Use of FGF19 amplification for evaluating and / or predicting sensitivity of esophageal cancer patients to radiotherapy

Abstract

The present invention relates to the use of FGF19 amplification as a biomarker for evaluating and / or predicting the sensitivity of esophageal cancer cells or esophageal cancer patients to radiotherapy. Specifically, the invention relates to application of a reagent for detecting an FGF19 gene or mRNA thereof or a protein or a protein fragment coded by the FGF19 gene in preparation of a kit for evaluating and / or predicting sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy. The invention also relates to a kit for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy.

Description

Technical Field

[0001] The present invention relates to evaluating and / or predicting the sensitivity of esophageal cancer cells or esophageal cancer patients to radiotherapy based on biomarkers. Specifically, the present invention relates to evaluating and / or predicting the sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy based on reagents based on gene markers or their mRNA or proteins or protein fragments encoded therein. Background Art

[0002] At present, clinical intervention measures for esophageal cancer mainly include surgery, radiotherapy, chemotherapy and drug therapy. Radiotherapy plays an important role in the comprehensive treatment of esophageal cancer. For patients with operable esophageal cancer, surgery after neoadjuvant chemoradiotherapy is currently a widely recognized standard treatment option; for patients with inoperable esophageal cancer, synchronous chemoradiotherapy is the only treatment option that may provide a chance of radical cure; for patients with postoperative indications for adjuvant radiotherapy, radiotherapy can improve the patient's local control rate and survival rate; for patients with advanced metastatic esophageal cancer, radiotherapy can improve the patient's local symptoms, improve the quality of life, and improve survival. However, among patients with esophageal cancer who receive radiotherapy, 40%-60% of patients will have residual tumors or recurrences.

[0003] Therefore, enhancing the radiotherapy sensitivity of esophageal cancer patients and improving the efficacy of radiotherapy are issues that urgently need to be addressed in clinical practice. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the inventors of the present application screened new targets for the treatment of esophageal cancer based on the gene sequencing results of clinical esophageal cancer patients; and screened out a suitable variant gene FGF19 through esophageal cancer gene sequencing data. Then, the inventors of the present application analyzed the characteristics of the FGF19 gene in esophageal cancer and its effect on prognosis through the database, and analyzed that FGF19 may have the potential to be a target. Finally, the inventors of the present application found through in vivo and in vitro experiments that FGF19 amplification causes esophageal cancer cells to resist radiotherapy. Based on this, the inventors of the present application found that FGF19 can be used as a key biomarker for clinical evaluation and / or prediction of reduced sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy, and has great clinical medical transformation value.

[0005] The present invention generally relates to the use of FGF19 amplification as a biomarker to evaluate and / or predict the sensitivity of esophageal cancer cells or esophageal cancer patients to radiotherapy.

[0006] In one aspect, the present invention relates to the use of a reagent for detecting the FGF19 gene or its mRNA or the protein or protein fragment it encodes in the preparation of a kit for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy, wherein compared with the control, an increased expression level and / or copy number amplification of the FGF19 gene indicates that the esophageal cancer cells or patients with esophageal cancer have a reduced sensitivity to radiotherapy.

[0007] As described in detail in the definition section below, a person skilled in the art will understand the meaning of a control and how to set a control. For example, a control may be a healthy esophageal cell without FGF19 gene amplification, or a diseased cell unrelated to FGF19 gene amplification, such as an esophageal cancer cell in the early stage of the disease or a patient with esophageal cancer whose sensitivity to fluorouracil chemotherapy is not reduced.

[0008] FGF19 amplification referred to herein has the same meaning as FGF19 gene amplification, covering FGF19 gene copy number amplification and increased expression level, etc. Those skilled in the art also understand how to judge "increase" and / or "amplification", as described in detail in the definition section below, the increase and / or amplification is statistically significant, such as at least about 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more increase and / or amplification, such as copy number amplification.

[0009] Furthermore, it is within the technical scope of those skilled in the art to design or select oligonucleotide probes, primers or antibodies for detecting FGF19 gene amplification and other reagents and methods for implementing the detection based on the FGF19 gene sequence (NM_005117) or the corresponding mRNA and protein sequences.

[0010] In some embodiments, the reagent for detecting the FGF19 gene or its mRNA or the protein or protein fragment encoded by it includes a binding agent that binds to the protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA.

[0011] In some embodiments, the binding agent that binds to the protein or protein fragment encoded by the FGF19 gene is an anti-FGF19 antibody.

[0012] In some embodiments, the substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA includes or is a probe or an oligonucleotide primer.

[0013] Based on the teachings of the present invention, those skilled in the art may also select any other technical means and reagents that can detect the FGF19 gene or its mRNA or its encoded protein or protein fragment.

[0014] In another aspect, the present invention relates to a kit for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy, wherein the kit comprises a reagent for detecting the FGF19 gene or its mRNA or the protein or protein fragment encoded therein.

[0015] In some embodiments, the reagent for detecting the FGF19 gene or its mRNA or the protein or protein fragment encoded by it includes a binding agent that binds to the protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA.

[0016] In some embodiments, the binding agent that binds to the protein or protein fragment encoded by the FGF19 gene is an antibody against FGF19. Those skilled in the art can reasonably determine any anti-FGF19 antibody that can achieve the purpose of detection, such as a monoclonal antibody, a polyclonal antibody, a single-chain antibody, etc. or an antibody fragment thereof, according to the purpose of detection and actual conditions; the antibody can be a mouse antibody, a human antibody, etc.

[0017] In some embodiments, the substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA includes or is a probe or an oligonucleotide primer.

[0018] In some embodiments, the esophageal cancer is esophageal squamous cell carcinoma or esophageal adenocarcinoma.

[0019] In some embodiments, the esophageal cancer is esophageal squamous cell carcinoma.

[0020] In some embodiments, the esophageal cancer is selected from recurrent esophageal cancer, metastatic esophageal cancer, recurrent metastatic esophageal cancer, and advanced metastatic esophageal cancer.

[0021] In yet another aspect, the present invention relates to a method for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy, comprising the following steps:

[0022] (1) obtaining a biological sample from a subject; and

[0023] (2) detecting the expression level and / or copy number of the FGF19 gene or its mRNA or its encoded protein or protein fragment in the sample;

[0024] Compared with the control, increased expression level and / or copy number amplification of the FGF19 gene indicates that the esophageal cancer cells or patients with esophageal cancer have reduced sensitivity to radiotherapy.

[0025] In one embodiment, the biological sample is ctDNA, tumor tissue, tumor circulating cells, or tissue from other sources in the human body.

[0026] Those skilled in the art can reasonably select and determine appropriate detection techniques according to the detection purpose and actual situation. Suitable for different detection targets, the detection techniques may include but are not limited to gene sequencing, PCR, FISH, immunohistochemistry, ELISA, Western or flow cytometry.

[0027] As shown in the following examples,

[0028] Anlotinib can reverse the reduced sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy caused by increased expression level and / or copy number amplification of the FGF19 gene. Thus, in another aspect of the present invention, the present invention relates to the use of anlotinib to prepare a drug for improving (compared with a control) the sensitivity of esophageal cancer cells or patients with esophageal cancer with FGF19 gene amplification (including increased expression level and / or copy number amplification of the FGF19 gene) to radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : The results of gene sequencing of 50 patients with esophageal cancer show the top 16 gene mutations and mutation frequencies. Among them, it shows that the mutation type of FGF19 gene in esophageal cancer is gene amplification, ranking third.

[0030] Figure 2 :Database analysis of FGF19-related features. A. cBioPortal website analysis of the proportion of FGF19 gene amplification in esophageal cancer datasets; B. GeneMANIA database analysis of 20 genes related to FGF19; C. STRING database analysis of 10 proteins related to FGF19; D. KEGG pathway enrichment analysis of FGF19+20 related genes; E. KEGG pathway enrichment analysis of FGF19+10 related proteins.

[0031] Figure 3 :FGF19 is differentially expressed in esophageal cancer tissues and normal tissues. A. TCGA pan-cancer analysis showed that FGF19 was highly expressed in a variety of cancer tissues; B. FGF19 was significantly highly expressed in esophageal cancer tissues; C. ROC curve of FGF19 expression predicted esophageal cancer tumor tissues drawn using TCGA database (AUC = 0.788); D. ROC curve of FGF19 expression predicted esophageal cancer tumor tissues drawn using TCGA + GTEx database (AUC = 0.914).

[0032] Figure 4:Successfully constructed esophageal cancer cell lines containing FGF19 gene amplification. A. qPCR experiments and protein immunoblotting experiments verified that FGF19 amplified esophageal cancer cells were successfully constructed (KYSE-410FGF19AM and KYSE-510FGF19AM, respectively); B. FGF19 amplification promotes the proliferation of esophageal cancer cells. The control group was the corresponding empty esophageal cancer cell lines (KYSE-410CON and KYSE-510CON).

[0033] Figure 5 :FGF19 amplification leads to a significant decrease in radiosensitivity of esophageal cancer cells.

[0034] Figure 6 : Anlotinib enhances the radiosensitivity of FGF19-amplified esophageal cancer cells.

[0035] Figure 7 : Exploration of the signaling pathway by which FGF19 amplification reduces the radiosensitivity of esophageal cancer cells. A. FGF19-amplified esophageal cancer cells activate the downstream ERK signaling pathway through FGF19-FGFR4 signaling, resulting in decreased radiosensitivity; B. FGF19-amplified esophageal cancer cells do not reduce radiosensitivity through the Wnt signaling pathway, but inhibiting the Wnt signaling pathway can enhance the radiosensitivity of esophageal cancer cells (LGK974 is a Wnt pathway inhibitor).

[0036] Figure 8 : Shows γ-H2AX immunofluorescence staining experiments during radiotherapy (4Gy) and radiotherapy in the presence of anlotinib (2μM) or FGF401 (10μM). Magnification 200 times. The groups represented from top to bottom in the legends in the lower figure are: control group / blank control, control group / radiotherapy (4Gy), FGF19 amplification group / blank control, FGF19 amplification group / radiotherapy (4Gy), FGF19 amplification group / radiotherapy (4Gy) + anlotinib (2μM), FGF19 amplification group / radiotherapy (4Gy) + FGF401 (10μM).

[0037] Fig. 9 :FGF19 amplification affects the sensitivity of esophageal cancer cell subcutaneous transplants to radiotherapy. A. The therapeutic response of esophageal cancer cells in the control group to radiotherapy (4Gy×3F). B. The therapeutic response of FGF19 amplified esophageal cancer cells to each treatment, where the treatments represented from top to bottom in each figure are: blank control group, radiotherapy group (4Gy×3F), anlotinib group (0.3mg / Kg / d), radiotherapy group (4Gy×3F) + anlotinib group (0.3mg / Kg / d).

[0038] Fig.10: Immunohistochemical detection of subcutaneous tumors, targeting Ki 67 and CD31. The legends in the bar graphs represent from top to bottom: control group / blank control, control group / radiotherapy, FGF19 amplification group / blank control, FGF19 amplification group / radiotherapy, FGF19 amplification group / anlotinib, FGF19 amplification group / radiotherapy+anlotinib.

[0039] Fig.11 : Map of plasmid pcDNA3.1-FGF19. DETAILED DESCRIPTION

[0040] The present invention relates to the relationship between the expression and / or copy number of a newly discovered biomarker (i.e., FGF19) and the sensitivity of esophageal cancer, especially esophageal squamous cell carcinoma to radiotherapy. The biomarkers described herein provide uses and methods for evaluating and / or predicting the effects of radiotherapy on the treatment of esophageal cancer. Therefore, one embodiment of the present invention represents an improvement in a biomarker, which is suitable for evaluating and / or predicting the effects of radiotherapy on the treatment of esophageal cancer. In another embodiment, the newly discovered biomarker (i.e., FGF19) of the present invention can be used in combination with one or more other cancer markers known in the art (e.g., CEA, CA 19-9, CA 125, CA 72-4, SCC, CF21-1, TSGF, P53-Ab, VEGFR2, VEGFA, CD24), for example, for evaluating and / or predicting the effects of radiotherapy on the treatment of esophageal cancer or for preparing a kit for this purpose.

[0041] The present invention discovered and confirmed that FGF19 amplification (e.g., increase in gene copy number) is a key biomarker for predicting radiotherapy sensitivity in esophageal squamous cells, which suggests that FGF19 has the potential to become a key biomarker for clinical evaluation and / or prediction of reduced sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy, and has huge exploration potential and application prospects.

[0042] The inventors hope to clarify some concepts here. For example, mutant genes detected in cancer cells, even mutant genes used as biomarkers for detection, are not necessarily related to the sensitivity of cancer or tumors to radiotherapy. There is no necessary connection between the two. As described in Example 1 of the present application, based on the frequency statistical analysis of the gene sequencing results of 50 patients with esophageal cancer, we found the top 16 mutant genes, among which the FGF19 mutant gene ranked only third, and the TP53 and CCND1 mutant genes ranked first and second respectively. The phenomenon that FGF19 gene amplification reduces the sensitivity of esophageal cancer cells to radiotherapy found in the present invention is unexpected.

[0043] definition

[0044] The term "sample" means a material known or suspected to express or contain a biomarker (i.e., FGF19) or a binding agent, such as an antibody specific to a biomarker (i.e., FGF19). The sample may be derived from a biological source ("biological sample"), such as a tissue (e.g., a biopsy sample), an extract, or a cell culture including a cell (e.g., a tumor cell), a cell lysate, and a biological or physiological fluid, such as whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, urine, milk, peritoneal fluid, etc. The sample obtained from the source or the sample after pretreatment to improve the sample characteristics (e.g., preparing plasma from blood, diluting mucus, etc.) can be used directly. In certain aspects of the present invention, the sample is a human physiological fluid, such as human serum. In certain aspects of the present invention, the sample is a biopsy sample, such as a tumor tissue or cell obtained by tissue examination. In certain aspects of the present invention, the sample is a malignant or normal tissue sample, such as a normal tissue sample adjacent to cancer.

[0045] The term "marker" as used herein refers to a molecule to be used as a target for analyzing a patient's experimental sample. Examples of such molecular targets are genes, proteins or polypeptides. Genes, proteins or polypeptides used as markers in the present invention are expected to include naturally occurring variants of the gene or protein and fragments of the gene or protein or the variant, in particular immunologically detectable fragments. Immunologically detectable fragments preferably contain at least 6, 7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. It will be appreciated by those skilled in the art that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cut into such fragments. Certain markers are synthesized in an inactive form, which may subsequently be activated by proteolysis. As will be appreciated by the skilled person, proteins or fragments thereof may also exist as part of a complex. Such a complex may also be used as a marker in the sense of the present invention. Variants of marker polypeptides may be encoded by the same gene, but may differ in their isoelectric point (=PI) or molecular weight (=MW) or both, for example as a result of optional mRNA or mRNA precursor processing. The amino acid sequence of the variant has 95%, 96%, 97%, 98%, 99% or higher identity with the corresponding marker sequence. In addition, or in the alternative, the marker polypeptide or its variant may carry a post-translational modification. Non-limiting examples of post-translational modifications are glycosylation, acylation and / or phosphorylation.

[0046] The expression of the marker can also be identified by detecting the translation of the marker (i.e., detection of the marker protein in the sample). Methods suitable for detecting marker proteins include any suitable method for detecting and / or measuring proteins derived from cells or cell extracts. Such methods include, but are not limited to immunoblotting (such as Western blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry and immunofluorescence. Particularly preferred methods for detecting proteins include any cell-based assays, including immunohistochemistry and immunofluorescence assays. Such methods are well known in the art.

[0047] The terms "subject", "patient" and "individual" are used interchangeably herein and refer to warm-blooded animals, such as mammals. The term includes, but is not limited to, livestock, rodents (such as rats and mice), primates and humans. Preferably, the term refers to humans.

[0048] Fibroblast growth factor 19 (FGF19) was first discovered in the human brain. Its coding gene is located on chromosome 11q13 and consists of 216 amino acids. It is highly expressed in ileal and gallbladder epithelial cells, but cannot be detected in normal liver. FGF15 is highly expressed in the ileum, jejunum and duodenum of adult mice. It is the mouse homolog of FGF19 and consists of 218 amino acids. It has 51% similarity with human FGF19. The FGF19 gene discussed in this article refers to the human FGF19 gene whose sequence is shown in NCBI No. NM_005117.

[0049] The term "control" should be understood according to the general understanding of those skilled in the art, and represents any useful reference for comparing gene copy number, protein or mRNA level. The control can be any sample, standard, standard curve or level for comparison purposes. The control can be a normal reference sample or reference standard or level. The control can be a blood control sample of the patient himself; the expression of an internal reference gene (such as FGF19) of esophageal cancer such as esophageal squamous cell carcinoma tumor cells themselves; predetermined cells that are relatively insensitive to radiotherapy (e.g., primary esophageal squamous cell carcinoma cells that are relatively insensitive to radiotherapy), such as "normal control" or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample from a subject without the disease (e.g., a cell or tissue); or a sample of a purified protein or RNA at a known normal concentration. "Reference standard or level" refers to a value or number derived from a reference sample. "Normal control value" is a predetermined value indicating a non-disease state, for example, a value expected in a healthy control subject. Typically, normal control values ​​are expressed as a range ("between X and Y"), a high threshold ("not higher than X"), or a low threshold ("not lower than X"). For a particular biomarker, a subject with a measurement value within the normal control value is often referred to as being "within normal limits" for the biomarker. A normal reference standard or level can be a normal subject who has never suffered from a disease or condition (e.g., cancer). The empty esophageal cancer cell lines (KYSE-410CON and KYSE-510CON) constructed in the examples herein are the control groups of the constructed FGF19 amplified stably transfected esophageal cancer cell lines (KYSE-410FGF19AM / KYSE-510FGF19AM).

[0050] Compared to a control or standard (e.g., normal levels from healthy esophageal cells, levels at different stages of the disease or levels in other samples from patients, levels of esophageal cancer cells from previous stages of the disease or levels in patients with esophageal cancer that have maintained undiminished sensitivity to radiotherapy), an "increase" or "decrease" in the expression level of a marker in a patient sample or an "amplification" or "deletion" in the copy number can represent a level that is higher or lower than the standard error of the detection assay, preferably the level or copy number is at least about 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more of the control or standard, or at most about 1 / 1.1, 1 / 1.25, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9 or 1 / 10 or less of the control or standard. Copy number amplification or deletion can be detected by techniques well known in the art, such as high-throughput sequencing as described in the examples, or whole genome sequencing, whole transcriptome sequencing, other second-generation sequencing detection methods, chip detection, droplet digital polymerase chain reaction (ddPCR), etc. as known in the art. FGF19 gene amplification referred to herein means that the copy number of the FGF19 gene is increased or the expression level is increased compared to the above-mentioned control or standard that does not contain amplification, preferably increased or increased by at least about 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or more.

[0051] The terms "polypeptide" and "protein" or "protein" are used interchangeably herein to refer to at least one molecular chain of amino acids linked by covalent and / or non-covalent bonds. The term includes peptides, oligopeptides and post-translational modifications of proteins and polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Protein fragments, analogs, mutant proteins or variant proteins, fusion proteins, etc. are also included in the meaning of the term.

[0052] In the present invention, protein fragment refers to a polypeptide having an amino terminal deletion, a carboxyl terminal deletion and / or an intermediate deletion compared to the full-length native protein. The fragment may also contain modified amino acids compared to the native protein. In certain embodiments, the fragment length is about 5-215 amino acids. For example, the fragment length may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150 or 200 amino acids. In one embodiment, the fragment is an immunologically detectable fragment, which preferably comprises at least 6, 7, 8, 10, 12, 15 or 20 consecutive amino acids of a marker polypeptide. The change of protein expression level refers to the expression level compared with the control or standard, at least at least about 1.1,1.25,1.5,2,3,4,5,6,7,8,9 or 10 times or more times of the expression level of the control or standard or at most about 1 / 1.1,1 / 1.25,1 / 1.5,1 / 2,1 / 3,1 / 4,1 / 5,1 / 6,1 / 7,1 / 8,1 / 9 or 1 / 10 or less of the expression level of the control or standard.In this article, due to the amplification of FGF19 gene, its corresponding RNA (such as mRNA) and protein expression or level can also be compared to control group and increase, for example, increase at least about 1.1,1.25,1.5,2,3,4,5,6,7,8,9 or 10 times or more times.In certain embodiments, as used herein, mensuration " protein expression level ", " gene expression " or " gene expression level " include but not limited to mensuration corresponding RNA, albumen or peptide level (or its combination). The present invention is not limited to specific methods and reagents for measuring protein, peptide or RNA levels, all of which are well known in the art.

[0053] Methods for determining the amount or concentration of a protein in a sample are known to the skilled artisan. Such methods include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays. For methods using antibodies, both monoclonal and polyclonal antibodies are suitable. The antibodies may be immunologically specific for a protein, protein epitope, or protein fragment.

[0054] The term "oligonucleotide" refers to a polymeric form of nucleotides of any length, which is a ribonucleotide or a deoxyribonucleotide. The term includes double-stranded and single-stranded DNA and RNA, such as modified forms and unmodified forms such as methylation or capping of polynucleotides. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. Oligonucleotides may include but are not necessarily other coding or non-coding sequences, or they may be but are not necessarily connected to other molecules and / or carriers or support materials. The oligonucleotides used in the methods of the present invention or kits may have any length suitable for a specific method. In certain applications, the term refers to an antisense nucleic acid molecule (e.g., an mRNA or DNA chain in the opposite direction of a sense polynucleotide encoding a cancer marker of the present invention (e.g., FGF19)).

[0055] Oligonucleotides used in the present invention include complementary nucleic acid sequences and nucleic acids substantially identical to these sequences, and also include sequences that are different from nucleic acid sequences due to genetic code degeneracy. Oligonucleotides that can be used in the present invention also include nucleic acids that hybridize to oligonucleotide cancer marker nucleic acid sequences under stringent conditions, preferably under high stringency conditions.

[0056] Nucleotide hybridization assays are well known in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected based on known general binding methods, see, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. Science Press, 2002); and Young and Davis, PNAS, 80:1194 (1983). Methods and apparatus for performing repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996, 6,386,749 and 6,391,623, each of which is incorporated herein by reference.

[0057] In some cases, it may be necessary to amplify the sample. Genomic samples can be amplified by various mechanisms, some of which can use PCR. Samples can be amplified on an array. See, for example, U.S. Patent No. 6,300,070 and U.S. Patent Application Serial No. 09 / 513,300.

[0058] Other suitable amplification methods include ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al. Science 241, 1077 (1988), and Barringer et al. Gene 89: 117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88 / 10315), self-sustaining sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90 / 06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence-primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245), and nucleic acid-based sequence amplification (NABSA) (see U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference).

[0059] Reagents that can be used to detect the expression level and / or copy number of FGF19 are well known in the art. Such reagents suitable for use in the present invention can be purchased commercially or routinely prepared by methods well known to those skilled in the art.

[0060] The term "binding agent" refers to substances such as polypeptides, antibodies, ribosomes or aptamers that specifically bind to the biomarkers of the present invention (FGF19). If a substance reacts with the biomarkers of the present invention at a detectable level, but does not detectably react with peptides containing unrelated sequences or sequences of different polypeptides, it "specifically binds" to the biomarkers of the present invention. Binding properties can be evaluated by methods that can be easily performed by those skilled in the art, such as ELISA.

[0061] The binding agent may be a ribosome, RNA or DNA molecule or a polypeptide with or without a peptide component. The binding agent may be a polypeptide comprising a polypeptide biomarker sequence, a peptide variant thereof, or a non-peptide mimetic of such a sequence.

[0062] Aptamers include DNA or RNA molecules that bind to nucleic acids and proteins. Aptamers that bind to the markers of the present invention can be produced using conventional techniques without undue experimentation. [See, for example, the following publications describing in vitro selection of aptamers: Klug et al., Mol. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429 (1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al., Mol. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996)].

[0063] Antibodies for use in the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2), dAbs (domain antibodies; see Ward et al., 1989, Nature, 341:544-546), antibody heavy chains, intracellular antibodies, humanized antibodies, human antibodies, antibody light chains, single-chain Fvs (scFvs) (e.g., including monospecific, bispecific, etc.), anti-idiotypic (ant-Id) antibodies, proteins comprising antibody portions, chimeric antibodies (e.g., antibodies containing the binding specificity of a mouse antibody but in which the remainder is of human origin), derivatives such as enzyme conjugates or labeled derivatives, double-chain antibodies, linear antibodies, disulfide-linked Fvs (sdFvs), multispecific antibodies (e.g., bispecific antibodies), epitope-binding fragments of any of the above, and any other modified configurations of immunoglobulin molecules comprising an antigen recognition site of the desired specificity. Antibodies include antibodies of any type (e.g., IgA, IgD, IgE, IgG, IgM, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b), and the antibodies need not be of any particular type, class, or subclass. In certain embodiments of the invention, the antibody is an IgG antibody or a class or subclass thereof. The antibody may be from any animal source, including birds and mammals (e.g., humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens).

[0064] For example, antibodies for use in the present invention are commercially available from, for example, BioVision (e.g., catalog number 5542R-100), Abcam (e.g., catalog number ab172545), etc. Alternatively, the antibodies can be prepared by recombinant methods known in the art. In some embodiments, the antibodies are monoclonal antibodies. For the preparation of monoclonal antibodies, see, for example, Kohler et al. (1975) Nature 256: 495-497; Kozbor et al. (1985) J. Immunol Methods 81: 31-42; Cote et al. (1983) Proc Natl Acad Sci 80: 2026-2030 and Cole et al. (1984) Mol Cell Biol 62: 109-120.

[0065] The kit according to the present disclosure can be prepared by conventional methods in the art. The kit may contain materials or reagents required for implementing the method or use of the present invention (including reagents for detecting the FGF19 gene or its mRNA or its encoded protein or protein fragment). The kit may include containers for storing reaction reagents (e.g., primers, dNTPs, enzymes, etc. in suitable containers) and / or supporting materials (e.g., buffers, instructions for implementing detection, etc.). For example, the kit may include one or more containers (e.g., boxes) containing corresponding reaction reagents and / or supporting materials. Such contents can be applied to the sample to be detected together or separately. As an example, the kit may contain reagents, buffers, and instructions for use for detecting the FGF19 gene or its mRNA or its encoded protein or protein fragment. The kit may also contain polymerase and dTNP, etc. The kit may also contain internal standards, positive and negative controls, etc. for quality control. The kit may also contain reagents for preparing nucleic acids such as DNA from samples. The above examples are only used to more clearly illustrate the present invention, and do not constitute any limitation on the kits and contents thereof applicable to the present invention.

[0066] Example

[0067] In order to explain the content of the present invention more clearly, it will be described in detail below with reference to the accompanying drawings and embodiments.

[0068] Example 1 Screening of new therapeutic targets for esophageal cancer based on gene sequencing results of clinical esophageal cancer patients

[0069] In clinical work, we collected clinical data of esophageal cancer patients who underwent gene sequencing. We statistically analyzed the whole genome sequencing results of 50 esophageal cancer patients collected by the Animal Experiment Center of Nanfang Hospital and screened out new therapeutic targets.

[0070] The frequency statistical analysis of the gene sequencing results of 50 patients with esophageal cancer found that the top 16 variant genes were: TP53, CCND1, FGF19, NOTCH1, LRP1B, FAT1, MYC, CDKN2A, NSD1, EP300, CHEK2, ZNF217, PTCH1, DICER1, CBLB, GNAS. The types of gene mutations included: gene mutation, gene amplification, single copy number deletion, and gene fusion. The top three gene mutations and their proportions were: TP53 gene mutation accounted for 88%, CCND1 gene mutation accounted for 48% (among which amplification accounted for 46% and mutation accounted for 2%), and FGF19 gene amplification accounted for 46%. Figure 1 shown.

[0071] We screened out FGF19 as a new target for research.

[0072] Example 2 Database Analysis of FGF19 Gene Characteristics in Esophageal Cancer and Its Effect on Prognosis

[0073] Although the research results based on Example 1 show that FGF19 amplification has the potential to be a new target for precision treatment of esophageal cancer, it is necessary to further clarify whether the new target is significantly highly expressed in tumor tissues and whether it affects the prognosis of esophageal cancer patients. Therefore, this example further analyzes the characteristics of FGF19 in esophageal cancer patients and its effect on the prognosis of esophageal cancer patients through the sequencing results of a large sample size in an online database.

[0074] The proportion of FGF19 amplification in esophageal cancer was analyzed using the esophageal cancer dataset (TCGA, FirehoseLegacy) from the cBioPortal website. Gene-gene and protein-protein interaction network analysis of FGF19 was performed using the GeneMANIA and STRING websites, and pathway enrichment analysis of FGF19 gene-related genes and proteins was performed using KEGG. The differential expression of FGF19 in esophageal cancer tumor tissues and normal tissues was analyzed using the esophageal cancer dataset from the TCGA database. Genes positively and negatively correlated with FGF19 expression in esophageal cancer were screened using the LinkedOmics database, and the biological function enrichment analysis of FGF19 was performed. The clinical characteristics, prognosis analysis, and visualization of the esophageal cancer dataset from the TCGA database were performed using the R language.

[0075] R software was used for analysis. Independent sample t-test was used to analyze the differential expression level of FGF19 mRNA between esophageal cancer tissues and normal tissues in the TCGA database. Pearson chi-square test was used to analyze the correlation between FGF19 expression and clinicopathological characteristics. Kaplan-Meier plot was used for disease-specific survival (DSS) analysis, and log-rank test was used to compare differences. Cox proportional hazards regression model was used for univariate and multivariate analysis. Two-tailed P < 0.05 was considered statistically significant.

[0076] First, we analyzed the mutation of FGF19 gene in esophageal cancer through the database to understand its characteristics in esophageal cancer. We analyzed the TCGA database esophageal cancer data set (TCGA, Firehose Legacy) through the cBioPortal website and found that FGF19 amplification accounted for 35% in esophageal cancer ( Figure 2A). Gene-gene and protein-protein interaction network analysis was performed using the GeneMANIA website and the STRING website, and 20 potential target genes interacting with the FGF19 gene were found (FGFR4 / KLB / KL / SDC2 / FGFR1 / FGF20 / FGF5 / FGF17 / FGF22 / FGF16 / FGF23 / FGF8 / FGF18 / FGF6 / FGF9 / FGF3 / FGF7 / FGF4 / FGF10 / FGF1)( Figure 2 B) and 10 potential target proteins (KLB / FGFR1 / FGFR3 / FGFR2 / FGF23 / FGF10 / FGF7 / FGF2 / PLCG1 / FGFR4) ( Figure 2 C) FGF19 and 20 related genes ( Figure 2 D) and 10 related protein-coding genes ( Figure 2 E) KEGG pathway enrichment analysis was performed separately, and it was found that the main enriched signaling pathways related to FGF19 at the gene and protein levels were PI3K-Akt signaling pathway and MAPK signaling pathway.

[0077] We analyzed the expression of FGF19 in pan-cancer in the TCGA database and found that FGF19 was differentially expressed in various tumor tissues and normal tissues ( Figure 3 A), the expression of FGF19 in esophageal cancer tissues was significantly higher than that in normal tissues ( Figure 3 B). In the process of tumor development, high expression of FGF19 can predict tumor tissue (AUC = 0.788) ( Figure 3 C), TCGA+GTEx database showed that high expression of FGF19 had a high accuracy in predicting tumor tissue (AUC = 0.914) ( Figure 3 D). The above results suggest that FGF19 is significantly overexpressed in esophageal cancer tumor tissues and may serve as a new target for the treatment of esophageal cancer.

[0078] Example 3 Effect of FGF19 amplification on radiosensitivity of esophageal cancer cells

[0079] Using human esophageal cancer cell lines KYSE-410 and KYSE-510 purchased from Shanghai Meixuan Biotechnology Co., Ltd., we successfully constructed a stably transfected esophageal cancer cell line containing FGF19 amplification (KYSE-410FGF19 AM / KYSE-510FGF19 AM ), and empty vector esophageal cancer cell lines (KYSE-410CON / KYSE-510CON). The plasmid named pcDNA3.1-FGF19 ( Fig.11) was constructed, the plasmid contained the FGF19 gene inserted downstream of the CMV promoter and carried the puromycin resistance gene as a selection marker. The sequence of the FGF19 gene is the gene sequence shown in NCBI No.: NM_005117, and the gene fragment was synthesized by Jikai Biotechnology Co., Ltd. The empty plasmid is pcDNA3.1-empty, which contains the CMV promoter and the puromycin resistance gene, but no FGF19 gene is inserted. When the cells are cultured to 30-50% confluence, 50ul of virus solution and 8μg / ml polybrene are used for transfection. Fresh culture medium is replaced after 24-48 hours of culture. KYSE-410 cells and KYSE-510 cells are placed in complete culture medium containing 250μg / ml and 450μg / ml G418 for 5-7 days respectively after transfection 48-96 hours to obtain cells with G418 resistance, and then monoclonal stable transfection cell lines are screened and cultured.

[0080] Real-time quantitative PCR (qPCR) and Western blot were used to verify the mRNA level and protein level, respectively. Figure 4 As shown in A, the stably transfected esophageal cancer cell line containing FGF19 amplification had significantly higher FGF19 expression than the control group.

[0081] It was also found through clone formation experiments that FGF19 amplification promoted the proliferation of esophageal cancer cells. Stably transfected cells were seeded into 6-well culture dishes at a low density (500 cells / well) and cultured for two weeks, during which the culture medium was changed every 3 days. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of clones formed in each culture dish was counted and compared. The results are shown in Figure 2. Figure 4 B. The experimental comparison results were statistically significant based on the t-test, all P values ​​were two-sided, and P < 0.05 was considered statistically significant. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001.

[0082] 3.1 Cloning experiments confirmed that FGF19 amplification reduces the sensitivity of esophageal cancer cells to radiation

[0083] The esophageal cancer cell lines (KYSE-410CON, KYSE-510CON) and the single gene stable transfection cell lines (KYSE-410FGF19 AM KYSE-510FGF19 AM) and made into a single cell suspension, centrifuged at 1000rpm for 3 minutes, washed with PBS buffer once, removed pancreatin, and adjusted the cell concentration after cell counting using a cell counter. The number of cells inoculated in a 6-well plate and the corresponding radiotherapy dose were: 0Gy-200 cells, 2Gy-400 cells, 4Gy-1000 cells, 6Gy-2000 cells, 8Gy-4000 cells, 3 replicates. After inoculation, the cells were cultured at a constant temperature to adhere to the wall, and irradiated within 24 hours of inoculation. The 6-well plate was sealed with adhesive tape during irradiation, and the sealing adhesive tape was removed after irradiation. After irradiation, the cells were statically cultured in a constant temperature box, and the cells were observed from time to time. If there were too many cell floaters, the liquid was replaced, and the process was gentle. After 10-14 days of culture, when the number of clones visible to the naked eye appeared in the culture dish of the 0Gy dose, and the number of cells was about 50, the culture was terminated and the number of clones was calculated by staining. The process is as follows: discard the culture medium, wash 3 times with PBS, fix with methanol for 15 minutes, wash twice with PBS, stain with crystal violet for 30 minutes, carefully rinse the dye from the edge with running water, take pictures and count after the culture dish is air-dried, calculate the number of clones and clone formation rate. Draw a clone formation curve, such as Figure 5 The experimental comparison results were statistically significant based on the t-test, all P values ​​were two-sided, and P < 0.05 was considered statistically significant.

[0084] The results showed that FGF19 amplified esophageal cancer cells (KYES-410FGF19 AM / KYES-510FGF19 AM ) showed a significantly lower radiosensitivity than that of the esophageal cancer cells in the control group (KYES-410CON / KYES-510CON). Figure 5 shown.

[0085] 3.2 Anlotinib enhances the radiosensitivity of FGF19-amplified esophageal cancer cells

[0086] The above clone formation experiment confirmed that FGF19 amplification reduced the sensitivity of esophageal cancer cells to radiotherapy. Next, we further explored whether anlotinib could enhance the radiotherapy sensitivity of FGF19 amplified esophageal cancer cells. Three control groups and FGF19 amplification groups were set up with inoculation amounts of 500, 2000 and 2000 cells / well, respectively. After inoculation, the cells were cultured at a constant temperature to allow them to adhere to the wall, and 4Gy radiation treatment was performed within 24 hours of inoculation. We found that the radiotherapy sensitivity of FGF19 amplified esophageal cancer cells was significantly lower than that of the control group. The combined use of anlotinib can significantly enhance the radiotherapy sensitivity of FGF19 amplified esophageal cancer cells, while there was no significant change in the control group. Figure 6 As shown in the figure, the experimental comparison results were statistically significant based on the t-test, all P values ​​were two-sided, and P < 0.05 was considered statistically significant.

[0087] The results of this part indicate that anlotinib enhances the radiosensitivity of FGF19-amplified esophageal cancer cells, but has no significant effect on the radiosensitivity of the esophageal cancer cell lines in the control group.

[0088] 3.3FGF19 amplification affects the sensitivity of esophageal cancer cells to radiotherapy through the FGF19-FGFR4-ERK signaling pathway

[0089] Through previous experiments, it has been confirmed that FGF19 amplification reduces the radiosensitivity of esophageal cancer cells, and anlotinib can enhance the radiosensitivity of FGF19 amplified esophageal cancer cells. Next, we further explored the signaling pathways through which FGF19 affects the radiosensitivity of esophageal cancer cells.

[0090] First, a protein immunoblot (Western blot) experiment was performed to explore the expression and activation of related proteins that affect the radiosensitivity of esophageal cancer cells after FGF19 amplification. The antibodies used in the protein immunoblot included p-AKT (Cell Signaling Technology, #4060, 1:1000 dilution), AKT (Cell Signaling Technology, #9272, 1:1000 dilution), p-ERK1 / 2 (Cell Signaling Technology, #4370, 1:1000 dilution), ERK1 / 2 (Cell Signaling Technology, #9102, 1:1000 dilution), p-FGFR4 (phospho Y642) (Abcam, ab192589, 1:1000 dilution), FGFR4 (Abcam, ab178296, 1:1000 dilution), and β-actin (Cell Signaling Technology, #4970, 1:5000 dilution) as internal reference antibodies. Figure 7 As shown in Figure A, after radiotherapy (4Gy), the ERK1 / 2 signal of FGF19-amplified esophageal cancer cells was significantly activated compared with the control group, and the expression of p-ERK1 / 2 protein was significantly increased. When the FGFR4-specific inhibitor FGF401 (10μM) was added, the phosphorylated ERK1 / 2 was significantly inhibited. At the same time, we also used the Wnt pathway inhibitor LGK974 (3μM) to explore whether FGF19 amplification affects the Wnt signaling pathway during radiotherapy. Through CCK8 experiments, we found that radiotherapy combined with LGK-974 significantly enhanced the radiosensitivity of esophageal cancer cells, but it was not related to whether FGF19 was amplified or not. Figure 7As shown in Figure B. The main parameters of the CCK8 experiment are as follows: the number of cells inoculated is 2000 per well, the experimental materials used include CCK8 reagent, the culture conditions are 37°C, 5% CO2, and the experimental time is 72 hours. The experimental comparison results are expressed based on mean ± standard deviation (Mean ± SD), and the inter-group comparison uses t-test or one-way analysis of variance (ANOVA), and the significance level is set at P < 0.05.

[0091] The above results show that radiotherapy leads to the activation of ERK1 / 2 signaling in FGF19-amplified esophageal cancer cells, but radiotherapy combined with FGF401 blocking the FGF19-FGFR4 signaling pathway can inhibit the activation of ERK1 / 2 signaling, and ERK1 / 2 may be the downstream molecules of the FGF19-FGFR4 signaling pathway. At the same time, inhibiting the Wnt signaling pathway can enhance the radiosensitivity of esophageal cancer cells, but the Wnt signal is not a downstream participating signal of FGF19-FGFR4 leading to radiation resistance.

[0092] 3.4 FGF19 amplification leads to reduced DNA fragmentation after radiotherapy

[0093] We used γ-H2AX immunofluorescence assay to detect double-stranded DNA damage in esophageal cancer cells after radiotherapy. We selected exponentially growing esophageal cancer cells (KYSE-410CON / KYSE-410FGF19 AM ,KYSE-510CON / KYSE-510FGF19 AM ) were used for the experiment. First, the concentration of the cell suspension was adjusted to 3000 cells / well. After mixing, 100 μl was inoculated into each well of a 96-well plate. After standing at room temperature for 15 minutes, it was placed in a 37°C carbon dioxide incubator for culture. The next day, the groups were treated: control group, radiotherapy (4Gy) group, radiotherapy + anlotinib (2μM) group, radiotherapy + FGF401 (10μM) group, with 3 replicates in each group. The drug was added 3-6 hours before radiotherapy, and the test was performed 2 hours, 6 hours, and 24 hours after radiotherapy. The DNA damage detection kit (γ-H2AX immunofluorescence method) (Biyuntian, C2035S) was used for detection.

[0094] The results showed that the nuclear γ-H2AX immunofluorescence staining of esophageal cancer cells in the control group (KYSE-410CON / KYSE-510CON) after radiotherapy was significantly higher than that in the esophageal cancer cells in the FGF19 amplification group (KYSE-410FGF19 AM / KYSE-510FGF19 AM ), but after the FGF19 amplification group esophageal cancer cells were treated with anlotinib combined with radiotherapy, the γ-H2AX immunofluorescence staining in the nucleus was significantly enhanced ( Figure 8The experimental comparison results were statistically significant based on the t-test, all P values ​​were two-sided, and P < 0.05 was considered statistically significant.

[0095] The above results show that compared with the esophageal cancer cells in the control group, the DNA double-strand breaks in FGF19 amplified esophageal cancer cells after radiotherapy were significantly reduced; however, anlotinib can significantly increase the nuclear DNA double-strand breaks in FGF19 amplified esophageal cancer cells after radiotherapy.

[0096] 3.5 In vivo experiments verify the effect of FGF19 amplification on radiosensitivity of esophageal cancer cells

[0097] The effect of FGF19 amplification on the radiosensitivity of esophageal cancer cells was further verified by in vivo animal experiments. A total of 60 female BALB / c nude mice aged 3-4 weeks (purchased from the Animal Experiment Center of Southern Medical University, Guangzhou, Guangdong Province, and raised in the SPF environment of the Animal Experiment Center of Southern Hospital) were selected and KYSE-410CON and KYSE-410FGF19AM cells were inoculated subcutaneously on the back of nude mice, with 5×10 cells per nude mouse. 5 cells. After injection, the nude mice were observed for subcutaneous tumor formation and growth of subcutaneous transplanted tumors every 2-5 days, and their body weight was monitored. When the average volume of subcutaneous tumors reached 150-250mm 3 At about 14:00 pm, the nude mice were randomly divided into groups (10 in each group). KYSE-410CON was divided into blank control group, radiotherapy group (4Gy×3F); KYSE-410FGF19 AM The patients were divided into blank control group, radiotherapy group (4 Gy×3F), anlotinib group (0.3 mg / Kg / d×12d, oral gavage), radiotherapy group (4 Gy×3F) + anlotinib group (0.3 mg / Kg / d×12d, oral gavage). The tumor volume was measured every 2-3 days, and the tumor volume change graph was drawn.

[0098] The results showed that the tumor growth rate of the control group slowed down after 1 week of radiotherapy, and the volume after radiotherapy on the 12th day was significantly smaller than that of the blank control group ( p =0.0062). In the FGF19 amplification group, the tumor did not shrink significantly after radiotherapy compared with the blank control group ( p =0.18, and the anlotinib treatment group did not significantly reduce the blank control group ( p =0.25), but after radiotherapy combined with anlotinib treatment, the tumor was significantly smaller than that of the blank control ( p =0.0099), and was significantly better than the radiotherapy group ( p =0.0149) and anlotinib-treated group ( p =0.01), such as Fig. 9 shown.

[0099] The subcutaneous tumor was further stained with immunohistochemistry, and the antibodies used in immunohistochemistry included Ki67 antibody (CellSignaling Technology, #9027, 1:200 dilution) and CD31 antibody (Abcam, ab28364, 1:100 dilution). Fig.10 As shown in the figure, the Ki67 staining positive rate in the FGF19 amplification group was significantly higher than that in the control group (p=0.0386). The Ki67 staining positive rate in the control group decreased significantly after radiotherapy (R), and there was no significant change in the Ki67 staining positive rate in the amplification group after radiotherapy (R). After anlotinib (A) treatment, the Ki67 staining positive rate also did not change significantly, but after radiotherapy combined with anlotinib (R+A) treatment, the Ki67 staining positive rate decreased significantly, indicating that: FGF19 amplification significantly inhibits the sensitivity of esophageal cancer cells to radiotherapy, radiotherapy combined with anlotinib significantly inhibits tumor growth, and radiotherapy and anlotinib have a synergistic effect. There was no significant difference in CD31 staining between the control group and the amplification group and each treatment group. The experimental comparison results were statistically significant based on the t-test, all P were two-sided tests, and P<0.05 was considered statistically significant. * represents P<0.05; ** represents P<0.01; *** represents P<0.001; ns represents no statistical significance.

[0100] 3.6 Conclusion

[0101] Through the above studies, we determined that FGF19 gene amplification reduced the sensitivity of esophageal cancer cells to radiotherapy, and anlotinib could reverse the resistance of FGF19 amplified esophageal cancer cells to radiotherapy.

[0102] Anlotinib had no significant effect on the radiosensitivity of the control group ( Figure 6 ), which further illustrates that anlotinib reverses radiotherapy resistance by inhibiting the FGF19-FGFR4 signaling pathway, rather than other targets of anlotinib. It was also found that the ERK1 / 2 signaling pathway downstream of FGF19-FGFR4 was also involved in this process, and the Wnt signaling pathway was not involved in the downstream pathway of the FGF19-FGFR4 signaling pathway. In clinical work, for patients with esophageal cancer undergoing radiotherapy with FGF19 amplification, the combined use of anlotinib can be considered to enhance radiotherapy sensitivity and improve radiotherapy efficacy.

[0103] According to the present disclosure, all uses, products and methods disclosed and claimed herein can be prepared and implemented without excessive experimentation. Those skilled in the art can modify and change the technical solutions and contents disclosed in the present invention according to practical needs, but these modifications and changes do not deviate from the concept, spirit and scope of the present disclosure. All such modifications and changes are obvious to those skilled in the art and are within the scope of protection defined by the claims of the present application.

Claims

1. Use of a reagent for detecting the FGF19 gene or its mRNA or its encoded protein or protein fragment in the preparation of a kit for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients with esophageal cancer to radiotherapy, wherein compared with a control, an increased expression level and / or amplified copy number of the FGF19 gene indicates that the esophageal cancer cells or patients with esophageal cancer have a reduced sensitivity to radiotherapy.

2. The use according to claim 1, wherein the reagent for detecting the FGF19 gene or its mRNA or its encoded protein or protein fragment comprises a binding agent that binds to the protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA.

3. The use according to claim 2, wherein the binding agent that binds to the protein or protein fragment encoded by the FGF19 gene is an anti-FGF19 antibody.

4. The use according to claim 2, wherein the substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA is a probe or an oligonucleotide primer.

5. A kit for evaluating and / or predicting the sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy, wherein the kit comprises a reagent for detecting the FGF19 gene or its mRNA or the protein or protein fragment encoded by it.

6. A kit according to claim 5, wherein the reagent for detecting the FGF19 gene or its mRNA or its encoded protein or protein fragment comprises a binding agent that binds to the protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA.

7. The kit according to claim 6, wherein the binding agent that binds to the protein or protein fragment encoded by the FGF19 gene is an anti-FGF19 antibody.

8. The kit according to claim 6, wherein the substance that hybridizes with the FGF19 gene or its mRNA or amplifies the FGF19 gene or its mRNA is an oligonucleotide primer or a probe.

9. Use of anlotinib in the preparation of a drug for improving the sensitivity of FGF19 gene amplified esophageal cancer cells or patients with esophageal cancer to radiotherapy.

10. The use according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 8 or the use according to claim 9, wherein the esophageal cancer is esophageal squamous cell carcinoma or esophageal adenocarcinoma.

11. The use according to any one of claims 1-4 or the kit according to any one of claims 5-8 or the use according to claim 9, wherein the esophageal cancer is selected from recurrent esophageal cancer, metastatic esophageal cancer, recurrent metastatic esophageal cancer and advanced metastatic esophageal cancer.

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