Method for measuring distribution of drug in cancer tissue

Abstract

The present invention develops a method with which it is possible to assess the extent to which a drug reaches cancer tissue or cancer cells, and how much of the drug reaches the cancer tissue or cancer cells, in assessing the effect of the drug on cancer.　A method for measuring the distribution of a drug and / or a metabolite thereof in a solid cancer tissue section comprises: (A) measuring, with an imaging mass spectrometry microscope, a solid cancer tissue section prepared specimen derived from a subject to which the drug has been administered; (B) obtaining an image in which a cancer region and a non-cancer region are identified in the solid cancer tissue section prepared specimen; and (C) comparing the image measured in (a) and the image obtained in (B).

Description

Method for measuring drug distribution in cancer tissue

[0001] The present disclosure relates to a method for measuring drug distribution in cancer tissue, a method for determining whether to continue administering the drug based on the measured drug distribution, etc. The contents of all documents described in this specification are incorporated herein by reference.

[0002] Breast cancer has been rapidly increasing in recent years, with approximately 2 million people diagnosed with the disease worldwide each year and approximately 90,000 in Japan. In breast cancer, preoperative chemotherapy is often used to shrink the tumor before surgery. If the preoperative drug is determined to be effective (sensitive), the same drug is used postoperatively; if it is determined to be ineffective, a different drug is used postoperatively. Conventional methods for assessing drug efficacy have been to observe tumor size reduction or growth using diagnostic imaging (ultrasound, MRI), or to assess efficacy through pathological diagnosis (microscopic diagnosis) of surgically resected sections.

[0003] JP 2018-077253 A JP 2014-206389 A

[0004] Clin Trans Med (2016) 5:10 “Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging”Breast Cancer Research (2017) 19:107 “Heterogeneous drug penetrance of veliparib and carboplatin measured in triple negative breast tumors”Scientific Reports | 6:39284 | DOI: 10.1038 / srep39284 (2016) “Heterogeneity of paclitaxel distribution in different tumor models assessed by MALDI mass spectrometry imaging”Scientific Reports volume 12, Article number: 4513 (2022)Scientific Reports | 3 3050 | DOI: 10.1038 / srep03050 (2013) “The significance of microscopic mass spectrometry with high resolution in the visualisation of drug distribution”Int. J. Biol. Sci. 2025, 21(1): 363-381. doi: 10.7150 / ijbs.102744Cancer Science. 2022;113:2916-2925. DOI: 10.1111 / cas.15428Nature Communications (2023) 14:5967Theranostics 2020, 10(16): 7070-7082. doi: 10.7150 / thno.45543

[0005] In imaging diagnosis, the effectiveness of drugs is judged by the reduction or increase in tumor size, but tumor size alone does not necessarily reflect drug efficacy. In pathological diagnosis, the effectiveness of drugs is judged by microscopic observation of tissue sections stained with hematoxylin and eosin (HE) staining or immunostaining, but it may be difficult to judge drug efficacy based on stained images alone.

[0006] After surgery, the removal of cancer tissue removes visible tissue from the patient, making it difficult to re-sample cancer tissue and assess drug efficacy. Therefore, the timing for assessing drug efficacy is generally limited to before and after surgery, especially in the case of resectable breast cancer. If drug efficacy cannot be properly assessed, for example, if a drug is administered because it is deemed effective when in fact it is not, or if the drug's efficacy is unknown, there is a risk of patients having to take an ineffective drug for a long period of time. In particular, in clinical practice, efficacy assessments for individual patients are not performed for postoperative endocrine therapy.

[0007] The purpose of the present disclosure is to provide a method for analyzing and evaluating drug distribution and drug amount in cancer tissue at the molecular level in order to more directly determine the efficacy of drugs on cancer (particularly breast cancer).

[0008] The present inventors have discovered that, particularly for cancer (breast cancer) tissue slice preparations, it is possible to detect drug distribution in cancer tissue with very high resolution by measuring the drug distribution in the cancer tissue using an imaging mass spectrometry microscope. Note that in this disclosure, a cancer tissue slice preparation refers to a cancer tissue slice that has been thinly sliced ​​and mounted on a glass slide.

[0009] Furthermore, although the correlation between efficacy and distribution of a drug and / or its metabolites at a target site has not generally been clarified, the present inventors have found that there is a certain degree of correlation between efficacy and distribution of a drug and / or its metabolites at a target site. They have also found that the efficacy of a drug can be determined based on this distribution. In particular, when the disease is breast cancer and the therapeutic agent is tamoxifen, they have found that the efficacy of tamoxifen can be determined based on whether tamoxifen and / or its metabolites are distributed at a relatively high intensity in breast cancer tissue.

[0010] The present disclosure includes, for example, the subject matter described in the following sections: Item A. A method for measuring the distribution of a drug and / or its metabolites in a solid cancer tissue section, comprising: (A) measuring a solid cancer tissue section prepared from a subject to which a drug has been administered using an imaging mass spectrometry microscope; (B) obtaining an image that identifies cancerous and non-cancerous regions in the solid cancer tissue section preparation; and (C) comparing the image measured in (a) with the image obtained in (B). Item B. A method for treating a solid cancer, comprising: (A) measuring a solid cancer tissue section prepared from a subject to which a drug has been administered using an imaging mass spectrometry microscope; (B) obtaining an image that identifies cancerous and non-cancerous regions in the solid cancer tissue section preparation; and (C) comparing the image measured in (a) with the image obtained in (B), and (D) administering the drug or a drug other than the drug to the subject based on the comparison. Item C. Item 1. A method for measuring tamoxifen distribution in a breast cancer tissue section, comprising: (a) measuring a breast cancer tissue section prepared from a subject administered tamoxifen using an imaging mass spectrometry microscope; (b) removing the matrix from the breast cancer tissue section after measurement and staining it with hematoxylin and eosin (HE staining); and (c) comparing the measurement image obtained in (a) with the HE-stained image obtained in (b).Item 1a. A method for treating breast cancer, comprising: (a) measuring a breast cancer tissue section preparation from a subject to which tamoxifen has been administered using an imaging mass spectrometry microscope; (b) removing the matrix from the breast cancer tissue section preparation after measurement and staining it with hematoxylin and eosin (HE staining); and (c) comparing the measurement image in (a) with the HE-stained image in (b); and (d) administering tamoxifen or a drug other than tamoxifen (an anti-breast cancer drug) to the subject based on the comparison. Item 1b. The method of Item 1 or 1a, wherein the image measured using an imaging mass spectrometry microscope is an image showing the distribution of tamoxifen and / or tamoxifen metabolites. Item 1c. The method of Item 1, 1a, or 1b, wherein the HE-stained image is an HE-stained image in which cancerous and non-cancerous regions are identified. Item 1d. The method of any one of Items 1, 1a, 1b, or 1c, wherein the comparison measures the distribution of tamoxifen and / or tamoxifen metabolites in cancerous and non-cancerous regions. Item 2. The method of any one of the above items (Items 1 to 1d), wherein the subject is a human breast cancer patient. Item 3. The method of Items 1 to 1d or 2, wherein the breast cancer tissue section preparation is a breast cancer tissue section fixed to a substrate coated with a conductive coating and a hydrophilic anti-peeling coating. Item 4. The method of any one of Items 1 to 1d and 2 to 3, wherein the matrix is ​​removed from the breast cancer tissue section preparation with methanol and / or 70 to 100% ethanol. Item 5. A method for determining whether to continue administering tamoxifen to the subject, based on the distribution of tamoxifen in the breast cancer tissue section measured by the method of any one of Items 1 to 1d and 2 to 4.Item 6a. (a) measuring a breast cancer tissue section preparation from a subject administered tamoxifen using an imaging mass spectrometry microscope, (b) removing the matrix from the breast cancer tissue section preparation after measurement and staining it with hematoxylin and eosin (HE staining), and (c) comparing the measurement image in (a) with the HE-stained image in (b), (d) administering tamoxifen or a drug other than tamoxifen (an anti-breast cancer drug) to the subject based on the comparison, wherein the image measured using the imaging mass spectrometry microscope is an image showing the distribution of tamoxifen and / or tamoxifen metabolites, and the HE-stained image is an HE-stained image in which cancerous regions and non-cancerous regions are identified, and the distribution of tamoxifen and / or tamoxifen metabolites in cancerous regions and non-cancerous regions is measured by the comparison, and (i) the signal intensity of tamoxifen and / or tamoxifen metabolites in cancerous regions is (i) if the signal intensity of tamoxifen and / or tamoxifen metabolites in the cancerous region is higher than that of tamoxifen and / or tamoxifen metabolites in the non-cancerous region, continue administering tamoxifen to the subject; and (ii) if the signal intensity of tamoxifen and / or tamoxifen metabolites in the cancerous region is equal to or lower than that of tamoxifen and / or tamoxifen metabolites in the non-cancerous region, administer an anti-cancer drug other than tamoxifen (preferably an anti-breast cancer drug) to the subject.Item 6b. (a) measuring a breast cancer tissue section preparation from a subject administered tamoxifen using an imaging mass spectrometry microscope, (b) removing the matrix from the breast cancer tissue section preparation after measurement and staining it with hematoxylin and eosin (HE staining), and (c) comparing the measurement image in (a) with the HE-stained image in (b), (d) administering tamoxifen or a drug other than tamoxifen (an anti-breast cancer drug) to the subject based on the comparison, wherein the image measured using the imaging mass spectrometry microscope is an image showing the distribution of tamoxifen and / or tamoxifen metabolites, and the HE-stained image is an HE-stained image in which cancerous regions and non-cancerous regions are identified, and the distribution of tamoxifen and / or tamoxifen metabolites in cancerous regions and non-cancerous regions is measured by the comparison, and (i) the signal intensity of tamoxifen and / or tamoxifen metabolites in cancerous regions is A method for treating breast cancer, comprising: (i) continuing to administer tamoxifen to the subject when the signal intensity of tamoxifen and / or tamoxifen metabolites in the cancerous region is higher than the signal intensity of tamoxifen and / or tamoxifen metabolites in the non-cancerous region; and (ii) administering an anti-cancer drug other than tamoxifen (preferably an anti-breast cancer drug) to the subject when the signal intensity of tamoxifen and / or tamoxifen metabolites in the cancerous region is equal to or lower than the signal intensity of tamoxifen and / or tamoxifen metabolites in the non-cancerous region.

[0011] The distribution of a drug and / or its metabolites in cancer (particularly breast cancer) tissue can be detected at the tissue and cellular level. Furthermore, the inventors have found that the greater the distribution of the drug and / or its metabolites in cancer tissue (particularly the greater the distribution of tamoxifen and / or its metabolites in breast cancer tissue), the more effective (sensitive) the drug is. This allows the effectiveness of the drug to be determined at the molecular level. Furthermore, if the drug is effective, administration can be continued, and if it is ineffective, a different drug can be switched to, allowing for more appropriate treatment.

[0012] The mass-to-charge ratio (m / z) of various substances in breast cancer tissue detected by the iMScopeQT imaging mass spectrometry microscope is shown. The m / z value at the green triangle position perfectly matches the m / z value of tamoxifen powder measured previously, confirming that the drug was correctly detected in breast cancer tissue. The tamoxifen distribution image obtained by the imaging mass microscope, an HE-stained image, and an optical image of the specimen before matrix deposition are also shown. Also shown are a merged image of the tamoxifen distribution image and the optical image. The HE-stained image and a merged image of the HE-stained image and the tamoxifen distribution image obtained by the imaging mass microscope are shown. Each region of interest (ROI) was set to a 25-pixel range for each cancerous and non-cancerous region, and 10 regions of interest were randomly selected (10 ROIs for a total of 250 pixels). The results of a statistical analysis of tamoxifen signal intensity between these 10 ROIs are shown. An example of this analysis (images of seven specimens) is shown. Distribution images of each tamoxifen metabolite (N-desmethyltamoxifen (NDMTAM), 4-hydroxytamoxifen (4OHTAM), and endoxifen (ENX)) obtained using an imaging mass microscope are shown. Circled numbers 1 to 12 are images obtained from specimens derived from breast cancer patients for whom tamoxifen was pathologically ineffective (TAM ineffective), and circled numbers 13 to 16 are images obtained from specimens derived from breast cancer patients for whom tamoxifen was pathologically effective (TAM effective). The results of statistical analysis of the distribution images of each tamoxifen metabolite in Figure 4 are shown.

[0013] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a method for measuring the distribution of a drug and / or its metabolites in cancer tissue, and a method for determining whether to continue administering the drug based on the measured distribution of the drug and / or its metabolites. The present disclosure includes all of the methods disclosed herein and recognizable by a person skilled in the art.

[0014] A method for measuring the distribution of a drug and / or its metabolites in cancer tissue encompassed by the present disclosure includes (A) measuring a cancer tissue section prepared from a subject to which a drug has been administered using an imaging mass spectrometry microscope. The method for measuring the distribution of a drug and / or its metabolites in cancer tissue may be referred to as the drug distribution measurement method of the present disclosure.

[0015] The cancer may be any cancer for which the drug distribution measurement method of the present disclosure is effective, and solid cancers are preferred. Among solid cancers, epithelial cell cancers are preferred, such as breast cancer, uterine cancer, cervical cancer, ovarian cancer, gastric cancer, colon cancer, lung cancer, and liver cancer. Breast cancer is particularly preferred. Among breast cancers, luminal breast cancer is preferred.

[0016] The subject includes not only human cancer patients but also other mammals suffering from cancer, such as pets and livestock, including dogs, cats, monkeys, mice, rats, horses, cows, sheep, goats, and camels.

[0017] Furthermore, any drug may be used as long as it can exert the effects of the drug distribution measurement method of the present disclosure. Drugs known to have anticancer effects against specific solid cancers can be appropriately selected and used. For example, for breast cancer, hormone drugs (e.g., tamoxifen and toremifene) are preferred, with tamoxifen being more preferred. Among breast cancers, luminal-type breast cancer is preferred, and it is particularly preferred to use hormone drugs for luminal-type breast cancer.

[0018] Cancer tissue slice preparations can be prepared by known methods or methods that can be easily derived from known methods. For example, a 1 cm square portion is taken from cancer tissue, frozen on dry ice, and the frozen specimen is sliced ​​using a cryostat to a thickness of, for example, 5 to 20 μm (preferably 7 to 15 μm, more preferably 8 to 12 μm, and even more preferably 9.5 to 10.5 μm), and then placed on a substrate (e.g., a glass slide). A preferred substrate is, for example, a glass plate (particularly a glass slide).

[0019] Furthermore, the substrate preferably has a conductive coating on its surface, and more preferably has an ITO (Indium Tin Oxide) coating. Furthermore, the substrate preferably has a peel-resistant coating, and examples of such coatings include hydrophobic peel-resistant coatings (e.g., poly-L-lysine (PLL) coating, 3-aminopropyltriethoxysilane (APS) coating, etc.) and hydrophilic peel-resistant coatings, with hydrophilic peel-resistant coatings being particularly preferred. A preferred example of a substrate (glass slide) with a hydrophilic peel-resistant coating is an MAS-coated glass slide (Matsunami Glass Industry Co., Ltd.). Incidentally, MAS coating is a type of hydrophilic peel-resistant technology that imparts amino groups to the glass surface and forms electrostatic bonds between the positive charges of the amino groups and the negative charges of cells / tissues, thereby preventing peeling.

[0020] More preferred substrates (e.g., glass plates, particularly glass slides) are those coated with both a conductive coating and an anti-release coating. For example, a particularly preferred example of such a substrate is a glass slide in which a conductive coating (particularly an ITO coating) is applied to the top surface (the surface on which the tissue section is attached) of the glass slide, and a hydrophilic anti-release coating (particularly an MAS coating) is applied to both sides of the glass slide. Such glass slides can be purchased commercially, and examples of such commercially available products include ITO-coated glass slides, 100Ω, MAS coated (product number: SI0100M) (Matsunami Glass Industry Co., Ltd.).

[0021] The cancer tissue section preparation is preferably a preparation having a structure in which a solid cancer tissue section (5-20 μm), particularly a breast cancer tissue section (5-20 μm), is fixed to a glass slide. It is desirable to use the preferred glass slides described above. In particular, glass slides coated with both a conductive coating and an anti-peeling coating are particularly preferred.

[0022] Cancer tissue (particularly breast cancer tissue) slice preparations are measured using an imaging mass spectrometry microscope. Measurements using an imaging mass spectrometry microscope involve applying a matrix treatment to the tissue slices on the preparation, irradiating them with a laser to simultaneously ionize multiple molecules present at various locations in the tissue, and performing mass analysis (MALDI-TOF MS), thereby obtaining an image of their distribution. An example of an imaging mass spectrometry microscope that can be used is the iMScopeQT (Shimadzu Corporation).

[0023] Known matrices can be used, and preferably CHCA (α-cyano-4-hydroxycinnamic acid) can be used. The matrix treatment can be preferably carried out by, for example, evaporating the matrix onto the slice. For this purpose, a matrix deposition device (e.g., iMLayer (Shimadzu Corporation)) can be used. Although not particularly limited, the thickness of the matrix layer formed on the slice is preferably, for example, about 0.5 to 1 μm.

[0024] MALDI-TOFMS analysis conditions can be set appropriately. For example, for analysis with a spatial resolution of 10 μm, the following conditions can be used: detector voltage of 2.12 kV, laser repetition frequency of 1000 Hz, laser diameter of approximately 10 μm, and laser intensity of 40.

[0025] The drug distribution measurement method of the present disclosure further includes (B) obtaining an image in which cancer and non-cancerous regions are identified in the cancer tissue section preparation. As described below, the drug distribution measurement method of the present disclosure preferably further includes comparing the image with the image measured in step (A), and therefore preferably obtaining an image in which cancer and non-cancerous regions are identified in the same region in the cancer tissue section preparation as the region measured in step (A).

[0026] The method for obtaining such an image that identifies cancerous and non-cancerous regions is not particularly limited, and any known method or method that can be easily derived from known methods can be used. Examples of such methods include (i) hematoxylin-eosin staining (HE staining), (ii) label-free multiphoton microscopy (more specifically, label-free two-photon microscopy) and a method using this in combination with machine learning (e.g., deep learning), (iii) multimodal imaging and a method using this in combination with machine learning (e.g., deep learning) (more specifically, virtual HE staining using ultraviolet photoacoustic remote sensing (Uv-PARS) and scattering microscopy, and machine learning (particularly deep learning)), and (iv) MS imaging. For example, with regard to the method (ii), specific examples are reported in Non-Patent Document 6 (Int. J. Biol. Sci. 2025, 21(1): 363-381. doi: 10.7150 / ijbs.102744) and Non-Patent Document 7 (Cancer Science. 2022;113:2916-2925. DOI: 10.1111 / cas.15428). Specific examples are reported for the method (iii) in Non-Patent Document 8 (Nature Communications (2023) 14:5967). Specific examples are reported for the method (iv) in Non-Patent Document 9 (Theranostics 2020, 10(16): 7070-7082. doi: 10.7150 / thno.45543). These can be used alone or in combination of two or more. When a technique using a combination of machine learning techniques is adopted, either supervised learning or unsupervised learning can be adopted, and when supervised learning is adopted, data obtained by a different technique (e.g., (i)) can be used as supervised learning data.

[0027] Among these, it is preferable to include HE staining of the cancer tissue section preparation, and more preferably to include removing the matrix from the cancer tissue section preparation after measurement and then HE staining.

[0028] To identify cancerous and non-cancerous regions in an image of an HE-stained cancer tissue section, a person (e.g., a pathologist) may input the cancerous and non-cancerous regions into the image based on the HE staining results, or image processing may be used to identify the cancerous and non-cancerous regions based on information such as the color of the HE staining. Machine learning (e.g., deep learning) may also be used.

[0029] It is also possible to HE-stain a cancer tissue section preparation prepared using a section adjacent to the section used to prepare the cancer tissue section preparation subjected to imaging mass spectrometry microscopy, and compare the HE-stained image obtained with the distribution image of the drug and / or its metabolites obtained by imaging mass spectrometry microscopy. However, in this case, images of parts shifted by the thickness of the section are compared, making high-precision analysis at the cellular level difficult (it is difficult to observe the same cells). However, by HE-staining the cancer tissue section preparation subjected to imaging mass spectrometry microscopy, it is possible to compare the HE-stained image of the same tissue / cell with the drug distribution image obtained by imaging mass spectrometry microscopy, enabling high-precision analysis at the cellular level.

[0030] To remove the matrix from the cancer tissue slice preparation after imaging mass spectrometry microscopy, for example, methanol, ethanol, or a mixture of these with water can be used, and in particular, methanol, ethanol, or aqueous ethanol (70 to 100% ethanol) can be used preferably. The amount of these solvents used is not particularly limited, and an amount that can remove the matrix can be appropriately set.

[0031] Furthermore, when matrix removal is performed, it is preferable that the cancer tissue slice preparation be prepared using a substrate (especially a glass slide) that has been coated with an anti-peeling agent. Otherwise, the tissue slice itself may be peeled off and removed during the matrix removal process. Details of the substrate with the anti-peeling agent are as described above.

[0032] The drug distribution measurement method of the present disclosure preferably further includes (C) comparing the image measured by the imaging mass spectrometry microscope in step (A) (sometimes referred to as the image acquired in step (A)) with the image obtained in step (B) (sometimes referred to as the image acquired in step (B)). Note that the image acquired in step (A) and the image acquired in step (B) used for comparison are images acquired from the same region of the cancer tissue section.

[0033] The image comparison can be performed using, for example, known software. Examples of such software include IMAGEREVEAL ＴＭ (Shimadzu Corporation) can be used.

[0034] The image acquired in step (B) can be used to measure cancerous regions (and non-cancerous regions) in tissue sections, and the image acquired in step (A) can be used to measure the distribution of a drug and / or its metabolites in tissue sections, so by comparing these images, the distribution of a drug and / or its metabolites in cancerous regions (and non-cancerous regions) can be measured.

[0035] More specifically, the measurement can be performed, for example, as follows. That is, (I) the distribution of a drug and / or its metabolites in each of the cancer and non-cancerous regions is determined, and (II) the amounts of the drug and / or its metabolites in each of the cancer and non-cancerous regions are compared, thereby measuring the distribution of the drug and / or its metabolites in the cancer (and non-cancerous regions). In this step, for example, the average signal intensity (concentration) per unit area of ​​the drug and / or its metabolites in each of the cancer and non-cancerous regions can be determined and compared. Alternatively, the cancer and non-cancerous regions may each be divided into several regions, and the highest (average) signal intensities may be compared.

[0036] More specifically, for example, a range of specific pixels (e.g., 25 pixels) selected from, for example, 10 to 50 pixels is set per target region (1 ROI) for each cancer region and non-cancerous region, and, for example, 5 to 20 target regions (e.g., 10 target regions) are randomly selected (e.g., for 10 target regions with 25 pixels each, a total of 250 pixels). If a statistical analysis of the signal intensities of the drug and / or its metabolites between these same number of target regions reveals a significant difference (the signal intensities of the drug and / or its metabolites in the cancer region are significantly higher), it can be said that the drug and / or its metabolites are significantly distributed in the cancer region.

[0037] Furthermore, it is preferable that the components compared between cancer and non-cancerous regions are the same compound. For example, when a drug and two of its metabolites (more specifically, drug X, metabolite α of drug X, and metabolite β of drug X) are the measurement target compounds, it is preferable to compare the average signal intensity (concentration) of drug X in the cancerous region with the average signal intensity (concentration) of drug X in the non-cancerous region. It is also preferable to compare the average signal intensity (concentration) of metabolite α in the cancerous region with the average signal intensity (concentration) of metabolite α in the non-cancerous region. It is also preferable to compare the average signal intensity (concentration) of metabolite β in the cancerous region with the average signal intensity (concentration) of metabolite β in the non-cancerous region.

[0038] Furthermore, the inventors have also found that, with regard to the distribution of drugs and / or their metabolites in cancer tissue measured by the drug distribution measurement method of the present disclosure, images from cancer patients in whom the administered drug is barely effective show almost no distribution of the drug and / or its metabolites in the cancer tissue, whereas images from cancer patients in whom the administered drug is highly effective show a high distribution of the drug and / or its metabolites in the cancer tissue. Based on this, it is possible to determine whether to continue administering the drug to the subject based on the distribution of the drug and / or its metabolites in cancer tissue sections measured by the drug distribution measurement method of the present disclosure. In other words, the greater the distribution of the drug and / or its metabolites in the cancer tissue, the more effective the drug is, and it can be determined that administration of the drug should be continued. Furthermore, the less the drug and / or its metabolites are distributed in the cancer tissue, the less effective the drug is, and it can be determined that administration of the drug should not be continued (a different drug (anticancer drug) should be administered). The present disclosure also preferably encompasses such determination methods.

[0039] Specifically, the distribution of a drug and / or its metabolites in cancerous and non-cancerous regions is compared, and if the drug and / or its metabolites are distributed more abundantly in cancerous regions, the drug is determined to have a pathological effect (efficacy). More specifically, for example, if the signal intensity (concentration) of tamoxifen is higher in cancerous regions than in non-cancerous regions in image analysis (more specifically, for example, if it is 1.2 times or more, 1.5 times or more, 2 times or more, 5 times or more, or 10 times or more higher), the drug is determined to be effective. In such a signal intensity comparison, for example, a range of specific pixels (e.g., 25 pixels) selected from, for example, 10 to 50 pixels per region of interest (ROI) is set for each of the cancerous and non-cancerous regions, and the total signal intensity of, for example, 5 to 20 target regions (e.g., 10 target regions) is randomly selected (e.g., 250 pixels in total for 10 target regions with 25 pixels each) can be compared. For example, for each region of interest (ROI) in cancer and non-cancer areas, a specific pixel range (e.g., 25 pixels) selected from 10 to 50 pixels is set, and 5 to 20 target regions (e.g., 10 target regions) are randomly selected (e.g., 10 target regions with 25 pixels each, for a total of 250 pixels). The signal intensities of the drug and / or its metabolites in these equal number of target regions (e.g., 10 target regions in the cancer region and non-cancer regions) are statistically analyzed (e.g., t-test) to determine whether there is a significant difference (signal intensity of the drug and / or its metabolites in the cancer region is significantly higher). Significance here refers to, for example, a p-value of less than 0.01, less than 0.02, less than 0.05, or less than 0.1. Whether the signal intensity is (significantly) high may be determined, for example, by image processing, machine learning, or the like.

[0040] Furthermore, the drug and its metabolite distributions in cancer tissue measured by the drug distribution measurement method of the present disclosure can be determined by combining multiple distributions. Drug distributions and their metabolite distributions can be used in combination. One type of drug metabolite distribution can be used alone, or two or more types can be used in combination. For example, when the distribution of drug X (X distribution), the distribution of drug X's metabolite α (α distribution), and the distribution of drug X's metabolite β (β distribution) are measured, the determination of whether to continue administering the drug to the subject can be based on only the X distribution, only the α distribution, or only the β distribution. Alternatively, the determination of whether to continue administering the drug to the subject can be based on any two or three of the X distribution, α distribution, and β distribution. In particular, when there is no clear difference in the distribution of a particular drug or its metabolites between cancerous and non-cancerous regions, the accuracy of the determination can be improved by making a determination based on the distribution of the drug and / or its metabolites in multiple cancer tissue sections.

[0041] Furthermore, the distribution of a drug and / or its metabolites that does not show a clear difference between cancer and non-cancerous regions can be excluded from the distribution used for assessment (not used for assessment). Drugs and / or their metabolites that show such a distribution can be easily selected by confirming the distribution using the above-mentioned method. Furthermore, when a metabolite is the compound itself that exhibits pharmacological activity (in other words, when the drug is a prodrug), it is preferable to use the distribution of the metabolite as the distribution for assessment.

[0042] In particular, a more detailed explanation will be given using as an example a case where the cancer tissue is breast cancer tissue and the administered drug is tamoxifen.

[0043] For the above-mentioned determination, the distribution of tamoxifen and / or the distribution of tamoxifen metabolites may be used alone or in combination of two or more. Tamoxifen has several known metabolites, and any of the known metabolites may be used as the measurement target. Among them, 4-hydroxytamoxifen (4OHTAM) and endoxifen (ENX) are preferably used. Endoxifen is an active metabolite of tamoxifen. For the above-mentioned determination, it is preferable to use at least one distribution selected from the group consisting of the distribution of tamoxifen, the distribution of 4-hydroxytamoxifen (4OHTAM), and the distribution of endoxifen (ENX).

[0044] The present disclosure also encompasses a method for treating cancer, comprising administering to the subject the drug or a drug other than the drug based on the comparison. If the comparison determines that administration of the drug should be continued, the drug is administered, and if the comparison determines that administration of the drug should not be continued (a different drug should be administered), a drug other than the drug is preferably administered. Note that the drug other than the drug is preferably a drug other than the drug (anticancer drug) known to be effective against the target cancer.

[0045] For example, when it is determined that administration of tamoxifen should not be continued, it is preferable to administer an anticancer drug (particularly an anti-breast cancer drug) other than tamoxifen.

[0046] It should be noted that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses any and all combinations of the constituent elements described in this specification.

[0047] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0048] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0049] 1. Investigation of tamoxifen distribution in breast cancer tissue using imaging mass spectrometry microscopy. A breast cancer (luminal type) patient who had been taking 20 mg of tamoxifen orally once daily from before surgery until the day of surgery underwent radical breast tumor surgery. The surgical tissue specimens obtained from the surgery were analyzed. A 1 cm cube was taken from the breast cancer tissue excised during surgery and frozen on dry ice as quickly as possible. To prevent air bubbles from forming inside the tissue, the specimens were frozen from one side.

[0050] Fresh-frozen specimens were sliced ​​into 10-μm-thick slices using a cryostat and placed on conductive ITO-coated slides. For the HE staining analysis of the same specimens described below, ITO-coated slides, 100Ω MAS-coated (product number SI0100M) (Matsunami Glass Industry Co., Ltd.), were used.

[0051] The prepared specimen (breast cancer tissue section preparation) was uniformly deposited with CHCA (α-cyano-4-hydroxycinnamic acid), a matrix for MALDI-TOFMS, to a thickness of 0.7 μm using a matrix deposition system, iMLayer. The CHCA-deposited specimen was then analyzed by MALDI-TOFMS using an imaging mass microscope, iMScopeQT (Shimadzu Corporation). For analysis with a spatial resolution of 10 μm, the detector voltage was set to 2.12 kV, the laser repetition rate to 1000 Hz, the laser diameter to approximately 10 μm, and the laser intensity to 40.

[0052] Next, the measured tamoxifen distribution was compared with the breast cancer cell distribution in the tissue. First, a specimen (breast cancer tissue section preparation) prepared using a section adjacent to the section used to prepare the specimen (breast cancer tissue section preparation) was stained with hematoxylin and eosin (HE stain).

[0053] These results are shown in Figures 1 and 2.

[0054] Figure 1 shows the mass-to-charge ratio (m / z) of various substances in breast cancer tissue as detected by the imaging mass spectrometry microscope iMScopeQT. The m / z values ​​at the positions indicated by downward triangles (▼) perfectly matched the m / z values ​​of tamoxifen powder measured in advance, confirming that the drug was correctly detected in breast cancer tissue.

[0055] Figure 2 shows an optical image of the specimen after matrix deposition, a tamoxifen distribution image obtained by imaging mass microscopy, and an HE-stained image obtained as described above. Also shown is a merged image of the optical image and the tamoxifen distribution image. Image analysis was performed using IMAGEREVEAL. ＴＭ (Shimadzu Corporation) was used. In Fig. 2, SA to SG indicate analyses of different specimens (breast cancer tissue section preparations).

[0056] The yellow areas in the MS image (tamoxifen distribution image) indicate the distribution of tamoxifen administered preoperatively to breast cancer patients within the tissue (the white areas in the image converted to a black-and-white image indicate the distribution of tamoxifen administered preoperatively to breast cancer patients within the tissue). The distribution of tamoxifen within the tissue closely matched the cancer tissue area in the HE-stained image (the areas identified as cancerous tissue in the HE-stained image closely matched the areas where tamoxifen was distributed in the MS image). Furthermore, it was observed that a large amount of tamoxifen was distributed within the cancer tissue of cases in which tamoxifen was effective (tumor size reduction), while only a small amount was distributed within the cancer tissue of cases in which tamoxifen was ineffective (tumor size increase). From this, it was inferred that the greater the distribution of tamoxifen within the cancer tissue, the greater the effectiveness of tamoxifen.

[0057] 2. Investigation of the Acquisition of Tamoxifen Distribution Images and HE Images from the Same Specimen. Furthermore, to perform analysis at even higher resolution, we attempted to perform HE staining analysis on the same specimen used for imaging mass spectrometry microscopy analysis. To this end, we first attempted to remove the deposited matrix (CHCA) from the specimen (breast cancer tissue section preparation) used for imaging mass spectrometry microscopy analysis. When we investigated removal by washing with 100% ethanol, we found that in specimens (breast cancer tissue section preparations) prepared using standard glass slides, not only the matrix but also the main tissue samples were removed. Therefore, after extensive investigation, we found that specimens (breast cancer tissue section preparations) prepared using ITO-coated glass slides 100Ω MAS Coat (product number: SI0100M) (Matsunami Glass Industry Co., Ltd.), which were coated with a hydrophilic anti-stripping coating, MAS Coat, could be gently removed without tissue peeling by using 100% methanol and ethanol diluted stepwise from 100% to 70%. This enabled HE staining to be performed after matrix removal. The measured tamoxifen distribution image and HE stained image were then imaged using IMAGEREVEAL. ＴＭ(Shimadzu Corporation) and the analysis results are shown in Figure 3A.

[0058] Figure 3A shows an HE-stained image and an image obtained by superimposing (merging) the HE-stained image with a tamoxifen distribution image obtained using an imaging mass microscope. Because the tamoxifen distribution image and the HE image were obtained from the same specimen, when they were superimposed, it became possible to analyze with high accuracy whether tamoxifen was distributed in the cancer tissue. (As mentioned above, when HE staining was performed on a specimen prepared using a section adjacent to the section used for the specimen subjected to imaging mass microscope analysis, there was a problem that analysis at the cellular level was difficult (it was difficult to observe the same cells) due to the difference in thickness of the section. However, this method made it possible to analyze the same cells.)

[0059] Furthermore, in images from breast cancer patients in whom tamoxifen was pathologically ineffective (TAM ineffective), tamoxifen was hardly distributed in the breast cancer tissue, whereas in images from breast cancer patients in whom tamoxifen was pathologically effective (TAM effective), tamoxifen was distributed in large amounts in the breast cancer tissue. More specifically, in the analysis of images from TAM-ineffective breast cancer patients, tamoxifen was hardly observed in the merged image (HE staining + tamoxifen distribution) in both areas of the HE-stained image that were judged to be cancerous tissue (cancer area) and areas that were judged not to be cancerous tissue (non-cancerous area). On the other hand, in the analysis of images from TAM-effective breast cancer patients, it was observed that in the areas of the HE-stained image judged to be cancerous tissue, a large amount of tamoxifen was distributed, even in the merged image (HE-stained + tamoxifen distribution) (the areas shown in yellow are the areas where tamoxifen is distributed. In the case of black and white images, the areas shown in black in the HE-stained image are roughly the areas judged to be cancerous tissue in this analysis, and in the merged image, it is observed that a large amount of white areas are distributed in those areas, and this distribution indicates the distribution of tamoxifen.) From this, it was thought that there was a correlation between the distribution of tamoxifen in cancerous areas and the pathological effects of tamoxifen.

[0060] The analysis of this distribution is described in more detail below. The determination of cancerous and non-cancerous regions in the HE-stained images was performed by a pathologist. Furthermore, the pathologist determined that the treatment was effective when morphological changes were pathologically observed in the cancer cells or surrounding stroma in the HE-stained images. (However, these determinations can also be made using known methods other than those performed by a pathologist. For example, label-free multiphoton microscopy, multimodal imaging, MS imaging, etc. can be used, and these methods can also be combined with machine learning (e.g., deep learning).)

[0061] In addition, a 25-pixel region of interest (ROI) was set for each cancer and non-cancerous region, and 10 regions of interest were randomly selected (10 ROIs, a total of 250 pixels). The tamoxifen signal intensity between these 10 ROIs was statistically analyzed. Significant differences (t-test: p<0.05) were determined to indicate distribution of tamoxifen in the cancerous region. Examples of this analysis and determination (images of seven specimens) are shown in Figure 3B. Each of the seven specimens (a-g) was analyzed, and in each analysis, significant differences were found between the cancerous and non-cancerous regions (the left side of the graph showing the statistical analysis is a graph plotting 10 ROIs from the cancerous region, and the right side is a graph plotting 10 ROIs from the non-cancerous region). Therefore, tamoxifen was determined to be distributed in the cancerous region.

[0062] In addition, as mentioned above, the distribution of tamoxifen in cancerous regions is considered to be correlated with the pathological effects of tamoxifen, so it is also considered possible to determine the pathological effects of tamoxifen from the distribution of tamoxifen in cancerous regions.In other words, by comparing the distribution of tamoxifen in cancerous regions and non-cancerous regions, it is considered possible to determine that tamoxifen has a pathological effect if tamoxifen is distributed more in cancerous regions than in non-cancerous regions.More specifically, for example, in image analysis, if the signal intensity (concentration) of tamoxifen in cancerous regions is higher than that in non-cancerous regions (more specifically, for example, 1.2 times, 1.5 times, 2 times, 5 times, or 10 times or more), it is considered possible to determine that tamoxifen is effective.In addition, for example, the above-mentioned analysis method for the distribution of tamoxifen in cancerous regions and non-cancerous regions can be used as is for such image analysis.In this analysis method, it is considered possible to determine that tamoxifen is effective if the signal intensity (concentration) of tamoxifen in cancerous regions is significantly higher than that in non-cancerous regions. Here, significance refers to, for example, a p-value of less than 0.01, less than 0.02, less than 0.05, or less than 0.1. Whether the signal intensity is (significantly) high may be determined by, for example, image processing or machine learning. Furthermore, an example of statistical processing is a t-test.

[0063] Based on the above, it became possible to confirm the distribution of tamoxifen with high accuracy (at the same cell level) and determine the effectiveness of tamoxifen.

[0064] 3. Investigation into the acquisition of tamoxifen metabolite distribution images and HE images from the same specimen In the above-mentioned imaging mass spectrometry microscope analysis, for tamoxifen metabolites, similar to tamoxifen shown in Figure 1, peaks whose mass / charge ratio (m / z) matched the mass / charge ratio (m / z) of the tamoxifen metabolites were confirmed, and then a distribution image of the tamoxifen metabolites was acquired.

[0065] The specific tamoxifen metabolites analyzed were the known tamoxifen metabolites N-desmethyltamoxifen (NDMTAM), 4-hydroxytamoxifen (4OHTAM), and endoxifen (ENX).

[0066] For each tamoxifen metabolite distribution, images were acquired for 16 specimens. Specifically, for each tamoxifen metabolite distribution, images were acquired from 12 specimens from breast cancer patients with pathologically ineffective tamoxifen (TAM ineffective) and images were acquired from 4 specimens from breast cancer patients with pathologically effective tamoxifen (TAM effective). These images are shown in Figure 4.

[0067] NDMTAM was detected regardless of whether pathological tamoxifen was effective or not, and its distribution was consistent with that of cancer, whereas 4OHTAM and ENX were detected only in the pathological tamoxifen effective group, and their distribution was consistent with that of cancer.

[0068] Figure 5 shows the results of statistical analysis of the images obtained from these 16 specimens. Specifically, for each tamoxifen metabolite, the number of specimens in which distribution (accumulation) in the cancer region was observed (+) and the number of specimens in which accumulation was not observed (-) are shown as bar graphs. Each bar in the graph indicates the number of specimens in which pathological tamoxifen was effective (Effect(+)) and the number of specimens in which pathological tamoxifen was not effective (Effect(-)). The results (p-values) of statistical analysis (Fisher's exact test) for the correlation between the pathological effect of tamoxifen and the distribution intensity of each tamoxifen metabolite are also shown.

[0069] The determination of cancerous and non-cancerous areas, the determination of whether the treatment was effective, and the determination of whether tamoxifen metabolites were distributed in the cancerous area were performed in the same manner as in the analysis for tamoxifen described above.

[0070] Based on the above, for example, by dividing the obtained image into cancerous and non-cancerous regions based on HE staining, determining the distribution of tamoxifen or its metabolites in each of the cancerous and non-cancerous regions, and comparing the amounts of tamoxifen or its metabolites in each of the cancerous and non-cancerous regions, it is expected that the effect of tamoxifen can be measured based on tamoxifen or its metabolites (particularly 4OHTAM and ENX).

[0071] For example, the average signal intensity (concentration) per unit area of ​​tamoxifen or its metabolites in the cancerous and non-cancerous regions can be determined and compared. Alternatively, the cancerous and non-cancerous regions can be divided into several regions, and the highest (average) signal intensities can be compared.

Claims

1. A method for measuring the distribution of a drug and / or its metabolites in a solid cancer tissue slice, comprising: (A) measuring a solid cancer tissue slice preparation from a subject to which a drug has been administered using an imaging mass spectrometry microscope; (B) obtaining an image that identifies cancerous and non-cancerous regions in the solid cancer tissue slice preparation; and (C) comparing the image measured in (a) with the image obtained in (B).

2. A method for measuring the distribution of tamoxifen and / or its metabolites in breast cancer tissue sections, comprising: (a) measuring a breast cancer tissue section preparation from a subject administered tamoxifen using an imaging mass spectrometry microscope; (b) removing the matrix from the breast cancer tissue section preparation after measurement and staining with hematoxylin and eosin (HE staining); and (c) comparing the measurement image in (a) with the HE stained image in (b).

3. The method according to claim 2, wherein the image measured by the imaging mass spectrometry microscope is an image showing the distribution of tamoxifen and / or tamoxifen metabolites.

4. The method according to claim 2 or 3, wherein the HE-stained image is an HE-stained image in which cancerous and non-cancerous regions are identified.

5. The method of claim 2 or 3, wherein the comparison measures the distribution of tamoxifen and / or tamoxifen metabolites in cancerous and non-cancerous areas.

6. The method of claim 2 or 3, wherein the subject is a human breast cancer patient.

7. The method according to claim 2 or 3, wherein the breast cancer tissue section preparation is a breast cancer tissue section fixed to a substrate having a conductive coating and a hydrophilic anti-peeling coating.

8. The method according to claim 2 or 3, wherein the matrix is ​​removed from the breast cancer tissue section preparation using methanol and / or 70 to 100% ethanol.

9. A method for determining whether to continue administering tamoxifen to a subject based on the distribution of tamoxifen and / or its metabolites in a breast cancer tissue section measured by the method of claim 2 or 3.

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