WO2018025869A1 - Biomarker for predicting therapeutic effect of fstl1 inhibitor in cancer patient - Google Patents

Abstract

A method for predicting the therapeutic effect of a FSTL1 inhibitor in a cancer patient, said method being characterized by comprising the steps of: (1) measuring the expression level of FSTL1 and/or DIP2A in a sample collected from the patient; (2) comparing a measurement value with a reference value; and (3) determining that the therapeutic effect is promised when the measurement value is higher than the reference value. For example, according to the present invention, a companion diagnosis for predicting the efficacy or the like of a treatment with a FSTL1 inhibitor can be performed suitably.

Description

Biomarkers for predicting therapeutic effects of FSTL1 inhibitors in cancer patients

The present invention relates to a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient, a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient, and a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient. It relates to the kit.

The purpose of companion diagnosis is to assist the optimal medication by examining biomarkers related to the efficacy and side effects of the drug before the drug is administered and predicting the patient's individual responsiveness to the drug before the treatment. . There are no limitations on the testing method, and genetic abnormality testing, gene expression testing, tissue testing of proteins and metabolites, blood component testing, and the like are used. Pharmaceutical companies around the world are developing companion diagnostics using biomarkers at the same time as developing new drugs. In the oncology field, the development of innovative therapies such as cancer immunotherapy and cancer vaccine therapy using immune checkpoint inhibitors is progressing, and the effectiveness of specific drugs and the individual differences in the occurrence of side effects are ascertained. The establishment of an appropriate companion diagnostic method that assists in determining drug validity and dosage will continue to be an increasingly important issue for research and development of new anticancer drugs.

Immunosuppression has become known as the cause of cancer deterioration, and releasing immunosuppression leads to effective treatment of cancer. Research and development of cancer immunotherapy with immune checkpoint inhibitors has greatly advanced. ing. In Patent Document 1, FSTL1 (Follistatin-like protein 1) was identified as one of the molecules involved in immunosuppression, and it was reported that an inhibitor of FSTL1 could be a useful anticancer agent. According to previous studies, FSTL1 is secreted from cancer cells and cancer-related mesenchymal stem cells (hereinafter referred to as “MSC”), acts on MSCs in autocrine, and promotes MSC proliferation. However, it has been clarified that immunosuppression is induced through the T cell immune control system and causes cancer deterioration, and that FSTL1 also induces metastasis of cancer cells, particularly acquisition of bone metastasis ( Non-patent document 1).

FSTL1 is secreted mainly in the arthritic joint matrix, and it has been reported that FSTL1 secretion is stimulated by IL-1β and that FSTL1 can be a useful biomarker for systemic juvenile rheumatoid arthritis (non- Patent Document 2) and FSTL1 as a biomarker for inflammatory diseases such as juvenile rheumatoid arthritis and Kawasaki disease have been filed (Patent Documents 2 and 3).

FSTL1 binds to various receptors through the cell membrane (FSTL1 receptor) and affects various physiological actions (Non-patent Document 3). Among them, DIP2A (disco-interacting protein 2 homolog A) is an FSTL1 receptor present in the endothelial cell membranes of various tissues (Non-patent Document 4), and according to the human protein expression information database (Human Protein Inatlas), In normal tissues, expression is observed in some tissues such as smooth muscle, female genital organs, gastrointestinal tract, and in cancer tissues, it is detected in colorectal cancer, breast cancer, prostate cancer, lung cancer, liver cancer and the like.

One of the challenges of cancer treatment is to identify whether the tumor is a malignant tumor with high metastasis by biopsy, and to determine the appropriate treatment policy based not only on the primary lesion but also on the presence or possibility of metastasis. Is to decide. An anti-FSTL1 antibody that is an FSTL1 inhibitor not only inhibits MSC that induces immune disruption, but also inhibits metastasis of cancer cells, and thus may be an effective anticancer agent against malignant tumors that are prone to metastasis. Expected. Furthermore, with the development of anti-FSTL1 antibody cancer therapeutics, the development of biomarkers useful for companion diagnosis, such as the effectiveness of anti-FSTL1 antibody treatment, is strongly desired.

International Publication No. 2009/028411 International Publication No. 2009/097424 International Publication No. 2012/019099

The present invention finds a biomarker useful for companion diagnosis for selecting a cancer patient that can be expected to have a therapeutic effect by an FSTL1 inhibitor, a method for predicting the therapeutic effect by an FSTL1 inhibitor in a cancer patient, It is an object of the present invention to provide a method for determining the start of administration of an FSTL1 inhibitor and a kit for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for predicting a therapeutic effect of an FSTL1 inhibitor in a cancer patient,
(1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient;
(2) a step of comparing the measured value with a reference value, and (3) a step of determining that a therapeutic effect can be expected when the measured value is higher than the reference value,
A method comprising the steps of:
[2] The method according to [1], wherein in the step (1), the expression levels of both FSTL1 and DIP2A are measured.
[3] The method according to [1] or [2], wherein the sample is a sample containing cancer cells of a patient or blood of the patient.
[4] The method according to [3] above, wherein the sample containing cancer cells of the patient is a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
[5] In the step (1), the expression level of FSTL1 and / or DIP2A and the blood FSTL1 concentration in a biopsy cancer tissue or a cancer tissue obtained by excision are measured and the blood FSTL1 concentration is measured. ] Method.
[6] The method according to any one of [1] to [5], wherein the expression level is measured immunohistochemically in the step (1).
[7] A method for determining the start of administration of an FSTL1 inhibitor to a cancer patient,
(1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient;
(2) comparing the measured value with a reference value, and (3 ′) determining the start of administration of the FSTL1 inhibitor when the measured value is higher than the reference value,
A method comprising the steps of:
[8] The method according to [7], wherein in the step (1), the expression levels of both FSTL1 and DIP2A are measured.
[9] The method according to [7] or [8] above, wherein the sample is a sample containing cancer cells of the patient or the blood of the patient.
[10] The method according to [9] above, wherein the sample containing cancer cells of the patient is a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
[11] In the step (1), the expression level of FSTL1 and / or DIP2A and the blood FSTL1 concentration in a biopsy cancer tissue or a cancer tissue obtained by excision are measured, and the blood FSTL1 concentration is measured [7] ] Method.
[12] In the step (1), the expression level of FSTL1 and / or DIP2A and the blood DIP2A soluble type concentration in a biopsy cancer tissue or a cancer tissue obtained by excision are measured. The method according to [7] above.
[13] The method according to any one of [7] to [12], wherein the expression level is measured immunohistochemically in the step (1).
[14] A kit for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient, comprising a reagent for measuring the expression level of FSTL1 and / or DIP2A in a sample collected from the patient.
[15] The above-mentioned [14], wherein the reagent contains one or more selected from anti-FSTL1 antibody, anti-DIP2A antibody, FSTL1 RT-PCR primer set, and DIP2A RT-PCR primer set Kit.

The present invention also includes the following aspects.
[16] Use of FSTL1 and / or DIP2A for predicting a therapeutic effect by an FSTL1 inhibitor in a cancer patient.
[17] Use of FSTL1 and / or DIP2A to determine the start of administration of an FSTL1 inhibitor to a cancer patient.
[18] A method for providing information on the therapeutic effect of an FSTL1 inhibitor against cancer, comprising measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a cancer patient.
[19] A method for providing the cancer patient with information on when to start administration of an FSTL1 inhibitor, which comprises measuring the expression level of FSTL1 and / or DIP2A in a sample collected from the cancer patient.

According to the present invention, a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient, a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient, and a method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient Kits can be provided.

Further, according to the present invention, it becomes possible to predict the therapeutic effect of an FSTL1 inhibitor in a cancer patient, so that the therapeutic effect in a cancer-related disease can also be predicted. Examples of cancer-related diseases include diseases that are highly likely to become cancer and diseases that exacerbate cancer. Examples include obesity, diabetes, and rheumatism.

Human pancreatic cancer cell line Panc1 empty vector introduced cell line (Panc1-mock), human pancreatic cancer cell line Panc1 Snail forced expression cell line (Panc1-snail +), human breast cancer low metastatic cell line MCF7 and human breast cancer high It is a figure which shows the result of having analyzed the expression of FSTL1 and DIP2A in the bone metastatic cell line MDA231 by immunohistochemical staining. FSTL1 in mouse melanoma B16-F10 empty vector-introduced cell line (F10-mock), mouse melanoma B16-F10 Snail forced expression cell line (F10-snail +), mouse colon cancer cell line CT26 and mouse lung cancer cell line 3LL It is a figure which shows the result of having analyzed the expression of DIP2A by immunohistochemical fluorescence dyeing | staining. It is a figure which shows the result of having administered the anti-FSTL1 antibody to the Snail forced expression cell transplant bone transplantation model of mouse melanoma B16-F10, and evaluating the inhibitory effect with respect to subcutaneous tumor growth. Results of administration of anti-FSTL1 antibody to mouse melanoma B16-F10 Snail forced expression cell transplanted bone metastasis model, and evaluation of bone metastasis effect (left), MSC increase effect (middle) and weight loss suppression effect (right) FIG. It is a figure which shows the result of having administered anti-FSTL1 antibody to the mouse | mouth colorectal cancer CT26 cell transplant bone metastasis model, and evaluating the inhibitory effect with respect to subcutaneous tumor growth. It is a figure which shows the result of having administered the anti-FSTL1 antibody to the mouse | mouth colorectal cancer CT26 cell transplant bone metastasis model, and evaluating the inhibitory effect with respect to lung metastasis. It is a figure which shows the result of having administered the anti-FSTL1 antibody to the mouse | mouth lung cancer 3LL cell transplant bone metastasis model, and evaluating the inhibitory effect with respect to subcutaneous tumor growth. It is a figure which shows the result of having administered the anti-FSTL1 antibody to the mouse | mouth lung cancer 3LL cell transplant bone metastasis model, and evaluating the inhibitory effect with respect to a weight loss. The figure which shows the result of having analyzed the expression level of FSTL1 and DIP2A in the tissue section derived from the patient of a breast cancer, a liver cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, and a prostate cancer by immunohistochemical fluorescent staining and pixel count. is there. It is a figure which shows the result of having analyzed the correlation of the FSTL1 expression level and the DIP2A expression level in the tissue slice derived from the patient of a breast cancer, a liver cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, and a prostate cancer. FSTL1 expression level, DIP2A expression level and FSTL1 / DIP2A co-expression level in tissue sections derived from patients with breast cancer, liver cancer, bladder cancer, ovarian cancer, pancreatic cancer, prostate cancer according to the stage of cancer progression It is a figure which shows the result of having analyzed. It is a figure which shows the result of having analyzed the expression level of FSTL1 and DIP2A in the tumor tissue derived from an advanced stage melanoma patient by immunohistochemical fluorescence staining and pixel count. It is a figure which shows the result of having analyzed the correlation of the FSTL1 expression level and DIP2A expression level in the tumor tissue derived from an advanced stage melanoma patient. It is a figure which shows the result of having measured the FSTL1 density | concentration in the serum of various terminal cancer patients (stage IV). It is a figure which shows the result of having administered the anti-FSTL1 antibody to the mouse | mouth osteosarcoma cell transplant model, and evaluating the inhibitory effect with respect to tumor growth. It is a figure which shows the result of having administered the anti-FSTL1 antibody to the mouse | mouth osteosarcoma cell transplant model, and measuring the FSTL1 density | concentration in serum.

As a result of analyzing the expression of FSTL1 and DIP2A in the high metastasis human cancer cell line and the low metastasis human cancer cell line, the present inventors strongly expressed both FSTL1 and DIP2A in the high metastasis human cancer cell line. I found out. Therefore, it has been found that the expression of FSTL1 and / or DIP2A can be used as an index as a biomarker for detecting highly metastatic cancer. Furthermore, both FSTL1 and DIP2A are strongly expressed in mouse cancer cell lines (B16-F10, CT26 and 3LL) in which anti-FSTL1 antibody has been confirmed to be successful in a tumor-bearing mouse model. confirmed. Therefore, it was thought that the therapeutic effect by an anti-FSTL1 antibody can be expected with respect to a cancer patient who develops cancer that highly expresses either or both of FSTL1 and DIP2A. Furthermore, when the serum FSTL1 concentration of various cancer patients was measured, it was shown that the serum FSTL1 concentration of end-stage skin cancer patients, particularly melanoma patients, was high. From these findings, the DIP2A expression level and the FSTL1 expression level in the cancer tissue of the cancer patient and the serum FSTL1 concentration predict the therapeutic effect by the FSTL1 inhibitor in the cancer patient, and the therapeutic effect by the FSTL1 inhibitor can be expected. It was revealed to be a useful biomarker for companion diagnosis to select cancer patients.

[Method for predicting therapeutic effect of FSTL1 inhibitor in cancer patients]
The present invention provides a method for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients. The therapeutic effect prediction method of the present invention only needs to include the following steps (1) to (3).
(1) A step of measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient (2) A step of comparing the measured value with a reference value (3) A therapeutic effect can be expected when the measured value is higher than the reference value The therapeutic effect prediction method of the present invention may include steps other than the above steps (1) to (3) as long as the object of the present invention can be achieved, and the content thereof is not limited.

There are no particular limitations on the type of cancer of the cancer patient for which the therapeutic effect is predicted, including skin cancer, colon cancer, breast cancer, lymphoma, lung cancer, prostate cancer, kidney cancer, head and neck cancer, and esophagus. Cancer, stomach cancer, pancreatic cancer, liver cancer, biliary tract cancer, spleen cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, brain central nervous system tumor, sarcoma, leukemia, multiple Myeloma and the like. Preferred are skin cancer (particularly melanoma), colon cancer, breast cancer, lung cancer, liver cancer, bladder cancer, ovarian cancer and pancreatic cancer.

The FSTL1 inhibitor is not particularly limited as long as it reduces the biological activity of FSTL1, and examples thereof include a substance that inhibits the expression of FSTL1, a substance that inhibits the binding between FSTL1 and its receptor, and the like. Examples of the substance that inhibits the expression of FSTL1 include a nucleic acid that inhibits the expression of FSTL1. Examples of such a nucleic acid include siRNA (shortｓinterfering RNA), shRNA (short hairpin RNA), antisense oligonucleotide and the like of the FSTL1 gene. Examples of the substance that inhibits the binding between FSTL1 and its receptor include an antibody (anti-FSTL1 antibody) or peptide that specifically binds to FSTL1, or an antibody or peptide that specifically binds to the FSTL1 receptor. A substance that inhibits the binding between FSTL1 and its receptor is preferable, and an anti-FSTL1 antibody is more preferable.

In step (1), the expression level of FSTL1 and / or DIP2A in a sample collected from a patient is measured. The sample collected from the patient is not particularly limited as long as it can measure the expression level of FSTL1 or the expression level of DIP2A. For example, tissues obtained by biopsy or surgery; blood, tissue fluid, lymph fluid, bone marrow, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, ascites, pleural effusion, etc .; nasal cavity, bronchial, skin, Examples of the washing liquid after washing various organs and bones; A sample containing cancer cells or blood is preferred. The sample containing cancer cells is preferably a biopsy cancer tissue or a cancer tissue obtained by excision. Moreover, when measuring the expression level of both FSTL1 and DIP2A in the step (1), the sample for measuring the expression level of FSTL1 and the sample for measuring the expression level of DIP2A may be the same or different. If it is a sample which can measure an expression level, it will not specifically limit. For example, an embodiment in which the sample for measuring the expression level of FSTL1 is blood containing cancer cells, and the sample for measuring the expression level of DIP2A is a biopsy cancer tissue or a cancer tissue obtained by excision surgery. It is done.

In step (1), only the expression level of FSTL1 may be measured or only the expression level of DIP2A may be measured, but it is preferable to measure the expression levels of both FSTL1 and DIP2A. It has been confirmed that the accuracy of predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients is improved by judging the combination of both expression levels. Moreover, since FSTL1 is a secreted protein, the expression level of FSTL1 can be evaluated based on the blood FSTL1 concentration. Therefore, as an aspect of measuring the expression level of FSTL1, only the expression level of FSTL1 in a sample containing cancer cells (cancer tissue) may be measured, or only the FSTL1 concentration in blood may be measured. May be measured. Preferably, both the expression level of FSTL1 and the blood FSTL1 concentration in a sample containing cancer cells (cancer tissue) are measured. In this specification, the measurement of the expression level of FSTL1 and / or DIP2A may be an aspect of measuring the level of FSTL1 and DIP2A itself, or an aspect of measuring their soluble type levels. . Therefore, as an aspect of the step (1), an aspect of measuring only the expression level of FSTL1, an aspect of measuring only the blood FSTL1 concentration, an aspect of measuring only the expression level of DIP2A; the expression level of FSTL1 and the blood FSTL1 concentration An embodiment for measuring both FSTL1 expression level and DIP2A expression level, an embodiment for measuring blood FSTL1 concentration and DIP2A expression level; an FSTL1 expression level, DIP2A expression level and blood FSTL1 concentration The aspect which measures 3 types, the aspect which measures 3 types, the expression level of FSTL1, the expression level of DIP2A, and a blood DIP2A soluble type density | concentration are mentioned.

A method for measuring the expression level in a sample containing cancer cells is not particularly limited, a method for measuring the amount of target protein in the sample, and a method for measuring the amount of mRNA (target mRNA) of a gene encoding the target protein in the sample. A method for measuring the expression level using an immunohistochemical method can be preferably used. As a sample containing cancer cells, it is preferable to use a biopsy cancer tissue or a cancer tissue obtained by excision surgery. For example, the amount of target protein can be measured by extracting a protein from a sample by a known method and using a known protein measurement method such as Western blotting, EIA, ELISA, or RIA. The target mRNA amount is measured by extracting RNA from a sample by a known method and using a known mRNA amount measuring method such as Northern blotting, RT-PCR, quantitative RT-PCR, or RNase protection assay. be able to. The RT-PCR method or the quantitative RT-PCR method is preferable, and the quantitative RT-PCR method is more preferable.

When measuring the expression level of a target protein using an immunohistochemical method, a method of preparing a tissue specimen according to a conventional method and performing immunostaining using an antibody that specifically binds to the target protein can be suitably used. . The sample is preferably a biopsy cancer tissue or a cancer tissue obtained by excision surgery. For immunostaining, any of the fluorescent antibody method, the enzyme antibody method, and the metal-labeled antibody method may be used, but the fluorescent antibody method is preferred. The fluorescent antibody method may be performed by a direct method or an indirect method. When the target protein is stained by the fluorescent antibody method, the fluorescence intensity of the antibody on the specimen indicates the expression level of the target protein. The fluorescence intensity can be measured using a commercially available pixel counter or the like. Therefore, the expression level of FSTL1 can be measured by immunostaining using an anti-FSTL1 antibody, and the expression level of DIP2A can be measured by immunostaining using an anti-DIP2A antibody. In one specimen, FSTL1 and DIP2A can also be immunofluorescently stained using antibodies labeled with different fluorescent substances (double staining). The in situ hybidization method can be used to measure the amount of target mRNA in the tissue specimen.

The blood FSTL1 concentration can be measured, for example, by separating serum from blood collected from a patient and quantifying the serum FSTL1 concentration using a known method such as ELISA. It has been confirmed that the blood FSTL1 concentration is high in patients suffering from highly metastatic cancer and advanced in degree of progression. Therefore, it can be expected that the therapeutic effect of the FSTL1 inhibitor in the metastatic lesion is predicted by performing the determination by combining the measured values of the blood FSTL1 concentration. Specifically, for example, in a cancer patient whose primary lesion has been completely removed, when the FSTL1 and / or DIP2A expression level of the resected cancer tissue is high and the blood FSTL1 concentration is also high, the primary lesion has already been removed. Even if it is done, it can be expected to predict the therapeutic effect of the FSTL1 inhibitor on the metastatic lesion. Therefore, it is considered that the accuracy of predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients is further improved by evaluating the combination of the expression level of FSTL1 and / or DIP2A and the blood FSTL1 concentration.

The amino acid sequences of human FSTL1 and human DIP2A and the base sequence of the encoding gene can be obtained from a known database such as GenBank. The NCBI accession number of the base sequence of the gene encoding human FSTL1 is NM_007085, and the NCBI accession number of the amino acid sequence of human FSTL1 is NP_009016. In addition, the NCBI accession number of the nucleotide sequence of the gene encoding human DIP2A is NM_015151, and the NCBI accession number of the amino acid sequence of human DIP2A is NP_055966. Based on the obtained base sequence information, a primer set for RT-PCR can be designed.

In step (2), the measured value of the expression level of FSTL1 and / or DIP2A obtained in step (1) is compared with each reference value. As the reference value, the measured value of the expression level of FSTL1 and / or DIP2A in the healthy subject sample measured simultaneously in the step (1) can be used. Moreover, you may set arbitrary reference values based on the accumulated measurement value (health person accumulation data) of the expression level of FSTL1 and / or DIP2A in a healthy person sample. Therefore, the reference value in the present specification may be a measurement value obtained by simultaneously measuring the expression level of FSTL1 and / or DIP2A in a healthy subject sample or an accumulated measurement value, and the blood FSTL1 concentration of a healthy subject may be measured simultaneously. It may be a measured value or an accumulated measured value. Preferably, a reference value set based on healthy person accumulated data is used. A healthy person is preferably an adult who does not suffer from a chronic disease, regardless of gender. It is more preferable that the patient is not old (for example, 20s to 40s). Moreover, as a healthy person sample, it is preferable that it is the sample extract | collected from the healthy person similarly to the sample extract | collected from the patient used at process (1).

In step (3), when the measured value of the expression level of FSTL1 and / or DIP2A obtained in step (1) is higher than the reference value, it is determined that the cancer patient can expect a therapeutic effect by the FSTL1 inhibitor. . For example, in the case of the blood FSTL1 concentration, it is preferable to determine that the therapeutic effect can be expected when the measured value is five times higher than the reference value, and the therapeutic effect is expected when the measured value is ten times higher than the reference value. It is more preferable to determine that it is possible. For example, in the case of FSTL1 or DIP2A mRNA level in a sample containing cancer cells (cancer tissue), it is preferable to determine that a therapeutic effect can be expected when the measured value is 1.5 times higher than the reference value, It is more preferable to determine that a therapeutic effect can be expected when the measured value is twice or more higher than the reference value. For example, when the expression level of FSTL1 or DIP2A in a sample containing cancer cells (cancer tissue) is evaluated by the fluorescence intensity in immunohistochemical fluorescence staining, when the numerical value measured by the pixel counter is three times higher than the reference value It is preferable to determine that the therapeutic effect can be expected, and it is more preferable to determine that the therapeutic effect can be expected when the therapeutic effect is 6 times higher than the reference value. Therefore, the therapeutic effect prediction method of the present invention is based on the determination that the therapeutic effect in step (3) can be expected. Here, “the therapeutic effect (by the FSTL1 inhibitor) can be expected” means This means that there is a possibility that the cancer can be treated with the FSTL1 inhibitor, and if it is clear that the measured value is higher than the reference value, this includes determining the possibility more accurately. Thus, the therapeutic effect prediction method of the present invention is a method of providing information to a sample provider as to whether or not cancer treatment with an FSTL1 inhibitor is an effective method. When it is clear that it is higher than the reference value, the accuracy of information that administration of the FSTL1 inhibitor is an effective method is improved. In addition, when the sample provider is a patient whose primary lesion has been excised, by measuring the expression level of FSTL1 and / or DIP2A in the provided sample, early detection of metastasis and early treatment with an FSTL1 inhibitor Information can be provided. Furthermore, since the expression of FSTL1 and / or DIP2A is significantly enhanced in the early stage cancer, it is possible to select an early stage cancer patient who can expect a therapeutic effect by the FSTL1 inhibitor with high accuracy. Further, in the present invention, if the sample provider's sample before and after administration of the anticancer agent is used and the expression level of FSTL1 and / or DIP2A in the sample after administration is higher than that of the sample before administration, FSTL1 inhibition It is possible to determine that there is a possibility of being treated by the agent, and as a result, it is possible to evaluate the therapeutic effectiveness of the administered anticancer agent.

Moreover, two or more selected from the expression level of FSTL1 in a sample containing cancer cells (cancer tissue), the expression level of DIP2A in a sample containing cancer cells (cancer tissue), and the blood FSTL1 concentration were measured. In some cases, if at least one measurement value is higher than the reference value, the cancer patient can be determined to be able to expect a therapeutic effect by the FSTL1 inhibitor, but patients whose two measurement values are higher than the reference value are more likely. A high therapeutic effect can be expected, and a patient with three measured values higher than the reference value can expect a higher therapeutic effect. That is, patients with at least one measured value higher than the reference value may be able to treat cancer with an FSTL1 inhibitor, and patients with two measured values higher than the reference value may be able to treat cancer with an FSTL1 inhibitor Patients with higher and three measured values above the reference value are more likely to be able to treat the cancer with an FSTL1 inhibitor. Therefore, this invention also provides the parameter | index which selects a FSTL1 inhibitor as an anticancer agent optimal for the cancer treatment which the said patient suffers from among many anticancer agents.

[Method for Determining Start of Administration of FSTL1 Inhibitor to Cancer Patient]
The present invention provides a method for determining the start of administration of an FSTL1 inhibitor to a cancer patient. The administration start determination method of the present invention only needs to include the following steps (1), (2) and (3 ′).
(1) A step of measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient (2) A step of comparing the measured value with a reference value (3 ′) When the measured value is higher than the reference value, the FSTL1 inhibitor Step of determining the start of administration

In the administration start determination method of the present invention, when it is determined in step (3 ′) that the measured value is higher than the reference value, it is preferable to start administration of the FSTL1 inhibitor as soon as possible, and administration of the FSTL1 inhibitor immediately It is more preferable to start. The administration start determination method of the present invention can be carried out in substantially the same manner as the therapeutic effect prediction method of the present invention. Therefore, in order to avoid duplication, description of the administration start determination method of the present invention is omitted.

〔kit〕
The present invention provides a kit for predicting the therapeutic effect of an FSTL1 inhibitor in cancer patients. Since the kit of the present invention measures the expression level of FSTL1 and / or DIP2A, it contains either or both of a reagent for measuring the expression level of FSTL1 and a reagent for measuring the expression level of DIP2A. What is necessary is just to include the reagent for measuring the expression level of DIP2A preferably. Examples of the reagent for measuring the expression level of DIP2A include an anti-DIP2A antibody and a primer set for RT-PCR of DIP2A. The kit of the present invention preferably contains one or both of these. The anti-DIP2A antibody can be used for immunohistochemical staining, and the primer set can be used for quantification of DIP2A mRNA.

When the kit of the present invention contains an anti-DIP2A antibody, it is preferable to include a secondary antibody, a blocking reagent, a washing buffer, an instruction manual, etc. in addition to this. When the kit of the present invention contains a primer set for DIP2A RT-PCR, in addition to this, a DNA synthase, a nucleotide serving as a substrate for the DNA synthase, an amplification reaction solution, a reaction tube, a sample preparation reagent, It is preferable to include an instruction manual and the like.

The kit of the present invention preferably further contains a reagent for measuring the expression level of FSTL1 in cancer cells or cancer tissues and / or a reagent for measuring blood FSTL1 concentration. Examples of such a reagent include an anti-FSTL1 antibody and a primer set for RT-PCR of FSTL1. The kit of the present invention preferably contains one or both of these in addition to the reagent for measuring the expression level of DIP2A. The anti-DIP2A antibody can be used for immunohistochemical staining and measurement of blood FSTL1 concentration, and the primer set can be used for quantification of DIP2A mRNA.

The kit of the present invention can also be suitably used for carrying out the method for determining the start of administration of the FSTL1 inhibitor to the cancer patient of the present invention. Therefore, the kit of the present invention can be rephrased as a kit for determining the start of administration of an FSTL1 inhibitor to cancer patients.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Example 1: Expression analysis of FSTL1 and DIP2A in high metastasis human cancer cell line and low metastasis human cancer cell line]
(1) Experimental method Using the PE-labeled FSTL1 antibody and the DIP2A antibody (Santa Cruz; combined with FITC-labeled secondary antibody), the following four types of human tumor cell lines are immunostained to determine whether FSTL1 and DIP2A are expressed: And the expression distribution was analyzed immunohistochemically.
1. Empty vector introduced cell line of human pancreatic cancer cell line Panc1 (Panc1-mock)
2. Snail forced expression cell line of human pancreatic cancer cell line Panc1 (Panc1-snail +)
3. Human breast cancer low metastatic cell line MCF7
4). Human breast cancer high bone metastatic cell line MDA231

First, using a microslide chamber (Nippon Genetics, product number ib-80821), 2 × 10 ⁴ cells / 300 μL / well of tumor cells were seeded and cultured in DMEM medium containing 10% FCS, and cultured 2 days later. The supernatant was discarded, and BD Cytofix / Cytoperm solution (BD Pharmingen, product number 554714) was added to stain FSTL1 present in the cells, and the membrane was treated at room temperature for 20 minutes to increase the permeability of the cell membrane. After discarding this, 200 μL / well of 1 μg / mL anti-DIP2A antibody (Santa Cruz, product number sc-67556) diluted with BD Perm / Wash buffer (BD Pharmingen, product number 554723) was added and reacted at room temperature for 1 hour. I let you. Thereafter, the antibody solution was discarded, PBS was added to wash the cells, and 1 μg / mL PE-labeled FSTL1 antibody diluted with BD Perm / Wash buffer (anti-FSTL1 antibody was converted to DOJINDO's R-Phycoerythrin Labeling Kit-NH2, 200 μL / well of a PE-labeled antibody using product number LK23) was added and reacted at room temperature for 1 hour. Thereafter, the antibody solution is discarded, and PBS is added to wash the cells. Then, 4% PFA / PBS is added at 200 μL / well and treated at room temperature for 15 minutes to fix the cells. The cells are discarded and discarded at 200 μL / well. Vectashield Mounting Medium (Vector Laboratories, product number H-1000) was placed and sealed. The stained cells were observed under a confocal laser scanning microscope (Carl Zeiss, product number LSM700) and photographed.

(2) Results FIG. 1 shows double-stained images of FSTL1 and DIP2A excluding DAPI nuclear staining. Panc1-mock (upper left) contained a small amount of DIP2A positive cells, but FSTL1 showed only faint staining in the nuclei of some cells. On the other hand, in Panc1-Snail + (upper right), both FSTL1 and DIP2A were strongly positive in almost all cells. DIP2A was basically expressed on the cell membrane, but accumulated with FSTL1 in the periphery of the nucleus, and this was also observed in the nucleus in some cells. This is presumed that FSTL1 secreted outside the cell binds to DIP2A, is taken into the cell and moves to the periphery of the nucleus, and is considered to be a very important phenomenon in terms of signal transduction via DIP2A.

On the other hand, in low-metastatic MCF7 (lower left), FSTL1 was slightly positive in the nuclei of some cells, but DIP2A was hardly observed. On the other hand, MDA231 with high bone metastasis (lower right) was slightly weaker than the Panc1 Snail forced expression cell line, but both FSTL1 and DIP2A were strongly positive in almost all cells. However, although double-staining molecules of FSTL1 and DIP2A were accumulated in the periphery of the nucleus, they do not show a dense staining pattern on one side of the nucleus as in the Panc1 Snail forced expression cell line, but on one side or the periphery of the nucleus. The dots were scattered evenly. This is presumably because MDA231 produces less FSTL1 than Panc1 Snail forced expression cell line.

From the above results, FSTL1 and DIP2A are both highly expressed in cancer malignant traits (or refractory traits) characterized by high proliferation and metastasis, such as metastasis including bone metastasis. It was suggested that it is important in controlling.

[Example 2: Expression analysis of FSTL1 and DIP2A of a mouse cancer cell line in which anti-FSTL1 antibody is successful in a tumor-bearing model]
(1) Experimental method The expression of FSTL1 and DIP2A in the following four types of mouse tumor cell lines used for in vivo drug efficacy evaluation (see Reference Examples) was analyzed immunohistochemically.
1. Mouse melanoma B16-F10 empty vector-introduced cell line (F10-mock)
2. Snail forced expression cell line of mouse melanoma B16-F10 (F10-snail +)
3. Mouse colon cancer cell line CT26
4). Mouse lung cancer cell line 3LL
Using the same PE-labeled FSTL1 antibody and DIP2A antibody (Santa Cruz; combined with FITC-labeled secondary antibody) as in Example 1, immunostaining and observation were performed in the same manner as in Example 1.

(2) Results The results are shown in FIG. F10-mock was observed to have very few levels of DIP2A and a small number of cells expressing FSTL1 near the nucleus. On the other hand, in F10-Snail +, it was observed that both FSTL1 and DIP2A were highly expressed in all cells. It was also confirmed for the first time that both FSTL1 and DIP2A were highly expressed in CT26 and 3LL. In particular, it was speculated that the 3LL cytoplasm was filled with DIP2A, which was considered to be incorporated into the cell by binding to FSTL1, and that the signal transduction system downstream of DIP2A was activated.

[Reference Example 1: Evaluation of drug efficacy of anti-FSTL1 antibody using Snail forced expression cell transplanted bone metastasis model of mouse melanoma B16-F10]
(1) Experimental Method Antitumor effect and immune suppression release effect were analyzed using a bone metastasis model in which Snail forced expression cells of mouse melanoma B16-F10 were transplanted subcutaneously and intravenously into C57BL / 6N mice.
(1-1) Experimental group (n = 5)
1. No treatment (0.9% NaCl as a sham)
2. Control IgG (anti-DNP)
3. Anti-FSTL1 Clone # 6-55
4. Anti-FSTL1 Clone # 7-34
5. Anti-FSTL1 Clone # 8-1
(1-2) Experimental procedure When Snail-positive tumor cells were transplanted subcutaneously and intravenously in mice, they metastasized not only to various organs but also to the bone marrow, which increased mesenchymal stem cells (MSC) starting from the bone marrow. It is known that the induction of anti-tumor immunity is strongly suppressed systemically (Cancer Research 73: 6185, 2013). Therefore, 5 × 10 ⁵ GFP positive Snail positive B16-F10 tumor cells introduced by forced introduction of GFP gene and mouse Snail gene were subcutaneously injected into C57BL / 6N mice, and 1 × 10 ⁵ cells were injected into the tail vein. Transplanted (Day 0), 5 days later (Day 5) and 10 days later (Day 10) anti-FSTL1 antibody (# 6-55, # 7-34, # 8-1) prepared to 1 mg / mL with physiological saline or the Mouse IgG (anti-DNP antibody), which is an isotype, was intraperitoneally administered at a dose of 10 mg / kg as a control antibody. Saline was intraperitoneally administered to the untreated group. Various immunological assays were performed 14 days after cell transplantation (Day 14). Subcutaneous tumor diameters were measured 7 days, 11 days, and 14 days after cell transplantation to calculate the tumor volume, and the inhibitory effect on subcutaneous tumor growth was evaluated. As an assay, the number of GFP positive Snail positive B16-F10 tumor cells in bone marrow cells, the number of CD45 negative cells in bone marrow cells, and body weight were measured.

(2) Results FIG. 3 shows the results of the antitumor effect of the anti-FSTL1 antibody. All three anti-FSTL1 antibodies used significantly suppressed the growth of subcutaneous tumors relative to the control antibody. In the figure, the numerical value in parentheses is the P value when compared with the untreated group, and the numerical value shown in the horizontal line is the cell transplantation day 14 between the group with one end of the horizontal line and the group with the other end. P value when compared later is shown.
FIG. 4 shows the effect on bone metastasis (left: GFP-positive Snail-positive B16-F10 tumor cells in the bone marrow), the effect on MSC increase (middle: the number of CD45-negative cells in the bone marrow), the effect on weight loss ( Right). Anti-FSTL1 antibodies # 6-55 and # 8-1 showed an effect of suppressing weight loss. In addition, all of the three types of anti-FSTL1 antibodies decreased MSC and showed bone metastasis inhibitory effect.

[Reference Example 2: Drug efficacy evaluation of anti-FSTL1 antibody using mouse colon cancer CT26 cell transplanted lung metastasis model]
(1) Experimental Method Antitumor effect and antimetastatic effect were analyzed using a bone metastasis model in which mouse colon cancer CT26 cells were transplanted subcutaneously and intravenously in BALB / c mice. Clone # 6-55 was used as an anti-FSTL1 antibody. The experiment was performed in the same procedure as in Reference Example 1. Evaluation was made on subcutaneous tumor growth and lung metastasis. Assessment of lung metastasis was performed by macroscopically counting the number of tumor nodules in the lung.

(2) Results FIG. 5 shows the results of the antitumor effect, and FIG. 6 shows the results of the antimetastatic effect. Anti-FSTL1 antibody (# 6-55) suppressed CT26 subcutaneous tumor growth and lung metastasis very strongly. Specifically, the solid tumor disappeared in 3 out of 5 mice, and the number of lung metastases was very small (the average number of nodules in the control antibody group was 14 and the number of nodules in the anti-FSTL1 antibody group was 0). ~ 3). In addition, the numerical value shown in the place of a horizontal line in FIG. 5 shows P value at the time of a cell transplant 14 days after a cell transplantation between the group with the one end of a horizontal line, and the group with the other end.

[Reference Example 3: Drug efficacy evaluation of anti-FSTL1 antibody using mouse lung cancer 3LL cell transplantation lung metastasis model]
(1) Experimental method The antitumor effect and the antimetastatic effect were analyzed using a bone metastasis model in which mouse lung cancer 3LL cells were transplanted subcutaneously and intravenously in BALB / c mice. Clone # 6-55 was used as an anti-FSTL1 antibody. The experiment was performed in the same procedure as in Reference Example 1. Evaluation was made on subcutaneous tumor growth and body weight. In addition, the presence or absence of metastasis to tumor organs was visually observed.

(2) Results FIG. 7 shows the results of the antitumor effect, and FIG. 8 shows the results of the effect on weight loss. Anti-FSTL1 antibody (# 6-55) strongly suppressed the growth of 3LL subcutaneous tumors. Specifically, solid tumors disappeared in 2 out of 5 mice. In addition, no metastasis was observed in any organ. In the anti-FSTL1 antibody group, weight loss, thinning, and fluffing were not observed at all, and all mice were healthy.

From the results of Example 2 and Reference Examples 1, 2, and 3, it can be confirmed that the anti-FSTL1 antibody has an antitumor effect against a mouse model transplanted with mouse cancer cells highly expressing FSTL1 and DIP2A. It was. In Example 1, since it was confirmed that both FSTL1 and DIP2A are highly expressed in human cancer cells with high metastasis, such a cancer that highly expresses FSTL1 and DIP2A was developed. The anti-FSTL1 antibody can be expected to have a therapeutic effect on existing cancer patients.

[Example 3: Expression analysis of FSTL1 and DIP2A in tumor tissues derived from patients of various cancer types]
(1) Experimental method In order to examine the cancer types to which the anti-FSTL1 antibody is applied, immunohistochemistry is used to determine the expression levels of FSTL1 and DIP2A using commercially available patient tissue sections (see below) of various cancer types. Analysis. Tissue sections were deparaffinized with xylene and ethanol, then treated with 0.1% trypsin for 30 minutes at 37 ° C. to activate the antigen, and then 20% ImmunoBlock to suppress non-specific staining. (DS Pharma, product number CTKN001) was treated for 15 minutes at room temperature. Thereafter, the entire section was covered with 5 μg / mL anti-DIP2A antibody solution (Santa Cruz, product number sc-67556) diluted with 1% BSA / PBS, and allowed to react at 4 ° C. for 24 hours in a wet box. Thereafter, the section was washed thoroughly with PBS, and 5 μg / mL PE-labeled FSTL1 antibody solution (anti-FSTL1 antibody was diluted with 1% BSA / PBS) using DOJINDO's R-Phycoerythrin Labeling Kit-NH2, product number LK23. The whole section was covered with a PE-labeled antibody) and reacted in a wet box at 4 ° C. for 24 hours. Thereafter, it was dehydrated and fixed with ethanol and xylene, the section was sealed with Vectashield Mounting Medium (Vector Laboratories, product number H-1000), and observed under a confocal laser scanning microscope (Carl Zeiss, product number LSM700). The expression levels of FSTL1 and DIP2A were analyzed by measuring the pixel count using the fluorescence intensity automatic measurement function built in the microscope.
<Paraffin section set (Super Bio Chips)>
MB4 (Human common cancers-2): breast cancer, liver cancer, bladder cancer, ovarian cancer, pancreatic cancer, prostate cancer (n = 9-10)

(2) Results The results are shown in Table 1, FIG. 9 and FIG. Expression of FSTL1 (PE / Red staining) and DIP2A (FITC / Green staining) for 6 types of breast cancer, stage I-IV breast cancer, liver cancer, bladder cancer, ovarian cancer, pancreatic cancer, prostate cancer As a result of analysis, the expression of FSTL1 was higher in the order of liver cancer, breast cancer, bladder cancer, ovarian cancer, pancreatic cancer and prostate cancer. On the other hand, the expression of DIP2A was higher in the order of breast cancer, liver cancer, pancreatic cancer, ovarian cancer, prostate cancer, and bladder cancer. In four types (breast cancer, liver cancer, ovarian cancer, pancreatic cancer) with relatively high expression of both FSTL1 and DIP2A, many FSTL1 / DIP2A co-positive small Snail-induced MSC (sMSC) -like cells infiltrate In addition, in ovarian cancer and pancreatic cancer, large numbers of FSTL1 / DIP2A co-positive large cancer stem cell (CSC) -like tumor cells were also increased. The vertical axis is the FSTL1 expression level and the horizontal axis is the DIP2A expression level, and the correlation between the expression in each tumor tissue has been confirmed. Statistics among breast cancer, liver cancer, bladder cancer, ovarian cancer, pancreatic cancer A scientifically significant correlation was observed (Figure 10). From the above results, it was suggested that anti-FSTL1 antibody treatment could be applied to at least liver cancer, breast cancer, bladder cancer, ovarian cancer and pancreatic cancer.

Further, FIG. 11 shows the results of analyzing the expression levels of FSTL1 and DIP2A in the above 6 cancer types according to the progression stage (stage I-IV). In the figure, black circles (●) indicate average values of expression levels in each stage, and white circles (◯) indicate individual values. From this analysis result, it was revealed that both FSTL1 and DIP2A were enhanced in expression in stages I to III, and FSTL1 was particularly enhanced in stage I. In stage I, the co-expression of FSTL1 and DIP2A at the same time is significantly enhanced compared to the other stages, and both molecules regulate cancer metastasis, so cancer cells are exactly It is speculated that FSTL1 and DIP2A expression is enhanced in the primary lesion to be detached, and that cancer progression and metastasis are promoted. From these results, it was suggested at the clinical level that detecting the expression of FSTL1 and DIP2A, particularly the co-expression of both molecules and their correlation, may lead to early detection and early treatment of cancer metastasis.

[Example 4: Expression analysis of FSTL1 and DIP2A in tumor tissue derived from patients with advanced stage melanoma]
(1) Experimental method As a tumor tissue derived from an advanced stage melanoma patient, a section set (Human malignant melanoma, product number CK2) containing a primary tumor tissue and a metastatic tumor tissue was purchased from Super Bio Chip, and the same method as in Example 3 was used. Immunostaining of FSTL1 and DIP2A was performed, and the expression intensity was analyzed by measuring the pixel count.

(2) Results The results are shown in FIG. 12 and FIG. As can be seen from the pixel count numbers in FIG. 12, there was no statistically significant difference in the expression levels of both molecules in the primary tumor tissue and metastatic tumor tissue (mainly lymph nodes), but there was a tendency to be relatively high in the primary tumor. It was. On the other hand, from the figure using the FSTL1 expression level on the vertical axis and the DIP2A expression level on the horizontal axis, unlike the metastatic tumor tissue, many expression patterns of FSTL1 / DIP2A co-expression are seen in the primary tumor tissue, and between FSTL1 and DIP2A There was a correlation. That is, as discussed in Example 3 above, it was suggested that detecting the expression of FSTL1 and DIP2A in the primary lesion and the correlation thereof may lead to early detection and early treatment of cancer metastasis.

FIG. 13 shows the analysis of the expression levels of FSTL1 and DIP2A in the primary lesion by various classifications, but no significant correlation was found in age, sex, patient survival month, and the like. However, in the case of patients expressing DIP2A higher than the overall average value, the survival time is short (low expression 41.0 months vs high expression 34.0 months; P = 0.3103), and DIP2A is higher than the average value. Patients with high expression and high expression of FSTL1 were also observed to have a shorter survival period (high expression of F 21.8 months vs low expression of 39.4 months; P = 0.1608). On the other hand, as shown in FIG. 13, when FSTL1 and DIP2A expression in the primary lesions were compared by grouping on the basis of the average survival period of “38 months” of all patients, no difference was found in the FSTL1 expression level. However, it was found that the levels of DIP2A expression (short-term survivor 17408 vs long-term survivor 7938) and FSTL1 and DIP2A co-expression (short-term survivor 7635 vs long-term survivor 3288) were enhanced in short-term survivors. In addition, the correlation between FSTL1 expression and DIP2A expression is seen only in male patients, and even when grouped on the basis of the average survival period of “36 months”, the expression of both molecules is observed only in short-lived male patients. It turns out that it correlates. From the above results, it was suggested that in melanoma patients, the expression level of both FSTL1 and DIP2A may be a factor that defines the prognosis of patients, particularly the prognosis of male patients.

[Example 5: Measurement of serum FSTL1 concentration in various cancer patients]
(1) Experimental method Serum of various types (skin cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, breast cancer) terminal cancer patients (stage IV) is purchased (ProMedDx), and FSTL1 concentration in the serum Was measured using an R & D Systems DuoSet ELISA Human FSTL1 (R & D Systems, Cat # DY1694). The measurement was performed according to the product protocol.

(2) Results The results are shown in FIG. Black circles (●) in the figure indicate the average value of serum FSTL1 concentration of each cancer patient. The FSTL1 concentration in serum of various terminal cancer patients was higher in the order of skin cancer, lung cancer, ovarian cancer, colon cancer, prostate cancer, and breast cancer. Terminal cancer patients generally have a high possibility of metastasis, and in particular, terminal cancer patients such as melanoma are known to have a high frequency of metastasis. The average serum FSTL1 concentration of skin cancer patients was 77.7 ng / mL, of which 2 were melanoma patients, and 1 of them had a high serum FSTL1 concentration of 285.9 ng / mL. Although it has been reported that the FSTL1 concentration in normal human serum is approximately 10 ng / mL or less (Arthritis Res Ther. 2011 Feb), the average FSTL1 concentration in melanoma patient serum was 77.7 ng / mL From the above, the difference from the normal person can be clearly identified. From this result, it was suggested that anti-FSTL1 antibody treatment can be expected to be successful for patients with high serum FSTL1 concentration.

[Example 6: Efficacy evaluation of anti-FSTL1 antibody using mouse osteosarcoma cell transplantation model]
(1) Experimental method The antitumor effect was analyzed using a model in which NHOS cells, which are mouse osteosarcoma cell lines, were transplanted subcutaneously into BALB / c mice. Clone # 6-55 was used as an anti-FSTL1 antibody. The experiment was performed in the same procedure as in Reference Example 1 except that the antibody administration day was 7 days after NHOS cell transplantation. Evaluation was performed on subcutaneous tumor growth and FSTL1 concentration in serum. Serum FSTL1 concentration was measured using a sandwich ELISA system using two anti-FSTL1 antibodies previously obtained by the inventors. Specifically, 5 μg / mL # 6-55 antibody was added to a 50 μL plate and immobilized at 4 ° C. overnight. After blocking, washing was performed, each sample was added, and incubated at room temperature for 2 hours. After washing, biotin-labeled # 33 antibody was reacted at room temperature for 1 hour. After washing, streptavidin-PolyHRP80 complex was added at a 1000-fold dilution, and allowed to react at room temperature for 30 minutes in the dark. After washing, a coloring substrate was added and reacted for 30 minutes, and then the absorbance was measured.

(2) Results FIG. 15 shows the results of the antitumor effect, and FIG. 16 shows the results of measuring the FSTL1 concentration in serum. The anti-FSTL1 antibody (# 6-55) suppressed the growth of subcutaneous tumors and decreased the serum FSTL1 concentration to support the antitumor effect. From this, it was suggested that anti-FSTL1 antibody may be effective for osteosarcoma.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (15)

1. A method for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient,
   (1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient;
   (2) a step of comparing the measured value with a reference value, and (3) a step of determining that a therapeutic effect can be expected when the measured value is higher than the reference value,
   A method comprising the steps of:
2. The method according to claim 1, wherein in the step (1), the expression levels of both FSTL1 and DIP2A are measured.
3. 3. The method according to claim 1 or 2, wherein the sample is a sample containing a patient's cancer cells or a patient's blood.
4. 4. The method according to claim 3, wherein the sample containing cancer cells of the patient is a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
5. The FSTL1 and / or DIP2A expression level and blood FSTL1 concentration in a biopsy cancer tissue or a cancer tissue obtained by excision surgery and blood FSTL1 concentration are measured in the step (1). Method.
6. The method according to any one of claims 1 to 5, wherein in the step (1), the expression level is measured immunohistochemically.
7. A method for determining the start of administration of an FSTL1 inhibitor to a cancer patient comprising:
   (1) measuring the expression level of FSTL1 and / or DIP2A in a sample collected from a patient;
   (2) comparing the measured value with a reference value, and (3 ′) determining the start of administration of the FSTL1 inhibitor when the measured value is higher than the reference value,
   A method comprising the steps of:
8. The method according to claim 7, wherein the expression levels of both FSTL1 and DIP2A are measured in step (1).
9. 9. The method according to claim 7 or 8, wherein the sample is a sample containing cancer cells of a patient or blood of the patient.
10. The method according to claim 9, wherein the sample containing cancer cells of the patient is a biopsy cancer tissue or a cancer tissue obtained by excision surgery.
11. The step (1) comprises measuring the expression level of FSTL1 and / or DIP2A and the blood FSTL1 concentration in a biopsy cancer tissue or a cancer tissue obtained by excision surgery, Method.
12. In the step (1), the expression level of FSTL1 and / or DIP2A and the blood DIP2A soluble type concentration in a biopsy cancer tissue or a cancer tissue obtained by excision surgery are measured. The method described in 1.
13. The method according to any one of claims 7 to 12, wherein in the step (1), the expression level is measured immunohistochemically.
14. A kit for predicting the therapeutic effect of an FSTL1 inhibitor in a cancer patient, comprising a reagent for measuring the expression level of FSTL1 and / or DIP2A in a sample collected from the patient.
15. The kit according to claim 14, wherein the reagent comprises one or more selected from anti-FSTL1 antibody, anti-DIP2A antibody, FSTL1 RT-PCR primer set, and DIP2A RT-PCR primer set.

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