CN114763573A - FGF19 amplification as biomarker for predicting sensitivity of FGFR inhibitor in esophageal squamous carcinoma and potential application thereof - Google Patents

FGF19 amplification as biomarker for predicting sensitivity of FGFR inhibitor in esophageal squamous carcinoma and potential application thereof Download PDF

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CN114763573A
CN114763573A CN202110034776.5A CN202110034776A CN114763573A CN 114763573 A CN114763573 A CN 114763573A CN 202110034776 A CN202110034776 A CN 202110034776A CN 114763573 A CN114763573 A CN 114763573A
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fgf19
gene
squamous carcinoma
protein
esophageal
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苏丹
应莉莎
金娇悦
张付闯
许晓雅
张大东
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Shanghai Siludi Medical Laboratory Co ltd
Zhejiang Cancer Hospital
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Shanghai Siludi Medical Laboratory Co ltd
Zhejiang Cancer Hospital
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Abstract

The invention relates toFGF19Application of amplification in esophageal squamous carcinoma. The invention discloses and useIn detectingFGF19Use of a gene or mRNA thereof or a protein or protein fragment encoded thereby in the manufacture of a kit or microarray for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or patients suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors. The invention also discloses a kit or microarray for evaluating and/or predicting the sensitivity of esophageal cancer cells, such as esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, such as esophageal squamous carcinoma, to FGFR inhibitors.

Description

FGF19Biomarker for predicting sensitivity of FGFR inhibitor in esophageal squamous carcinoma through amplification and potential application of biomarker
Technical Field
The invention relates to the technical field of medicaments, in particular toFGF19Application of amplification in esophageal squamous carcinoma.
Background
The incidence and mortality of esophageal cancer are ranked at 6 th and 4 th in all cancer species in China, respectively. Based on the histopathological classification statistics of tumors, the proportion of esophageal squamous carcinoma in all esophageal cancers in China is over 90 percent. The risk factors for inducing esophageal squamous carcinoma mainly include poor diet and life style such as smoking and drinking, and exposure to carcinogens such as nitrosamine and fungi. Early clinical symptoms of esophageal cancer are not obvious, most patients are in the local advanced stage of esophageal cancer or have cancer cell metastasis at the time of diagnosis, and therefore the esophageal cancer generally belongs to tumors with higher malignancy degree.
Aiming at the high-incidence area and high-risk population of the esophageal cancer, the risk of suffering from esophageal cancer can be effectively reduced by publicizing and popularizing the esophageal cancer prevention health knowledge, changing the bad dietary behavior and habit of life and reducing the contact with carcinogenic and toxic substances. In addition, by carrying out medical detection and screening on people, discovering esophageal cancer as soon as possible and treating the esophageal cancer in time, more people can be locked in the early stage of the disease, so that a good opportunity is provided for the treatment of the esophageal cancer, and the survival quality and the prognosis effect of patients with esophageal cancer (ESCC) can be greatly improved. Generally, esophageal cancer has a better treatment window stage at the early stage, and the curative effect is obvious after clinical treatment. However, if not screened early, esophageal cancer is often accompanied by cancer cell metastasis when it progresses to the middle and advanced stage of the disease, which brings great challenges to clinical therapeutic intervention; meanwhile, great economic and psychological burden is brought to patients, and the national government medical burden is also greatly increased.
The current clinical intervention measures aiming at the esophageal cancer mainly comprise operations, radiotherapy, chemotherapy and drug treatment. The precise personalized treatment scheme of the drug targeting represented by molecular targeting and immunotherapy is accepted by more and more medical experts, brings new hope and possibility for the treatment of patients, effectively relieves the disease symptoms of the patients and improves the prognosis life quality. Small molecule antitumor targeted drugs represented by FGFR inhibitors, such as 7 classes of FGFR inhibitor drugs like Sorafenib (Sorafenib), Sunitinib (Sunitinib), Nintedanib (Nintedanib), and Lenvatinib (Lenvatinib), have been approved by the FDA in the united states for various tumor interventions. Meanwhile, the application of various FGFR inhibitors such as LY2874455, AZD4547, Cediranib and the like in the aspect of tumor treatment is in the research state. Aberrant changes in downstream pathways caused by aberrant genes such as FGFR activation mutations, translocations, amplifications, and fusions can lead to tumorigenesis. The FGFR inhibitor can effectively inhibit the malignant characteristics of tumor cells mainly by inhibiting cancer driving genes and related signal paths, thereby achieving the effects of killing the tumor cells and treating tumors. However, the current lack of effective in vitro and in vivo models for researching biomarkers for target drug sensitivity prediction results in the fact that many drugs cannot be accurately screened out of sensitive people, and the advance of drug clinical tests is greatly influenced.
Disclosure of Invention
In order to solve the defects of the prior art, the invention takes the primary cells of the cancer tissue source of the esophageal squamous carcinoma patient as a model, and the experiment verification of in vitro and in vivo tumor transplantation proves that about 30 percent of esophageal squamous carcinoma cells are obtainedThe presence of cancer in patientsFGF19Amplifying gene copy number in high frequency;FGF19has the potential to become a key biomarker for identifying whether esophageal squamous carcinoma patients can respond to FGFR inhibitor treatment. The present invention has foundFGF19The expanded esophageal squamous carcinoma is more sensitive to FGFR inhibitors; the FGFR inhibitor can remarkably inhibitFGF19The tumor forming capability of primary cells derived from cancer tissues of patients with esophageal squamous carcinoma is expanded. The invention discovers the FGFR inhibitor inFGF19Has a wide application prospect in the treatment aspect of amplifying esophageal squamous carcinoma patients,FGF19has the potential to become a key biomarker for responding to the FGFR inhibitor treatment and benefiting esophageal squamous cell carcinoma patients in clinical diagnosis, and has larger clinical medical transformation value. The method is beneficial to better screening the FGFR inhibitor-responsive population in the esophageal squamous carcinoma treatment process and improvingFGFRThe accuracy of the inhibitor administration. .
In one aspect, the invention relates to methods for detectingFGF19Use of a gene or its mRNA or its encoded protein or protein fragment in the manufacture of a kit or microarray for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g., esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, e.g., esophageal squamous carcinoma, to FGFR inhibitors.
In some embodiments, the composition is administered to a subject in need thereof, as compared to a control,FGF19a high expression level and/or copy number amplification of the gene indicates that the cancer cell or patient is sensitive to the FGFR inhibitor, anFGF19A low expression level and/or no change or deletion in copy number of the gene indicates that the cancer cell or patient is not susceptible to FGFR inhibitors. In some embodiments, the FGFR inhibitor is selected from sorafenib, sunitinib, Pazopanib (Pazopanib), panatinib (Ponatinib), Regorafenib (Regorafenib), Nintedanib (Nintedanib), lenvatinib, LY2874455, AZD4547, BGJ398 (also known as infiratinib), BMS-582664 (also known as alanine brianib alaninate), AZD2171 (also known as Cediranib), Debio1347 (also known as CH-5183284), polyviranib (also known as TKI258/CHIR258), inccb 054828, erdalitinib (also known as tkj-42756493), PD173074, and Cediranib (Cediranib).
In some embodiments, the method is used forDetection ofFGF19Reagents for genes or their mRNAs or their encoded proteins or protein fragments includeFGF19Binding agents which bind to a protein or protein fragment encoded by a gene, or toFGF19Hybridization or amplification of genes or their mRNAsFGF19A gene or its mRNA. In some embodiments, the reaction is withFGF19The binding agent to which the gene-encoded protein or protein fragment binds is an antibody against FGF 19. In some embodiments, the reaction is withFGF19Hybridization or amplification of genes or their mRNAsFGF19The substance of the gene or its mRNA is an oligonucleotide primer or probe.
In another aspect, the invention relates to a kit or microarray for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors, wherein the kit or microarray comprises means for detectingFGF19A gene or its mRNA or its encoded protein or protein fragment.
In some embodiments, the composition is administered to a subject in need thereof, as compared to a control,FGF19a high expression level and/or copy number amplification of the gene indicates that the cancer cell or patient is sensitive to the FGFR inhibitor, anFGF19A low expression level and/or no change or deletion in copy number of the gene indicates that the cancer cell or patient is not susceptible to the FGFR inhibitor. In some embodiments, the FGFR inhibitor is selected from sorafenib, sunitinib, pazopanib, panatinib, regorafenib, nidanib, lenvatinib, LY2874455, AZD4547, BGJ398, BMS-582664, AZD2171, Debio1347, doviranib, INCB054828, erdifitinib, PD173074, and cediranib.
In some embodiments, the method is for detectingFGF19Reagents for genes or their mRNAs or their encoded proteins or protein fragments includeFGF19Binding agents bound to a gene-encoded protein or protein fragment, or toFGF19Hybridization or amplification of genes or their mRNAsFGF19A gene or its mRNA. In some embodiments, the reaction is withFGF19The binding agent to which the gene-encoded protein or protein fragment binds is an antibody directed against FGF 19. In some embodiments, the reaction is withFGF19Hybridization or amplification of genes or their mRNAsFGF19GeneOr mRNA thereof is an oligonucleotide primer or probe.
In a further aspect, the invention relates to the use of an FGFR inhibitor for the manufacture of a medicament for the treatment of esophageal cancer, e.g. esophageal squamous carcinoma, wherein the esophageal cancer, e.g. esophageal squamous carcinomaFGF19High expression levels of the gene and/or copy number amplification. In some embodiments, the FGFR inhibitor is selected from sorafenib, sunitinib, pazopanib, panatinib, regorafenib, nidanib, lenvatinib, LY2874455, AZD4547, BGJ398, BMS-582664, AZD2171, Debio1347, doviranib, INCB054828, erdifitinib, PD173074, and cediranib. In some embodiments, the amount of the FGFR inhibitor is an effective amount
In yet another aspect, the invention relates toFGF19Use of a gene or mRNA thereof or an encoded protein or protein fragment thereof as a biomarker for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or patients suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors.
In a further aspect, the present invention relates to a method for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors, comprising the steps of:
(1) obtaining a biological sample from a subject; and
(2) detecting said sampleFGF19The level of expression and/or copy number of a gene or its mRNA or its encoded protein or protein fragment;
wherein the concentration of the active ingredient in the composition is compared with a control,FGF19a high expression level and/or copy number amplification of the gene indicates that the cancer cell or patient is sensitive to the FGFR inhibitor, anFGF19A low expression level and/or no change or deletion in copy number of the gene indicates that the cancer cell or patient is not susceptible to the FGFR inhibitor.
In one embodiment, the biological sample is ctDNA, tumor tissue, tumor circulating cells, or tissue of other origin in a human. In one embodiment, the detection is by gene sequencing, PCR, FISH, immunohistochemistry, ELISA, Western or flow cytometry.
Drawings
FIG. 1 is a drawing ofFGF19 Graph of relative expression values of mRNA in the ZEC061 cells, ZEC145 cells, ZEC157 cells and ZEC219 cells of the present invention.
FIG. 2 shows the present inventionFGF19Schematic representation of the evaluation of esophageal squamous carcinoma drug sensitivity using expanded and non-expanded patient-derived cancer cell line (PDC) cells as a model.
FIG. 3 is a graphical representation of the IC50 measurements of LY2874455 drug by cells of the invention in vitro.
FIG. 4 shows the present inventionFGF19Schematic representation of in vivo neoplasia experimental results of an expanded Patient Derived Xenograft (PDX) model.
FIG. 5 is a schematic representation of the present inventionFGF19Schematic representation of the results of in vivo neoplasia experiments in PDX model without amplification.
Detailed Description
Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One skilled in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods.
The present invention relates to newly discovered biomarkers (i.e.FGF19) And the sensitivity of esophageal cancer, particularly esophageal squamous carcinoma, to FGFR inhibitors. The biomarkers described herein provide methods for assessing and/or predicting the effect of FGFR inhibitors on the treatment of esophageal cancer, e.g., esophageal squamous carcinoma. Accordingly, one embodiment of the present invention represents an improvement of biomarkers suitable for assessing and/or predicting the effect of FGFR inhibitors on the treatment of esophageal cancer, such as esophageal squamous carcinoma. In yet another embodiment, the newly discovered biomarkers of the invention (i.e., theFGF19) 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), e.g., for evaluating and/or predicting FGFR inhibitorsEffect on the treatment of esophageal cancer, such as esophageal squamous carcinoma, or for the preparation of kits and/or microarrays for this purpose.
The invention discovers and confirmsFGF19Amplification is a potential key biomarker for predicting FGFR inhibitor sensitivity in esophageal scales, suggesting that FGFR inhibitors are inFGF19Has huge exploration potential and application prospect in the aspect of expanding the clinical treatment of esophageal squamous carcinoma patients.
The term "sample" means known or suspected to express or contain a biomarker (i.e., a biological marker)FGF19) Or a material of a binding agent, e.g., for a biomarker (i.e., for a biological marker)FGF19) Antibodies with specificity. The sample may be derived from a biological source ("biological sample"), such as tissue (e.g., biopsy sample), 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., preparation of plasma from blood, dilution of mucus, etc.) can 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 histological examination. In certain aspects of the invention, the sample is a malignant or normal tissue sample, such as a paracancerous normal tissue sample.
Samples that can be analyzed according to the present invention include polynucleotides of clinical origin. As will be appreciated by those skilled in the art, the target polynucleotide may comprise RNA, including without limitation cellular total RNA, poly (a) + messenger RNA (mRNA) or portions thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (i.e., cRNA).
The target polynucleotide or a substance that hybridizes or amplifies with the target polynucleotide (e.g., an oligonucleotide primer or probe) can be detectably labeled on one or more nucleotides using methods known in the art. The detectable label may be, without limitation, a luminescent label, a fluorescent label, a bioluminescent label, a chemiluminescent label, a radioactive label, and a colorimetric label.
The term "marker" as used herein refers to a molecule to be used as a target for the analysis of a patient test 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 of 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. One 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 an inactive form, which can be subsequently activated by proteolysis. As will be appreciated by the skilled artisan, proteins or fragments thereof may also be present as part of a complex. Such complexes may 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 a 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 post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation, and/or phosphorylation.
Expression of the marker can 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, livestock, rodents (e.g., rats and mice), primates, and humans. Preferably the term refers to a human.
The term "effective amount" is used in the broadest sense to refer to a non-toxic but sufficient amount of an active agent (FGFR inhibitor) to provide a desired effect or benefit.
Human Fibroblast growth factor 19 (Fibroblast growth factor 19,FGF19) The gene is located on chromosome 11q13.3, is about 6.1 kb in length, and consists of three exons and two introns. Of a human beingFGF19The gene-encoded protein consists of 216 amino acids, and is related to mouseFGF15The gene encodes a protein which is capable of specifically binding to the fibroblast growth factor receptor 4 (FGFR4) gene.FGF15Can be regarded asFGF19The homologous gene of (1).FGF19Genes are conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, chickens, zebrafish, and frogs. In the case of a human being,FGF19mainly expressed in the intestinal tract, but also in fetal cartilage, skin, retina, adult gallbladder and colon-derived cellsFGF19Expression (Song Qian and Fang morning, FGF 19-New metabolic regulatory factor, medical review, Vol.18, vol.21, 2012, 3553-3556).
The term "control" is to be understood according to the general understanding of a person skilled in the art and denotes any useful reference for comparing gene copy number, protein or mRNA levels. The control can be any sample, standard curve or level used for comparison purposes. The control can be a normal reference sample or reference standard or level. The control may be a patient's own blood control sample; expression of reference genes (e.g., TBP) of esophageal cancer such as esophageal squamous carcinoma tumor cells themselves; predetermined cells that are relatively insensitive to FGFR inhibitor (e.g., esophageal squamous carcinoma primary cells that are relatively insensitive to FGFR inhibitor), such as a "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 (e.g., a cell or tissue) from a subject not suffering from a disease; 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 a healthy control subject. Typically, the normal control value is 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 having a measurement value within a normal control value is generally referred to as "within normal limits" for that biomarker. The normal reference standard or level can be a normal subject who has never suffered a disease or disorder (e.g., cancer).
A "high" or "low" level of marker expression or a "amplification" or "deletion" of copy number in a patient sample compared to a control or standard (e.g., a normal level, a level at a different disease stage, or a level in another sample of the patient) may represent a level that is higher or lower than the standard error of the detection assay, preferably a level or copy number that is at least about 1.1, 1.25, 1.5, 2, 3, 4,5, 6,7, 8, 9, or 10-fold or more times 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, respectively, 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 two-generation sequencing detection methods, chip detection, droplet digital polymerase chain reaction (ddPCR), and the like, as known in the art.
The references to "sensitive" and "insensitive" to inhibitors are relative concepts, generally based onFGF19The relative judgment between the groups is made for different groups of different group-entering people by the presence or absence of copy number amplification. In other words, withFGF19Low gene expression level and/or no change or deletion of copy number, compared to the groupFGF19Groups with high gene expression levels and/or copy number amplification have a more sensitive response to FGFR inhibitors and thus can be usedFGF19Differentiation of high or low levels of gene expression and/or absence of copy number amplification for susceptibility to FGFR inhibitorsGroup of feelings or insensitivity. The sensitivity degree of the FGFR inhibitor can be measured by using an IC50 value, wherein the lower IC50 value reflects that the esophageal squamous carcinoma cells are more sensitive to the FGFR inhibitor treatment, and the higher IC50 value reflects that the esophageal squamous carcinoma cells are less sensitive to the FGFR inhibitor treatment. The terms "polypeptide" and "protein" are used interchangeably herein to denote at least one molecular chain of amino acids linked by covalent and/or non-covalent bonds. The term includes peptides, oligopeptides, and proteins and post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and the like. Protein fragments, analogs, mutant or variant proteins, fusion proteins, and the like are also included within the meaning of this 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 compared to the native protein. In certain embodiments, fragments are 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. A change in the level of protein expression refers to an expression level that is at least about 1.1, 1.25, 1.5, 2, 3, 4,5, 6,7, 8, 9, or 10-fold or more greater than the expression level of a 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 than the expression level of a control or standard.
In certain embodiments, determining a "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 particular 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 person. The methods include radioimmunoassays, competitive binding assays, western blot analysis and ELISA assays. For methods using antibodies, both monoclonal and polyclonal antibodies are suitable. The antibody may be immunologically specific for a protein, protein epitope or protein fragment.
The term "oligonucleotide" refers to a polymeric 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, but need not, include other coding or non-coding sequences, or it may, but need not, be linked to other molecules and/or carriers or support materials. The oligonucleotides used in the methods or kits of the invention may be of any length suitable for the particular method. In certain applications, the term refers to antisense nucleic acid molecules (e.g., in a nucleic acid molecule that encodes a cancer marker of the invention)FGF19) The opposite sense polynucleotide of (a) or a DNA strand).
Oligonucleotides for use in the present invention include complementary nucleic acid sequences and nucleic acids substantially identical to these sequences, and also include sequences that differ from the nucleic acid sequence by the degeneracy of the genetic code. Oligonucleotides useful in the 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 are selected according to known general binding methods, see, e.g., j. sambrook et al, molecular cloning: experimental guidelines (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 can be amplified by various mechanisms, some of which can employ PCR. The sample may be amplified on the 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), Landegren 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 WO88/10315), self-sustaining sequence replication (Guatelli et al, Proc. nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of a target polynucleotide sequence (U.S. patent No. 6,410,276), consensus-primed polymerase chain reaction (CP-PCR) (U.S. patent No. 4,437,975), random-primed polymerase chain reaction (AP-PCR) (U.S. patent nos. 5,413,909, 5,861,245), and nucleic acid-based sequence amplification (NABSA) (see U.S. patent nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference).
Can be used for detectingFGF19Reagents for expression levels and/or copy number are well known in the art. Such agents 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" means, for example, a compound that binds to a biomarker of the invention (A)FGF19) Specifically binding polypeptide, antibody, ribosome or aptamer. 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 the sequence of the unrelated sequence or a different polypeptide. The binding properties can be assessed using an ELISA, which can be readily performed by a person skilled in the art.
The binding agent may be a ribosome, RNA or DNA molecule or polypeptide with or without a peptide component. The binding agent can 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 generated using conventional techniques without undue experimentation. [ see, for example, the following publications describing aptamer selection in vitro: klug et al, mol. biol. Reports 20:97-107 (1994); wallis et al, chem. biol. 2: 543-; 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) ].
Antibodies useful in the 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- Decorating the configuration. Antibodies include antibodies of any class (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 antibodies need not be of any particular class, or subclass. In certain embodiments of the invention, the antibody is an IgG antibody or class or subclass thereof. The antibody may be from any animal source, including avian and mammalian (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken).
For example, antibodies for use in the present invention are commercially available from, e.g., BioVision (e.g., cat No. 5542R-100), Abcam (e.g., cat No. ab172545), and the like. Alternatively, the antibodies can be prepared by recombinant methods well known in the art. In some embodiments, the antibody is a monoclonal antibody. For monoclonal antibody preparation see, e.g., 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-.
The kit of the present invention can be prepared by a method conventional in the art. The kit may comprise materials or reagents for carrying out the method of the invention (including for detectionFGF19A gene or its mRNA or its encoded protein or protein fragment). The kit may include storage 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, a kit may comprise one or more containers (e.g., cassettes) containing the corresponding reaction reagents and/or support materials. Such contents may be delivered to the intended recipient together or separately. As an example, the kit may contain a reagent for detectionFGF19Reagents, buffers, and instructions for use of the gene or its mRNA or its encoded protein or protein fragment. The kit may further contain polymerase, dTNP, and the like. The kit can also contain internal standards for quality control, positive and negative controls and the like. The kit may further comprise reagents for preparing nucleic acids, e.g., DNA, from the sample. The above examples are not to be construed as limiting the kits and their contents suitable for use in the present invention.
A microarray refers to a solid support having a flat surface with an array of nucleic acids, each member of the array comprising the same copy of an oligonucleotide or polynucleotide immobilized at a spatially defined region or site that does not overlap with regions or sites of other members of the array; that is, the regions or sites are spatially discrete. In addition, a spatially defined hybridization site can be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined (e.g., known or predetermined prior to its use). Typically, the oligonucleotide or polynucleotide is single stranded and is covalently attached to the solid support, typically from the 5 '-end or the 3' -end. The density of nucleic acids comprising non-overlapping regions in a microarray is typically greater than 100/cm2More preferably more than 1000/cm2. Microarray technology is disclosed, for example, in the following references: microarray edited by Schena A Practical Aproach (IRL Press, Oxford, 2000); southern, Current Opin. chem. biol., 2:404- "410, 1998, the entire contents of which are incorporated herein by reference.
The invention disclosesFGF19The gene application can be realized by appropriately modifying the process parameters by the skilled person with reference to the contents in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations and modifications, as appropriate, may be made to the disclosed use of the technology herein described to practice and use the technology herein without departing from the spirit and scope of the invention.
Examples
For a clearer understanding of the contents of the present invention, reference will be made to the accompanying drawings and examples.
All patient tumor tissue samples are collected in a tumor hospital tissue bank in Zhejiang province. All tumor patients have signed paper-version informed consent, and the collection, post-processing and analysis of all patient tissues all meet the ethical specifications. The study was approved by the review board of the cancer research institute in Zhejiang province and strictly followed the requirements of the world medical Association Helsinki declaration.
Example 1 Experimental protocol
1.1 construction of esophageal squamous carcinoma PDC model
The 123 ESCC patient pathology information used to construct PDC (i.e., patient-derived cancer cell line) models were confirmed by a qualified pathologist. Fresh ESCC patient tumor tissue was washed with cold PBS 3 times and then rapidly dissected into 0.5-1mm pieces3The fragments were transferred to cell culture dishes and cultured by adding Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 Xnonessential amino acids, 50U/mL penicillin and 50. mu.g/mL streptomycin, respectively. The culture conditions of the incubator were set at 37 ℃ and 5% CO2. And after 5 to 7 days of culture, performing gradient adherent culture on the digested cells to purify the tumor cells, marking the adherent cells as 0 th generation, and performing STR identification when the purified cells are subcultured to the fifth generation. After the identification is finished, the cells are subjected to amplification culture to obtain enough cells, related tests are carried out, and redundant cells are frozen and stored in liquid nitrogenIn (1).
1.2 in vitro cell viability assay
Primary cells in good growth status were digested, resuspended, counted, and plated in 96-well plates in quadruplicate at 4000 cell density per well, with the cell culture conditions being standard medium. After the cells adhere to the wall, the appropriate concentration of FGFR inhibitor LY2874455 is added. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay 72 hours after drug treatment. The concentration of drug that resulted in 50% inhibition of cell viability (IC 50) was calculated by four parameter curve analysis, and the IC50 values and standard deviations for FGFR inhibitors were calculated from four independent experiments, providing a data reference for further in vivo validation.
1.3 real-time fluorescent quantitative PCR reaction
Primers (Forward: 5'-GCTGCTTCAGTACTCGGAGGAA-3', reverse: 5'-CTTCTCGGATCGGTACACATTG-3') required for the real-time fluorescent quantitative PCR reaction were synthesized by Shanghai Bioengineering Co., Ltd. And (3) amplifying a target gene fragment by using cDNA synthesized by reverse transcription as a template and TBP as an internal reference and using a SYBR Green PCR Master Mix kit. The reaction conditions were as follows:
s1: heating to 95 ℃ for 15 seconds;
s2: cooling to 60 ℃ for 30 seconds;
s3: heating to 72 ℃ for 30 seconds;
the above reaction is one cycle, totaling 40 cycles.
TBP is used as an internal reference of the gene to be detected, and a relative mRNA expression value is calculated according to a delta CT method. Then, primary cells of patient esophageal squamous carcinoma tumor tissue source with the number of ZEC219 are selected as a control group, the relative mRNA expression value of the calibration gene is 1, and the relative mRNA expression value of the target gene of the experimental group is calculated.
1.4 construction of esophageal squamous carcinoma PDX model
Fresh ESCC patient tumor tissue was washed with cold PBS 3 times and then rapidly dissected into 1mm volume3The pieces of (4) -week-old male BALB/c nude mice were implanted and randomly divided into four groups, and the four groups of male BALB/c nude mice were individually subjected to control (saline), 1mg/kg LY2874455, and 3mg/kg LY2874455 and 6mg/kg cis-platin, and subcultured, after 25 days, when the transplanted tumor grows to 2cm in diameter in the nude mice of the control group, all mice were sacrificed, and tumor tissues and blood samples were taken out for storage.
1.5 high throughput sequencing and Gene copy number Change analysis
The high-throughput sequencing and analysis of samples are completed by Shanghai thought Didi medical inspection institute limited company, and the company has the capability of detecting the gene mutation of the NGS solid tumor tissue of international first-class standard. The screening aspect of the drug sensitive gene is as follows: the targeted genome includes 365 cancer-associated genes and a highly rearranged gene among 25 cancers. The DNA sample is extracted from the tumor tissue contained in the FFPE slide glass, and simultaneously, a fresh tumor tissue sample of the constructed PDX model and a blood sample of a corresponding patient source are also obtained (when the copy number of the tumor tissue or tumor primary cell gene is judged to be changed, the blood sample is used as a control). IDTX gen hybridization buffer was used to prepare and prepare the library required for analysis, IlluminaNextSeq 500 was used for information capture and sequencing. The quality of the sequencing data was assessed using FastQC software (http:// www.bioinformatics.bbsrc.ac.uk/projects/FastQC /). Mapping sequences read from the genome to the human genome using BWA-MEM 50; the bam file is further processed via Picard (http:// branched. github. io/Picard /) to order the sequence and delete duplicate reads. Tumor coverage was normalized by matching to normal tissues and further calibrated by nucleotide composition, and fractional and logarithmic ratio estimates. Segment level CNV is defined as log ratio > 0.7 or < -0.7. Gene level CNV is defined as a gene with exon > 75% overlapping gain/deletion fragments.
Example 2 analysis of the results of the experiment
2.1 in esophageal squamous carcinoma Primary cellsFGF19Amplification assay
Carrying of cells numbered ZEC061 and cells numbered ZEC145 in esophageal squamous carcinoma primary cells (PDC) by adopting high-throughput sequencing detectionFGF19Expanded in ZEC157 cell and ZEC219 cellFGF19There was no copy number change. Further using real-time fluorescent quantitative PCR detectionFGF19In ZEC061 cells, ZEC145 cells, ZEC157 cells and ZEC219 cells, and the results showed that the relative expression values of mRNA in these cells were found to beFGF19The relative expression value of mRNA in ZEC061 cell and ZEC145 cell was significantly (p < 0.05) higher than that of ZEC157 cell and ZEC219 cell, and the results are shown in FIG. 1.
2.2 toFGF19Evaluation of sensitivity of amplified and non-amplified PDC as model to esophageal squamous carcinoma drug
And (3) screening the small-molecule inhibitor drug sensitivity of ZEC061 cells, ZEC145 cells, ZEC157 cells and ZEC219 cells by using a CellTiter-Glo luminous cell viability assay. Small molecule inhibitors include c-Myc inhibitor (10058-F4), AKT inhibitor (Afureertib/MK-22062 Hcl), FGFR inhibitor LY2874455, EGFR inhibitor (AST-1306/Dacomitinib/Lapatinib), histone methyltransferase inhibitor (MM-102), PARP inhibitor (Olaparib), PI3K inhibitor (PI-103), CDK4/6 inhibitor (Palbociclib), SIRT1/2 inhibitor (Tenovin-6), and B-rafV600E inhibitor (Vemurafenib), FGF 19-amplified esophageal squamous carcinoma primary cells were found to be more sensitive to FGFR inhibitor 28LY 74455, as shown in FIG. 2.
2.3 in vitro cell viability assay
FGFR inhibitor LY2874455 drug IC50 measurements were performed on ZEC061 cells, ZEC145 cells, ZEC157 cells and ZEC219 cells using in vitro cell viability assay techniques, and the data quantification process was based on the most sensitive ZEC145 cell IC50, as shown by the data results shown in fig. 3, which show that ZEC061 cells and ZEC145 cells are more sensitive than ZEC157 cells and ZEC219 cells.
2.4 authenticationFGF19Amplification of differences in sensitivity to FGFR inhibitors in response to esophageal squamous carcinoma primary cells
For further verificationFGF19Amplifying sensitivity difference of primary esophageal squamous carcinoma cell response FGFR inhibitor LY2874455, constructing an in vivo primary cell (PDX) model for further verification,FGF19the result of the amplified PDX model tumor forming experiment shows that the pair of FGFR inhibitors LY2874455FGF19The expanded primary cells were more effective and significantly inhibited tumor growth, as shown in fig. 4. For theFGF19The experimental result of the PDX model tumor without amplification shows that FGFR inhibitsPreparation LY2874455 pairFGF19The tumor growth of primary cells without expansion had no significant inhibitory effect, as shown in fig. 5. The above results confirm that,FGF19the expanded esophageal squamous carcinoma primary cells have sensitivity to the FGFR inhibitor LY 2874455.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. For detectingFGF19Use of a gene or its mRNA or an encoded protein or protein fragment thereof in the manufacture of a kit or microarray for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors, preferably as compared to a control,FGF19a high expression level and/or copy number amplification of the gene indicates that the cancer cell or patient is sensitive to the FGFR inhibitor, anFGF19A low expression level and/or no change or deletion in copy number of the gene indicates that the cancer cell or patient is not susceptible to the FGFR inhibitor.
2. Use according to claim 1, characterized in that the FGFR inhibitor is selected from sorafenib, sunitinib, pazopanib, panatinib, regorafenib, nidanib, lenvatinib, LY2874455, AZD4547, BGJ398, BMS-582664, AZD2171, Debio1347, doviranib, INCB054828, erdapitinib, PD173074 and cedanib.
3. Use according to claim 1, characterized in that said method is used for detectingFGF19Reagents for genes or their mRNAs or their encoded proteins or protein fragments includeFGF19Binding agents bound to a gene-encoded protein or protein fragment, or toFGF19Hybridization or amplification of genes or their mRNAsFGF19A gene or its mRNA.
4. According toUse according to claim 3, characterized in that the reaction withFGF19The binding agent to which the gene-encoded protein or protein fragment binds is an antibody directed against FGF 19.
5. Use according to claim 3, characterized in that said andFGF19hybridization or amplification of genes or their mRNAsFGF19The substance of the gene or its mRNA is an oligonucleotide primer or probe.
6. A kit or microarray for assessing and/or predicting the sensitivity of esophageal cancer cells, e.g. esophageal squamous carcinoma cells, or a patient suffering from esophageal cancer, e.g. esophageal squamous carcinoma, to FGFR inhibitors, characterized in that the kit or microarray comprises means for detectingFGF19A gene or its mRNA or its encoded protein or protein fragment.
7. The kit or microarray of claim 6, wherein the control comprises, in comparison to a control,FGF19a high expression level and/or copy number amplification of the gene indicates that the cancer cell or patient is sensitive to the FGFR inhibitor, anFGF19A low expression level and/or no change or deletion in copy number of the gene indicates that the cancer cell or patient is not susceptible to FGFR inhibitor; preferably the FGFR inhibitor is selected from sorafenib, sunitinib, pazopanib, panatinib, regorafenib, nidanib, lenvatinib, LY2874455, AZD4547, BGJ398, BMS-582664, AZD2171, Debio1347, dorvatinib, INCB054828, erdifitinib, PD173074 and cediranib.
8. The kit or microarray of claim 6, wherein the kit or microarray is used for detectionFGF19Reagents for genes or their mRNAs or their encoded proteins or protein fragments includeFGF19Binding agents bound to a gene-encoded protein or protein fragment, or toFGF19Hybridization or amplification of genes or their mRNAsFGF19A gene or its mRNA.
9. The kit or microarray of claim 8, which isCharacterized in that the reaction is carried out withFGF19The binding agent to which the gene-encoded protein or protein fragment binds is an antibody directed against FGF19, and/or
The above-mentioned andFGF19hybridization or amplification of genes or their mRNAsFGF19The substance of the gene or its mRNA is an oligonucleotide primer or probe.
10. Use of an FGFR inhibitor for the preparation of a medicament for the treatment of esophageal cancer, e.g., esophageal squamous carcinoma, in whichFGF19High expression level and/or copy number amplification of the gene; preferably the FGFR inhibitor is selected from sorafenib, sunitinib, pazopanib, panatinib, regorafenib, nidanib, lenvatinib, LY2874455, AZD4547, BGJ398, BMS-582664, AZD2171, Debio1347, dorvatinib, INCB054828, erdifitinib, PD173074 and cediranib; more preferably, the amount of FGFR inhibitor is an effective amount.
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EP1918376A1 (en) * 2006-11-03 2008-05-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. FGFR4 promotes cancer cell resistance in response to chemotherapeutic drugs
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EP1918376A1 (en) * 2006-11-03 2008-05-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. FGFR4 promotes cancer cell resistance in response to chemotherapeutic drugs
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