JP7477872B2 - Novel cancer-related biomarkers - Google Patents

Description

The present disclosure relates to novel biomarkers that can be used as indicators of the malignancy of proliferative diseases such as cancer, indicators of the effectiveness of FGF19/FGFR4 pathway targeting drugs, indicators of FGF19 expression, and the like. The present disclosure also relates to novel uses of FGF19/FGFR4 pathway targeting drugs.

Fibroblast growth factor 19 (FGF19) is a hormone protein expressed in humans and is known to be involved in bile acid biosynthesis, but it has also been suggested that it may be associated with tumors such as liver cancer (Non-Patent Document 1). FGF19 binds to fibroblast growth factor receptor 4 (FGFR4) and exerts its function via the FGF19/FGFR4 signaling pathway. The functions of FGF19 and FGFR4 in vivo have not been fully elucidated, and there is a demand for means that can be used directly or indirectly to analyze their functions.

Several drugs targeting the FGF19/FGFR4 pathway have been developed (Non-Patent Document 2), but since there may be subjects for whom such drugs are effective and subjects for whom they are not effective, there is a need for indicators for selecting appropriate subjects.

Cells 2019, 8(6), 536 Cancer Discov 2019; 9(12):1696-1707

The inventors have found that evaluation of FGF19 expression in cancer and the like can be difficult without direct measurement using tissue samples and the like, and have further found a new biomarker that correlates with FGF19 expression. Based on this new knowledge, the present disclosure provides the use of this biomarker as an indicator of the malignancy of proliferative diseases such as cancer, an indicator of the effectiveness of FGF19/FGFR4 pathway targeting drugs, an indicator of FGF19 expression, and the use of FGF19/FGFR4 pathway targeting drugs.

Thus, the present disclosure provides:
(Item 1)
1. A method for determining in a subject having a proliferative disease whether expression of a biomarker selected from group (A) biomarkers and group (B) biomarkers is indicative of the malignancy of the proliferative disease, comprising:
measuring expression of said biomarker in a sample from said subject;
When the expression level of a biomarker selected from the biomarkers in group (A) measured exceeds a reference value, or when the expression level of a biomarker selected from the biomarkers in group (B) measured is below a reference value, the possibility that the proliferative disease in the subject is highly malignant is indicated.
Method.
(Item 2)
A method for determining in a subject having a disease whether expression of a biomarker selected from group (A) biomarkers and group (B) biomarkers is an indicator of efficacy of an FGF19/FGFR4 pathway targeting drug, comprising:
measuring expression of said biomarker in a sample from said subject;
When the expression level of a biomarker selected from the biomarkers in group (A) measured exceeds a reference value, or when the expression level of a biomarker selected from the biomarkers in group (B) measured is below a reference value, the subject is shown to be in an effective group for the FGF19/FGFR4 pathway targeting drug.
Method.
(Item 3)
The method of any of the preceding items, wherein the sample comprises a blood sample.
(Item 4)
The method of any of the preceding items, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1.
(Item 5)
The method of any of the preceding items, wherein the biomarker is ST6GAL1.
(Item 6)
The method of any of the above items, wherein the disease is cancer.
(Item 7)
The method of any of the preceding items, wherein the cancer comprises liver cancer, breast cancer, pancreatic cancer or colon cancer.
(Item 8)
The method of any of the preceding items, wherein the FGF19/FGFR4 pathway targeting drug is an anti-cancer drug.
(Item 9)
The method of any of the preceding items, wherein the FGF19/FGFR4 pathway targeting drug is selected from the group consisting of lenvatinib, fisogatinib, FGF401, H3B-6527 and BLU9931.
(Item 10)
The method of any of the preceding items, wherein the FGF19/FGFR4 pathway targeting drug comprises lenvatinib.
(Item 11)
11. A method of testing for expression of FGF19 in a subject comprising the step of measuring expression of a biomarker selected from the biomarkers of group (A) and the biomarkers of group (B) in a sample from said subject.
(Item 12)
The method of any of the preceding items, wherein a disease in which FGF19 is abnormally expressed is indicated based on the measured expression of the biomarker.
(Item 13)
The method of any of the preceding items, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1.
(Item 14)
The method of any of the preceding items, wherein the biomarker is ST6GAL1.
(Item 15)
A kit for use in any of the methods described above, comprising a detection agent for the biomarker.

In the present disclosure, it is contemplated that one or more of the above features may be provided in combinations in addition to those combinations explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present disclosure provides a biomarker that correlates with the expression of FGF19. By using such a biomarker, the condition of cancer or the like (e.g., malignancy, drug sensitivity, etc.) can be evaluated without placing an excessive burden on the patient.

1 is an overview of stable isotope labeling with amino acids in cell culture medium (SILAC)-based cellular proteomics and secretomics. Proteins that showed more than 0.5-fold downregulation in FGF19 siRNA transfected cells compared to NC siRNA transfected cells were selected and compared between Hep3B cells and Huh7 cells. Six proteins in the supernatant and 37 proteins in the cell lysate were common between Hep3B cells and Huh7 cells. Six proteins identified in both the supernatant and cell lysate are described. Hep3B and HuH7 cells were transfected with NC or FGF19 siRNA. CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1 mRNA levels were analyzed by qPCR 72 hours after transfection. n=4 each; *, p<0.05 vs. NC siRNA; AU represents arbitrary units. (A) ST6GAL1 and GDF15 mRNA levels (n=3 each) in 9 hepatoma cell lines (left) and correlation with FGF19 mRNA levels (right) (ns, not significant; AU, arbitrary units). (B) Correlation between FGF19 and ST6GAL1 mRNA levels (top) and correlation between FGF19 and GDF15 mRNA levels (bottom) in 373 HCC patients registered in the TCGA database portal. Sixty-two surgically resected HCC tissue sections were stained for FGF19, and tumor grade (A) and recurrence-free survival (RFS) (B) assessed by the TNM staging system between FGF19-high (n=15) and FGF19-low (n=47) groups classified based on FGF19 expression are shown. Sixty-two surgically resected HCC tissue sections were stained for FGF19, and serum ST6GAL1 levels measured by ELISA between FGF19-high (n=15) and FGF19-low (n=47) groups were classified based on FGF19 expression. *, p<0.05. Receiver operating characteristic (ROC) curves for the diagnosis of FGF19-high HCC patients by serum ST6GAL1 levels are shown. AUC, area under the curve. The cutoff value of each ROC curve was determined by Youden's J statistic (gray dots). Correlation between serum ST6GAL1 levels and tumor size (left) and between serum ST6GAL1 levels and tumor grade according to the TNM staging system (right) in 79 HCC patients who underwent surgical resection. 1 shows the RFS time of HCC patients with high and low serum ST6GAL1 levels. (A) 62 surgically resected HCC tissue sections were stained with FGF19, and serum FGF19 levels measured by ELISA between FGF19-high (n=15) and FGF19-low (n=47) groups were classified based on FGF19 expression. n.s., not significant. (B) Receiver operating characteristic (ROC) curve for the diagnosis of FGF19-high HCC patients by serum FGF19 levels. AUC, area under the curve. (A) Correlation between serum FGF19 levels and tumor size (left) and between serum FGF19 levels and tumor grade according to the TNM staging system (right) in 77 HCC patients who underwent surgical resection. (B) Recurrence-free survival (RFS) of HCC patients with high and low levels of serum FGF19. n.s., not significant. 1 shows baseline serum ST6GAL1 levels measured by ELISA in 96 advanced HCC patients treated with either lenvatinib or sorafenib. n=31 (sorafenib), n=65 (lenvatinib); ns, not significant. (A) Overall survival (OS) of 96 patients with advanced HCC treated with either lenvatinib (n=65) or sorafenib (n=31) as assessed by the Kaplan-Meier method. (B) For ST6GAL1 high and ST6GAL1 low groups categorized based on the cutoff value of serum ST6GAL1 level to identify FGF19-driven HCC, OS time of patients in the serum ST6GAL1 low group (62 patients) treated with either lenvatinib (43 patients) or sorafenib (19 patients) (left) and OS time of patients in the serum ST6GAL1 high group (34 patients) treated with either lenvatinib (22 patients) or sorafenib (12 patients) (right) are shown.

Hereinafter, the present disclosure will be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified. Thus, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.

The following provides definitions of terms and/or basic technical content that are particularly used in this specification.

(Definition)
As used herein, "fibroblast growth factor 19 (FGF19)" refers to a hormone protein that is known to be involved in the inhibition of bile acid biosynthesis through downregulation of CYP7A1 expression and to stimulate glucose uptake in adipocytes. FGF15 in mice is known as an orthologous protein of human FGF19. It is known that the expression of FGF19 can be improved in tumors such as breast cancer and liver cancer. In addition, as a relationship between FGF19 and other diseases, it is known that an increase in FGF19 levels can be observed in the liver of patients with cholestasis, and a decrease in FGF19 levels can be observed in patients with chronic diarrhea due to bile acid malabsorption, metabolic syndrome, nonalcoholic fatty liver, and insulin resistance. Representative sequences of human FGF19 are nucleic acid sequence: NM_005117.2 and amino acid sequence: NP_005108.1.

In this specification, "fibroblast growth factor receptor 4 (FGFR4)" is known to be a receptor protein that recognizes FGF19. Therefore, the FGF19/FGFR4 pathway refers to a signal transduction pathway involving FGF19 and FGFR4. In addition, the FGF19/FGFR4 pathway targeting drug refers to a drug that affects the FGF19/FGFR4 pathway by directly or indirectly regulating (increasing or decreasing) the expression and/or function of FGF19 and/or FGFR4. Representative examples of FGFR4 in humans include isoform 1 (nucleic acid sequence: NM_213647.2, amino acid sequence: NP_998812.1), isoform 2 (nucleic acid sequence: NM_022963.3, amino acid sequence: NP_075252.2), and isoform 3 (nucleic acid sequence: NM_001291980.1, amino acid sequence: NP_001278909.1).

As used herein, "β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1)" is known to be an enzyme that catalyzes the glycosylation of proteins. In humans, representative isoforms include isoform a (nucleic acid sequence: NM_173216.2, amino acid sequence: NP_775323.1) and isoform b (nucleic acid sequence: NM_173217.2, amino acid sequence: NP_775324.1).

Unless otherwise specified, it is understood that references herein to each protein contemplate not only the protein having the amino acid sequence set forth in the particular accession number (or the nucleic acid encoding it), but also its functional equivalents.

As used herein, a "functional equivalent" of a molecule is understood to include mutants or variants of the molecule (e.g., amino acid sequence variants, etc.) that have the function of a characteristic (e.g., a marker) described herein, that exhibit a similar biological function (not necessarily to the same extent) as the molecule, and that can change to the molecule itself at the time of acting. As functional equivalents of the present disclosure, insertions, substitutions, or deletions of one or more amino acids in the amino acid sequence, or additions to one or both termini, can be used.

As used herein, a "functional variant" of a molecule includes a substance in which the molecule has been modified while maintaining (although the degree of) its function is altered. For example, functional variants of binding molecules such as antibodies include those conjugated with other moieties (such as labels, other functional molecules (such as proteins)) or fragmented so as to maintain the binding function to a target molecule. Thus, as used herein, a functional variant is one that maintains the basic function prior to modification.

As used herein, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body. In this specification, a biological function may be exerted by "biological activity". As used herein, "biological activity" refers to an activity that a certain factor (e.g., a polynucleotide, a protein, etc.) may have in a living body, and includes, for example, an activity in which a certain molecule is activated or inactivated by interaction with another molecule. When two factors interact with each other, the biological activity can be determined by the binding between the two molecules and the biological change that occurs as a result. For example, when one molecule is precipitated with an antibody and the other molecule is also co-precipitated, the two molecules are considered to be bound. For example, when a certain factor is an enzyme, the biological activity includes its enzymatic activity. In another example, when a certain factor is a ligand, the biological activity includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art.

As used herein, a "derivative" or "analog" or "variant" of a protein or nucleic acid includes, but is not limited to, a molecule that contains a region that is substantially homologous to the protein or nucleic acid, and in various embodiments, is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical over the same size of the amino acid or nucleic acid sequence, or when compared to sequences aligned by computer homology programs known in the art, or such a nucleic acid "derivative" or "analog" or "variant" is hybridizable to the original nucleic acid under stringent, moderately stringent or non-stringent conditions. Typically, a "derivative" or "analog" or "variant" of a protein refers to a product of modifications, such as amino acid substitutions, deletions and additions, of a naturally occurring protein, yet still exhibits the biological function of the naturally occurring protein, although not necessarily to the same degree. For example, the biological function of such proteins can be examined by suitable available in vitro assays described herein or known in the art. While this disclosure primarily discusses humans, it is understood that other species, such as other species within primates or other genera of animals, also apply, and that these mammals are also within the scope of this disclosure.

As used herein, a "functionally active" protein, polypeptide, fragment or derivative has a structural, regulatory, or biochemical function of a protein, such as biological activity.

In this specification, "gene" refers to a factor that determines a genetic trait. It is usually arranged in a certain order on a chromosome. A gene that determines the primary structure of a protein is called a structural gene, and a gene that controls its expression is called a regulatory gene. In this specification, "gene" can refer to "polynucleotide," "oligonucleotide," and "nucleic acid" (including DNA, RNA, etc.). "Gene product" refers to a substance produced based on a gene, such as protein or mRNA. Therefore, mRNA can be included in the concept of a gene, and can also correspond to a gene product. Furthermore, when referring to "gene expression," it often refers to the transcription level of mRNA, etc.

As used herein, "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also include those assembled into complexes of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art.

As used herein, "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length.

As used herein, the "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are compared directly, the genes have homology if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. Thus, as used herein, "homolog" or "homologous gene product" refers to a protein in another species, preferably a mammal, that exerts the same biological function as a protein component of a complex as further described herein. Such a homolog may also be referred to as an "orthologous gene product."

Amino acids may be referred to herein by either their commonly known three letter symbols or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.2.9 (published 12 May 2004). The identity value in this specification usually refers to the value obtained when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them shall be regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

As used herein, a "purified" substance or biological agent (e.g., a nucleic acid or protein, etc.) refers to a biological agent from which at least a portion of the factors naturally associated with the biological agent have been removed. Thus, typically, the purity of the biological agent in a purified biological agent is greater (i.e., concentrated) than the state in which the biological agent is normally present. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological agent is present. The substances used in this disclosure are preferably "purified" substances.

As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has or is predicted to have the same action as a specific amino acid or nucleotide in a polypeptide or polynucleotide that is used as a reference for comparison, and in particular in an enzyme molecule, refers to an amino acid that is located at a similar position in the active site and contributes to catalytic activity in a similar manner. For example, in the case of an antisense molecule, it may be a similar portion in an orthologue that corresponds to a specific portion of the antisense molecule. The corresponding amino acid may be, for example, a specific amino acid that is cysteinized, glutathionylated, S-S bond formed, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, the corresponding amino acid may be an amino acid that is responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain that spans a certain range. In such cases, it is referred to as a "corresponding" region or domain in this specification.

In the present specification, the "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. being subjected to a certain action in vivo and becoming a different form. Preferably, it refers to the gene, polynucleotide, etc. being transcribed and translated into the form of a polypeptide, but the transcription to produce mRNA can also be one aspect of expression. More preferably, the form of such a polypeptide can be one that has been subjected to post-translational processing (a derivative as referred to in the present specification). For example, the expression level of a molecule can be determined by any method. Specifically, the expression level can be known by evaluating the amount of mRNA, the amount of protein, or the biological activity of the protein of this molecule. Such expression levels include the expression level at the protein level of the polypeptide of the present disclosure evaluated by any suitable method, including immunological measurement methods such as ELISA, RIA, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present disclosure, or the expression level at the mRNA level of the polypeptide used in the present disclosure evaluated by any suitable method, including molecular biological measurement methods such as Northern blot method, dot blot method, and PCR method. "Change in expression level" means an increase or decrease in the expression level at the protein level or mRNA level of the polypeptide used in the present disclosure, as assessed by any appropriate method, including the above-mentioned immunological measurement method or molecular biological measurement method. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.

As used herein, a "marker (substance, protein, or gene (nucleic acid))" refers to a substance that serves as an indicator for tracking whether or not a person has a certain condition (e.g., a disease) or is at risk of having such a condition. Examples of such markers include genes (nucleic acid = DNA level), gene products (mRNA, proteins, etc.), and metabolic substances. In this disclosure, detection, diagnosis, preliminary detection, prediction, or advance diagnosis of a certain condition can be achieved using a drug, agent, factor, or means specific to a marker associated with the condition, or a composition, kit, or system containing the same.

In this specification, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eradicating the worsening of a disease when that condition occurs, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents for this purpose are sometimes called "companion diagnostic agents."

As used herein, "prevention" refers to preventing a certain disease from occurring before it occurs.

In this specification, "diagnosis" refers to identifying various parameters related to a condition (e.g., a disease) in a subject and judging the current or future state of such a condition. By using the method, composition, and system of the present disclosure, the internal state can be examined, and such information can be used to select various parameters such as the condition in the subject, a formulation or method for treatment or prevention to be administered, etc. In the narrow sense of the present specification, "diagnosis" refers to diagnosing the current state, but in the broad sense, it includes "early diagnosis," "predictive diagnosis," "pre-diagnosis," etc. The diagnostic method of the present disclosure is, in principle, industrially useful because it can utilize something that comes out of the body and can be performed without the hands of a medical professional such as a doctor. In this specification, in order to clarify that it can be performed without the hands of a medical professional such as a doctor, it is sometimes referred to as "assisting" "predictive diagnosis, pre-diagnosis, or diagnosis." The technology of the present disclosure is applicable to such diagnostic technology.

In the present specification, the term "antibody" broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab') ₂ and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFVs, diabodies, sc(Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc). Furthermore, such antibodies may be covalently bound or recombinantly fused to enzymes, such as alkaline phosphatase, horseradish peroxidase, alpha-galactosidase, and the like. The antibodies used in the present disclosure are not limited to their origin, type, shape, and the like. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present disclosure, either monoclonal or polyclonal antibodies can be used as the antibody, but monoclonal antibodies are preferred. Binding of the antibody to the target protein is preferably specific.

As used herein, "means" refers to any tool that can achieve a certain purpose (e.g., detection, diagnosis, treatment, prevention, migration), and in particular, as used herein, "means for selective recognition (detection)" refers to a means that can recognize (detect) a certain object differently from others.

In the present specification, "detection" or "quantification" of polynucleotide or polypeptide expression can be achieved using suitable methods, including, for example, measurement of mRNA and immunological measurement methods, including binding or interaction with a marker detection agent. Examples of molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR. Examples of immunological measurement methods include, for example, ELISA, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining using microtiter plates. Examples of quantification methods include ELISA or RIA. Genetic analysis can also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are widely reviewed in (Shujunsha, Cell Engineering Special Issue "DNA Microarrays and the Latest PCR Methods"). Protein arrays are reviewed in Nat Genet. 2002 Dec;32 Suppl:526-32. In addition to the above, gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods: Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all descriptions therein are incorporated by reference.

The detection agent or detection means of the present disclosure may be a complex or a complex molecule in which another substance (e.g., a label, etc.) is bound to a detectable moiety (e.g., an antibody, etc.). As used herein, a "complex" or a "complex molecule" refers to any construct containing two or more moieties. For example, if one moiety is a polypeptide, the other moiety may be a polypeptide or may be another substance (e.g., a substrate, a sugar, a lipid, a nucleic acid, another carbohydrate, etc.). In this specification, the two or more moieties constituting the complex may be bound by a covalent bond or by another bond (e.g., hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). When the two or more moieties are polypeptides, they may also be referred to as chimeric polypeptides. Thus, in this specification, a "complex" includes a molecule formed by linking multiple types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules.

As used herein, the term "probe" refers to a substance used as a search tool in biological experiments such as in vitro and/or in vivo screening, and examples of such a substance include, but are not limited to, a nucleic acid molecule containing a specific base sequence, a peptide containing a specific amino acid sequence, a specific antibody or a fragment thereof, etc. In the present specification, a probe is used as a marker detection tool.

In the present specification, the term "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) for distinguishing a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When a plurality of markers of the present disclosure or factors or means for capturing them are labeled by a fluorescence method, the labeling is performed with fluorescent substances having different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but Alexa ^™ Fluor is preferable as the fluorescent substance. Alexa ^™ Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa ^™ 555 and Alexa ^™ 633, and a combination of Alexa ^™ 488 and Alexa ^™ 555. When labeling a nucleic acid, any dye that can bind to the base portion of the nucleic acid can be used, but it is preferable to use cyanine dyes (e.g., Cy3, Cy5, etc. of the CyDye ^™ series), rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc. Examples of fluorescent substances having a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present disclosure, such labels can be used to modify the target of interest so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the intended target.

As used herein, a "tag" refers to a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a substance that acts as a binding partner for binding a specific substance (e.g., having a relationship such as biotin-avidin or biotin-streptavidin), and may be included in the category of a "label". Thus, for example, a specific substance to which a tag is bound can be selected by contacting the specific substance with a substrate to which a binding partner of the tag sequence is bound. Such tags or labels are well known in the art. Representative tag sequences include, but are not limited to, myc tags, His tags, HA, Avi tags, and the like. Such tags may be bound to the markers or marker detection agents of the present disclosure.

In this specification, the terms "drug", "agent" or "factor" (all of which correspond to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, but are not limited to, cells (e.g., T cells), proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof.

As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., diagnostic agents, therapeutic agents, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit advantageously includes instructions or instructions that describe how to use the parts to be provided (e.g., testing agents, diagnostic agents, therapeutic agents) or how to handle the reagents.

In this specification, "instructions" refers to instructions for a physician or other user on how to use the present disclosure. The instructions include instructions on how to use the present disclosure, how to use a diagnostic agent, or how to administer a medicine. The instructions may also include instructions on the site or method of administration. The instructions are prepared in accordance with a format prescribed by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

The term "about" as used herein refers to the indicated value plus or minus 10%. Even if "about" is not explicitly stated, it can be interpreted as having the same meaning.

Preferred Embodiments
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

In one aspect, the disclosure provides for identification (e.g., diagnosis) of a subject's condition based on a biomarker found to correlate with expression of FGF19. This may be accomplished by any means, including performing a method, providing a composition, combination, kit, or system for this purpose, etc. For example, even if not explicitly stated, a description of a method using a biomarker to determine a condition also contemplates embodiments reflecting other means, such as a detection agent for the biomarker to determine the condition, a method or agent for modulating the condition determined based on the biomarker, etc.

(Biomarkers Correlated with FGF19 Expression)
In one embodiment, the present disclosure provides biomarkers (which may be referred to herein as "biomarkers of the present disclosure") that correlate with expression of FGF19. The biomarkers of the present disclosure include biomarkers of group (A) and biomarkers of group (B).

The following group of genes was found to be biomarkers that positively correlate with FGF19 expression, and this group of biomarkers is referred to herein as group (A) biomarkers or group (A) genes.
Group (A) genes A1BG, A2M, ABCC2, ABCF3, ABT1, ACSL4, ACSL5, ACTN3, ADAM10, ADCK3, AFM, AFP, AGR2, AGRN, AGT, AHR, AHSG, AKR1C2, ALDH16A1, ALG3, AMBP, AMDHD1, ANG, ANGPTL3, ANKRD17, ANLN, ANXA1, APIP, APLP2, APOA2, APOC1, A PP, ARID1B, ARMCX1, ARSB, ASGR2, ASNS, ATP1A4, ATP2C1, ATP6AP1, ATP6AP2, ATP6V1H, B2M, B3GAT2, B4GALT4, BAP1, BGN, BPGM, BTD, C11orf98, C14orf1, C19orf80, C1GALT1C1, C2, C3, C4BPA, C5, C5orf42, C6, C8G, CAP1, CAP2, C ASC3, CBR1 (CBR3), CCDC137, CCDC59, CCL15, CCL20, CCR6, CD63, CDC25C, CDH1, CDK6, CDKL1 (CDKL3; CDKL2; PRKD2; PRKD3; CDKL5; STK36), CDO1, CEBPB, CETN2, CFI, CGGBP1, CHTF8, CIB1, CLN5, CLSTN1, CLSTN3, CLU, COBLL1, COC H, COPS6, CORO6, COX15, COX17, COX6B1, CP, CPB2, CPD, CPN1, CPN2, CPSF7, CPVL, CRLF1, CRLF3, CST3, CTGF, CTH, CTNNA2, CTSC, CUTC, CXADR, CXCL1, CXCL3 (CXCL2), CXCL5, CXCL8, CYHR1, DAB2, DCAF16, DDX5, DHFRL1, DIAPH3, DI EXF, DIMT1, DKK1, DMXL1, DNAH9, DNAJB11, DPH5, DSC2, DTNA, DTYMK, DYNLL2, EFNA1, EHBP1, EIF1, EIF2B3, EMC1, ENPP2, ENPP4, ENY2, EPB41L2, EPHA2, EPS8, ERCC6L2, EXTL2, EYA3, F13B, F2, F5, FAM134C, FAM169A, FAM3C, FAM73 A, FAM96A, FBLN1, FBXL12, FBXO22, FEN1, FGA, FGB, FGF19, FGFR1, FGFR4, FGG, FGL1, FHOD1, FIBP, FKBP3, FLCN, FLRT3, FRAS1, FRRS1, FST, FTL, FUCA2, FURIN, FYTTD1, G3BP2, GAA, GABPA, GAK, GALNT2, GALNT4, GBA, GC, GCNT2, GC NT3, GDA, GDF15, GFM2, GJA1, GLA, GLS, GM2A, GNPTG, GOLM1, GOLPH3L, GOT1, GPC1, GPR126, GPT2, GYG1, H2AFX, HAL, HBB, HEXA, HINT1, HLA-A, HMCES, HMGN2 (HMGN3), HNRNPA3, HS6ST2, HSPA13, HYAL1, ICAM1, IFI30, IFRD1, IFRD2 , IGFBP3, INPP1, IPO13, IPO4, IREB2, ISOC1, ITGA2, JAG1, KALRN, KCNN4, KHNYN, KIF2B, KLC4, KMT2D, KNTC1, KYNU, LAMA4, LAMP1, LDLR, LENG1, LGALS3BP, LGMN, LIPC, LRG1, LSR, LUM, LYN, LYRM7, LYZ, MAFF (MAFK; MAFG), MAGI1, M, AN1A1, MAN1B1, MAN2A1, MAP2K3, MASP1, MDC1, MDK, MEP1A, METTL7B, MGA, MGAT4B, MGAT5, MIPEP, MKRN2, MMAA, MMP15, MTAP, MTG2, MTHFD2, MTTP, NANS, NAT8, NDEL1 (NDE1), NGEF, NME1, NOLC1, NOM1, NOMO2 (NOMO1; NOMO3), NPC2, NPTX2, NRXN3, NT5E, NTS, NUCB2, OGFOD1, OLA1, ORM1, ORM2, OS9, OSBPL2, OTUD7B, PAM, PARD6G (PARD6A), PARS2, PCDHB3 (PCDHB8; PCDHB16; PCDHB7; PCDHB4; PCDHB11; PCDHB13; PCDHB10), PCOLCE2, PCSK1N, PCSK5, PCSK6, PDE4D IP(CDK5RAP2), PDGFD, PDGFRL, PDXP, PEX13, PFKM, PIAS1, PISD, PLA2G15, PLA2G7, PLD1, PLD3, PLG, PLOD3, PLS1, PON2, POP5, POP7, PPAN, PPT1, PRCP, PROC, PROM1, PROS1, PSAT1, PSMD7, PSMD9, PSME4, PTMS, PTP4A2, PTPN12, PT PRK, PTRH1, PVR, QSOX2, RAB21, RANBP2, RAVER1, RBBP6, RBL1, RBP4, RCOR1 (RCOR3), RFFL, RIOK2, RNF126, RNF170, RPIA, RPL11, RPL29, RPL6, RPRD1A, RPS27L, RPS6KB2 (RPS6KB1), RPS8, SAA4, SCG2, SCPEP1, SCYL1, SDC1, SDC2, S DC4, SDCBP, SDF4, SEC24D, SEMA3A, SEMA4G, SEPT9, SERINC1, SERPINA10, SERPINA3, SERPINA7, SERPIND1, SERPINE1, SERPINI1, SGCE, SGK3, SH3GL1, SIAE, SIL1, SLAIN1, SLC1A4, SLC1A5, SLC38A1, SLC39A10, SLC39A14, SLC3A2 , SLC6A11, SLC7A1, SLC7A11, SLC7A2, SLIT1, SMG6, SMOC1, SMPD1, SNTB1, SORT1, SOWAHC, SPAG5, SPCS1, SPINK1, SPOCK2, SPON2, SPP1, SPTB, SPTBN4, SQSTM1, SRSF3, ST6GAL1, STC2, STK32C, STK4, STRIP2, STX18, STX8, STXBP5, SUGP2, SULT1A4 (SULT1A3), SUMF1, SUMO3, SVIP, SYF2, TARS2, TBC1D8B, TBC1D9, TFPI, TFRC, TGOLN2, THBS1, THBS4, THOC7, TMBIM6, TMCO5A, TMEM109, TMF1, TMPO, TMPRSS13, TNFRSF11B, TOP3B, TOR1B, TOR3A, TP53RK, TPD52L1, TPP1, TRIO, TSG101, TSPAN4, TSPYL1, TSPYL2, TTR, TUBE1, UBA52, UBAC2, UBE2B, UBE2S, UBE2T, UBE3C, UBFD1, UBL5, UBL7, UPF1, URB2, UXS1, VAMP3 (VAMP2), VBP1, VCAN, VPS39, VPS54, VWA1, WDR36, WDR55, WRAP53, YIPF4, ZC3H13

The following group of genes was found to be biomarkers that negatively correlate with FGF19 expression in multiple cell lines, and this group of biomarkers is referred to herein as group (B) biomarkers or group (B) genes.
Group (B) genes ABI3BP, ACE, ACHE, ACTA1 (ACTC1;ACTG2;ACTA2), ACTG1, ACTN1, ACTN2, ADAMTS13, AIFM1, ALAD, ALCAM, ALDH9A1, ALDOB, ALYREF, ANPEP, AOC3, AP1B1, AP2B1, APMAP, APOC3, APRT, ARF1 (ARF3;ARF5), ARHGDIB, ARMT1, ARPC4, BHMT, C1Q TNF3, C3P1, C4A (C4B), C8A, C8B, CAB39, CACNA2D1, CADM1, CAMK2D, CAND1, CAPN1, CAPN2, CAPNS1, CAPRIN1, CAT, CBLN4, CBS, CBX5, CCT2, CCT3, CCT5, CD109, CD276, CD44, CDC42, CDH11, CDH13, CDH5, CDH6, CFD, CHAD, CHST3, CILP2, CLEC11A, CLIC 4, CNN2, CNTFR, CNTN1, CNTRL, COL11A1, COL11A2, COL12A1, COL18A1, COL1A1, COL1A2, COL21A1, COL2A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL9A1, COMP, COPB2, COPE, COPS2, COPS5, CORO1A, COTL1, CPNE1, CPOX, CPPED1, CREG1, CROCC, C SPG4, CSTB, CUL1, CUTA, CYCS, DCD, DCUN1D1, DDAH2, DDX42, DDX6, DEK, DKK3, DMBT1, DPYS, DSC1, DSG1, DSP, DYNC1I2, EEA1, EFEMP1, EFEMP2, EGFR, EIF2S2, EIF3A, EIF3F, EIF3M, EIF4A1, EIF4A3, EML2, ENOPH1, ENPP1, ERAP1, ERP29, EWSR1, EXOSC 9, EXT1, EZR, F10, F11, F13A1, F9, FAH, FAP, FAT4, FBN1, FERMT3, FH, FHL1, FLNA, FLNB, FLNC, FMOD, FSCN1, FSTL1, FUBP1, G6PD, GCLC, GCLM, GDI1, GDI2, GGCT, GLIPR2, GLO1, GRHPR, GSN, GSS, GSTM2, GSTM3, H2AFY, H3F3A, HARS, HGFAC, HIST1H1B, HIST1H2AJ (HIST1H2AH; H2AFJ; HIST2H2AC; HIST2H2AA3; HIST1H2AD; HIST1H2AG), HIST1H4A, HIST2H3A, HMCN2, HNRNPR, HNRNPUL2, HPD, HPX, HRSP12, HSP90B1, HSPB1, HSPG2, HUWE1, IGFALS, IGJ, IL1RAP, IL6ST, ILF2, ILF3, IMPDH2, IPO9, IQGAP 1, ITGB1, JUP, KARS, KATNAL2, KLKB1, KPNA2, KPNA3, KPNA4, KPRP, KRT18, LAMA2, LAMB1, LCAT, LCP1, LDHB, LECT2, LGALS3, LMNA, LMNB2, LPL, LRP1, LTA4H, LTBP4, LTF, LUC7L2 (LUC7L), MAGOHB (MAGOH), MAPRE2, MCM2, MCM3, MFAP4, MGAT2, MMP2, MR C2, MSN, MST1, MYH10, MYH9, MYL6, MYLK, NACA, NAGA, NAP1L1, NAP1L4, NCAM1, NELL2, NEO1, NES, NME2, NONO, NOP58, NOTCH1, NOTCH2, NOTCH3, NPM1, NRP2, NT5C2, NUTF2, OGN, OMD, P4HB, PAIP1, PARP1, PARVB, PCDH17, PCDH18, PCDHGB5, PCDHGC3, PD IA3, PDIA4, PDIA6, PEPD, PFKL, PGAM4, PGM1, PGM2, PKHD1L1, PLRG1, PLS3, PLXDC2, PLXNA1, POSTN, PPP1CB, PPP1R12A, PPP2R4, PPP4C, PPP6C, PRDX2, PRDX4, PRKACG, PRKAR2A (PRKAR2B), PRPF19, PRSS3, PSMA1, PSMA3, PSMA4, PSMB1, PSMB3, PSMB 4, PSMB5, PSMC5, PSMC6, PSMD14, PSMD6, PSMD8, PSME1, PTPN11, PTPRD, PTPRG, PTPRM, PTPRS, PVRL1, PWP1, PYGB, PYGL, RAP1B (RAP1A), RBBP4, RCN1, RCN3, RDX, RGN, RPLP2, RPS15A, RPS6KA3, RPSA, RSF1, RSU1, RUVBL2, S100A1, S100A12, S100A7, S 100A8, S100A9, SAFB, SAR1B (SAR1A), SART3, SEC23A, SEC24C, SEP11, SEPT2, SEPT7, SEPT9, SERPINB1, SERPINH1, SF3B3, SH3BGRL3, SMARCA1, SMARCC1, SMS, SNRNP200, SNRPD2, SNRPF, SPARC, SPARCL1, SPON1, SPP2, SRRM1, SRSF1, SRSF5, SSRP1, T AGLN2, TCP1, TECPR2, TG, TGFBR3, THBS3, THRAP3, THUMPD1, TIE1, TLN1, TMEM132C, TNC, TNXB, TPM1, TPM3, TPM4, TPP2, TPR, TSPAN14, TSPAN31, TSPYL2, TUBA4A, TWF2, TXNDC5, TXNL1, UBA3, UBE2K, UBE2L3, UBE2NL, UBE2V1 (UBE2V2), UGP2, VASN, V AT1, VCAM1, VCL, VCP, VNN1, VNN2, VPS35, VTA1, VWF, WDR77, XPNPEP1, XRN2, YWHAE, YWHAH, ABHD17A, ACSS2, AHNAK, ANGPTL3, ANKRD46, ANO10, AP1G2, APOA1, APOC3, APOE, ASRGL1, BIN1, C3orf58, CASK, CDS2, CHML, COX5A, CPS1, CRIP1, CTIF, CTS H, DUSP9, EEF1A1 (EEF1A1P5), ENO3, EPB41L3, EPHX1, EPPK1, EPS8L2, FDPS, FTL, FUCA1, FUNDC2, GALNT1, GCN1L1, GLUD1, GM2A, GNG5, GPX7, HECTD3, HMOX1, HSPA6 (HSPA7), IGF2, IQSEC1, ITPKA, KIAA1161, KLHL25, L3HYPDH, LPCAT1, LSM14A, MANB A, MFGE8, NDRG1, NDUFS4, NHLRC3, NID1, NPEPL1, NT5DC2, OPA3, OPLAH, P4HA2, PALMD, PBX1, PEA15, PGRMC2, PHLDA1, PLCG1, PLIN2, PLTP, POLR3B, PPL, PPM1A, PPM1H, PPP1R18, PRKAR2A, PRRC2B, QKI, QSOX1, RBP1, REEP6, RELL1, S100A10, SAMHD1, SAR1B, SCAMP1, SEC24A, SERPINA5, SERPINF2, SFN, SLC12A2, SLC30A10, SLC46A1, SNAP25, SNX27, SPATS2L, STAT3, SWAP70, TCAF1, TGFBI, TLN2, TM7SF2, TMEM177, TMEM245, TMEM41A, TMEM63A, TRIM3, TSPAN3, TTYH3, TUBB2B, TUBB8, VTN, ZFYVE19

As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes were commonly found in the secretomes of multiple cell lines and therefore may be particularly preferably used: A1BG, A2M, ACSL4, AGRN, AHSG, APLP2, APOA2, APOC1, APP, ARID1B, ATP6AP2, B3GAT2, CCL15, CCL20, CLSTN3, CLU, CP, CTGF, DNAJB11, EXTL2, F2, F5, FGA, FGL1, FST, and FURIN. , GBA, GCNT2, GDF15, GPR126, HNRNPA3, ICAM1, LDLR, LGALS3BP, LRG1, LYZ, MAN1A1, MEP1A, NTS, ORM1, PCOLCE2, PPT1, PROS1, SDC4, SDCBP, SERPINA10, SERPINA3, SERPINE1, SERPINI1, SPOCK2, SPTBN4, ST6GAL1, STC2, TFPI, TFRC, TNFRSF11B, TOR1B, TTR, UXS1, VWA1. As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes were commonly found in the whole proteomes of multiple cell lines and therefore may be particularly preferably used: ANLN, ANXA1, ARMCX1, ATP1A4, CCL20, CTGF, DYNLL2, FIBP, FYTTD1, GDF15, H2AFX, HMCES, HSPA1. 3, IFRD1, INPP1, IREB2, ITGA2, MAP2K3, NT5E, ORM1, PARS2, PSME4, RNF126, RNF170, SERPINI1, SIL1, SLC7A1, SLC7A11, SMG6, ST6GAL1, SUGP2, THBS4, TMPO, TUBE1, UBE2B, VAMP3 (VAMP2), YIPF4. As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes were commonly found in the whole proteome and secretome of multiple cell lines and therefore may be more preferably used: CTGF, CCL20, GDF15, ST6GAL1, ORM1, SERPINI1. In the most preferred embodiment, the biomarker of the present disclosure is ST6GAL1.

As the biomarkers of the present disclosure, among the biomarkers of group (B), the following genes were commonly found in the whole proteome and secretome of multiple cell lines and therefore may be more preferably used: ACTG1, ALDH9A1, ANPEP, CDH6, COL18A1, DCD, FLNC, FSCN1, GSTM2, GSTM3, HIST1H4A, HIST2H3A, HPX, HSPB1, IL1RAP, S100A7, TLN1, and YWHAH.

In one embodiment, the biomarker correlated with FGF19 expression is one or more genes selected from group (A) and group (B) or transcription or translation products thereof. In one embodiment, the biomarker correlated with FGF19 expression is a gene selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1 or transcription or translation products thereof. These proteins have been shown to correlate with FGF19 expression at expression levels both in cells and in culture supernatants. In a particularly preferred embodiment, the biomarker correlated with FGF19 expression is the ST6GAL1 gene or transcription or translation products thereof. It has been found that serum ST6GAL1 levels robustly correlate with FGF19 expression in tumor tissues and are good biomarkers for tumor drug sensitivity. In one embodiment, the biomarker of the present disclosure is a protein. In one embodiment, the biomarker of the present disclosure is a nucleic acid (e.g., cell-free DNA).

In one embodiment, the biomarkers of the present disclosure may be evaluated at expression levels in a subject or in a sample obtained from the subject. In one embodiment, the biomarkers of the present disclosure may be evaluated at expression levels (e.g., secreted protein, amount of cell-free DNA) in blood or a blood sample (e.g., serum sample). The inventors have found that the FGF19 level in blood is poorly correlated with the FGF19 level in tissues at disease (e.g., cancer) sites, but in contrast, they have unexpectedly found that the level of the biomarkers of the present disclosure (particularly ST6GAL1) in blood is highly correlated with the FGF19 level in tissues at disease (e.g., cancer) sites and with the malignancy of the disease. Therefore, the identification of the condition of a subject using the biomarkers of the present disclosure can be performed without placing a large burden on the subject.

Identifying a Subject's Status Based on Biomarkers
In one aspect, the present disclosure provides for the identification (e.g., diagnosis) of a subject's condition using the biomarkers of the present disclosure as an index. In one embodiment, the identified condition of a subject includes the presence or absence of a disease in the subject, the state of the disease in the subject (such as malignancy, stage, etc.), the risk of disease in the subject, and drug sensitivity of the subject. The biomarkers of the present disclosure can be correlated with the expression of FGF19, and can therefore be used to identify any disease that may be associated with the expression of FGF19, such as proliferative diseases such as cancer, cholestasis, abnormal bile acid synthesis, chronic diarrhea due to bile acid malabsorption, metabolic syndrome, nonalcoholic fatty liver, and insulin resistance. In one embodiment, the proliferative disease whose condition can be identified by the biomarkers of the present disclosure can be a cancer (e.g., liver cancer, breast cancer) whose high expression of FGF19 correlates with its high malignancy. A high malignancy of a cancer can mean that the cancer is highly likely to recur, has a poor prognosis, and/or is metastatic. In one embodiment, the biomarkers of the present disclosure can be used to identify the aggressiveness of a proliferative disease. In one embodiment, the biomarkers of the present disclosure can be used to identify the stage of a proliferative disease. The biomarkers of the present disclosure have been found to correlate with the expression of FGF19, but are not necessarily associated with the expression of FGF19, and the biomarkers of the present disclosure alone can be used to identify (e.g., diagnose) a condition described herein. In one embodiment, the subject whose condition is to be identified using the biomarkers of the present disclosure has been diagnosed with a disease (e.g., cancer).

Proliferative diseases whose condition (e.g., malignancy, drug sensitivity) can be identified by the biomarkers disclosed herein include breast cancer, prostate cancer, pancreatic cancer, gastric cancer, lung cancer, colorectal cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer), brain tumors, schwannoma, neuroblastoma, glioma, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (uterine cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangioma, malignant lymphoma, malignant melanoma, bone tumor, angiofibroma, retinal sarcoma, penile cancer, pediatric solid tumors, Kaposi's sarcoma, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine fibroids, osteoblastoma, osteosarcoma, chondrosarcoma, mesothelial tumor, leukemia, and the like. In certain embodiments, the proliferative disease whose condition can be identified by the biomarkers of the present disclosure can be liver cancer.

In one embodiment, the biomarkers of the present disclosure can be used to identify drug sensitivity in subjects with a disease (e.g., cancer). In one embodiment, the biomarkers of the present disclosure can be correlated with the expression of FGF19, and can therefore be used to identify subjects for whom an FGF19/FGFR4 pathway targeting drug is effective. An FGF19/FGFR4 pathway targeting drug refers to a drug that regulates the function or expression of any biomolecule (mRNA, protein, etc.) in a signal transduction pathway involving FGF19 and FGFR4. In one embodiment, the FGF19/FGFR4 pathway targeting drug regulates the function or expression of FGF19 and/or FGFR4. In one embodiment, the FGF19/FGFR4 pathway targeting drug is an inhibitor of the FGF19/FGFR4 pathway. In one embodiment, the FGF19/FGFR4 pathway targeting drug reduces the expression of FGF19 or FGFR4. In one embodiment, the FGF19/FGFR4 pathway targeting drug inhibits the binding of FGF19 to FGFR4. In one embodiment, the FGF19/FGFR4 pathway targeting drug inhibits the kinase activity of FGFR4, where the inhibition may be selective for FGFR4 or may inhibit other FGFRs in addition to FGFR4 (e.g., FGFR1, FGFR2 and/or FGFR3). In one embodiment, the FGF19/FGFR4 pathway targeting drug is an anti-cancer drug. In one embodiment, the FGF19/FGFR4 pathway targeting drug is a small molecule compound, a protein (e.g., any protein in the FGF19/FGFR4 pathway, or a functional variant thereof), an antibody, or a nucleic acid (e.g., a nucleic acid that suppresses the mRNA encoding any protein in the FGF19/FGFR4 pathway). For example, small molecule compounds that target the FGF19/FGFR4 pathway include, but are not limited to, lenvatinib, fisogatinib (Cancer Discov 2019;9(12):1696-1707), FGF401 (Mol Cancer Ther;18(12) December 2019), H3B-6527 (Cancer Res; 77(24) December 15, 2017), and BLU9931 (Cancer Discov. 2015 Apr;5(4):424-37), and any known small molecule compound having one or more of the above functions can be used.

In one embodiment, the biomarkers disclosed herein can be used to identify expression levels (e.g., tissue expression levels) of FGF19 in a subject. The biomarkers disclosed herein can be used for any purpose, including research purposes, as they can be surrogate markers for tissue expression of FGF19.

In one embodiment, the biomarkers of the present disclosure may be combined with other indicators. In one embodiment, the biomarkers of the present disclosure may be used to identify a subject's condition, followed by identifying the expression level (e.g., tissue expression level) of FGF19 in the subject.

(Reference values for biomarkers)
In one embodiment, if the expression level of the biomarker of the present disclosure falls outside a predetermined range, the subject is identified (e.g., diagnosed) as having a particular condition. The predetermined range of the biomarker of the present disclosure may vary depending on the type of biomarker used and the type of condition of the subject to be identified, and a person skilled in the art can appropriately set a specific numerical range as the predetermined range of the biomarker of the present disclosure. In one embodiment, if the expression level of the biomarker of group (A) of the biomarkers of the present disclosure exceeds a predetermined reference value, the subject is identified (e.g., diagnosed) as having a particular condition. In one embodiment, if the expression level of the biomarker of group (B) of the biomarkers of the present disclosure falls below a predetermined reference value, the subject is identified (e.g., diagnosed) as having a particular condition. In one embodiment, the predetermined range of the biomarker of the present disclosure can be determined based on the correlation between the biomarker and the presence or absence of the condition of the subject to be identified. For example, when subjects are divided into two groups according to the presence or absence of a condition (presence or absence of a disease, presence or absence of drug sensitivity, presence or absence of FGF19 expression in tissues exceeding a predetermined level, etc.), the expression level of the biomarker of the present disclosure that achieves a particular sensitivity, specificity, and/or false positive rate can be set as the reference value. In one embodiment, the predetermined range of the biomarker of the present disclosure is set so as to achieve a particular sensitivity, specificity, and/or false positive rate when subjects with a particular disease (e.g., cancer) are divided into two groups based on the amount of FGF19 expression in diseased tissues. By making a judgment based on the reference value of the biomarker of the present disclosure set in this manner, it may be possible to further stratify subjects with a disease (e.g., cancer), and provide more appropriate treatment (e.g., drug selection). In one embodiment, the reference value of the expression level of the biomarker of the present disclosure can be determined by the Youden index, and a ROC curve is created for each cutoff value to calculate the true positive rate (TRP)-false positive rate (FRP), and a cutoff value that maximizes the true positive rate (TRP)-false positive rate (FRP) can be selected as the reference value.

In one embodiment, the criteria for identifying subjects who are sensitive to FGF19/FGFR4 pathway targeted drugs and/or have aggressive cancer using serum ST6GAL1 levels can be serum ST6GAL1 levels of about 15 ng/ml or more, about 16 ng/ml or more, about 17 ng/ml or more, about 18 ng/ml or more, about 19 ng/ml or more, about 20 ng/ml or more, about 21 ng/ml or more, about 22 ng/ml or more, about 23 ng/ml or more, about 24 ng/ml or more, or about 25 ng/ml or more, and in a specific embodiment, about 19.1 ng/ml or more.

(Measurement of biomarkers)
Each of the biomarkers of the present disclosure can be detected and the expression level can be measured using any suitable detection agent. In one embodiment, the detection agent is a molecule that specifically binds to the biomarker of the present disclosure, and can be, for example, an antibody, a protein or nucleic acid known to bind in vivo, a complementary nucleic acid, or a small molecule compound. In one embodiment, the detection agent may include a tag or label for performing functions such as purification, quantification, and signal generation. When the target biomolecule is known, the specific method and means for its detection and measurement can be appropriately selected by one skilled in the art.

(Treatment and Prevention)
In one aspect, the present disclosure provides treatment and/or prevention of a subject based on the identification of the subject's condition using the biomarker of the present disclosure. The biomarker of the present disclosure can identify the presence or absence of a disease in a subject, the state of the disease in a subject (such as malignancy, stage, etc.), the risk of the disease in a subject, the drug sensitivity of a subject, etc., and therefore can provide appropriate information for selecting a method for treating and/or preventing a subject. In particular, the biomarker of the present disclosure can identify a subject for whom an FGF19/FGFR4 pathway targeting drug may be effective, and therefore the present disclosure provides treatment and/or prevention of such a subject with an FGF19/FGFR4 pathway targeting drug. Here, the subject may have any disease that may be associated with the expression of FGF19, such as a proliferative disease described herein, cholestasis, bile acid synthesis disorder, chronic diarrhea due to bile acid malabsorption, metabolic syndrome, nonalcoholic fatty liver, or insulin resistance, or may have been diagnosed as having these diseases. In particularly preferred embodiments, the present invention may provide FGF19/FGFR4 pathway targeted drugs (such as lenvatinib) for use in patients (eg, cancer patients) determined to have high ST6GAL1 levels.

The treatment and/or prevention of the present disclosure may be combined with any known therapeutic and/or preventive treatment or procedure (e.g., anti-cancer treatment).

The therapeutic, prophylactic and/or diagnostic methods of the present disclosure can be performed in any subject. In one embodiment, the subject is a mammal, such as a human, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee. In a specific embodiment, the subject is a human.

(Drugs, dosage forms, etc.)
The FGF19/FGFR4 pathway targeting drug and the biomarker detection agent of the present disclosure described herein can be provided as a composition or medicine in various forms. In one embodiment, the present disclosure provides a composition comprising an FGF19/FGFR4 pathway targeting drug for treating a subject who has been shown to have a high possibility of malignancy of a proliferative disease by the method described herein, or for treating a subject who has been shown to be in an effective group by the method described herein. In one embodiment, the FGF19/FGFR4 pathway targeting drug is an anticancer drug (e.g., lenvatinib).

The administration route of the FGF19/FGFR4 pathway targeting drug (e.g., lenvatinib, etc.) and the biomarker detection agent of the present disclosure described herein is preferably one that is effective in treating, preventing, or detecting the diseases described herein, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, granule, etc. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. The aqueous solution for injection may also be mixed with, for example, physiological saline, sugar (e.g., trehalose), NaCl, NaOH, etc. The therapeutic agent may also be mixed with, for example, a buffer (e.g., phosphate buffer), a stabilizer, etc.

Generally, the compositions, medicaments, detection agents, etc. of the present disclosure include a therapeutically effective amount of an active ingredient or a detectable amount of a detection means, and a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic or detection agent is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When medicaments are administered orally, water is the preferred carrier. When pharmaceutical compositions are administered intravenously, saline and aqueous dextrose are the preferred carriers. Preferably, saline solutions, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The composition can also contain a small amount of a wetting or emulsifying agent, or a pH buffer, if desired. These compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. The composition can also be formulated as a suppository, using traditional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide a form that is properly administered to the patient. The formulation should be suitable for the mode of administration. In addition to these, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavorings, etc. may be included.

In one embodiment of the present disclosure, the term "salt" includes, for example, an anionic salt formed with any acidic (e.g., carboxyl) group, or a cationic salt formed with any basic (e.g., amino) group. Salts include inorganic or organic salts, such as those described in Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Examples of salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, and salts with organic acids. In one embodiment of the present disclosure, a "solvate" is a compound formed by a solute and a solvent. For details of solvates, see, for example, J. Honig et al., The Van Nostrand Chemist's Dictionary, P650(1953). If the solvent is water, the solvate formed is a hydrate. It is preferable that the solvent does not interfere with the biological activity of the solute. Examples of such preferable solvents include, but are not limited to, water or various buffers.

In the present disclosure, when administering the medicament, various delivery systems can be used together, and such systems can be used to administer the inhibitors and/or migration agents of the present disclosure to the appropriate site. Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules; using receptor-mediated endocytosis; constructing the therapeutic nucleic acid as part of a retroviral or other vector. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The medicament can be administered by any suitable route, for example, by injection, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), using an inhaler or nebulizer with an aerosolizing agent if necessary, and can be administered together with other biologically active agents. Administration can be systemic or local (e.g., intratumoral).

In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans, according to known methods. Such compositions can be administered by injection. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

The compositions, pharmaceuticals, therapeutic agents, or prophylactic agents of the present disclosure may be formulated in neutral or salt form or as other prodrugs (e.g., esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxide, etc.

The amount of the composition, medicament, therapeutic or prophylactic agent of the present disclosure may vary depending on the nature of the disease, but can be determined by one of skill in the art using standard clinical techniques based on the description herein. In addition, in vitro assays can be used in some cases to help identify optimal dosage ranges. The exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dose, or within any two of these values. The administration interval is not particularly limited, and may be, for example, once or twice per 1, 7, 14, 21, or 28 days, or once or twice per two of these values. The dosage, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. In addition, the therapeutic agent preferably contains a therapeutically effective amount, or an effective amount of the active ingredient that exerts the desired effect. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems.

The compositions, therapeutic agents, preventive agents, or detection agents of the present disclosure can be provided as kits.

In certain embodiments, the present disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the compositions, therapeutic agents, prophylactic agents, or detection agents of the present disclosure. Optionally, such containers may bear information associated therewith indicating approval by a government agency for manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products.

Procedures for formulating the presently disclosed therapeutic agents, prophylactic agents, and other pharmaceuticals are known in the art and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, and the Pharmacopoeias of other countries. Thus, given the description herein, a person skilled in the art can determine the embodiments, such as the amount to be used, without undue experimentation.

(Animal Model)
In one aspect, the present disclosure provides a new animal model (e.g., mouse, rat) for cancer research, a method for producing the same, or a means for producing the same. In one embodiment, the present disclosure provides a nucleic acid (e.g., a plasmid) containing a cancer gene and a unique barcode. In one embodiment, the present disclosure provides a population of multiple types of nucleic acids, each of which contains multiple types of cancer genes and corresponding unique barcodes. By introducing such a population of nucleic acids into an animal, it is possible to create an animal into which various cancer genes have been randomly introduced. Since animals into which various cancer genes have been randomly introduced may show different responsiveness to treatment with drugs or the like depending on the cancer genes introduced, they are useful for studying the relationship between cancer genes and treatment (e.g., drug sensitivity). When an animal into which a population of multiple types of nucleic acids, each of which contains multiple types of cancer genes and corresponding unique barcodes, is used, the expression level of each cancer gene can be estimated according to the number of unique barcodes read using cDNA based on mRNA extracted from an animal-derived sample (e.g., tumor tissue), so that the relationship between the quantitative information of each cancer gene and the responsiveness of the animal to the treatment can be easily analyzed.

In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "alternative." In this specification, when it is stated that "within the range of" two values, the range includes the two values themselves.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.

When necessary, animals used in the following examples were handled in accordance with the Shiga University of Medical Science guidelines for animal experiments. Specific reagents used were those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

In the following examples, the abbreviations have the following meanings:
ANOVA: Analysis of variance AUC: Area under the curve CT: Computed tomography DMSO: Dimethyl sulfoxide ELISA: Enzyme-linked immunosorbent assay FGF19: Fibroblast growth factor 19
FGFR: fibroblast growth factor receptor HCC: hepatocellular carcinoma IC50: 50% inhibitory concentration KD: knockdown OS: overall survival PFS: progression-free survival PB: piggyBac
PDGFR: Platelet-derived growth factor receptor RFS: Relapse-free survival ROC: Receiver operating characteristic SILAC: Stable isotope labeling with amino acids in cell culture medium TKI: Tyrosine kinase inhibitor VEGFR: Vascular endothelial growth factor receptor

(material and method)
Human serum and histological analysis Serum samples were obtained from 76 HCC patients who underwent therapeutic hepatectomy at Osaka University and 96 advanced HCC patients who received TKI treatment at Osaka University and other institutions participating in the Osaka Liver Forum. HCC tissues were obtained from 62 of the 76 HCC patients who underwent surgical resection. All patients provided written informed consent, and the study design was consistent with the principles of the Declaration of Helsinki. The protocol of the study using patient serum and resected tissues was approved by the Institutional Review Board (IRB) Committee of Osaka University Hospital (IRB No. 19438). Serum FGF19 and ST6GAL1 levels were examined by enzyme-linked immunosorbent assay (ELISA) using human FGF19 (DF1900, R&D Systems, Minneapolis, MN) and human ST6GAL1 (ab243669, Abcam, Cambridge, UK) ELISA kits, respectively, according to the manufacturer's protocols. Tumor tissues were fixed in 10% neutral buffered formaldehyde solution, paraffin embedded, and thin sectioned. Liver sections were stained with hematoxylin-eosin and primary FGF19 antibody (HPA036082, 1:300, Sigma-Aldrich, St. Louis, MO). Heat-mediated antigen retrieval was performed using citrate retrieval buffer (Agilent) and Decloaking Chamber NxGen (Biocare Medical, Pacheco, CA), and FGF19 staining intensity in liver tumor sections was quantified with Hybrid Cell Count software (Keyence, Osaka, Japan). Tumors with cytoplasmic FGF19 staining in ≥30% of tumor cells in ≥2 of 4 high-power fields were classified as FGF19-high group. The remaining tumors were classified as FGF19-low group.

Statistical analysis All data are presented in bar graphs as mean ± standard deviation (SD) or in dot plots with the central horizontal bar indicating the mean. Statistical analysis was performed using Student's t-test or Mann-Whitney U-test to evaluate differences between unpaired groups with parametric or nonparametric distribution, respectively. One-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc test or Kruskal-Wallis test was performed for parametric or nonparametric multiple comparisons, respectively. Chi-square test or Fisher's exact test was used to analyze categorical data. Correlation was evaluated using Pearson's product-moment correlation coefficient. Kaplan-Meier method and log-rank test were used to analyze differences in OS, progression-free survival (PFS), and recurrence-free survival (RFS). Univariate Cox proportional hazards regression model was used to analyze factors associated with improved OS. To evaluate the diagnostic performance of serum biomarkers, receiver operating characteristic (ROC) curve analysis was performed and predictive power was evaluated using area under the curve (AUC). Cut-off values were determined with Youden's J statistic (Cancer 1950;3:32-35). Otherwise, the statistical analysis used is indicated in the figure legend. A p value <0.05 was considered to indicate statistical significance. Analysis was performed using Prism ver.8.4.2 for Windows (GraphPad Software, San Diego, CA) and JMP® 13 (SAS Institute Inc., Cary, NC).

Cell lines Human hepatoma cell line Hep3B was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Huh-7 human hepatoma cell line was obtained from the Japanese Cancer Research Resource Bank (JCRB, Ibaraki, Japan). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. All cells were confirmed to be free of pathogens and mycoplasma.

Mass spectrometry of cell lines Hep3B and HuH7 cells were cultured for 10 passages in DMEM containing 10% dialyzed FBS and heavy isotope 13C6-L-lysine-2HCl and 13C6-L-arginine-2HCl or light isotope L-lysine-2HCl and L-arginine-2HCl according to standard stable isotope labeling with amino acids in cell culture (SILAC) protocols (Nat Protoc 2006;1:2650-60). Cells were transfected with siRNA at a final concentration of 10 nM using RNAiMAX according to the manufacturer's instructions. The following siRNAs were used: siControl (Thermo Fisher Scientific, 4390844) and siFGF19 (Thermo Fisher Scientific, s19354). 48 hours after siRNA transfection, the medium was replaced with fresh medium containing 0.1% FBS. Conditioned media were collected after 48 h of incubation, centrifuged at 1,500 rpm to remove cell debris, filtered through a 0.22 μm filter (Promega, Madison, WI), and concentrated approximately 10-fold with an Amicon Ultra-15 (nominal molecular weight cut-off (NMWL): 3,000, Merck, Hessen, Germany). To obtain whole-cell extracts, ² × 107 cells were lysed in 1 ml of PBS containing 1% sodium dodecyl sulfate (SDS) and 50 mM ammonium bicarbonate (ABC) supplemented with cOmplete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland) and boiled at 95 °C for 5 min. Protein concentrations in conditioned media and whole-cell extracts were determined by detergent-responsive (DC) protein assay (Bio-Rad Laboratories, Berkeley, CA).

Equal amounts of light and heavy isotope-labeled samples were mixed and precipitated with methanol-chloroform. The samples were then redissolved in 8 M urea and sonicated for 15 min. One hundred and fifty micrograms of each sample were diluted four-fold with 50 mM ABC and digested with trypsin (trypsin amount: protein weight, 1:50) at 37°C for 18 h. Tryptic peptides were acidified with 1% trifluoroacetic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and desalted using an Oasis® HLB column (Waters, Milford, MA).

Peptides (100 μg) were fractionated by high pH reversed-phase liquid chromatography (RPLC) on an LC-20AB instrument (Shimadzu, Kyoto, Japan). Peptide separation was performed on an L-column3 C18 analytical column (CERI) containing 5 μm particles at a flow rate of 0.2 μml/min. The solvent was prepared as mobile phase A with 2% acetonitrile and 5 mM ABC (pH 10) and as mobile phase B with 90% acetonitrile and 5 mM ABC (pH 10). Separation was performed with the following linear gradient: 1% B to 7% B in 1 min, 7% B to 27% B in 22 min, 27% B to 45% B in 6 min, 45% B to 95% B in 1 min. The sample was divided into 34 fractions and finally grouped into 15 pools in a non-continuous manner.

The pools were analyzed by LC-MS/MS using a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific). A nano HPLC capillary column (150 mm i.d. × 75 μm; Nikkyo Technos, Tokyo, Japan) was used as the analytical column with a nanoelectrospray ion source. RPLC was performed with a linear gradient of 5% to 40% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. Full scan spectra of MS1 surveys were acquired in the range of 400–1600 m/z. The 20 most intense ions per MS1 scan were selected for fragmentation by high-energy collision dissociation in the collision cell.

The acquired mass spectrometry data were processed using MaxQuant version 1.5.7.0 supported by the Andromeda search engine (Nat Biotechnol 2008;26:1367-72). Tandem mass spectra were searched against a database of human proteins (released April 2019) obtained from UniProt combining 262 common impurities. To account for the incorporation of heavy isotopes, a fixed modification of 6.020129 additional mass units for lysine and arginine residues was used in the database search. For peptide identification, the search criteria were set as follows: trypsin specificity, allowance of cleavage at the C-terminus of arginine or lysine at a proline bond, allowance of up to two missed cleavages, carbamidomethylation of cysteine residues as fixed modification, and methionine oxidation as variable modification. The false discovery rate for protein and peptide identification was less than 0.01. Peptides identified in the reverse database and the potential impurity database were excluded.

siRNA transfection. FGF19 was knocked down in HCC cells using siRNA (s19353, s19354, or s19355, Thermo Fisher Scientific) or negative control siRNA (4404020, Thermo Fisher Scientific) with Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) as previously described (Suemura et al., CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma. Cancers (Basel) 2019;11).

Real-time PCR analysis Total RNA was isolated from tissues or cells and reverse transcribed into cDNA as previously described (Cell Death Differ 2019;26:470-86). mRNA expression was measured by quantitative real-time reverse transcription PCR (RT-qPCR) using THUNDERBIRD RT-qPCR master mix (Toyobo, Osaka, Japan) and TaqMan probes (Thermo Fisher Scientific). Target gene expression was normalized to beta-actin expression and presented as fold increase compared to the level of the control group.

(result)
ST6GAL1 is a tumor-derived secreted protein positively regulated by FGF19 in HCC We next investigated tumor-derived secreted proteins to identify serum biomarkers of HCC driven by FGF19. To this end, we performed stable isotope labeling with amino acids in cell culture medium (SILAC)-based cellular proteomic and secretomic analyses using FGF19-expressing Hep3B and Huh7 HCC cells with or without FGF19 knockdown (Figure 1).

As a result, the proteins whose expression was decreased or increased (2-fold or more) by FGF19 knockdown are shown below. Proteins that were not detected in the FGF19 knockdown sample but were detected in the corresponding non-knockdown sample were considered to be decreased, and proteins that were detected in the FGF19 knockdown sample but not in the corresponding non-knockdown sample were considered to be increased.

The following proteins were commonly downregulated by FGF19 knockdown in secretome analysis of both Hep3B and Huh7 cells.
A1BG, A2M, ACSL4, AGRN, AHSG, APLP2, APOA2, APOC1, APP, ARID1B, ATP6AP2, B3GAT2, CCL15, CCL20, CLSTN3, CLU, CP, CTGF, DNAJB11, EXTL2, F2, F5, FGA, FGL1, FST, FURIN, GBA, GCNT2, GDF15, GPR126, HNRNPA3, ICAM1, L DLR, LGALS3BP, LRG1, LYZ, MAN1A1, MEP1A, NTS, ORM1, PCOLCE2, PPT1, PROS1, SDC4, SDCBP, SERPINA10, SERPINA3, SERPINE1, SERPINI1, SPOCK2, SPTBN4, ST6GAL1, STC2, TFPI, TFRC, TNFRSF11B, TOR1B, TTR, UXS1, VWA1

The following proteins were commonly down-regulated by FGF19 knockdown in whole proteome analysis of both Hep3B and Huh7 cells.
ANLN, ANXA1, ARMCX1, ATP1A4, CCL20, CTGF, DYNLL2, FIBP, FYTTD1, GDF15, H2AFX, HMCES, HSPA13, IFRD1, INPP1, IREB2, ITGA2, MAP2K3, NT5E, ORM1, PARS2, PSME4, RNF126, RNF170, SERPINI1, SIL1, SLC7A1, SLC7A11, SMG6, ST6GAL1, SUGP2, THBS4, TMPO, TUBE1, UBE2B, VAMP3 (VAMP2), YIPF4

The expression of the following proteins was increased by FGF19 knockdown in all four samples.
ACTG1, ALDH9A1, ANPEP, CDH6, COL18A1, DCD, FLNC, FSCN1, GSTM2, GSTM3, HIST1H4A, HIST2H3A, HPX, HSPB1, IL1RAP, S100A7, TLN1, YWHAH

CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1 were identified as proteins whose expression was commonly decreased by FGF19 knockdown in all four samples. The expression of these proteins was decreased in both the lysates and the supernatants of the two cell lines when FGF19 was silenced with siRNA (Figure 2). Next, we evaluated the expression levels of each of the six genes when FGF19 was inhibited with siRNA, and confirmed that CTGF, GDF15, and ST6GAL1 were significantly downregulated by FGF19 knockdown with two types of siRNA (Figure 3). We further evaluated the correlation between the gene expression of FGF19 and the gene expression of the two secreted proteins in HCC cell lines and human HCC tissues. FGF19 levels were significantly correlated with ST6GAL1 levels, but not with GDF15 levels, in nine human hepatoma cell lines (Figure 4) and HCC tissues from the TCGA cohort (Figure 5). In summary, the secreted protein ST6GAL1 was shown to be the most positively correlated protein with FGF19 in HCC. Therefore, ST6GAL1 may be a surrogate serum marker for FGF19-driven HCC.

Serum ST6GAL1 is a biomarker for FGF19-driven high-grade HCC We next investigated the association between serum ST6GAL1 levels and tumor FGF19 expression in 62 HCC patients who underwent curative surgical resection.

Immunohistochemical analysis showed that approximately one-quarter of HCC patients showed high levels of FGF19 expression in tumor cells. FGF19-high HCC patients showed significantly more advanced disease and shorter RFS after curative resection (Fig. 5), suggesting that FGF19-high HCC is highly malignant, consistent with previous reports (Dig Dis Sci 2013;58:1916-1922). Serum ST6GAL1 levels were significantly higher in FGF19-high HCC patients than in FGF19-low HCC patients (Fig. 6). Furthermore, using a cutoff serum ST6GAL1 value of 19.1 ng/ml determined by the Youden index, patients with FGF19-high HCC were identified with high sensitivity and specificity (Fig. 7). Furthermore, serum ST6GAL1 levels were positively correlated with tumor size and stage (Fig. 8), and patients with high serum ST6GAL1 levels had significantly shorter RFS than those with low levels (Fig. 9). In contrast, serum FGF19 levels at the tumor site did not differ between FGF19-high and FGF19-low HCC patients and could not be used to discriminate between groups (Figure 10). Furthermore, serum FGF19 levels did not correlate with either stage or RFS of HCC patients (Figure 11). These results demonstrate that ST6GAL1 is a reliable serum biomarker for identifying aggressive FGF19-expressing HCC patients.

Serum ST6GAL1 is a biomarker for lenvatinib-sensitive HCC Based on experimental evidence that FGF19-driven HCC shows high lenvatinib sensitivity, we hypothesized that baseline serum ST6GAL1 levels would also be a useful biomarker for predicting lenvatinib sensitivity in HCC patients. To test this hypothesis, we examined pretreatment serum ST6GAL1 levels in 96 advanced HCC patients who subsequently received lenvatinib or sorafenib treatment and analyzed their association with prognosis. There were no differences in serum ST6GAL1 levels (Figure 12) or OS (Figure 13A) between lenvatinib-treated and sorafenib-treated patients. Patients were then divided into ST6GAL1-high and ST6GAL1-low groups based on the cutoff serum ST6GAL1 level used to identify FGF19-high HCC. In 62 HCC patients with low serum ST6GAL1 levels, OS did not differ between lenvatinib-treated and sorafenib-treated patients (Figure 13B). On the other hand, among 34 HCC patients with high serum ST6GAL1 levels, lenvatinib-treated patients showed significantly longer OS than sorafenib-treated patients (Figure 13C). In addition to Child-Pugh score, Barcelona Clinical Liver Cancer (BCLC) stage, and serum alpha-fetoprotein (AFP) level, lenvatinib treatment was also identified as one of the predictors of improved OS in the ST6GAL1-high group by univariate Cox proportional hazards analysis (Table 7).

These results suggest that clinical observations may help select HCC patients for whom lenvatinib therapy provides superior survival benefits over sorafenib therapy. It is expected that the efficacy of lenvatinib in subjects stratified based on ST6GAL1 expression is mainly due to the FGFR4 inhibitory activity of lenvatinib.

The present disclosure provides a biomarker that correlates with the expression of FGF 19. Based on this finding, a new method for evaluating conditions such as cancer (e.g., drug sensitivity) without placing an excessive burden on patients is provided.

Claims (5)

1. A method of using expression of a biomarker in a subject having a proliferative disease as an indication of the aggressiveness of the proliferative disease, comprising:
measuring expression of said biomarker in a sample from said subject;
When the expression level of the biomarker exceeds a reference value, the possibility that the proliferative disease in the subject is highly malignant is indicated ;
The biomarkers include ST6GAL1;
The method , wherein said proliferative disease is liver cancer . The method of claim 1, wherein the aggressiveness of the proliferative disease comprises high expression of FGF19. The method of claim 1 or 2, wherein the sample comprises a blood sample. The method of any one of claims 1 to 3, wherein the biomarkers comprise a further biomarker selected from the group consisting of CTGF, CCL20, GDF15 , ORM1, and SERPINI1. A kit for use in the method according to any one of claims 1 to 3 , comprising a detection agent for said biomarker.