CN119101741A

CN119101741A - Use of FGF19 amplification for evaluating and/or predicting the sensitivity of esophageal cancer patients to radiotherapy

Info

Publication number: CN119101741A
Application number: CN202410984971.8A
Authority: CN
Inventors: 王玮; 林水苗; 曾钦; 褚帅
Original assignee: Nanfang Hospital of Southern Medical University
Current assignee: Nanfang Hospital of Southern Medical University
Priority date: 2024-07-22
Filing date: 2024-07-22
Publication date: 2024-12-10

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 patients with esophageal cancer to radiation therapy. In particular, the invention relates to the use of a reagent for detecting the FGF19 gene or an mRNA thereof or a protein or protein fragment encoded thereby, for the preparation of a kit for evaluating and/or predicting the sensitivity of esophageal cancer cells or a patient suffering from esophageal cancer to radiation therapy. 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 radiation therapy.

Description

Use of FGF19 amplification for evaluating and/or predicting the sensitivity of a patient with esophageal cancer to radiation therapy

Technical Field

The present invention relates to evaluating and/or predicting sensitivity of esophageal cancer cells or esophageal cancer patients to radiation therapy based on biomarkers. In particular, the invention relates to agents based on gene markers or mRNA thereof or encoded proteins or protein fragments thereof to evaluate and/or predict the sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiation therapy.

Background

Current clinical interventions for esophageal cancer mainly include surgery, radiation therapy, chemotherapy, and drug therapy. The radiotherapy occupies an important position in the comprehensive treatment of the esophageal cancer, can operate esophageal cancer patients, is a widely accepted standard treatment scheme after new auxiliary radiotherapy and chemotherapy, can not operate esophageal cancer patients, is the only treatment scheme which can possibly obtain radical treatment opportunities, has auxiliary radiotherapy indication after operation, can improve the local control rate and survival rate of patients after receiving radiotherapy, and can improve the local symptoms, life quality and survival of patients after advanced metastatic esophageal cancer patients. However, 40% -60% of patients with esophageal cancer that receive radiation therapy may have tumor residues or recurrence.

Therefore, the method for improving the sensitivity and the curative effect of the radiotherapy of the patients with the esophageal cancer is a problem which is needed to be solved in clinic.

Disclosure of Invention

In order to solve the defects in the prior art, the inventor screens out a new esophageal cancer treatment target point based on the clinical esophageal cancer patient gene sequencing result, and screens out a proper variant gene FGF19 through esophageal cancer gene sequencing data. The inventors then analyzed FGF19 gene characteristics and the effect on prognosis in esophageal cancer by database, and analyzed FGF19 might have potential as a target. Finally, the present inventors found through in vitro and in vivo experiments that FGF19 expansion resulted in esophageal cancer cells resistant to radiotherapy. Based on this, the present inventors found that FGF19 can be a key biomarker for clinical evaluation and/or prediction of reduced sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy, and has great clinical medical transformation value.

The present invention relates generally to the use of FGF19 amplification as a biomarker to evaluate and/or predict the sensitivity of esophageal cancer cells or patients with esophageal cancer to radiation therapy.

In one aspect, the invention relates to the use of an agent for detecting FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby, for the manufacture of a kit for evaluating and/or predicting the susceptibility of an esophageal cancer cell or a patient suffering from esophageal cancer to radiation therapy, wherein an increased expression level and/or copy number amplification of the FGF19 gene compared to a control is indicative for a decreased susceptibility of the esophageal cancer cell or the patient suffering from esophageal cancer to radiation therapy.

As described in detail in the definition section below, one skilled in the art can understand the meaning of the control and understand how to set the control. For example, the control may be healthy esophageal cells without amplification of the FGF19 gene, or diseased cells unrelated to the amplification of the FGF19 gene, such as pre-disease esophageal cancer cells that maintain no decreased sensitivity to fluorouracil chemotherapy, or patients with esophageal cancer.

FGF19 amplification as referred to herein has the same meaning as FGF19 gene amplification and encompasses FGF19 gene copy number amplification, elevated levels of expression, and the like. Those skilled in the art will also know how to judge "elevated" and/or "amplified" as described in detail in the definition section below, which is statistically significant, e.g., at least about 1.1, 1.25, 1.5, 2,3, 4, 5, 6, 7, 8, 9, or 10-fold or more elevated and/or amplified, e.g., copy number amplified.

Also, it is within the skill of those in the art to design or select oligonucleotide probes, primers or antibodies for detecting FGF19 gene amplification, as well as other reagents and methods for performing such detection, based on the FGF19 gene sequence (nm_ 005117) or the corresponding mRNA and protein sequences.

In some embodiments, the reagent for detecting the FGF19 gene or mRNA thereof, or a protein or protein fragment encoded by the FGF19 gene, comprises a binding agent that binds to a protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes to or amplifies the FGF19 gene or mRNA thereof.

In some embodiments, the binding agent that binds to a protein or protein fragment encoded by the FGF19 gene is an antibody to FGF 19.

In some embodiments, the substance that hybridizes to or amplifies the FGF19 gene or mRNA thereof comprises or is a probe or oligonucleotide primer.

Based on the teachings of the present invention, one skilled in the art may also select any other technical means and reagents capable of achieving the detection of the FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby.

In another aspect, the invention relates to a kit for evaluating and/or predicting the susceptibility of esophageal cancer cells or patients suffering from esophageal cancer to radiation therapy, wherein the kit comprises a reagent for detecting the FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby.

In some embodiments, the binding agent that binds to a protein or protein fragment encoded by the FGF19 gene is an antibody to FGF 19. Any antibody capable of achieving the purpose of detection, such as a monoclonal antibody, a polyclonal antibody, a single chain antibody, etc. or an antibody fragment thereof, which may be a murine antibody, a humanized antibody, etc., can be reasonably determined by those skilled in the art according to the purpose of detection and the actual situation.

In some embodiments, the esophageal cancer is esophageal squamous carcinoma or esophageal adenocarcinoma.

In some embodiments, the esophageal cancer is esophageal squamous carcinoma.

In some embodiments, the esophageal cancer is selected from the group consisting of recurrent esophageal cancer, metastatic esophageal cancer, recurrent metastatic esophageal cancer, and advanced metastatic esophagus.

In yet another aspect, the invention relates to a method for evaluating and/or predicting the sensitivity of esophageal cancer cells or a patient suffering from esophageal cancer to radiation therapy, comprising the steps of:

(1) Obtaining a biological sample from a subject, and

(2) Detecting the expression level and/or copy number of FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby in the sample;

Wherein an increased expression level and/or copy number amplification of the FGF19 gene compared to the control is indicative of a decreased sensitivity of esophageal cancer cells or a patient suffering from esophageal cancer to radiation therapy.

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

The person skilled in the art is able to reasonably select and determine a suitable detection technique according to the purpose and the actual situation of the detection. Suitable for different detection targets, detection techniques may include, but are not limited to, genetic sequencing, PCR, FISH, immunohistochemistry, ELISA, western, or flow cytometry.

As will be apparent from the examples section below,

An Luoti can reverse the decreased sensitivity of esophageal cancer cells or patients with esophageal cancer to radiation therapy caused by increased expression levels and/or copy number expansion of the FGF19 gene. Thus, in a further aspect according to the present invention, the present invention relates to the use of erlotinib for the preparation of a medicament for increasing (compared to a control) the sensitivity of FGF19 gene-amplified (including increased expression level and/or copy number amplification of FGF19 gene) esophageal cancer cells or patients suffering from esophageal cancer to radiation therapy.

Drawings

FIG. 1 shows the results of gene sequencing of 50 patients with esophageal cancer, showing the genetic variation and the frequency of variation in the top 16 rows. Wherein the variation type of FGF19 gene in esophageal cancer is shown as gene amplification and is arranged at the 3 rd position.

FIG. 2 database analysis of FGF 19-related features. A, analyzing the gene amplification ratio of FGF19 in esophageal cancer data set by using a cBIoPortal website, B, analyzing 20 genes related to FGF19 by using a GeneMANIA database, analyzing 10 proteins related to FGF19 by using a C.STRING database, performing KEGG pathway enrichment analysis on the genes related to FGF19+20, and performing KEGG pathway enrichment analysis on the proteins related to FGF 19+10.

FIG. 3 differential expression of FGF19 in esophageal cancer tissue and normal tissue. Analysis of TCGA pan-carcinoma species showed high expression of FGF19 in various cancer tissues, b.fgf19 was significantly highly expressed in esophageal cancer tissues, c.fgf 19 expression using TCGA database predicted ROC profile of esophageal cancer tumor tissue (auc=0.788), d.fgf 19 expression using tcga+ GTEx database predicted ROC profile of esophageal cancer tumor tissue (auc=0.914).

FIG. 4 successfully constructs an esophageal cancer cell line containing FGF19 gene amplification. A qPCR experiment and a western blot experiment prove that the FGF19 amplification of esophageal cancer cells is successfully constructed (KYSE-410 FGF19AM and KYSE-510FGF19AM respectively), and B.FGF19 amplification promotes proliferation of esophageal cancer cells. Wherein the control group is corresponding empty esophageal cancer cell lines (KYSE-410 CON and KYSE-510 CON).

Figure 5 fgf19 expansion resulted in a significant decrease in the sensitivity of esophageal cancer cells to radiation therapy.

FIG. 6 An Luoti Nile enhances the sensitivity of FGF19 to radiation therapy for the expansion of esophageal cancer cells.

FIG. 7 exploration of signal pathways that reduce sensitivity to esophageal cancer cell radiotherapy by FGF19 amplification. FGF19 expanded esophageal cancer cells decreased radiation sensitivity by activating ERK signaling downstream of FGF19-FGFR4 signaling, b.fgf19 expanded esophageal cancer cells did not decrease radiation sensitivity by Wnt signaling, but inhibition of Wnt signaling could increase radiation sensitivity of esophageal cancer cells (LGK 974 is a Wnt pathway inhibitor).

FIG. 8 shows an immunofluorescent staining experiment with gamma-H2 AX in the presence of either radiation (4 Gy) or An Luoti Ni (2. Mu.M) or FGF401 (10. Mu.M). Magnification of 200 times. The groups indicated by the legends from top to bottom in the lower panel are control/blank, control/radiotherapy (4 Gy), FGF19 amplified/blank, FGF19 amplified/radiotherapy (4 Gy) + An Luoti ni (2 μm), FGF19 amplified/radiotherapy (4 Gy) +fgf401 (10 μm), respectively.

Figure 9 fgf19 expansion affects the sensitivity of esophageal cancer cell subcutaneous transplants to radiation therapy. A. The esophageal cancer cells of the control group respond to the therapeutic effect of radiotherapy (4 Gy×3F). The curative effect response of the FGF19 amplified esophagus cancer cells to each treatment is shown in the figures from top to bottom, wherein each treatment is respectively a blank control group, a radiotherapy group (4 Gy multiplied by 3F), a An Luoti Ni group (0.3 mg/Kg/d) and a radiotherapy group (4 Gy multiplied by 3F) + An Luoti Ni group (0.3 mg/Kg/d).

FIG. 10 immunohistochemical detection of subcutaneous tumors against Ki 67 and CD31. The legends in the column diagrams respectively show a control group/blank control group, a control group/radiotherapy, an FGF19 amplified group/blank control group, an FGF19 amplified group/radiotherapy, an FGF19 amplified group/An Luoti Ni and an FGF19 amplified group/radiotherapy + An Luoti Ni from top to bottom.

FIG. 11 shows a map of plasmid pcDNA3.1-FGF 19.

Detailed Description

The present invention relates to the relationship between the expression and/or copy number of the newly discovered biomarker (i.e., FGF 19) and the susceptibility of esophageal cancer, particularly esophageal squamous carcinoma, to radiation therapy. The biomarkers described herein provide uses and methods for evaluating and/or predicting the effect of radiation therapy on the treatment of esophageal cancer. Thus, one embodiment of the invention represents an improvement in biomarkers suitable for evaluating and/or predicting the effect of radiation therapy on the treatment of esophageal cancer. In yet another embodiment, the newly discovered biomarker (i.e., FGF 19) of the present invention may 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, CD 24), e.g., for evaluating and/or predicting the effect of radiation therapy on the treatment of esophageal cancer or for preparing a kit for such purpose.

The invention discovers and proves that FGF19 amplification (such as gene copy number increase) is a key biomarker for predicting the sensitivity of radiotherapy in esophageal scales, which suggests that FGF19 has potential to become a key biomarker for clinically evaluating and/or predicting the sensitivity of esophageal cancer cells or patients suffering from esophageal cancer to radiotherapy to be reduced, and has huge development potential and application prospect.

The inventors expect some concepts to be clarified here. For example, a variant gene detected in a cancer cell, even as a biomarker for detection, is not necessarily associated with the sensitivity of the cancer or tumor to radiation. There is no necessarily link between the two. As described in example 1 of the present application, we found the variant genes ranked at the top 16 based on the frequency statistical analysis of the gene sequencing results of 50 esophageal cancer patients, in which FGF19 variant genes were ranked only at the 3 rd position and TP53 and CCND1 variant genes were ranked at the 1 st and 2 nd positions, respectively. The phenomenon that the amplification of FGF19 gene found in the present application reduces the sensitivity of esophageal cancer cells to radiotherapy is unexpected.

Definition of the definition

The term "sample" means a material that is known or suspected to express or contain a biomarker (i.e., FGF 19) or a binding agent, e.g., an antibody specific for the biomarker (i.e., FGF 19). The sample may be derived from biological sources ("biological samples"), such as tissues (e.g., biopsy samples), extracts or cell cultures including cells (e.g., tumor cells), cell lysates, and biological or physiological fluids, such as whole blood, plasma, serum, saliva, cerebral spinal fluid, sweat, urine, milk, peritoneal fluid, and the like. Samples obtained from sources or after pretreatment to improve sample characteristics (e.g., plasma preparation from blood, diluted mucus, etc.) may be used directly. In certain aspects of the invention, the sample is a human physiological fluid, such as human serum. In certain aspects of the invention, the sample is a biopsy sample such as tumor tissue or cells obtained by a biopsy. In certain aspects of the invention, the sample is a malignant or normal tissue sample, such as a paracancerous normal tissue sample.

The term "marker" as used herein refers to a molecule to be used as a target for analysis of a patient experimental sample. Examples of such molecular targets are genes, proteins or polypeptides. Genes, proteins or polypeptides used as markers in the present invention are intended to include naturally occurring variants of said genes or proteins as well as fragments, in particular immunologically detectable fragments, of said genes or proteins or said variants. The immunologically detectable fragment preferably comprises at least 6,7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. Those skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cleaved into such fragments. Certain markers are synthesized in inactive form, which can be subsequently activated by proteolysis. As will be appreciated by the skilled artisan, the protein or fragment thereof may also be present as part of a complex. Such complexes can also be used as markers in the sense of the present invention. Variants of the marker polypeptide may be encoded by the same gene, but may differ in their isoelectric point (=pi) or molecular weight (=mw) or both, e.g. as a result of alternative mRNA or mRNA precursor processing. The amino acid sequence of the variant has 95%, 96%, 97%, 98%, 99% or more identity to the corresponding marker sequence. In addition, or in the alternative, the marker polypeptide or variant thereof may carry a post-translational modification. Non-limiting examples of post-translational modifications are glycosylation, acylation, and/or phosphorylation.

Expression of the marker may also be identified by detecting translation of the marker (i.e., detection of the marker protein in the sample). Suitable methods for detecting the marker protein include any suitable method for detecting and/or measuring a protein obtained from a cell or cell extract. Such methods include, but are not limited to, immunoblotting (e.g., western blotting), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry, and immunofluorescence. Particularly preferred methods for detecting proteins include any cell-based assay, including immunohistochemistry and immunofluorescence assays. Such methods are well known in the art.

The terms "subject," "patient," and "individual" are used interchangeably herein to refer to a warm-blooded animal, such as a mammal. The term includes, but is not limited to, domestic animals, rodents (e.g., rats and mice), primates, and humans. Preferably the term refers to a human.

Fibroblast growth factor 19 (Fibroblast growth factor, FGF 19) was first found in the human brain, whose coding gene is located on chromosome 11q13, consisting of 216 amino acids, and was highly expressed in ileum and gall bladder epithelial cells, but not in normal liver. High expression of FGF15 in the ileum, jejunum and duodenum of adult mice is a mouse homolog of FGF19, consisting of 218 amino acids, with 51% similarity to human FGF 19. The FGF19 gene discussed herein refers to the human FGF19 gene having the sequence set forth in NCBI number nm_ 005117.

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 levels. The control may be any sample, standard curve or level used for comparison purposes. The control may be a normal reference sample or a reference standard or level. The control may be a blood control sample from the patient himself, expression of a reference gene (e.g., FGF 19) from an esophageal cancer, such as esophageal squamous carcinoma tumor cells themselves, predetermined cells that are relatively insensitive to radiation (e.g., esophageal squamous carcinoma primary cells that are relatively insensitive to radiation), such as a "normal control" or a prior sample taken from the same subject, a sample from a normal healthy subject, such as normal cells or normal tissue, a sample from a subject not suffering from a disease (e.g., cells or tissue), or a sample of purified protein or RNA at a known normal concentration. "reference standard or level" refers to a value or number derived from a reference sample. A "normal control value" is a predetermined value indicative of a non-disease state, e.g., a value expected in healthy control subjects. Typically, the normal control value is expressed as a range ("between X and Y"), a high threshold ("no higher than X"), or a low threshold ("no lower than X"). For a particular biomarker, a subject having a measured value that is within a normal control value is generally referred to as "within normal limits" for that biomarker. The normal reference standard or level may be a normal subject who has never had a disease or disorder (e.g., cancer). The empty esophageal cancer cell lines (KYSE-410 CON and KYSE-510 CON) constructed in the examples herein are control groups of the constructed FGF19 expansion stable transesophageal cancer cell lines (KYSE-410 FGF19AM/KYSE-510FGF19 AM).

The "increased" or "decreased" or "amplified" or "deleted" in the marker expression level in the patient sample may indicate a level above or below the standard error of the detection assay, preferably at least about 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold 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, 1/3, 1/6, 1/7, 1/8, 1/9 or 1/10 or less of the control or standard, respectively, as compared to a control or standard (e.g., the normal level from healthy esophageal cells, the level of a different disease stage or the level of other sample of the patient, the level of esophageal cancer cells of a previous disease stage that has maintained the sensitivity to radiation therapy not decreased, or the patient having esophageal cancer). 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), and the like, as known in the art. FGF19 gene amplification as referred to herein refers to an increase in the copy number or level of expression of the FGF19 gene, preferably by at least about 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold or more, as compared to a control or standard described above that does not comprise amplification.

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

In the present invention, a protein fragment refers to a polypeptide having an amino terminal deletion, a carboxyl terminal deletion and/or an intermediate deletion as compared to the full-length native protein. The fragments may also contain modified amino acids as compared to the native protein. In certain embodiments, the fragment is about 5-215 amino acids in length. For example, a fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, or 200 amino acids in length. In one embodiment, the fragment is an immunologically detectable fragment, preferably comprising at least 6, 7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. By a change in the level of expression of a protein is meant at least about 1.1, 1.25, 1.5, 2,3, 4, 5, 6, 7, 8, 9 or 10-fold or more 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 level of expression of the control or standard as compared to the level of expression of the control or standard. In this context, due to the amplification of the FGF19 gene, the expression or level of its corresponding RNA (e.g., mRNA) and protein can also be increased, e.g., by at least about 1.1, 1.25, 1.5, 2,3, 4, 5, 6, 7, 8, 9, or 10-fold or more, compared to the control group. In certain embodiments, determining "protein expression level", "gene expression" or "gene expression level" as used herein includes, but is not limited to, determining the corresponding RNA, protein or peptide level (or a combination thereof). The present invention is not limited to specific methods and reagents for determining protein, peptide or RNA levels, all of which are well known in the art.

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.

The term "oligonucleotide" refers to a multimeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term includes double and single stranded DNA and RNA, modified and unmodified forms such as methylation or capping of polynucleotides. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. An oligonucleotide may include, but need not include, other coding or non-coding sequences, or it may be, but need not be, linked to other molecules and/or carriers or support materials. Oligonucleotides used in the methods or kits of the invention can be of any length suitable for the particular method. In certain applications, the term refers to an antisense nucleic acid molecule (e.g., an mRNA or DNA strand in the opposite direction to the sense polynucleotide encoding a cancer marker of the invention (e.g., FGF 19)).

Oligonucleotides for use in the present invention include complementary nucleic acid sequences and nucleic acids that are substantially identical to those sequences, and also include sequences that differ from the nucleic acid sequence by the degeneracy of the genetic code. Oligonucleotides useful in the present invention also include nucleic acids that hybridize under stringent conditions, preferably high stringency conditions, to oligonucleotide cancer marker nucleic acid sequences.

Nucleotide hybridization assays are well known in the art. Hybridization assay procedures and conditions will vary depending on the application and will be selected according to known general binding methods, see, e.g., J. Sambrook et al, molecular cloning: guidelines for experiments (third edition. Scientific Press, 2002), and Young and Davis, P.N.A.S., 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.

In some cases, it may be desirable to amplify the sample. Genomic samples may be amplified by a variety of mechanisms, some of which may employ PCR. The sample may 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.

Other suitable amplification methods include Ligase Chain Reaction (LCR) (e.g., wu and Wallace, genomics 4,560 (1989), landeren et al Science 241, 1077 (1988) and Barringer et al Gene 89:117 (1990)), transcriptional amplification (Kwoh et al, proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO 88/10315), self-sustained sequence replication (Guatelli et al, proc. Nat. Acad. Sci. USA,87,1874 (1990) and WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus-initiated polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily-initiated polymerase chain reaction (AP-PCR) (U.S. Pat. No. 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 are incorporated herein by reference).

Reagents useful for detecting FGF19 expression levels and/or copy numbers are well known in the art. Such reagents suitable for use in the present invention are commercially available or are routinely prepared by methods well known to those skilled in the art.

The term "binding agent" refers to a substance such as a polypeptide, antibody, ribosome or aptamer that specifically binds to a biomarker (FGF 19) of the present invention. A substance "specifically binds" to a biomarker of the invention if it reacts at a detectable level with the biomarker, but not with a peptide containing an unrelated sequence or a sequence of a different polypeptide. Binding properties can be assessed using methods readily available to those skilled in the art, such as ELISA.

The binding agent may be a ribosome, RNA or DNA molecule or 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.

Aptamers include DNA or RNA molecules that bind to nucleic acids and proteins. Aptamers that bind to the markers of the invention can be produced using conventional techniques without undue experimentation. [ see, for example, publications describing aptamer selection in vitro, 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. Structure. Biol.6:281-287 (1996) ].

Antibodies useful in the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments (e.g., 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), anti-idiotype (ant-Id) antibodies, proteins comprising an antibody moiety, chimeric antibodies (e.g., antibodies comprising the binding specificity of murine antibodies but wherein the remainder is of human origin), derivatives such as enzyme conjugates or labeled derivatives, diabodies, linear antibodies, disulfide-linked Fv (sdFv), multispecific antibodies (e.g., bispecific antibodies), epitope-binding fragments of any of the foregoing, and any other modified configuration of immunoglobulin molecules comprising the antigen recognition site of the desired specificity. Antibodies include antibodies of any type (e.g., igA, igD, igE, igG, igM and IgY), of any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or of any subclass (e.g., igG2a and IgG2 b), 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 class or subclass thereof. Antibodies may be from any animal source, including birds and mammals (e.g., humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens).

For example, antibodies for use in the present invention are commercially available from, for example, bioVision (e.g., cat# 5542R-100), abcam (e.g., cat# ab 172545), and the like. Alternatively, the antibodies may be prepared by recombinant methods well known in the art. In some embodiments, the antibody is a monoclonal antibody. For the preparation of monoclonal antibodies see, for example, kohler et al (1975) Nature256:495-497; 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.

Kits according to the present disclosure may be prepared by methods conventional in the art. The kit may comprise materials or reagents (including reagents for detecting FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby) as required for performing the methods or uses of the present invention. The kit may include a container storing the reaction reagents (e.g., primers, dntps, enzymes, etc. in a suitable container) and/or support materials (e.g., buffers, instructions for performing the assay, etc.). For example, the kit may comprise one or more containers (e.g., cassettes) containing the respective reagents and/or support materials. Such contents may be applied together or separately to the sample to be tested. As an example, the kit may contain reagents, buffers, and instructions for use for detecting the FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby. The kit may further contain a polymerase, dTNP, etc. The kit may also contain internal standards for quality control, positive and negative controls, and the like. The kit may also comprise reagents for preparing nucleic acids, such as DNA, from the sample. The above examples are only intended to more clearly illustrate the invention and do not constitute any limitation on the kits and their contents suitable for use in the invention.

Examples

For a clearer explanation of the content of the present invention, the following will be explained in detail with reference to the drawings and embodiments.

Example 1 screening for novel targets for esophageal cancer treatment based on clinical esophageal cancer patient Gene sequencing results

In clinical work, we collected clinical data from esophageal cancer patients undergoing gene sequencing. And carrying out statistical analysis on the collected complete genome sequencing results of 50 esophageal cancer patients in the animal experiment center of the southern hospital, and screening out new treatment targets.

The frequency statistics analysis is carried out on the gene sequencing results of 50 esophageal cancer patients, and the mutation genes arranged at the first 16 positions are TP53, CCND1, FGF19, NOTCH1, LRP1B, FAT1, MYC, CDKN2A, NSD1, EP300, CHEK2, ZNF217, PTCH1, DICER1, CBLB and GNAS in sequence, wherein the gene mutation types comprise gene mutation, gene amplification, single copy number deletion and gene fusion, the gene mutation of the first three positions is respectively that the TP53 gene mutation accounts for 88%, the CCND1 gene mutation accounts for 48% (wherein the amplification accounts for 46%, the mutation accounts for 2%), and the FGF19 gene amplification accounts for 46%, as shown in figure 1.

We screened FGF19 as a new target for the study.

EXAMPLE 2 analysis of the database of FGF19 Gene characteristics in esophageal cancer and the Effect on prognosis

Although FGF19 has been found to be potentially useful as a novel target for accurate treatment of esophageal cancer based on the results of the study in example 1, it is further necessary to determine whether the novel target is significantly highly expressed in tumor tissue and has an effect on prognosis of patients with esophageal cancer. Thus, this example further analyzes FGF19 characteristics in and the effect on prognosis of esophageal cancer patients by measuring results from a large number of samples in an online database.

The ratio of FGF19 amplification in esophageal cancer was analyzed using cBioPortal website esophageal cancer dataset (TCGA, firehoseLegacy). Performing FGF19 related gene-gene and protein-protein interaction network analysis through GENEMANIA and STRING websites, and performing pathway enrichment analysis of FGF19 related gene and protein respectively by KEGG. Differential expression analysis of FGF19 in esophageal cancer tumor tissue and normal tissue was performed using the TCGA database esophageal cancer dataset. Screening the gene positively and negatively related to the expression of FGF19 in esophageal cancer through LinkedOmics database, and performing biological function enrichment analysis of FGF 19. Clinical characterization, prognostic analysis and visualization of the TCGA database esophageal cancer dataset were performed using the R language.

Analysis was performed using R software. Differential expression levels of FGF19 mRNA between esophageal cancer tissue and normal tissue in TCGA databases were analyzed using an independent sample t-test. FGF19 expression was analyzed for correlation with clinical pathology using the pearson chi-square test. Disease Specific Survival (DSS) analysis was performed using Kaplan-Meier plots and differences were compared using log-rank test. Single factor and multivariate analyses were performed using a Cox proportional hazards regression model. Double tail P <0.05 was considered statistically significant.

First, we analyze the variation of FGF19 gene in esophageal cancer through database to understand its characteristics in esophageal cancer. Analysis of the TCGA database esophageal cancer dataset (TCGA, firehose Legacy) by cBioPortal website found that FGF19 was amplified at 35% in esophageal cancer (fig. 2A). Gene-gene and protein-protein interaction network analysis by GENEMANIA website and STRING website found 20 potential target genes (FGFR4/KLB/KL/SDC2/FGFR1/FGF20/FGF5/FGF17/FGF22/FGF16/FGF23/FGF8/FGF18/FGF6/FGF9/FGF3/FGF7/FGF4/FGF10/FGF1)( interacting with FGF19 gene FIG. 2B) and 10 potential target proteins (KLB/FGFR 1/FGFR3/FGFR2/FGF23/FGF10/FGF7/FGF2/PLCG1/FGFR 4) (FIG. 2C). KEGG pathway enrichment analysis was performed on FGF19 and 20 related genes (fig. 2D) and 10 related protein-encoding genes (fig. 2E), respectively, and it was found that the major enrichment signal pathways associated with FGF19 from the gene and protein levels were PI3K-Akt signal pathway, MAPK signal pathway, and the like.

We analyzed FGF19 expression in pan-cancers in the TCGA database and found that FGF19 was differentially expressed in various tumor tissues compared to normal tissues (fig. 3A), with FGF19 expression significantly higher in esophageal cancer tissues than in normal tissues (fig. 3B). And in the course of tumorigenesis and development, FGF19 high expression can be used for predicting tumor tissues (auc=0.788) (fig. 3C), and tcga+ GTEx database shows that FGF19 high expression has higher accuracy in predicting tumor tissues (auc=0.914) (fig. 3D). The result shows that FGF19 is obviously and highly expressed in esophageal cancer tumor tissues, and is possible to be used as a new target point for esophageal cancer treatment.

EXAMPLE 3 Effect of FGF19 amplification on sensitivity to esophageal cancer cell radiation therapy

Using human esophageal cancer cell lines KYSE-410 and KYSE-510 from Shanghai Meixuang Biotechnology Co., ltd, we successfully constructed a stably transfected esophageal cancer cell line (KYSE-410 FGF19 ^AM/KYSE-510FGF19^AM) containing FGF19 expansion, and an empty esophageal cancer cell line (KYSE-410 CON/KYSE-510 CON). The construction was performed using a plasmid named pcDNA3.1-FGF19 (FIG. 11) containing the FGF19 gene inserted downstream of the CMV promoter and carrying the puromycin resistance gene as selection marker. The FGF19 gene has the sequence of NCBI number NM_005117 and the gene fragment is synthesized by Ji Kai Biotechnology Co. The empty plasmid was pcDNA3.1-empty, which contained the CMV promoter and puromycin resistance gene, but no insertion of the FGF19 gene. When the cells were cultured to 30-50% confluence, transfection was performed using 50ul of virus solution and 8. Mu.g/ml polybrene, fresh medium was changed after culturing for 24-48 hours, KYSE-410 cells and KYSE-510 cells were cultured in complete medium containing 250. Mu.g/ml and 450. Mu.g/ml G418 respectively for 5-7 days after 48-96 hours of transfection, cells with G418 resistance were obtained, and then the cultured monoclonal stable transgenic cell lines were selected.

Verification of mRNA levels and protein levels was performed by real-time quantitative PCR (qPCR) and western blot (Westernblot) experiments, respectively. As shown in fig. 4A, the stably transfected esophageal cancer cell line containing FGF19 expansion had significantly higher expression levels of FGF19 than the control group.

FGF19 was also found to promote proliferation of esophageal cancer cells by clonogenic experiments. Stably transfected cells were seeded at low density (500 cells/well) into 6-well plate dishes and cultured for two weeks with medium changes every 3 days. Cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of clones formed in each dish was counted and compared. The results are shown in FIG. 4B. Experimental comparison results significance statistics were performed based on t-test, all P were double-sided test, P <0.05 was considered statistically significant. * Represents P <0.05, P <0.01, P <0.001, and P <0.0001.

3.1 Clone formation experiments demonstrated that FGF19 expansion reduced sensitivity of esophageal cancer cells to radiation

The tumor cell lines of the esophagus cancer of the control group (KYSE-410 CON, KYSE-510 CON) and the single gene steady transfer cell lines (KYSE-410 FGF19 ^AM、KYSE-510FGF19^AM) which are prepared according to the above and are to be inoculated are digested by pancreatin, and are made into single cell suspension, the single cell suspension is centrifugated for 3 minutes at 1000rpm, PBS buffer is used for washing the cells for 1 time, pancreatin is removed, the cell concentration is regulated after the cell count is carried out by using a cell counter, and the cell number is inoculated by a 6-hole plate, and the corresponding radiotherapy dose is 0Gy-200 cells, 2Gy-400 cells, 4Gy-1000 cells, 6Gy-2000 cells, 8Gy-4000 cells and 3 multiple holes. Culturing at constant temperature after inoculation to adhere cells, performing irradiation treatment within 24 hours, sealing 6-hole plate with adhesive tape during irradiation, and removing sealing adhesive tape after irradiation. And (3) standing and culturing in a constant temperature box after irradiation, observing cells at random, and if the cell floats too much, carrying out liquid exchange treatment, wherein the process is gentle. Culturing for 10-14 days, when macroscopic clone number appears in the culture dish with 0Gy dose, and the cell number is about 50, stopping culturing and staining to calculate the clone number. The process comprises discarding culture medium, washing with PBS for 3 times, fixing with methanol for 15min, washing with PBS for 2 times, dyeing with crystal violet for 30min, washing dye with running water carefully from edge, air drying the culture dish, photographing, counting, and calculating clone number and clone formation rate. The clones were plotted to form a curve as shown in FIG. 5. Experimental comparison results significance statistics were performed based on t-test, all P were double-sided test, P <0.05 was considered statistically significant.

The results showed that the radiation sensitivity of FGF 19-expanded esophageal cancer cells (KYES-410 FGF19 ^AM/KYES-510FGF19^AM) was significantly reduced compared to control esophageal cancer cells (KYES-410 CON/KYES-510 CON), as shown in FIG. 5.

3.2 An Luoti Nile enhancing radiation sensitivity of FGF19 amplified esophageal cancer cells

FGF19 expansion was demonstrated to reduce sensitivity of esophageal cancer cells to radiotherapy by the above clone formation experiments. Next, it was further investigated whether An Luoti ni could enhance the radiation sensitivity of FGF 19-amplified esophageal cancer cells. Three control groups and FGF19 expansion group were set at inoculum sizes of 500, 2000 and 2000 cells/well, respectively, and after inoculation, cells were attached by constant temperature culture, and 4Gy irradiation treatment was performed within 24 hours of inoculation. We found that the radiation sensitivity of FGF 19-expanded esophageal cancer cells was significantly reduced compared to the control group, and that the radiation sensitivity of FGF 19-expanded esophageal cancer cells was significantly enhanced by the combination of An Luoti-Ni, whereas the control group was not significantly altered, and as shown in fig. 6, the experimental comparison results were statistically significant based on t-test, all P were double-sided tests, and P <0.05 was considered statistically significant.

The result shows that An Luoti Ni enhances the radiation sensitivity of FGF19 amplified esophageal cancer cells, but has no obvious influence on the radiation sensitivity of esophageal cancer cell strains in a control group.

3.3FGF19 amplification affecting sensitivity of esophageal cancer cells to radiation therapy through FGF19-FGFR4-ERK signaling pathway

Through previous experiments, it has been demonstrated that FGF19 expansion reduces the radiation sensitivity of esophageal cancer cells, and An Luoti ni enhances the radiation sensitivity of FGF19 expansion of esophageal cancer cells. Next, it was further explored what signaling pathway FGF19 affects the sensitivity of esophageal cancer cells to radiotherapy.

First, the protein expression and activation conditions related to the radiotherapy sensitivity of esophageal cancer cells are explored by the FGF19 amplification through western blot (Westernblot) experiments. Antibodies used in western blotting include 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 (phosphoY 642) (Abcam, ab192589,1:1000 dilution), FGFR4 (Abcam, ab178296,1:1000 dilution), and beta-actin (CELL SIGNALING Technology, #4970,1:5000 dilution) as reference antibodies. As shown in FIG. 7A, after radiotherapy (4 Gy), ERK1/2 signals of FGF 19-amplified esophageal cancer cells are significantly activated compared with the control group, the p-ERK1/2 protein expression amount is significantly increased, and when FGF401 (10 mu M) which is a FGF4 specific inhibitor is added, phosphorylated ERK1/2 is significantly inhibited. At the same time we also explored whether FGF19 amplification affected Wnt signaling in radiotherapy using Wnt pathway inhibitor LGK974 (3 μm). Through CCK8 experiments we found that the combination of radiotherapy with LGK-974 significantly enhanced the radiotherapy sensitivity of esophageal cancer cells, but was independent of FGF19 expansion or not, as shown in FIG. 7B. The main parameters of CCK8 experiments are as follows, the number of inoculated cells is 2000 per well, the used experimental materials comprise CCK8 reagent, the culture condition is 37 ℃ and 5% CO2, and the experimental time is 72 hours. Experimental comparison results are expressed based on Mean ± standard deviation (Mean ± SD), the comparison between groups using t-test or one-way analysis of variance (ANOVA), the significance level being set to P <0.05.

The result shows that the radiotherapy leads to the activation of the FGF19 for amplifying ERK1/2 signals in esophageal cancer cells, but the radiotherapy can inhibit the activation of ERK1/2 signals after being combined with FGF401 to block FGF19-FGFR4 signal paths, and ERK1/2 can be a downstream molecule of the FGF19-FGFR4 signal paths. Meanwhile, the inhibition of the Wnt signal pathway can enhance the radiation therapy sensitivity of esophageal cancer cells, but Wnt signals are not downstream participation signals of FGF19-FGFR4 for radiation resistance.

3.4FGF19 amplification leads to reduced DNA fragmentation after radiation therapy

The damage condition of double-stranded DNA in esophageal cancer cells after radiotherapy is detected by gamma-H2 AX immunofluorescence experiments. We select esophageal cancer cells in exponential growth phase (KYSE-410 CON/KYSE-410FGF19 ^AM、KYSE-510CON/KYSE-510FGF19^AM) for experiments, firstly, the concentration of cell suspension is adjusted to 3000 cells/well, 100 μl of the cell suspension is inoculated into a 96-well plate after uniform mixing, the cell suspension is placed in a 37 ℃ carbon dioxide incubator for 15 minutes at room temperature for culture, and the cell suspension is subjected to grouping treatment on the next day, namely a control group, a radiotherapy (4 Gy) group, a radiotherapy+ An Luoti Ni (2 μM) group and a radiotherapy+FGF401 (10 μM) group, wherein each group comprises 3 compound wells. The medicine is added 3-6 hours before radiotherapy, and the medicine is detected respectively at 2 hours, 6 hours and 24 hours after radiotherapy. The detection was performed using a DNA damage detection kit (γ -H2AX immunofluorescence) (bi yun, C2035S).

The results showed that the gamma-H2 AX immunofluorescence staining in the nuclei after radiotherapy of the control group esophageal cancer cells (KYSE-410 CON/KYSE-510 CON) was significantly higher than that of the FGF 19-amplified group esophageal cancer cells (KYSE-410 FGF19 ^AM/KYSE-510FGF19^AM), but that the gamma-H2 AX immunofluorescence staining in the nuclei was significantly enhanced after treatment of the FGF 19-amplified group esophageal cancer cells with An Luoti-Ni combined radiotherapy (FIG. 8). Experimental comparison results significance statistics were performed based on t-test, all P were double-sided test, P <0.05 was considered statistically significant.

The results show that compared with the esophageal cancer cells of the control group, the DNA double-strand break of the FGF19 amplified esophageal cancer cells after radiotherapy is obviously reduced, but An Luoti Ni can obviously increase the DNA double-strand break of the FGF19 amplified esophageal cancer cells after radiotherapy in the nucleus.

3.5 In vivo experiments verify the effect of FGF19 expansion on the sensitivity of esophageal cancer cell radiotherapy

Further, in vivo animal experiments prove the influence of FGF19 amplification on the sensitivity of esophageal cancer cell radiotherapy. Female BALB/c nude mice of 3-4 weeks old (purchased from the university of medical animal experiment center in southern medical science, guangzhou, guangdong, and fed to the SPF-class environment of the animal experiment center in southern hospital) were selected for 60 animals, KYSE-410CON and KYSE-410FGF19AM cells were inoculated subcutaneously on the backs of the nude mice, respectively, and each nude mouse was inoculated with 5X 10 ⁵ cells. Starting from the injection, nude mice were observed for subcutaneous neoplasia and monitored for subcutaneous graft growth every 2-5 days, and body weight was monitored. When the subcutaneous tumor volume was on average about 150-250mm ³, the nude mice were randomly grouped (10 per group). KYSE-410CON is divided into a blank control group and a radiotherapy group (4 Gy multiplied by 3F), KYSE-410FGF19 ^AM is divided into the blank control group, the radiotherapy group (4 Gy multiplied by 3F), an Luoti Ni group (0.3 mg/Kg/d multiplied by 12d, stomach irrigation), the radiotherapy group (4 Gy multiplied by 3F) + An Luoti Ni group (0.3 mg/Kg/d multiplied by 12d, stomach irrigation), tumor volume is measured every 2-3 days, and a tumor volume change chart is drawn.

The results showed that tumor acceleration slowed down after 1 week of control radiation, and the volume after radiation on day 12 was significantly smaller than the placebo (p=0.0062). FGF19 amplified group, tumor was not significantly reduced (p=0.18) after radiotherapy compared to the placebo group, an Luoti ni treated group was also not significantly reduced (p=0.25) compared to the placebo group, but after An Luoti ni treatment in combination with radiotherapy, tumor was significantly reduced (p=0.0099) compared to the placebo group and significantly better than the radiotherapy group (p=0.0149) and An Luoti ni treated group (p=0.01), as shown in fig. 9.

The subcutaneous tumors were further subjected to immunohistochemical staining using antibodies including Ki67 antibody (CELL SIGNALING Technology, #9027,1:200 dilution), CD31 antibody (Abcam, ab28364,1:100 dilution). As shown in fig. 10, the Ki67 staining positive rate of FGF19 amplified group was significantly higher than that of control group (p= 0.0386), ki67 staining positive rate was significantly reduced after radiotherapy (R) of control group, ki67 staining positive rate was not significantly changed after radiotherapy (R) of amplified group, ki67 staining positive rate was also not significantly changed after treatment with An Luoti ni (a), but Ki67 staining positive rate was significantly reduced after treatment with An Luoti ni (r+a) in combination with radiotherapy, which suggests that FGF19 amplification significantly inhibited sensitivity of esophageal cancer cells to radiotherapy, tumor growth was significantly inhibited with An Luoti ni in combination with radiotherapy, and radiotherapy had a synergistic effect with An Luoti ni. No significant difference was seen in CD31 staining for the control and amplification and treatment groups. Experimental comparison results significance statistics were performed based on t-test, all P were double-sided test, P <0.05 was considered statistically significant. * Represents P <0.05, P <0.01, P <0.001, ns no statistical significance.

3.6 Conclusion

From the above studies, we determined that FGF19 gene amplification reduced sensitivity of esophageal cancer cells to radiation therapy and An Luoti ni reversed resistance of FGF 19-amplified esophageal cancer cells to radiation therapy.

An Luoti n had no significant effect on the radiotherapy sensitivity of the control group (fig. 6), and it was further demonstrated that An Luoti n reversed radiotherapy resistance by inhibiting the FGF19-FGFR4 signaling pathway, but not other targets of An Luoti n, while the ERK1/2 signaling pathway downstream of FGF19-FGFR4 was also found to be involved in this process, and the Wnt signaling pathway was not involved in the downstream pathway of FGF19-FGFR4 signaling pathway. In clinical work, for patients with FGF19 amplified esophageal cancer radiotherapy, the joint application of An Luoti Ni can be considered to enhance the sensitivity of radiotherapy and improve the curative effect of radiotherapy.

All of the uses, products, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Modifications and variations may be made to the disclosed subject matter and aspects of the application by those skilled in the art without departing from the concept, spirit and scope of the disclosure, as desired by the practice of the application. All such modifications and variations will be apparent to a person skilled in the art and are intended to be within the scope of the application as defined in the following claims.

Claims

1. Use of an agent for detecting FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby, for the manufacture of a kit for evaluating and/or predicting the susceptibility of an esophageal cancer cell or a patient suffering from esophageal cancer to radiation therapy, wherein an increased expression level and/or copy number amplification of FGF19 gene compared to a control is indicative for a decreased susceptibility of said esophageal cancer cell or patient suffering from esophageal cancer to radiation therapy.

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

3. The use of claim 2, wherein the binding agent that binds to a protein or protein fragment encoded by the FGF19 gene is an antibody to FGF 19.

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

5. A kit for evaluating and/or predicting sensitivity of esophageal cancer cells or a patient suffering from esophageal cancer to radiation therapy, wherein the kit comprises reagents for detecting FGF19 gene or mRNA thereof or a protein or protein fragment encoded thereby.

6. The kit of claim 5, wherein the reagent for detecting FGF19 gene or mRNA thereof or a protein or protein fragment encoded by the same comprises a binding agent that binds to a protein or protein fragment encoded by the FGF19 gene, or a substance that hybridizes to or amplifies the FGF19 gene or mRNA thereof.

7. The kit of claim 6, wherein the binding agent that binds to a protein or protein fragment encoded by the FGF19 gene is an antibody to FGF 19.

8. The kit of claim 6, wherein the substance that hybridizes to or amplifies FGF19 gene or mRNA thereof is an oligonucleotide primer or probe.

9. Use of An Luoti ni in the manufacture of a medicament for increasing sensitivity of FGF19 gene-amplified esophageal cancer cells or a patient suffering from esophageal cancer to radiation therapy.

10. 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 esophageal squamous 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 esophagus.