the tumor cell is a circulating tumor cell (CTC) in the blood. It is the method in any one.

  A fifth aspect of the present invention is the method according to any one of the first to fourth aspects, wherein the anticancer agent is a B-Raf enzyme inhibitor and the gene mutation is a BRAF gene mutation.

  The sixth aspect of the present invention is the method according to any one of the first to fifth aspects, wherein the cancer is mesenchymal cancer.

  In the present invention, gene information analysis of tumor cells that are likely to undergo genetic mutation over time is performed on tumor cells collected from samples other than cancer primary tissues and cancer metastatic tissues, and tumors derived from cancer primary tissues or cancer metastatic tissues By using mutations that are not detected in cells or circulating free DNA or that are not major as an index of the effect of administration of an anticancer agent, it is possible to provide highly accurate information for predicting the effect of administration of the anticancer agent.

  In a preferred embodiment of the present invention, a step of measuring the number of tumor cells contained in a sample other than the cancer primary tissue and cancer metastatic tissue is also carried out, and the measured number of tumor cells is also added to the index of the anticancer drug administration effect. This makes it possible to provide more accurate information in predicting the effect of administering an anticancer drug.

  In addition, according to the present invention, it is possible to provide the doctor with information on the optimal medication index for the medical condition held by the cancer patient, the risk in the medical condition, and the probability of survival, so the doctor can select the optimal treatment method. As a result, it is possible to reduce the risk of unnecessary treatment (unnecessary anticancer drug administration) to the patient, and not only save money on unnecessary treatment, but also contribute to improving the prognosis of the patient through optimal treatment selection. it can.

  Furthermore, the present invention can be applied not only to the prediction of the administration effect of an anticancer agent on cancer patients, but also to the prediction of prognosis and the prediction of cancer metastasis. In cancer diagnosis, it can be applied to early detection of cancer.

It is the figure (exploded view) which showed an example of the cell collection | recovery apparatus which can be used at the process of collect | recovering tumor cells in this invention and can hold | maintain a tumor cell. It is a front view of the apparatus shown in FIG. It is the figure which showed an example of the process of collect | recovering tumor cells using the apparatus shown in FIG.

  Hereinafter, the present invention will be described in detail.

  In the present invention, there is no particular limitation on cancer, for example, hematopoietic cell malignancies such as leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, brain tumor, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, Esophageal cancer, stomach cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, oral floor cancer, gingival cancer, tongue cancer, buccal mucosa cancer, etc. Oral cancer, head and neck cancer, salivary gland cancer, sinus cancer, thyroid cancer, kidney cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, skin cancer, anal cancer, melanoma Is mentioned. It may be a primary cancer or a metastatic cancer.

  The cancer patient to which the present invention is applied may be a patient who has already received the anticancer agent for which the effect is to be predicted, a patient who is currently receiving the anticancer agent, or the administration of the anticancer agent. Although it may be a patient before receiving it, a patient before or during administration of an anticancer agent is preferable from the viewpoint of determining a dosing policy.

  The sample other than the cancer primary tissue and cancer metastatic tissue in the present invention is not particularly limited as long as it is a sample containing tumor cells. For example, urine, whole blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, Included in biological samples such as amniotic fluid, lymph, ascites, pleural effusion, and tissues and organs other than the primary cancer tissue and metastatic tissue (liver, lung, spleen, kidney, skin, lymph node, artery, etc.) Cell / tissue culture and culture fluid (culture sample). Note that samples other than the primary cancer tissue and cancer metastatic tissue may be subjected to treatments such as dilution, mixing, dispersion, and suspension in advance according to their properties.

  Among the biological samples, blood samples (for example, blood samples such as whole blood, plasma, and serum, and samples obtained by diluting the blood samples with physiological saline) are included in sample collection from cancer patients and samples. It is preferable as a sample other than the cancer primary tissue and cancer metastatic tissue in the present invention in that the tumor cells can be easily collected.

  In the present invention, first, a sample other than the aforementioned primary cancer tissue and cancer metastatic tissue is collected, and then tumor cells are collected from the collected sample. When the sample other than the cancer primary tissue and the cancer metastasis tissue is a blood sample such as whole blood, plasma, and serum, and the tumor cells are circulating tumor cells (CTC) in the blood, for example, density gradient centrifugation (specialized from the blood sample). A fraction containing CTC (concentrated with CTC) can be obtained by using a filter method (Japanese Unexamined Patent Publication No. 2015-006169) or a filter method (Japanese Patent Laid-Open No. 2014-233267), and CTC can be collected from the fraction.

  When a fraction containing tumor cells is obtained by density gradient centrifugation, the sample is overlaid on the density gradient solution and then centrifuged. Contaminant cells (red blood cells, leukocytes, etc. in the case of blood samples) contained in the sample move to the lower layer (density gradient solution side) while tumor cells remain in the upper layer (sample side). By collecting, a fraction containing tumor cells (enriched with tumor cells) can be obtained. If the above-described fraction containing tumor cells is obtained using a container (Japanese Patent Laid-Open No. 2015-006169) in which the upper layer and the lower layer can be separated, the fraction containing tumor cells can be easily obtained. This is preferable. Furthermore, when the sample is a blood sample, performing the step of hemolyzing the sample (hemolysis operation) before overlaying the sample on the density gradient solution may reduce the number of red blood cells that are contaminating cells. This is preferable because the number of red blood cells mixed into the upper layer is also reduced. In addition, the hemolysis operation may be performed after density gradient centrifugation, and in that case, an operation of removing contaminating cells by centrifugation or the like may be performed again.

  The fraction containing tumor cells obtained by the above-described method may be subjected to a preservation process by cryopreservation or chemical fixation. For example, in the case of cryopreservation, after replacing the solution in the cell preservation solution, it may be stored at a temperature of 0 ° C. or lower, preferably −20 ° C. or lower, more preferably −80 ° C. or lower. In this method, a cell stabilizer may be added to the cell suspension to insolubilize and / or inactivate the protein, thereby suppressing the deterioration of the cells for a long time. Examples of the stabilizer used for chemical fixation include solutions containing cell fixing agents such as aldehydes, acids, dehydrating agents / organic solvents, metal salts and the like.

  The fraction containing tumor cells obtained by the above-described method still contains a large amount of contaminating cells other than tumor cells (particularly leukocytes in the case of blood samples). Therefore, it is preferable to collect tumor cells after specifically detecting the tumor cells present in the fraction. In order to detect tumor cells, for example, first, a fraction containing tumor cells is applied to a slide, or a fraction containing tumor cells is introduced into a device capable of holding tumor cells, and the tumor cells are introduced into the device. Hold. Thereafter, the tumor cells may be detected based on the characteristics of the tumor cells using a microscope or an optical detector. Alternatively, tumor cells may be detected by introducing a fraction containing tumor cells into a flow cytometer.

  An example of a cell recovery apparatus capable of recovering tumor cells is shown in FIG. 1, and a front view thereof is shown in FIG.

The cell collection device 100 shown in FIG. 1 and FIG.
A flat light shielding member 11 having a through-hole 11a;
A flat insulator 12 having a through-hole 12a;
A flat spacer 20 having an introduction port 21, a discharge port 22 and a through-hole 23;
Electrode substrates 31 and 32 provided in close contact with the lower part of the light shielding member 11 and the upper part of the spacer 20,
A conductive wire 40 connecting the electrode substrates 31 and 32;
A signal generator 50 for applying signals to the electrode substrates 31 and 32;
It has.

  The through hole 11a included in the light shielding member 11 and the through hole 12a included in the insulator 12 have the same size and shape, and the light shielding member 11 and the insulator 12 are provided so that the positions of the respective through holes coincide with each other. . When the electrode substrate 31 provided in close contact with the lower part of the through hole 11a, the through hole 12a, and the light shielding member 11 constitutes the holding unit 60 in the cell recovery means 10, and when a liquid containing cells is introduced from the inlet 21, Cells are introduced into the holding unit 60 through the unit 23. The electrode substrate 32 is provided in close contact with the upper portion of the spacer 20 to prevent scattering and evaporation of the liquid containing cells introduced from the introduction port 21. The electrode substrate 32 has a structure that can be detached from the spacer 20 in order to facilitate the collection of the cells held in the holding unit 60. In addition, it is preferable to make the electrode substrates 31 and 32 transparent electrodes such as ITO (indium tin oxide) because the cells held in the holding unit 60 can be detected using a microscope or an optical detector.

  When the tumor cell is held in the cell collection device 100 shown in FIGS. 1 and 2, the fraction containing the tumor cell is introduced from the introduction port 21 provided in the spacer 20, and then the electrode from the signal generator 50 through the lead 40. A dielectrophoretic force may be generated by applying an alternating voltage to the substrates 31 and 32 to hold tumor cells. When introducing a fraction containing tumor cells into the cell collection device 100, a pellet containing tumor cells is obtained by centrifuging the fraction in advance, and then sugars such as mannitol, glucose and sucrose are added. It is preferable to suspend the pellet in the solution to be introduced and then introduce it into the cell collection apparatus 100 because damage to tumor cells is reduced. In addition to the sugars described above, the pellet suspension may further include proteins such as BSA and casein, and proteins bound with hydrophilic polymers. The concentration of the sugar contained in the suspension of the pellet may be a concentration that is isotonic with tumor cells. When mannitol is used as the sugar, the final concentration may be between 250 mM and 350 mM. Examples of the AC voltage applied to the electrode substrates 31 and 32 include a sine wave, a rectangular wave, a triangular wave, and a trapezoidal wave having a peak voltage of about 1 V to 20 V and a frequency of about 10 kHz to 10 MHz. As a specific example, when it is desired to hold live tumor cells one by one in the holding unit, it is preferable to use a rectangular wave with a frequency of 100 kHz to 3 MHz.

  As described above, tumor cells may be detected based on the characteristics of the tumor cells. For example, when detecting tumor cells (CTC) contained in a blood sample, it has a cell nucleus and does not substantially express a leukocyte marker (such as CD45) and / or a cancer cell-derived marker or epithelial system A mode in which a cell expressing a marker (such as cytokeratin (CK) or EpCAM (Epithelial cell adhesion molecule)) is detected as CTC is exemplified (SL Werner. Et al., J. Circ. Biomark., 4: 3, doi: 10.77772 / 60725 (2015)). Here, “substantially does not express the leukocyte marker” means that the expression of the leukocyte marker is hardly confirmed. Specifically, the expression level of the marker in the target cell is a cell that expresses the leukocyte marker ( A leukocyte marker is evaluated as “substantially does not express a leukocyte marker” when the expression level is less than half of the expression level of leukocytes, etc., preferably less than 1/3, more preferably less than 1/5, and even more preferably less than 1/10. sell. In addition, when the expression level of the marker in the target cell is equivalent to that of a negative control cell (eg, vascular endothelial cell, mesenchymal stem cell) that is known not to express the leukocyte marker, It can be evaluated as “substantially not expressed”. As cancer cell-derived markers, various proteins can be mentioned depending on the cancer type, and in the case of melanoma which is one of mesenchymal cancers, gp100 and MART-1 can be exemplified. In addition, although 20 types of proteins from CK1 to CK20 are known for CK, any of them can be used as the epithelial marker.

  When detecting tumor cells based on optical characteristics of the tumor cells, cell nuclei are detected with a cell nucleus staining reagent such as 4 ′, 6-diamidino-2-phenylindole (DAPI) or Hoechst 33342 (trade name). What is necessary is just to detect by dyeing | staining. The marker may be detected by directly staining the marker with a color reagent or fluorescent reagent, and using a labeled antibody against the marker or a primary antibody against the protein and a labeled secondary antibody against the primary antibody. Or may be detected by specifically amplifying the gene of the marker. Among them, the method of detecting a marker using a labeled antibody against the marker is preferably a method that can detect the marker simply, with high sensitivity, and specifically, and moreover, detection using a labeled secondary antibody is higher. It is particularly preferred because it can be detected with sensitivity and specificity. The substance that labels the antibody is not particularly limited, and examples thereof include fluorescent substances such as fluorescein isothiocyanate (FITC) and Alexa Fluor (trade name).

  As another aspect of tumor cell detection, there is an aspect in which detection is based on the size of cells. Many of the tumor cells are larger in diameter than red blood cells (disc shape having a diameter of 7 μm to 8 μm and a thickness of about 2 μm) and white blood cells (spherical diameters of about 6 μm to 15 μm except for macrophages) (in the case of CTC, the diameter is 10 μm). (Rostagno P. et al., Anticancer Res., 17 (4A), 2481-2485 (1997)), and using cells having a larger diameter as compared with erythrocytes and leukocytes as an index Thus, tumor cells (for example, mesenchymal cells derived from tumor tissue that has undergone epithelial-mesenchymal metastasis such as melanoma) can be detected with high accuracy.

  Still another embodiment of tumor cell detection is an embodiment in which detection is based on the size of cell nuclei. Many tumor cells are known to have larger cell nuclei compared to normal cells, and by extracting cells with larger cell nuclei compared to normal cells, even tumor cells of almost the same diameter as normal cells can be obtained. It becomes possible to detect with high precision (see Japanese Patent Application No. 2012-535345 and Japanese Patent No. 5138801). Specifically, the size of the cell nucleus may be measured by staining the cell nucleus region with a reagent capable of staining.

  The detection of tumor cells by a detector is performed, for example, by capturing an image (bright field image, fluorescent image, luminescent image, etc.) obtained by imaging with an imaging means such as a camera into a personal computer, etc., and then using a software What is necessary is just to discriminate | determine whether it is a cell. Whether or not it is a tumor cell can also be determined by visual observation without using software.

  Tumor cells detected by the above-described method may be collected using a collecting means capable of collecting cells. Examples of the collecting means include a collecting means by suction using a pump or an electroosmotic flow. When the tumor cells are relatively strongly adhered to the substrate by an adhesive substance such as poly-L-lysine and a syringe pump is used as a sampling means, it is necessary to suck at a high flow rate. It is preferably between 5.0 μL / s. Examples of the material of the thin tube used for cell suction include glass, metal, resin, and the like, and glass having high impact resistance and high light transmittance is preferable. The inner diameter of the thin tube is preferably larger than the diameter of the cells to be sucked in order to prevent clogging in the thin tube after the suction. For example, when a cell introduced into a holding part having a diameter of 30 μm (the distance between the holding parts is 50 μm) is sucked, the inner diameter of the capillary can be set between 25 μm and 35 μm.

  Tumor cells collected from samples other than cancer primary tissue and cancer metastatic tissue are subjected to gene mutation analysis. The tumor cells may be those collected by the detection and collection means described above, but tumor cells contained in a sample such as a suspension or culture sample of a biological sample other than the primary cancer tissue and cancer metastasis tissue may be used. When there are many cells and / or when there are few contaminating cells, the fraction containing tumor cells obtained by the above-mentioned density gradient centrifugation method or filter method (the fraction enriched with tumor cells) is directly used for gene mutation analysis. Also good. The gene mutation analysis technique is not particularly limited. For example, the gene analysis may be performed after extracting the gene from the collected tumor cells. The amplification reaction of the gene extracted at that time may be carried out. Examples of the gene amplification method include PCR (Polymerase Chain Reaction) method, TRC (Transcribion Reversion-translation reaction) method, LAMP (Loop-Mediated Reaction). (Amplification) method or the like can be used. In gene mutation analysis, for example, the base sequence of the extracted gene (or its amplification product) may be determined by techniques such as the Sanger method, cycle sequencing method, and next generation sequencing, and for example, the extracted gene May be analyzed qualitatively by performing a nucleic acid amplification reaction (such as the PCR method, TRC method, or LAMP method described above) using a primer set capable of detecting a specific mutation, and the absolute number of extracted genes is quantified. In order to achieve this, a digital PCR method may be performed.

  Based on the gene mutation information of tumor cells derived from samples other than cancer primary tissue and cancer metastatic tissue analyzed by the method described above, it is possible to predict the administration effect of the anticancer agent on the target patient (the patient from whom the sample was collected). .

  Among genes, there are genes whose mutations affect the response of anticancer drugs. In such a gene, for example, the mutation affects the structure and activity of the encoded protein, thereby changing the response and side effects of the anticancer drug to the patient. Therefore, the mutation of the gene is an index when administering an anticancer agent. For example, for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib, it is known that mutations in the EGFR gene are involved in their response. Regarding B-Raf enzyme inhibitors, it is known that BRAF gene mutations are involved in their response.

  In the present invention, specifically, among gene mutations detected in tumor cells derived from samples other than cancer primary tissue and cancer metastatic tissue, detection is performed in tumor cells derived from cancer primary tissue or cancer metastatic tissue or circulating free DNA. The anti-cancer agent administration effect is predicted using as an index a mutation that is not or is not major (hereinafter referred to as “target mutation of the present invention”). In the present invention, “circulating free DNA” means DNA that is released together with the contents of cells and flows into the blood when the cells die in the body. In addition, the “non-major mutation” means a frequency of 10% or less, for example, 5% or less, 3% or less, among genetic mutations detected in tumor cells derived from cancer primary tissue or cancer metastatic tissue or circulating free DNA. Mean 1% or less mutation. Mutation detection and frequency determination can be performed by the method described in the Examples of the present application.

  The target mutation of the present invention includes gene mutations detected in tumor cells derived from samples other than cancer primary tissues and cancer metastatic tissues, and genes detected in tumor cells derived from the cancer primary tissues or cancer metastatic tissues or circulating free DNA It can be identified by comparing with the mutation. Tumor cells derived from samples other than cancer primary tissue and cancer metastatic tissue and tumor cells derived from cancer primary tissue or cancer metastatic tissue or circulating free DNA used for comparison of gene mutations may be derived from the same person. preferable.

  In the comparison of gene mutations, gene mutations detected in tumor cells derived from samples other than cancer primary tissues and cancer metastatic tissues (hereinafter also referred to as tumor cells A) and tumor cells derived from the cancer primary tissues or cancer metastatic tissues (Hereinafter also referred to as tumor cell B) or gene mutation detected in circulating free DNA is compared based on differences in (i) gene mutation position, (ii) number of gene mutations, (iii) gene mutation type, etc. That's fine. In predicting the administration effect of the anticancer agent, not only the presence or absence of the target mutation of the present invention in tumor cells A but also the frequency thereof can be taken into consideration. Moreover, not only the situation of the target mutation of the present invention at a specific time point but also its change with time can be taken into consideration.

  For example, as a result of the comparison, when the target mutation of the present invention is present in the tumor cell A, when the target mutation is present above a certain threshold (number or ratio), or the tumor cell A in which the target mutation of the present invention is detected When the number or ratio increases, it can be predicted that administration of an anticancer agent corresponding to the gene is effective or ineffective. In cases where anticancer drug administration can be predicted to be effective, the degree of effectiveness (very effective, moderately effective, slightly effective, etc.) may be included in the evaluation.

  As a first aspect of the prediction, there is prediction based on the presence / absence of tumor cell A having a mutation at a position different from the gene mutation detected in tumor cell B or circulating free DNA. In addition, as a second aspect of the prediction, whether tumor cells A having a mutation at a position different from the gene mutation detected in tumor cell B or circulating free DNA are present above a certain threshold value (number or ratio) There is a prediction based on whether or not. Further, as a third aspect of the prediction, prediction based on an increase / decrease in the number or ratio of tumor cells A having a mutation at a position different from the mutation detected in tumor cells B or circulating free DNA can be mentioned. Among them, the prediction based on the third aspect is more preferable in that the administration effect prediction of the anticancer agent can be predicted earlier than the change in the disease state.

  The prediction of the anticancer drug administration effect by the method of the present invention can be performed by a medical assistant or the like without being performed by a doctor, or can be automatically associated on the apparatus and software. Therefore, it can be said that the method of the present invention is a preliminary method for determining the administration of an anticancer agent by a doctor or a method for obtaining information for predicting the effect of administering an anticancer agent by a doctor.

  The relationship between the gene mutation of the tumor cell and the prediction of the administration effect of the corresponding anticancer drug can also be determined by conducting a follow-up test. Such a relationship can also be determined according to the type and stage of the cancer disease the patient is suffering from. By determining in advance the association with an anticancer agent with high response or few side effects according to the genetic information of tumor cells contained in the patient blood before treatment, the administration policy of the anticancer agent to the patient can be determined. In addition, patients suffering from cancers that are prone to genetic mutations by pre-determining the association with anticancer agents with high response or few side effects according to the genetic information of tumor cells contained in samples from patients undergoing treatment It is also possible to determine an anticancer drug administration policy for the above. Furthermore, depending on the gene mutation information of the tumor cells contained in the patient-derived sample after treatment, it is possible to determine the administration policy of the anticancer agent by predetermining the association with a highly effective drug or an anticancer agent with few side effects. it can.

  As one of the preferred embodiments of the present invention, in addition to using the target mutation of the present invention as an index, by using the number of tumor cells derived from a sample other than the primary cancer tissue and cancer metastasis tissue as an index, the effect of administering an anticancer agent can be obtained. A prediction method is mentioned. The number of tumor cells derived from samples other than cancer primary tissue and cancer metastasis tissue and the prognosis are related, and the larger the number of tumor cells, the worse the prognosis (in the case of tumor cells contained in blood samples, Patent No. 5479355) reference). From this, it is possible to predict the prognosis of a patient by measuring the number of tumor cells derived from a sample other than the primary cancer tissue and cancer metastasis tissue, and when the patient is administered an anticancer drug, The effect can be predicted. Therefore, in addition to the step of analyzing gene mutations in tumor cells derived from samples other than cancer primary tissues and cancer metastatic tissues and identifying the target mutation of the present invention, tumors derived from samples other than cancer primary tissues and cancer metastatic tissues By measuring the number of cells, the effect of anticancer drug administration can be predicted with higher accuracy. In the case where the number of tumor cells is used as an index, it may be simply evaluated whether tumor cells having a certain threshold value or more are present in the collected sample. You may evaluate the time-dependent change of the number of tumor cells contained in a sample.

  Hereinafter, as an example of the prediction method of the present invention, the prediction method based on the target mutation of the present invention detected by tumor cells (CTC) contained in a blood sample using the cell recovery apparatus 100 shown in FIGS. 1 and 2 However, the present invention is not limited to the contents of this description.

  (1) Collect blood from patients suspected of having cancer or cancer patients. When blood is collected, an anticoagulant such as citric acid, heparin, ethylenediaminetetraacetic acid (EDTA) may be added. If necessary, the collected blood may be diluted with physiological saline or the like.

  (2) The collected blood (or diluted blood) is subjected to density gradient centrifugation to remove contaminating cells (red blood cells, white blood cells, etc.) contained in the blood. Density gradient centrifugation is a method of separating substances based on their specific gravity. After collecting blood (or diluted blood) on a medium that forms a density gradient (density gradient solution), centrifugation is performed. Thus, it is possible to remove contaminating cells and dust and collect a fraction (upper layer) containing CTC. Before performing the centrifugation, a binding agent (for example, RosetteSep (manufactured by StemCell Technologies)) that can bind to contaminating cells (red blood cells, white blood cells, etc.) is added to the collected blood (or diluted blood). You can also. The binding agent forms cell aggregates by binding to erythrocytes, leukocytes and / or surface antigens of these cells, and the density of these cells can be increased, so that separation of CTC by density gradient centrifugation is easy. To. The fraction containing CTC from which contaminating cells and dust have been removed by the density gradient centrifugation method is preferably subjected to the subsequent operation promptly. However, if the subsequent operation cannot be performed promptly, a preservation process by cryopreservation is performed. May be. When cryopreserving, a fraction containing CTC may be suspended in a cell preservation solution such as CELLBANKER2 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) and then cryopreserved at −80 ° C.

  (3) A solution containing ammonium chloride is added to the fraction containing CTC obtained in (2) and stirred to hemolyze red blood cells mixed in the fraction. This operation improves the observation of the separated and recovered CTC.

  (4) CTC is pelletized by removing blood components by centrifuging the solution containing CTC after hemolysis obtained in (3), and then suspending CTC using an appropriate solution .

  (5) The CTC-containing suspension prepared in (4) is centrifuged again, and the CTC-containing pellet is recovered. If necessary, a step of suspending the recovered pellet again in the solution and centrifuging it may be added.

  (6) After the CTC obtained in (5) is developed on the cell holding means 10 provided in the cell recovery apparatus 100 shown in FIG. 1, the cell 70 is held on the holding unit 60 by the dielectrophoretic force 80 (FIG. 3). (1)).

  (7) The adhesive substance 90 is introduced into the cell collection device 100, and the CTC is adhered to the holding unit 60 (FIG. 3 (2)). As the adhesive substance 90, for example, poly-L-lysine can be used, and its concentration is preferably 0.01 (w / v)% or less.

  (8) Preservation treatment agent and cell membrane permeation treatment agent are introduced into the cell recovery apparatus 100, and CTC preservation and membrane permeation treatment are performed. Examples of the preservative include formaldehyde, formaldehyde donor compounds (compounds capable of releasing formaldehyde upon hydrolysis), aldehydes such as glutaraldehyde, alcohols such as methanol and ethanol, and solutions containing heavy metals. Examples of the cell membrane permeabilizing agent include alcohols such as methanol and ethanol, and surfactants such as saponin.

  (9) In order to prevent a non-specific reaction caused by the antibody, a blocking treatment with a protein is applied to the holding portion holding the target cells after storage and membrane permeabilization treatment.

  (10) After the blocking treatment, a fluorescently labeled antibody against a protein expressed by leukocytes (leukocyte marker), a protein expressed by epithelial cells (epithelial marker), or a protein expressed by tumor cells (cancer cell-derived marker), Cells are labeled with a reagent that fluorescently stains cell nuclei (FIG. 3 (3)), and after washing, a fluorescence image and a bright field image of the cells are observed with a fluorescence microscope 200 or the like (FIG. 3 (4)). As an antibody against a protein expressed by leukocytes, an anti-CD45 antibody can be used. In addition, as an antibody against a protein expressed by epithelial cells, an anti-CK antibody, an anti-EpCAM antibody, or the like can be used. As an antibody against the protein expressed by tumor cells. When the tumor cell is a melanoma, an anti-gp100 antibody, an anti-MART-1 antibody, or the like can be used. As a reagent for fluorescently staining the cell nucleus, 4 ', 6-diamidino-2-phenyllinole (DAPI), Hoechst 33342 (trade name), or the like can be used.

  (11) CTC71 is detected based on the observed fluorescence image and bright field image (FIG. 3 (4)). In detection of CTC, for example, a cell nucleus is stained, not labeled with an anti-CD45 antibody, and an antibody against a protein expressed by epithelial cells (such as an anti-CK antibody or an anti-EpCAM antibody) or a protein expressed by a cancer cell What is necessary is just to detect the cell labeled with the antibody (in the case of melanoma, anti-gp100 antibody or anti-MART-1 antibody) as CTC. Alternatively, cells that are stained with cell nuclei, are not labeled with anti-CD45 antibodies, and have a larger cell shape in the bright field image than erythrocytes or leukocytes may be detected as CTCs.

  (12) In order to collect the CTC 71 detected by the fluorescence microscope 200, after removing the electrode substrate 32 from the spacer 20, the CTC 71 is collected by suction with the collecting device 300 (FIG. 3 (5)). When removing the electrode substrate 32, it is necessary to remove the spacer 20 so as not to peel off. This is because if the spacer 20 is peeled off from the insulator 12, the solution held in the apparatus flows out of the system and the CTC 71 is destroyed. The suction of the CTC 71 by the recovery device 300 is performed by moving the recovery device 300 to the holding unit 60 where the CTC 71 detected in (11) is held, and sucking the liquid by the recovery device 300 to recover the CTC 71. In addition, it is preferable that the suction position of the CTC 71 by the collection device 300 is a position shifted by a certain distance in the horizontal direction from the center of the holding unit 60 where the CTC 71 is sampled because the suction of the CTC 71 can be easily performed. Specifically, the suction position of the CTC 71 is set to a length that is 0.1 to 2 times the diameter of the holding unit 60 in the horizontal direction from the center of the holding unit 60 (however, two minutes of the distance between adjacent holding units 60). 1 or less) and a position that is 0.01 to 2 times as high as the height of the holding portion 60 in the vertical direction from the height of the holding portion 60 is preferable. Further, an operation of adding a solution containing an enzyme that weakens the adhesion between the CTC 71 and the holding unit 60 may be performed before the suction operation of the CTC 71 by the recovery device 300.

  (13) The CTC 71 recovered by the suction by the recovery device 300 is discharged to the recovery tube 400 (FIG. 3 (6)). The optical detector 200 may detect whether or not the CTC 71 is discharged to the collection tube 400.

  (14) A gene in CTC71 recovered in the recovery tube 400 is extracted, the gene is amplified by the PCR method, and then the gene mutation is analyzed by sequencing the gene by the Sanger method.

  (15) The analysis result of (14) is compared with the analysis result of gene mutation in tumor cells derived from cancer primary tissue or cancer metastasis tissue or circulating free DNA, and based on the presence or absence of the difference in these gene mutation positions, administration of an anticancer drug Predict the effect. In the case of performing effect prediction with higher accuracy, for example, a step of counting the CTC detected in the above (11) and observing the change with time may be added.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example and a comparative example, this invention is not limited to the said example.

[Example 1] Correlation between gene mutation in circulating tumor cells (CTC) and pathology caused by administration of anticancer agent (1) From stage IV melanoma patients who obtained informed consent, blood was collected a total of 4 times according to the course of treatment Collected. The time of blood collection is shown below.
(First time) Before administration of B-Raf enzyme inhibitor (Second time) Administration of B-Raf enzyme inhibitor and no change in pathological condition (stable pathological condition) (7 months after the first blood collection)
(3rd time) B-Raf enzyme inhibitor was administered, but lymph node metastasis and skin metastasis neoplasia were observed (pathological condition worsened), and before nivolumab administration (8 months after the second blood collection) rear)
(Fourth) Nivolumab administration (5 cycles) showed reduction of skin metastasis due to CT imaging (pathological improvement) (4 months after the third blood collection)
(2) 10 mL of blood collected in (1) is centrifuged at 20 × g for 10 minutes at room temperature, the supernatant is removed, and then suspended in 20 mL of PBS (Phosphate buffered saline) to prepare a diluted blood sample did.
(3) The diluted blood sample was layered on a density gradient solution having a density of 1.077 g / mL, and centrifuged at 800 × g for 20 minutes at room temperature, and then the fraction containing CTC in the supernatant was collected.
(4) 30 mL of PBS was added to the fraction containing CTC collected in (3), and the supernatant was removed by centrifugation at 600 × g for 10 minutes at room temperature to obtain a pellet containing CTC.
(5) The pellet containing CTC was resuspended in 20 mL of PBS, and the supernatant was removed by centrifugation at 300 × g for 8 minutes at room temperature to obtain a pellet containing CTC.
(6) The pellet containing CTC was resuspended in 20 mL of PBS again, and then centrifuged at 300 × g for 8 minutes at room temperature to remove the supernatant. (4), (5) and this operation are operations for removing blood components and concentrating desired cells.
(7) For the purpose of long-term storage of CTC, the pellet containing CTC was resuspended in 2 mL of a cell cryopreservation solution (CELLBANKER2, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) and stored frozen at -80 ° C.
(8) Thaw the suspension containing CTC cryopreserved in (7), suspend a part of it in 10 mL of a solution containing 300 mM mannitol, and then centrifuge at 300 × g for 5 minutes at room temperature. The supernatant was removed.
(9) The suspension was again suspended in 10 mL of a solution containing 300 mM mannitol, and then centrifuged at 300 × g for 5 minutes at room temperature to remove the supernatant. (8) and this operation are operations for removing the cell cryopreservation solution and concentrating the CTC.
(10) The suspension containing CTC from which the supernatant has been removed in (9) is introduced into the cell holding means 10 provided in the cell recovery apparatus 100 shown in FIG. The voltage (frequency 1 MHz) was applied for 3 minutes, and the cells including CTC were held in the holding unit 60 included in the means. The cell collection device 100 used in this example includes an insulator 12 having a plurality of fine holes with a diameter of 30 μm and a depth of 40 μm, and a light-shielding chromium film (light-shielding member 11) installed between the insulator 12 and the electrode substrate 31. ) And the electrode substrate 31, the spacer 20 having a thickness of 1 mm and the electrode substrate 32 are in close contact with the upper surface of the holder 60 provided in the cell holder 10.
(11) While applying an AC voltage under the conditions of (10), a 300 mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine was introduced, and the mixture was allowed to stand for 3 minutes. The application was stopped and the aqueous solution was removed by suction.
(12) An aqueous solution containing 50% (v / v) ethanol and 2% (w / v) formaldehyde (hereinafter referred to as “cell membrane permeation reagent”) is introduced and allowed to stand for 10 minutes to permeate the cell membrane. The cells containing CTC in the holding part were sampled.
(13) The cell membrane permeation reagent was removed by suction, and the remaining cell membrane permeation reagent was washed by introducing PBS.
(14) An aqueous solution (hereinafter referred to as labeling reagent A) containing a fluorescently labeled antibody capable of specifically binding to proteins inside and outside the cell membrane and a fluorescent reagent (DAPI: 4 ′, 6-diamidino-2-phenyllinole) for labeling the cell nucleus It was introduced and allowed to stand for 30 minutes. As the labeled antibody, an antibody against CD45 expressed on the surface of leukocytes, and an antibody against gp100 and MART-1 expressed in the cytoplasm of melanoma cells are used.
(15) The labeling reagent A was removed by suction, and the remaining labeling reagent A was removed by introducing PBS.
(16) After placing the cell holding means containing the CTC labeled in (15) on the stage of the fluorescence microscope, the entire holding unit was imaged in order to observe all the cells captured in the plurality of holding holes. . For this, a fluorescent microscope (IX83; manufactured by Olympus) equipped with a computer-controlled electric stage and a CMOS camera (ORCA-Flash 4.0; manufactured by Hamamatsu Photonics) was used. LabVIEW (manufactured by National Instruments) was used as image acquisition and analysis software.
(17) The cells imaged in (16) are stained with DAPI indicating that they have a cell nucleus (DAPI positive), and are not stained with an antibody against CD45 expressed in leukocytes (CD45 negative) ), Cells that were stained with antibodies against gp100 and MART-1 (gp100 / MART-1 positive) indicating that they have melanoma properties were detected as target tumor cells (melanoma-derived CTC).
(18) After removing the electrode substrate 32 from the spacer 20, the melanoma-derived CTCs detected in (17) were sucked one by one from an arbitrary holding part using a cylindrical thin tube under a fluorescence microscope. The cells were collected by discharging the sucked cells into a container.
(19) A gene was extracted from the cells collected in (18), and the BRAF gene region was amplified by PCR.
(20) The gene information was obtained from the BRAF gene amplified in (19) by sequence analysis by the Sanger method, and the presence or absence of mutation was analyzed.

[Example 2] Gene mutation in primary cancer tissue and metastatic tissue (1) From the cancer patient blood sampled in Example 1 (1), lymph nodes that are primary cancer tissue and cancer metastasis tissue before the first blood sampling, At the time of the third blood collection, neoplastic skin metastases that were cancer metastatic tissues were collected.
(2) The presence or absence of a gene mutation was analyzed for each collected tissue using a Covas BRAF V600 mutation detection kit (Roche Diagnostics).

  Table 1 shows the number of melanoma-derived CTCs for which genetic information was obtained in Example 1 and the results of gene mutation analysis. Table 2 shows the results of gene mutation analysis in the tissues subjected to gene analysis in Example 2.

  In the first blood collection (before administration of the B-Raf enzyme inhibitor), among the 13 melanoma-derived CTCs for which genetic information was obtained, CTCs with mutations in the BRAF V600 site that are highly effective with B-Raf enzyme inhibitors 2 detected (15.4% of CTCs with the mutation) and the mutation is different from the BRAF V600 site mutation (V600E) detected in the primary cancer tissue and lymph nodes (cancer metastasis tissue) (V600K). Since CTC having a BRAF V600K mutation was detected, it is predicted that administration of a B-Raf enzyme inhibitor is effective at this point.

  Even in the second blood collection (pathologically stable by administration of B-Raf enzyme inhibitor), one CTC having a BRAF V600K mutation was detected from 10 CTCs derived from melanoma from which genetic information was obtained (the ratio of CTCs having the mutation) 10.0%), and the results of gene mutation analysis show that administration of a B-Raf enzyme inhibitor is effective. In fact, the pathological condition is stabilized by administration of the B-Raf enzyme inhibitor. However, although no change in the pathological condition was observed in the second blood sampling, the number and ratio of CTCs having a BRAF V600K mutation were detected at the first blood sampling (two CTCs having a BRAF V600K mutation were detected. Since the ratio of CTC having the mutation is decreased compared to 18.2%), it is predicted that the B-Raf enzyme inhibitor will not be effective after the second blood collection.

  At the time of the third blood collection, although the B-Raf enzyme inhibitor administration was continued, the pathological condition was deteriorated, indicating that the efficacy of the B-Raf enzyme inhibitor was reduced. This is consistent with the prediction from the second blood collection result (that is, the prediction that the efficacy of the B-Raf enzyme inhibitor will decrease after the second blood collection). In the third blood collection, among the 9 melanoma-derived CTCs for which genetic information was obtained, no CTC having a BRAF V600K mutation was detected, while CTC having the same mutation (V600E) as the BRAF V600 site mutation in the skin metastatic tissue. 1 was detected (the ratio of CTC having the mutation was 11.1%). Therefore, when CTC having a mutation (V600K) different from the BRAF V600 mutation (V600E) detected in tumor cells derived from skin metastasis tissue is not detected, the effectiveness of the B-Raf enzyme inhibitor is reduced. (That is, the condition gets worse). On the other hand, since the BRAF V600 site mutation (V600E) remains in the tumor metastasis derived from the skin metastasis tissue, simply analyzing the mutation detected in the tumor cell derived from the primary cancer tissue or cancer metastasis tissue is an anticancer agent. It can be seen that it is difficult to predict the administration effect.

  Based on the above results, we observed changes in the pathological condition by observing changes in the number and proportion of CTCs with mutations (BRAF V600K) different from the gene mutations detected in tumor cells derived from cancer primary tissues and cancer metastatic tissues. It can be seen that the effect of administering the anticancer agent can be predicted at an earlier stage.

  In the fourth blood collection (improvement of the condition by administration of nivolumab), CTC having BRAF V600 mutation was not detected among 14 melanoma-derived CTCs for which genetic information was obtained.

  In addition, Table 3 shows the results of counting the number of melanoma-derived CTCs at the time of each blood collection detected in Example 1 (17).

  While the number of CTCs derived from melanoma increased significantly during the third blood collection when the condition worsened (104.2 / mL at the second blood collection → 1044 / mL at the third blood collection), the fourth time the condition improved The number of melanoma-derived CTCs decreased at the time of blood collection (1044 / mL at the time of the third blood collection → 765.8 / mL at the time of the fourth blood collection), and thus the melanoma-derived CTC contained in the blood sample derived from the melanoma patient. It can be seen that the number correlates with the patient's condition. Therefore, by observing the number of melanoma-derived CTCs contained in a melanoma patient-derived blood sample, it can be said that the prognosis of the patient, that is, the anticancer drug administration effect can be predicted.

[Comparative Example 1] Measurement of 5-S-SD (5-S-cysteinyldopa) in blood sample derived from melanoma patient Using blood collected in Example 1 (1), it has been conventionally used as a marker for melanoma diagnosis. The value of 5-S-CD was measured. The results are shown in Table 4.

  Regarding 5-S-CD, the value exceeded the reference value (2.5 to 6.1 nmol / L) indicating normality at the time of each blood collection. However, the amount of 5-S-SD was lower at the time of the third blood collection when the disease state worsened than at the time of the second blood collection (33.5 nmol / L at the time of the second blood collection → 27. 4 nmol / L). From this, it can be said that it is difficult to predict the prognosis of the patient even if the temporal change in the amount of 5-S-SD contained in the blood sample derived from the melanoma patient is observed.

[Example 3] Correlation between time course of CTC number using labeled secondary antibody and pathological condition (1) Informed consent was obtained from a stage IV melanoma patient different from Example 1 in accordance with the course of treatment. In total, blood was collected three times. The time of blood collection is shown below.
(1st) After dissection of lymph node metastasis (2nd) After appearance of lung metastasis (5 months after the first blood collection)
(3rd) Increased lung metastasis, appearance of liver metastasis, increased skin metastasis (pathological condition worsened) (3 months after the second blood collection)
(2) Except for using the blood collected in (1), the cells containing CTC were exposed to the cell membrane permeation reagent in the same manner as in Examples 1 (2) to (13), and then the remaining cell membrane permeation reagent was used. Washed.
(3) An aqueous solution containing a primary antibody capable of specifically binding to proteins inside and outside the cell membrane was introduced and allowed to stand for 30 minutes. As the primary antibody, antibodies against gp100 and MART-1 expressed in the cytoplasm of melanoma cells are used.
(4) The aqueous solution containing the primary antibody was removed by suction, and PBS was introduced to remove the remaining aqueous solution containing the primary antibody.
(5) a fluorescently labeled antibody that can specifically bind to proteins inside and outside the cell membrane, a fluorescently labeled secondary antibody that can specifically bind to the primary antibody, and a fluorescent reagent that labels the cell nucleus (DAPI: 4 ′, An aqueous solution containing 6-diamidino-2-phenylindole) (hereinafter referred to as labeling reagent B) was introduced and allowed to stand for 20 minutes. The fluorescently labeled antibody is an antibody against CD45 expressed on the surface of leukocytes, and the fluorescently labeled secondary antibody is an antibody capable of specifically binding to the primary antibody producing animal species and subclass, respectively. Used.
(6) The labeling reagent B was removed by suction, and PBS was introduced to remove the remaining labeling reagent B.
(7) CTC was detected by the same method as in Example 1 (16) and (17) using a cell holding means containing CTC labeled in (6).

  The results of counting the number of melanoma-derived CTCs at the time of each blood collection are shown in Table 5.

  The number of CTCs derived from melanoma increased significantly at the time of the third blood collection when the pathological condition worsened (110.6 / mL at the second blood collection → 565.8 / mL at the third blood collection), and a correlation with the pathological condition was observed. It was. In Example 1, the correlation between the CTC number and the disease state was detected using the labeled primary antibody. However, in this example, the correlation between the CTC number and the disease state can be obtained even when the labeled secondary antibody is used. There was found.

[Example 4] Genetic mutation in CTC (1) Blood was collected once from a patient with stage IV melanoma different from Examples 1 to 3 that obtained informed consent.
(2) CTC was detected by the same method as in Examples 3 (1) to (6) except that the blood collected in (1) was used.
(3) 33 randomly extracted from the melanoma-derived CTCs detected in (2) were collected one by one in the same manner as in Examples (18) to (20), and the BRAF gene region mutation was recovered. The presence or absence was analyzed.

[Example 5] Cancer primary tissue, cancer metastasis tissue and gene mutation in circulating free DNA (1) From cancer patients blood sampled in Example 4 (1), cancer primary tissue and cancer metastasis tissue were collected. Further, peripheral blood was collected, cells were removed, and DNA (circulating free DNA) contained in the peripheral blood was extracted.
(2) The presence or absence of mutations in the BRAF gene region was analyzed using the Cobas BRAF V600 mutation detection kit (Roche Diagnostics) and the Sanger method for the tissues collected in (1). . Analysis of the presence or absence of mutations in the BRAF gene region V600E, V600K, and K601E was performed on the extracted circulating free DNA using the digital droplet PCR method.

  Table 6 shows the results of gene mutation analysis of melanoma-derived CTCs for which genetic information was obtained in Example 4. Table 7 shows the results of gene mutation analysis in the primary cancer tissues, cancer metastasis tissues and circulating free DNA subjected to genetic analysis in Example 5.

  Of the 33 melanoma-derived CTCs for which genetic information was obtained in Example 4, 6 CTCs having mutations in the vicinity of BRAF V600 were detected, and among these mutations, BRAF was detected in cancer metastasis tissue and circulating free DNA. Mutations (V600E heterozygous mutant, V600A heterozygous mutant, K601E homozygous mutant) different from the mutation in the vicinity of V600 (K601E heterozygous mutant) were also detected. No mutation was detected in the primary cancer tissue, and the same mutation was detected in the cancer metastasis tissue and circulating free DNA, indicating that only the mutation that can be detected in the cancer metastatic tissue is also detected from circulating free DNA. On the other hand, since gene mutations that are not detected in primary cancer tissues, cancer metastatic tissues, and circulating free DNA were detected from CTC, information on the diversity of genetic mutations that cannot be detected in primary cancer tissues, cancer metastatic tissues, and circulating free DNA It can be seen that it can be obtained from CTC.

DESCRIPTION OF SYMBOLS 100: Cell collection | recovery apparatus 10: Cell holding means 11: Light-shielding member 12: Insulator 11a, 12a: Through-hole 20: Spacer 21: Inlet 22: Outlet 23: Through-portion 31/32: Electrode substrate 40: Conductor 50: Signal generator 60: holding unit 70: cell 71: target cell (CTC)
80: Dielectrophoretic force 90: Adhesive substance 200: Fluorescence microscope 300: Collection device 400: Collection tube

Claims (6)

A method for predicting the effect of anticancer drug administration,
(1) a step of collecting a sample other than a cancer primary tissue and a cancer metastatic tissue from a cancer patient;
(2) a step of recovering tumor cells from the sample collected in (1), and (3) analyzing a genetic mutation in the tumor cells recovered in (2) to obtain tumor cells derived from cancer primary tissue or cancer metastatic tissue or Identifying mutations that are not detected or are not major in circulating free DNA;
And using the mutation identified in (3) as an index of the effect of administering an anticancer agent. A method for predicting the effect of anticancer drug administration,
(1) a step of collecting a sample other than a cancer primary tissue and a cancer metastatic tissue from a cancer patient;
(2) a step of collecting tumor cells from the sample collected in (1), and (3) measuring the number of tumor cells collected in (2), and gene mutation in the tumor cells collected in (2) To identify mutations that are not detected or are not major in tumor cells or circulating free DNA from primary or metastatic tissue of cancer,
And the number of tumor cells measured in (3) and the mutation identified in (3) are used as an index of the anticancer drug administration effect. The step (2) is carried out using a cell recovery device comprising a cell holding means provided with a plurality of holding parts capable of holding tumor cells contained in a sample and a means for generating a dielectrophoretic force. Or the method of 2. The method according to any one of claims 1 to 3, wherein the sample other than the cancer primary tissue and the cancer metastatic tissue is a blood sample, and the tumor cells are circulating tumor cells (CTC) in the blood. The method according to any one of claims 1 to 4, wherein the anticancer agent is a B-Raf enzyme inhibitor and the gene mutation is a BRAF gene mutation. The method according to any one of claims 1 to 5, wherein the cancer is mesenchymal cancer.