WO2019065609A1 - Ｐｒｄｍ１４発現を確認する方法 - Google Patents

Ｐｒｄｍ１４発現を確認する方法 Download PDF

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WO2019065609A1
WO2019065609A1 PCT/JP2018/035395 JP2018035395W WO2019065609A1 WO 2019065609 A1 WO2019065609 A1 WO 2019065609A1 JP 2018035395 W JP2018035395 W JP 2018035395W WO 2019065609 A1 WO2019065609 A1 WO 2019065609A1
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prdm14
cancer
subject
gene
protein
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English (en)
French (fr)
Japanese (ja)
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今井　浩三
均 前佛
博昭 谷口
杏里 斎藤
洋平 宮城
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University of Tokyo NUC
Kanagawa Prefectural Hospital Organization
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University of Tokyo NUC
Kanagawa Prefectural Hospital Organization
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Priority to US16/650,415 priority Critical patent/US20210208148A1/en
Priority to EP18861512.4A priority patent/EP3690444A4/en
Publication of WO2019065609A1 publication Critical patent/WO2019065609A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7158—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for chemokines

Definitions

  • the present invention relates to a method for confirming the expression of PRDM14 gene from blood or lymph collected from a subject, and more specifically, (1) a peripheral blood circulating tumor cell biomarker for use in the confirmation method, a cancer Diagnostic method, method for determining the efficacy and prognosis of a therapeutic agent for cancer, kit containing the biomarker detection reagent, method for identifying peripheral blood circulating tumor cells, and method for diagnosing a disease associated with gene expression of PRDM14
  • the present invention relates to a method for evaluating the efficacy of a therapeutic agent for a disease, a method for determining a therapeutic course for the disease and / or a method for determining a prognosis after treatment of the disease, and a cancer metastasis marker and kit that can be used for these methods.
  • PRDM (PR domain-containing protein) 14 is identified as a nuclear transcription factor transiently expressed in ES cells (embryonic stem cells) and primordial germ cells, and contains PR domains with high homology with the SET domain and six It consists of a Zn finger domain. Although the expression of PRDM14 molecules is enhanced locally at tumor sites in breast cancer, pancreatic cancer, non-small cell lung cancer and the like, its expression is not observed in adult normal tissues (Non-patent documents 1 to 3).
  • the PRDM14 gene product as a biomarker for these cancers is not a secreted protein or a cell membrane protein, and is present in the nucleus, so it is difficult to handle it as a tumor marker that can be easily detected from blood.
  • the present invention relates to a method and biomarker for detecting invasively from breast cancer, pancreatic cancer or non-small cell lung cancer from a subject and a method of diagnosing as a disease accompanied by gene expression of PRDM14, PRDM14 targeted disease It is an object of the present invention to provide a method of evaluating the efficacy of a therapeutic agent, a method of determining a therapeutic course for the disease and / or a method of determining the prognosis after treatment of the disease.
  • CTC peripheral blood circulating tumor cells
  • PRDM14-positive CTCs can be detected, and furthermore, the concentrations of these secreted proteins in the sample are measured and compared with the reference range concentrations identified by the present inventors for the first time.
  • concentrations of these secreted proteins in the sample are measured and compared with the reference range concentrations identified by the present inventors for the first time.
  • the present invention relates to the following matters.
  • PRDM PR domain-containing protein
  • PRDM14-positive CTC peripheral blood circulating tumor cells
  • MDC macrophage-derived chemokine
  • IFN ⁇ interferon ⁇
  • the anti-PRDM14 antibody or a fragment thereof is a biomarker for judging that it is effective as a therapeutic agent for cancer, wherein the antibody and the fragment thereof bind to PRDM14 protein, according to [4] Biomarker.
  • a method of determining a cancer treatment policy comprising administering to a subject a pharmaceutical composition comprising an effective amount of an expression inhibitor of PRDM14 gene or an anti-PRDM14 antibody or a fragment thereof.
  • a pharmaceutical composition comprising an effective amount of an expression inhibitor of PRDM14 gene or an anti-PRDM14 antibody or a fragment thereof.
  • kits for determining the presence or absence of PRDM14-positive CTCs comprising a reagent for detecting a biomarker according to any of [2] to [8].
  • the reagent is a reagent for staining PRDM14 protein, cytokeratin, CD45 and cell nucleus, respectively.
  • a method for detecting PRDM14-positive CTCs from blood collected from a subject comprising the steps of: (I) collecting a band containing peripheral blood mononuclear cells (PBMC) from the blood by density gradient centrifugation; (Ii) isolating EpCAM positive cells from said band using an anti-EpCAM (CD326) antibody; (Iii) Staining PRDM14 protein, cytokeratin, CD45 and cell nuclei on EpCAM positive cells, The method wherein, in step (iii), the EpCAM cells in which PRDM14 protein, cytokeratin and cell nuclei are stained but not CD45 are identified as PRDM14-positive CTCs.
  • PBMC peripheral blood mononuclear cells
  • the therapeutic agent comprises an expression inhibitor of PRDM14 gene selected from antisense, siRNA or shRNA that inhibits expression of PRDM14 gene.
  • the therapeutic agent comprises an anti-PRDM 14 antibody or fragment thereof, The method according to [16] or [17], wherein said antibody and fragment thereof bind to PRDM14 protein.
  • the sample is blood, If the reference concentration range of LEPR is 4.3 ng / ml or more, The reference concentration range of MDC is 11.2 pg / ml or less, or The reference concentration range of IFN ⁇ is 3.2 pg / ml or less The method according to any one of [16] to [19].
  • a cancer metastasis marker comprising at least one protein selected from the group consisting of LEPR, MDC and IFN ⁇ , for use in the method described in [20].
  • the presence or presence of a cancer such as breast cancer or pancreatic cancer is less invasive to the subject It can be conveniently identified.
  • diagnosis of a disease accompanied by gene expression of PRDM14 can be easily and easily performed from a low invasive sample such as blood without directly collecting a target lesion tissue.
  • the efficacy of a therapeutic agent for a disease that can be performed and that targets PRDM 14 the disease can be treated without applying an unnecessary therapeutic agent to the subject. It is possible to determine the therapeutic course for the disease of administering a PRDM14-targeted therapeutic agent, and to determine the prognosis after treatment of the disease.
  • cancer metastasis markers and kits that can be used in those methods can also be provided.
  • the present inventors tried to detect CTCs from 36 breast cancer patients, and found CTCs to be detected from 29 cases (80%), of which about 65% of 19 cases were positive for PRDM14. (Example 1), the use of the biomarker according to the present invention also makes it possible to administer such a therapeutic agent as a therapeutic policy whether or not the PRDM 14 targeted therapeutic agent is effective. Also, it can be judged whether the prognosis after treatment is poor or not. As a result, for example, among breast cancer patients, breast cancer patients having PRDM14 positive or negative can be distinguished, so that more appropriate treatment can be given to each patient.
  • FIG. 1 is a view schematically showing one aspect of the PRDM14-positive CTC identification method according to the present invention.
  • FIG. 2 shows a graph comparing the amount of tumor marker CA15-3 contained in plasma collected from breast cancer patients with the number of CTCs for each patient.
  • FIG. 3 shows a graph comparing the amount of CA15-3 contained in plasma collected from breast cancer patients with the PRDM14 positive rate of CTC for each patient.
  • FIG. 4 is a diagram plotting the number of CTCs and the PRDM14 positive rate of CTCs, respectively, against the amount of CA15-3 contained in plasma collected from breast cancer patients.
  • FIG. 5-1 shows a photomicrograph of CTC isolated from the blood of a patient with pancreatic cancer.
  • FIG. 5-2 shows photomicrographs of PRDM14-positive CTCs isolated from blood of breast cancer patients and pancreatic cancer patients.
  • FIG. 6 shows a schematic diagram outlining a series of screening methods for the identification of serum biomarkers that correlate with the expression level of the PRDM14 gene.
  • FIG. 7 shows the results of experiments in which the correlation between the PRDM14 gene expression level and the LEPR gene expression level as an example was verified.
  • FIG. 8 is a box and whisker plot showing statistical significant differences in PRDM14 high expression and low expression groups of LEPR, MDC and IFN ⁇ identified as serum biomarkers correlated with the expression level of PRDM 14 gene.
  • FIG. 9 is a diagram outlining an index of the efficacy of a PRDM14-targeted therapeutic agent and a method of evaluating the efficacy.
  • PRDM14 means a protein that is PRDM14 (PR domain-containing protein 14) itself, and when described as “PRDM14 gene”, PRDM14
  • PRDM14 also refers to the gene encoding PRDM14.
  • PRDM14 used in the present invention is a protein having a PR domain and a zinc finger domain, called PR domain-containing protein 14 (alias PFM11 or MGC59730), for example, the amino acid represented by SEQ ID NO: 1
  • PR domain-containing protein 14 alias PFM11 or MGC59730
  • SEQ ID NO: 1 the amino acid represented by SEQ ID NO: 1
  • proteins that contain an amino acid sequence that is identical or substantially identical to the sequence are proteins that contain an amino acid sequence that is identical or substantially identical to the sequence.
  • an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 about 70% or more, preferably about 80% or more, more preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 1
  • An amino acid sequence having a homology of% or more, more preferably about 95% or more, and most preferably about 98% or more can be exemplified.
  • the PRDM14 gene used in the present invention is, for example, a DNA containing the nucleotide sequence shown in SEQ ID NO: 2 or a hybridization specifically under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2 Included are DNAs encoding proteins having substantially the same properties as the proteins containing the base sequence to be soyed and containing the amino acid sequence represented by SEQ ID NO: 1 above.
  • the DNA or the like contains a base sequence having a homology of about 70% or more, more preferably about 80% or more, still more preferably about 90% or more, particularly preferably about 95% or more, most preferably about 98% or more Can be used.
  • the “stringent conditions” are usually the conditions of 42 ° C., 2 ⁇ SSC and 0.1% SDS, preferably 65 ° C., 0.1 ⁇ SSC and 0.1% SDS. is there.
  • cancer is also used to mean “malignant tumor” which is not limited to “carcinoma”.
  • cancer is also used to mean "malignant tumor” which is not limited to “carcinoma”.
  • gene expression refers to a series of biological reactions that start from transcription of the gene and include translation.
  • known techniques such as hybridization techniques (Northern hybridization method, dot hybridization method, RNase protection assay, cDNA microarray, etc.), gene amplification techniques (reverse transcription polymerase chain reaction) (RT-PCR) (including competitive RT-PCR, real-time PCR, etc.) and the like can be used.
  • oligonucleotides or polynucleotides capable of hybridizing to a polynucleotide encoding PRDM14 or a part thereof can be used as a probe, and when using gene amplification techniques, such oligonucleotides Alternatively, polynucleotides can be used as primers.
  • the “polynucleotide encoding PRDM14 or a part thereof” includes both DNA and RNA, and includes, for example, mRNA, cDNA, cRNA and the like.
  • the nucleotides constituting the oligonucleotide or polynucleotide may be either deoxyribonucleotides or ribonucleotides.
  • the base length of the oligonucleotide to be used for quantifying the expression level of mRNA encoding PRDM14 in the present invention is not particularly limited, but is usually 15 to 100 bases, preferably 17 to 35 bases, and it is also possible to use
  • the base length of the nucleotide is not particularly limited, but is usually 50 to 1000 bases, preferably 150 to 500 bases.
  • the expression of the gene is significantly high” means that the expression level of the mRNA (expression amount) and / or the expression level of the polypeptide including the protein (expression amount) is statistically significant compared to the control. High (more), or preferably at least 1.5 times, more preferably at least 2 times, more preferably at least 3 times, most preferably at least 5 times higher (more).
  • the expression level of the PRDM14 gene is based on the expression level of the internal standard gene, using as an internal standard gene a gene whose expression level does not greatly fluctuate between tissues or individuals (for example, a housekeeping gene such as ⁇ -actin gene, GAPDH gene). It is preferable to correct.
  • LPR gene the protein which is a leptin receptor
  • macrophage origin chemokine macrophage-derived chemokine
  • interferon gamma interferon-gamma
  • Examples of LEPR, MDC and IFN ⁇ used in the present invention include, for example, proteins containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, 2 and 3.
  • the amino acid sequence substantially identical to the amino acid sequence represented by each of SEQ ID NOs: 1, 2 and 3 is about 70% or more of the amino acid sequence represented by each of SEQ ID NOs: 1, 2 and 3.
  • the amino acid sequence etc. which preferably have about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, most preferably about 98% or more of homology can be exemplified.
  • the present invention detects the expression of PRDM14 gene from blood or lymph collected from a subject, (1) detecting peripheral blood circulating tumor cells (PRDM14-positive CTC) expressing PRDM14 gene, or (2) leptin reception
  • PRDM14-positive CTC peripheral blood circulating tumor cells
  • MDC macrophage-derived chemokine
  • IFN ⁇ interferon ⁇
  • the present inventors have also investigated whether members other than 14 of the PRDM family, cancer markers and stem cell markers can be used for diagnosis of cancer etc. in place of PRDM 14 in the present invention, but more useful than PRDM 14 No indication of sex has yet been found.
  • the present invention also provides a biomarker for use in the confirmation method (1), including PRDM14 positive CTC.
  • PRDM14-positive CTCs may be cancer stem cells.
  • Cancer stem cells refer to cells having the stem cell property among cancer cells, and “stem cells” refer to cells maintaining differentiation ability even after cell division.
  • Cancer stem cells are stained with Hoechst fluorescent dye (Hoechst 33342), and flow cytometry is used for detection using a UV laser (wavelength about 350 nm) as excitation light, and the Side Population (SP) fraction Concentrated to The SP fraction refers to the fraction that is not stained by discharging the dye outside the cell via the ABC transporter or the like to the Main Population (MP) fraction that is stained by the Hoechst fluorescent dye.
  • Point to Stem cells can also be identified by marker molecules such as Oct3 / 4, Nanog, SSEA1 and SSEA4.
  • the present invention also provides, in one aspect, the above biomarker for diagnosing at least one type of cancer selected from the group consisting of breast cancer, pancreatic cancer and non-small cell lung cancer, and in another aspect, The present invention provides a method for diagnosing that the subject suffers from the cancer, or that the subject suffers from the cancer when the biomarker is detected from the subject, or provides a method for assisting the diagnosis thereof. .
  • the term "subject" refers to any living individual, preferably a vertebrate, more preferably a mammal, eg, a rodent, such as a mouse, a rat, a gerbil, a guinea pig, a feline such as a cat, a pumer, a tiger, etc.
  • a rodent such as a mouse, a rat, a gerbil, a guinea pig, a feline such as a cat, a pumer, a tiger, etc.
  • deer, deer animals such as deer, etc., rabbits, dogs, minks, sheep, goats, cattle, horses, monkeys, humans and the like, particularly preferably humans.
  • a human can be excluded from the subject.
  • the subject may be healthy or may suffer from any disease, but when treatment of the disease is intended, typically the disease is or is at risk of suffering from the disease Means an object.
  • the present invention provides, in one aspect, an antisense that inhibits expression of the PRDM14 gene, an expression inhibitor of the PRDM14 gene selected from siRNA or shRNA, in which the cancer is judged to be effective as a therapeutic agent.
  • the present invention provides a biomarker, and in another aspect, provides a method for determining that the PRDM14 gene expression inhibitor is effective as a therapeutic agent for cancer when the biomarker is detected from a subject. .
  • the present invention also provides, in one aspect, the above-mentioned biomarker for judging that an anti-PRDM 14 antibody or a fragment thereof is effective as a therapeutic agent for cancer, and in another aspect, the above-mentioned biomarker from a subject When detected, the anti-PRDM14 antibody or a fragment thereof provides a method for determining that it is effective as a therapeutic agent for cancer.
  • the “expression inhibitor of PRDM14 gene” and the “anti-PRDM14 antibody or fragment thereof” may be collectively referred to as “PRDM14-targeted therapeutic agent”.
  • the anti-PRDM14 antibody may be a monoclonal antibody or a polyclonal antibody, and a fragment of such an antibody can also bind to the PRDM14 protein to the same extent as the anti-PRDM14 antibody.
  • PRDM14 target therapeutic agent the thing of patent 5219818 can be used without a restriction
  • the present invention relates to the above-mentioned biomarker for determining a cancer treatment strategy of administering to a subject a pharmaceutical composition comprising an effective amount of an expression inhibitor of PRDM14 gene or an anti-PRDM14 antibody or a fragment thereof
  • the present invention provides a method of determining a cancer treatment policy of administering the pharmaceutical composition to a subject when the biomarker is detected from the subject.
  • the present invention is also expected in a further aspect, by comparing the number of PRDM14-positive CTCs in the blood before the start of chemotherapy and after the end of each cycle, when one or more cycles after starting such chemotherapy have ended. The therapeutic effect can also be determined.
  • the present invention provides the above biomarker for determining that the prognosis after cancer treatment is poor, and in other embodiments, when the above biomarker is detected from a subject, It provides a method to judge or assist the judgment that the prognosis after cancer treatment is poor.
  • the present invention provides a method for detecting PRDM14-positive CTCs from blood collected from a subject, comprising the following steps (i) to (iii), wherein in step (iii) PRDM 14 protein, site It provides a method to identify EpCAM cells in which keratin and cell nuclei are stained but not CD45 are identified as PRDM14 positive CTCs.
  • PRDM 14 protein site It provides a method to identify EpCAM cells in which keratin and cell nuclei are stained but not CD45 are identified as PRDM14 positive CTCs.
  • PBMC peripheral blood mononuclear cells
  • Ii isolating EpCAM positive cells from the band using an anti-EpCAM (CD326) antibody.
  • Step (i) is a step of collecting a band containing peripheral blood mononuclear cells (PBMC) from the blood collected from the subject by density gradient centrifugation.
  • the density gradient centrifugation and the density gradient centrifugation medium are not particularly limited as long as they are commonly performed as blood cell separation in the art.
  • the collected blood is appropriately diluted and density gradient centrifugation is performed using Histopaque®-1077 (10771, manufactured by SIGMA-ALDRICH) as a density gradient centrifugation medium.
  • Histopaque®-1077 10771, manufactured by SIGMA-ALDRICH
  • PBMCs form a band (or layer or buffy coat) at the interface between plasma and erythrocytes (Histopaque).
  • Bands containing PBMCs can include T cells, B cells, monocytes, granulocytes, dendritic cells, etc., as well as CTCs when the subject suffers from or has cancer.
  • Step (ii) is a step of isolating EpCAM-positive cells from the band recovered in step (i) using an anti-EpCAM (epithelial cell adhesion molecule) antibody or an anti-CD326 antibody .
  • EpCAM-positive cells are EpCAM (KA4439) immobilized on CytoChipNano (U0095, Abnova). , Abnova, Inc.).
  • Step (ii) has the effect of concentrating EpCAM-positive cells, since CTCs, if present, contain only a few to a few tens per 10 mL of blood.
  • Step (iii) is a step of staining PRDM14 protein, cytokeratin, CD45 and cell nuclei on the EpCAM-positive cells isolated in step (ii).
  • an anti-PRDM14 antibody labeled with a red fluorescent dye or the like such as Alexa Fluor (registered trademark) 647
  • cytokeratin (CK) for staining of cytokeratin (CK)
  • FITC Anti-cytokeratin antibody eg, anti-PanCK antibody
  • Anti-CD45 labeled with an orange fluorescent dye or the like such as PE (R-phycoerythrin) for staining with CD45
  • a blue fluorescent dye DAPI (4 ', 6-diamidino-2-phenylindole) which strongly binds to DNA can be used for staining of cell nuclei.
  • DAPI blue fluorescent dye
  • ⁇ DAPI negative and CD45 negative are cell debris and dust.
  • -DAPI positive and CD45 positive cells are non-cancer PBMC.
  • ⁇ DAPI positive and CD45 negative cells are CTCs, Furthermore, it can be identified as PRDM14 positive CTC as cytokeratin positive and PRDM14 positive.
  • the present invention also provides, in one aspect, a kit for determining the presence or absence of PRDM14-positive CTC from the blood of a subject, comprising a reagent for detecting the above-mentioned biomarker, and in a further aspect As reagents, PRDM14 protein, cytokeratin, CD45 and reagents for staining cell nuclei can be included, respectively.
  • the kit of the present invention may contain CytoChipNano (U0095, Abnova) or CytoQuestTM CR (Abnova), and may also include instructions for using the kit.
  • the present invention relates to (A) A method for assisting diagnosis of a disease associated with gene expression of PRDM14 (herein also simply referred to as “diagnostic method”), (B) A method for evaluating the efficacy of a therapeutic agent for the aforementioned disease targeting PRDM 14 (herein also referred to simply as “method for evaluating efficacy”), (C) A method for aiding in the determination of a therapeutic course for the above-mentioned disease (herein also referred to simply as a “method for determining a therapeutic course”) and / or (D) A method for aiding in the determination of prognosis after the treatment of the disease (herein also referred to simply as “a method of determination of prognosis”).
  • each of these methods involves the following steps (i) and (ii): (I) performing the confirmation method (2) according to the present invention, that is, measuring the concentration of at least one protein selected from the group consisting of LEPR, MDC and IFN ⁇ from blood or lymph collected from a subject . (Ii) When at least one of the concentrations obtained by measurement in step (i) satisfies the reference concentration range, In (A), the subject is diagnosed as having the disease, In (B), the therapeutic agent is evaluated to be effective for a subject having the disease, In (C), decide on a treatment policy of administering the therapeutic agent to the subject, In (D), it is determined that the prognosis after the treatment of the disease is not good.
  • the “diseases with gene expression of PRDM14” are diseases including lesions in which gene expression of PRDM14 is observed except for transient gene expression of PRDM14 in ES cells (embryonic stem cells) or primordial germ cells, Preferably, it is cancer, more preferably breast cancer, pancreatic cancer, or non-small cell lung cancer with a low degree of differentiation.
  • diagnosis includes, for example, identifying or classifying a disease or disorder associated with gene expression of PRDM14 and its stage.
  • One aspect of diagnosing is measuring a protein in a sample collected from a subject, and when the obtained concentration satisfies a reference concentration range, a method for aiding in the diagnosis that the subject has the disease, or a method for diagnosing such It is a method.
  • the term "therapeutic agent” may be any drug or agent for treating a disease, such as, but not limited to, for example, a drug, a pharmaceutical composition, a therapeutic agent, an internal agent, an external agent, etc. And further includes a therapeutic agent for cancer, and further a therapeutic agent for breast cancer or pancreatic cancer.
  • the therapeutic agent is an antisense that inhibits expression of the PRDM14 gene, an expression inhibitor of the PRDM14 gene selected from siRNA or shRNA, and the like.
  • such a therapeutic agent comprises an anti-PRDM14 antibody or fragment thereof, wherein a fragment of said antibody binds to PRDM14 protein as well as said antibody.
  • these therapeutic agents targeting PRDM14 may be simply referred to as "PRDM14-targeted therapeutic agents", and those described in Patent No. 5219818 can be used without limitation.
  • such a therapeutic agent may be used in companion diagnostics for targeted cancer molecule therapy.
  • the cancer to be targeted for the cancer molecule target treatment may include not only the cancer included in the disease accompanied by the gene expression of PRDM14 described above but also the cancer metastasized from the cancer accompanied by the gene expression of PRDM14 (but metastasized Cancer does not necessarily involve gene expression of PRDM14).
  • Such metastatic cancer includes, but is not limited to, for example, cancers such as lung, bone and brain where breast cancer is likely to metastasize, and cancers such as liver and peritoneum which easily metastasize from pancreatic cancer.
  • treatment includes treatment, and further, all medically acceptable types of prophylactic and / or therapeutic purpose for curing, temporary remission or prevention of disease etc. Includes interventions. Also, for example, “treatment” includes medically acceptable interventions of various purposes, including delaying or stopping the progression of the disease, regression or disappearance of the lesion, prevention of the onset of the disease or prevention of recurrence.
  • evaluating efficacy refers to determining whether or not there is a cure, temporary remission or prevention of a disease, or the like, and in the case of being or if there is an effect. Is meant to be effective. Note that one aspect of evaluating the efficacy is to measure the protein in the sample collected from the subject, and if the obtained concentration satisfies the reference concentration range, the therapeutic agent is effective for the subject having the disease. It is a method to evaluate.
  • determining the course of treatment means determining the direction of a treatable means in anticipation of a cure, a temporary remission or prevention of a disease.
  • One aspect of determining the therapeutic course is to measure the protein in the sample collected from the subject, and when the obtained concentration satisfies the reference concentration range, assist in determining the therapeutic course to administer the therapeutic agent to the subject Or a method of determining such a treatment strategy.
  • determining the prognosis after treatment means to predict whether the disease has an effect such as curing, temporary remission or prevention.
  • One aspect of determining the prognosis after treatment is to measure the protein in a sample collected from the subject, and if the obtained concentration satisfies the reference concentration range, it is determined that the prognosis after treatment of the disease is not good. It is a method to assist or such a determination method.
  • determining not good means determining that the cure, temporary remission or prevention of the disease can not be expected or that there is no effect.
  • sample collected from the subject is preferably blood or lymph, more preferably blood, but is not particularly limited as long as it is a body fluid having the same or substantially the same composition as serum.
  • the protein to be measured in the sample is preferably a secreted protein, more preferably LEPR, MDC and IFN ⁇ .
  • the number of proteins to be measured is at least one, and preferably a plurality (eg, two or three).
  • the protein to be measured may be, for example, at least one protein selected from the group consisting of LEPR, MDC and IFN ⁇ , and further at least two selected from the group consisting of LEPR, MDC and IFN ⁇ It may be a protein, and furthermore, each protein of LEPR, MDC and IFN ⁇ may be measured.
  • the diagnosis, efficacy evaluation, and treatment policy according to the method of the present invention It is possible to make decisions and make a prognosis decision. In this case, it is not necessary to measure the concentration for the other two types, but if the result is 2 or 3 as a result of the measurement, diagnosis, effectiveness evaluation, treatment policy determination, and prognosis judgment will be highly reliable. There is.
  • the reference concentration range of the measured protein is, for example, 4.3 ng / mL or more for LEPR, 11.2 pg / mL or less for MDC, or 3.2 pg / mL or less for IFN ⁇ .
  • another aspect of the present invention is a method for evaluating the effectiveness of the method of assisting diagnosis of the present invention by dividing measured values by the above-mentioned reference concentration cut-off value, scoring them, and then totaling those values. It may be used as a method, a method of aiding in the determination of a treatment course, or a method of aiding in the determination of prognosis.
  • Specific embodiments of the invention are, for example, LEPR cutoff values ⁇ 4.3 ng / ml,> 4.3 ng / ml, MDC cutoff values ⁇ 11.2 pg / ml, 11.211.2 pg / ml
  • the cutoff value of IFN ⁇ is ⁇ 3.2 pg / ml, 3.2 3.2 pg / ml, etc.
  • the score when the measured value of LEPR is ⁇ 4.3 ng / ml, the score is 0, when> 4.3 ng / ml, the score is 1 and the measured MDC is ⁇ 11.2 pg / ml When score is 1 when score is 11.211.2pg / When score is 0 when IFN ⁇ is ⁇ 3.2pg / ml, when score is ⁇ 3.2pg / when score is 1
  • the value of each score can be summed up with 0 as 0 and used in the present invention. If the total score is 1 or more, the subject can be diagnosed as (A) suffering from a disease accompanied by gene expression of PRDM14, and (B) it may be evaluated that the PRDM14-targeted therapeutic agent is effective.
  • the method of measuring the concentration of protein may be any method known in the art, preferably a method such as ELISA.
  • ⁇ Cancer metastasis marker> another aspect of the present invention is selected from the group consisting of LEPR, MDC and IFN ⁇ , for use in the aforementioned diagnostic method, efficacy evaluation method, treatment strategy determination method and / or prognosis determination method.
  • the present invention relates to a cancer metastasis marker comprising at least one protein.
  • kits for use in the aforementioned diagnostic method, efficacy evaluation method, treatment strategy determination method and / or prognosis determination method which is selected from the group consisting of LEPR, MDC and IFN ⁇
  • a kit comprising an antibody or fragment thereof that binds to at least one protein of interest.
  • the kit of the present invention is not limited to, for example, reagents (eg, LEPR detection reagents etc.) including antibodies or fragments thereof that bind to proteins such as LEPR, MDC and IFN ⁇ , instruments (eg, pipettes , Syringes, etc.), methods for assisting diagnosis, methods for evaluating efficacy, methods for assisting in the determination of treatment plan, or instructions on methods for assisting in the determination of prognosis (eg, instruction manual, information on usage)
  • Media such as a flexible disk, a CD, a DVD, a Blu-ray disk, a memory card, a USB memory, etc., may be included.
  • Example 1 In the method for detecting PRDM14-positive CTCs according to the present invention, CTCs are detected and counted in 36 samples of 2 mL blood collected from breast cancer patients according to the instruction manual attached to CytoQuest (registered trademark) CR of Abnova. Furthermore, the PRDM14 positive rate was calculated by immunostaining using an anti-PRDM14 antibody (product code: ab187881 and ab192411; Abcam). The results are shown in Table 1.
  • Example 2 Using the tumor marker CA15-3 that is said to be useful for tracking recurrence / metastasis of breast cancer and PRDM14, while measuring the number of CTCs as in Example 1, measure the amount of CA15-3 in plasma Then, the PRDM14 positive rate was calculated. The results are shown in FIGS. 2 to 4 respectively. There was no correlation between the amount of CA15-3 in the plasma and the number of CTCs and the rate of positive for PRDM14 (Fig. 4), but the number of specimens positive for PRDM14 and detectable for CTC was greater than that for CA15-3. . The following shows the stage of breast cancer, the number of CTCs, and the PRDM14 positive rate.
  • Fig. 5-1 shows a microscope image of CTC isolated from peripheral blood of a patient with pancreatic cancer by CytoQuest (registered trademark) CR (Abnova), and a microscope of PRDM14-positive CTC collected from a breast cancer / pancreatic cancer clinical specimen. The image is shown in Figure 5-2.
  • Example 3 Identification of biomarkers correlated with the expression level of PRDM14 gene >> A series of screening methods for identifying biomarkers correlated with the expression level of the PRDM14 gene is schematically outlined in FIG. First, in the primary screening, comprehensive gene expression information was analyzed by microarray for each of 29 breast cancer clinical tissues, and PRDM14 gene was found to be amplified at a high frequency (about 60%) in breast cancer.
  • the expression level is related to (1) a correlation coefficient of 0.7 or more, (2) the slope of the regression line is 0.5 to 2.0, and (3) it is known to be a secreted protein on the database (4) After narrowing down the candidate molecules under the condition that the ELISA kit is available, the results of the reproducibility verification using the comprehensive gene expression information by the microarray for another 50 breast cancer clinical tissues in the secondary screening , LEPR was found to be correlated with the expression level of PRDM14 gene.
  • FIG. 7 shows the correlation between the PRDM14 gene expression level and the LEPR gene expression level was examined with the PRDM14 gene expression level on the vertical axis and the LEPR gene expression level on the horizontal axis based on the above screening results.
  • the upper left figure in FIG. 7 shows the result of the primary screening, the upper right figure shows the result of the secondary screening, and the lower figure corresponds to the combination of the above.
  • the PRDM14 gene expression level and the LEPR gene expression level have a positive correlation.
  • the biomarker according to the present invention When the biomarker according to the present invention is detected from the subject, the presence or presence of cancer such as breast cancer or pancreatic cancer is identified, and the PRDM 14-targeted therapeutic drug is effective. It is possible to determine a therapeutic course of administering a pharmaceutical composition comprising an effective amount of a PRDM14-targeted therapeutic agent, and to judge that the prognosis after treatment is poor. Furthermore, for example, among breast cancer patients, breast cancer patients having PRDM14 positive or negative can be distinguished, so that more appropriate treatment can be given to each patient. In addition, according to one aspect of the present invention, although the efficacy of a PRDM14-targeted therapeutic agent can be evaluated, for example, not all subjects suffering from breast cancer express the PRDM14 gene. Prior to administering a PRDM14-targeted therapeutic agent, it can be determined whether the PRDM14-targeted treatment is effective or ineffective for the subject.

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