CN114763574A

CN114763574A - CDKN2A/2B deficiency as biomarker for predicting sensitivity of CDK4/6 inhibitor in esophageal squamous carcinoma

Info

Publication number: CN114763574A
Application number: CN202110034777.XA
Authority: CN
Inventors: 苏丹; 金娇悦; 应莉莎; 张付闯; 许晓雅; 张大东
Original assignee: Shanghai Siludi Medical Laboratory Co ltd; Zhejiang Cancer Hospital
Current assignee: Shanghai Siludi Medical Laboratory Co ltd; Zhejiang Cancer Hospital
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2022-07-19

Abstract

The invention relates toCDKN2A/2BUse of deletion in esophageal squamous carcinoma. The invention discloses a method for detectingCDKN2AGenes and/orCDKN2BUse of a reagent of genes or their mrnas or their encoded proteins or protein fragments for 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 a CDK4/6 inhibitor. Also disclosed is a kit or microarray for assessing and/or predicting the sensitivity of an esophageal cancer cell, e.g., an esophageal squamous carcinoma cell, or a patient suffering from an esophageal cancer, e.g., esophageal squamous carcinoma, to a CDK4/6 inhibitor.

Description

CDKN2A/2BDeletion as biomarker for predicting sensitivity of CDK4/6 inhibitor in esophageal squamous carcinoma

Technical Field

The invention relates to the technical field of medicaments, in particular toCDKN2A/2BUse of deletion 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 in the early stage, and the curative effect is obvious after clinical treatment. However, if the esophageal cancer is not screened early, the esophageal cancer is often accompanied by cancer cell metastasis when the esophageal cancer progresses to the middle and advanced stage of the disease, so that great challenges are brought to clinical treatment and 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. CDK4/6 is a key cell cycle regulatory kinase that is abnormally highly expressed in a variety of cancers, resulting in loss of normal regulation of the tumor cell cycle. The CDK4/6 inhibitor can inhibit CDK4/6 with high selectivity, block excessive division of tumor cells, effectively restore control on cell cycle and finally achieve the effect of inhibiting proliferation of the tumor cells. CDK4/6 shows good curative effect in the treatment of various cancers, including breast cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal squamous carcinoma and acute myelogenous leukemia. The cancer targeted drug represented by the CDK4/6 inhibitor shows the characteristics of high specificity and low toxicity in the tumor treatment process, can effectively target tumors, improves the life quality and survival period of tumor patients, and even can cure part of patients.

However, no biomarker is available for distinguishing tumor patients who are sensitive to treatment with the CDK4/6 inhibitor, which greatly limits the clinical use of the drug and also provides a great hindrance to tumor treatment. In recent years, with the rise of accurate medical treatment, the search for genome mutation after CDK4/6 inhibitor treatment and the screening of CDK4/6 inhibitor sensitive regulatory genes of CDK4/6 positive patients provide certain help for solving the problem of predicting sensitivity difference in the treatment process of tumor patients. However, no effective solution has been found for predicting the sensitivity of CDK4/6 inhibitors in esophageal squamous carcinoma.

Therefore, finding and exploring biological markers that can predict the sensitivity of esophageal squamous carcinoma to CDK4/6 inhibitors to more precisely select patients receiving targeted therapy has become an important scientific issue that must be considered in the field of esophageal squamous carcinoma basic medicine and in the clinical course of treatment of oncology patients.

Disclosure of Invention

To overcome the deficiencies of the prior art, the method comprises the steps of in vitro cell andin-vivo transplantation tumor experiment proves that about 27 percent of patients with esophageal squamous carcinoma have the esophageal squamous carcinomaCDKN2A/2BA deletion of gene copy number;CDKN2A/2Bhas the potential to become a key biomarker for identifying whether patients with esophageal squamous carcinoma can respond to CDK4/6 inhibitor treatment. The present invention has foundCDKN2A/2BDeficient esophageal squamous carcinoma is more sensitive to CDK4/6 inhibitors; CDK4/6 inhibitor can remarkably inhibitCDKN2A/2BThe tumor tissue-derived primary cells of the patients with the deficient esophageal squamous carcinoma have the tumor forming capability. The present invention finds out that CDK4/6 inhibitors areCDKN2A/2BHas a wide application prospect in the treatment of patients with esophageal squamous carcinoma,CDKN2A/2Bhas the potential to become a key biomarker for clinically diagnosing patients who can respond to CDK4/6 inhibitor treatment and benefit esophageal squamous carcinoma, and has larger clinical medical transformation value. The invention is helpful for better screening CDK4/6 inhibitor response population in the treatment process of the esophageal squamous carcinoma and improvingCDK4/6The accuracy of the inhibitor administration.

In one aspect, the invention relates to methods for detectingCDKN2AGenes and/orCDKN2BUse of a reagent of genes or their mrnas or their encoded proteins or protein fragments for 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 a CDK4/6 inhibitor.

In some embodiments, the composition is administered to a subject in need thereof, as compared to a control,CDKN2Agenes and/orCDKN2BA low expression level and/or a loss of copy number of the gene indicates that the cancer cell or patient is sensitive to a CDK4/6 inhibitor, anCDKN2AGenes and/orCDKN2BA high expression level and/or an unchanged copy number of the gene indicates that the cancer cell or patient is not sensitive to the CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is selected from Palbociclib (Palbociclib, trade name Ibrance), ribbociclib (Ribociclib, trade name Kisqali), abercinib (Abemaciclib, also known as LY2835219), abercinib mesylate (Abemaciclib mesylate), trilicib, G1T38, SHR-6390, FLX-925, alvocib (flavidiol); HMR-1275, Cdk4 inhibitor developed by Sanofi-Aventis as anticancer agent), voruciclib, AT-7519, INOC-005, and,BPI-1178, G1T30-1, GZ-38-1, P-276-00 (CDK 4-a selective inhibitor of cyclin D1, developed by Nicholas Piramal for the treatment of cancer), R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996, and PD-171851.

In some embodiments, the method is for detectingCDKN2AGenes and/orCDKN2BAgents for genes or their mRNAs or their encoded proteins or protein fragments includeCDKN2AGenes and/orCDKN2BGenes or their mRNAs. In some embodiments, the method is for detectingCDKN2AGenes and/orCDKN2BReagents for amplifying genes or their mRNAs or their encoded proteins or protein fragments includeCDKN2AGenes and/orCDKN2BGenes or their mRNA species. In some embodiments, the method is for detectingCDKN2AGenes and/orCDKN2BAgents for genes or their mRNAs or their encoded proteins or protein fragments includeCDKN2AGenes and/orCDKN2BA binding agent to which a gene-encoded protein or protein fragment binds. In some embodiments, the reaction is withCDKN2AGenes and/orCDKN2BThe binding agent to which the gene-encoded protein or protein fragment binds is an antibody against CDKN2A and/or CDKN 2B. In some embodiments, the reaction is withCDKN2AGenes and/orCDKN2BThe substance to which the genes or their mrnas hybridize is an oligonucleotide primer or probe. In some embodiments, the amplification isCDKN2AGenes and/orCDKN2BThe substance of the genes or their mrnas 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 an esophageal cancer cell, e.g. an esophageal squamous carcinoma cell, or a patient suffering from an esophageal cancer, e.g. an esophageal squamous carcinoma, to a CDK4/6 inhibitor, wherein said kit or microarray comprises a means for detectingCDKN2AGenes and/orCDKN2BGenes or their mrnas or their encoded proteins or protein fragments.

In some embodiments, the composition is administered to a subject in need thereof, as compared to a control,CDKN2Agenes and/orCDKN2BA low expression level and/or a loss of copy number of the gene is indicative of the cancer cell or patient pairCDK4/6 inhibitor sensitivity, andCDKN2Agenes and/orCDKN2BA high expression level and/or an unchanged copy number of the gene indicates that the cancer cell or patient is not sensitive to the CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is selected from the group consisting of palbociclib, ribbociclib, abbciclib mesylate, Trilaciclib, G1T38, SHR-6390, FLX-925, alvocidib, voruciclib, AT-7519, INOC-005, BPI-1178, G1T30-1, GZ-38-1, P-276-00, R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996, and PD-171851.

In a further aspect, the invention relates to the use of a CDK4/6 inhibitor in the manufacture of a medicament for the treatment of oesophageal cancer, such as oesophageal squamous carcinomaCDKN2AGenes and/orCDKN2BLow expression levels and/or copy number deletions of the gene. In some embodimentsWherein the CDK4/6 inhibitor is selected from the group consisting of Pabociclib, Ribociclib, Abbesiclib mesylate, Trilaciclib, G1T38, SHR-6390, FLX-925, alvocidib, voruciclib, AT-7519, INOC-005, BPI-1178, G1T30-1, GZ-38-1, P-276-00, R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996, and PD-171851. In some embodiments, the amount of the CDK4/6 inhibitor is an effective amount

In yet another aspect, the invention relates toCDKN2AGenes and/orCDKN2BUse of genes or their mrnas or their encoded proteins or protein fragments as biomarkers 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 CDK4/6 inhibitors.

In a further aspect, the invention relates to a method for assessing and/or predicting the sensitivity of an esophageal cancer cell, e.g. an esophageal squamous cancer cell, or a patient suffering from an esophageal cancer, e.g. an esophageal squamous cancer, to a CDK4/6 inhibitor, comprising the steps of:

(1) obtaining a biological sample from a subject; and

(2) detecting said sampleCDKN2AGenes and/orCDKN2BThe level of expression and/or copy number of genes or their mrnas or their encoded proteins or protein fragments;

wherein the concentration of the surfactant in the composition is compared with a control,CDKN2Agenes and/orCDKN2BA low expression level and/or a loss of copy number of the gene indicates that the cancer cell or patient is sensitive to a CDK4/6 inhibitor, anCDKN2AGenes and/orCDKN2BA high expression level and/or no change in copy number of the gene indicates that the cancer cell or patient is not sensitive to the CDK4/6 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

Figure 1 is a graph showing that in a population of patients with esophageal cancer,CDKN2A/2Bcopy number deletion andschematic illustration of unchanged scale.

FIG. 2 shows the CDK4/6 inhibitor palbociclibCDKN2A/2BSchematic representation of IC50 concentration in tumor tissue-derived primary cells of esophageal squamous carcinoma patients in the deletion group and the Copy Number Variation (CNV) -free group.

FIG. 3 shows the CDK4/6 inhibitor RibociclibCDKN2A/2BSchematic representation of IC50 concentrations of primary cells derived from tumor tissue of esophageal squamous carcinoma patients in the deletion group and in the no CNV group.

FIG. 4 shows the origin of the tumor tissue of patients with esophageal squamous carcinoma of the CDK4/6 inhibitor PabociclibCDKN2A/2BSchematic representation of inhibition of deleted primary cell ZEC 127.

FIG. 5 shows the origin of the CDK4/6 inhibitor palbociclib in tumor tissue from patients with esophageal squamous cell carcinomaCDKN2A/2BSchematic representation of the inhibition of primary cell ZEC118 without copy number variation.

FIG. 6 shows the origin of the CDK4/6 inhibitor Ribociclib in tumor tissue from patients with esophageal squamous cell carcinomaCDKN2A/2BSchematic representation of inhibition of deleted primary cells ZEC 127.

FIG. 7 shows the origin of the CDK4/6 inhibitor Ribociclib in tumor tissue from patients with esophageal squamous cell carcinomaCDKN2A/2BSchematic representation of the inhibition of primary cell ZEC118 without copy number variation.

FIG. 8 is a graph showing that on days 0 (panel A) and 28 (panel B) of transplantation,CDKN2A/2Bschematic representation of tumor volume for xenograft (PDCX) model of deletion patient derived cancer cell lines.

FIG. 9 is a graph showing that on days 0 (panel A) and 17 (panel B) of transplantation,CDKN2A/2Bschematic representation of tumor volume for xenograft (PDCX) model of patient derived cancer cell lines without copy number variation.

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.CDKN2A/2B) And esophageal cancer, particularly esophageal squamous carcinoma, to CDK4/6 inhibitors. The biomarkers described herein provide methods for assessing and/or predicting the effect of CDK4/6 inhibitors on the treatment of esophageal cancer, e.g., esophageal squamous carcinoma. Accordingly, one embodiment of the present invention represents an improvement in biomarkers suitable for use in assessing and/or predicting the effect of CDK4/6 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., theCDKN2A/2B) 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, CD24), e.g., for assessing and/or predicting the effect of CDK4/6 inhibitors on, or for preparing kits and/or microarrays for, the treatment of esophageal cancers, e.g., esophageal squamous cell carcinoma.

The invention discovers and confirmsCDKN2A/2BDeletion is a potential key biomarker for predicting CDK4/6 inhibitor sensitivity in esophageal squamous carcinoma, which suggests that CDK4/6 inhibitors are inCDKN2A/2BHas huge exploration potential and application prospect in the aspect of clinical treatment of patients with deficient esophageal squamous carcinoma.

The term "sample" means known or suspected to express or contain a biomarker (i.e., a biological marker)CDKN2A/2B) Or a material of a binding agent, e.g., for a biomarker (i.e., for a biological marker)CDKN2A/2B) 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 human physiological fluid, such as human serum. In certain aspects of the invention, the sample isBiopsy samples are, for example, 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, mRNAs, proteins or polypeptides (e.g.CDKN2AGenes and/orCDKN2BGenes or their mrnas or their encoded proteins or protein fragments). Genes, mrnas, proteins or polypeptides for use as markers in the present invention are intended to include naturally occurring variants of said genes, mrnas or proteins as well as fragments, in particular immunologically detectable fragments, of said genes, mrnas 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. In the present invention, the marker polypeptide also includes variants of the marker polypeptide. Alteration of marker polypeptideThe entities may be encoded by the same gene, but may differ in their isoelectric point (= PI) or molecular weight (= MW) or both, for example as a result of 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 (CDK4/6 inhibitor) to provide a desired effect or benefit.

CDKN2ALocated on chromosome 9, encodes cyclin-dependent kinase inhibitor 2a, which is an oncogene. At least three different splice variants of this gene have been reported, two of which are believed to be CDK4 kinase inhibitors. The other variant has an ARF structural domain and can stabilize the P53 protein, and the three variants can regulate the G1 cell cycle in a combined action mode. And withCDKN2ARelated diseases include malignant melanoma, ovarian cancer, bladder cancer, familial pancreatic cancer, esophageal cancer, and lung cancer.

CDKN2BAlso located on chromosome 9, encodes cyclin-dependent kinase inhibitor 2B, also an oncogene.CDKN2BCan bind with CDK4 or CDK6 to form a complex, inhibit the activity of CDK kinase, and control the progression of cell cycle G1.CDKN2BInactivation of (b) may lead to uncontrolled cell growth and proliferation, leading to carcinogenesis.CDKN2A/2BIs a necessary event for Rb protein phosphorylation and oncogenes to induce cellular senescence.CDKN2BThe loss of expression of (a) is frequently seen in various tumors such as ovarian cancer, breast cancer, lung cancer, pancreatic endocrine tumor, lung cancer, esophageal cancer, etc.

Terms used herein "CDKN2A/2BThe gene refers toCDKN2AGenes and/orCDKN2BA gene.CDKN2A/ 2BDeletion refers toCDKN2ADeletion and/orCDKN2BDeletions, i.e., including both homozygous and heterozygous deletions.CDKN2A/2BNo copy number change meansCDKN2AAndCDKN2Bthere was no copy number change.

The term "CDK 4/6 inhibitor" as used herein refers to an inhibitor of CDK4 and/or an inhibitor of CDK 6.

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; the expression of the internal reference gene (such as TBP) of esophageal cancer such as esophageal squamous cell carcinoma tumor cells; a predetermined cell that is relatively insensitive to CDK4/6 inhibitor (e.g., a primary esophageal squamous carcinoma cell that is relatively insensitive to CDK4/6 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.

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 onCDKN2A/2BAnd (3) judging the relative judgment among different groups of different grouped people by judging whether the copy number is missing or not. In other words, withCDKN2A/ 2BHigh level of gene expression and/or unchanged copy numberCDKN2A/2BGroups with low gene expression levels and/or copy number deletions have a more sensitive response to CDK4/6 inhibitors and are therefore usedCDKN2A/2BHigh or low gene expression levels and/or copy number amplification or lack thereof, to distinguish between groups sensitive or insensitive to CDK4/6 inhibitors. The degree of sensitivity to CDK4/6 inhibitors can be measured by the IC50 value, with lower IC50 values reflecting greater sensitivity of esophageal squamous carcinoma cells to CDK4/6 inhibitor treatment and higher IC50 values reflecting lesser sensitivity of esophageal squamous carcinoma cells to CDK4/6 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, which preferably comprises at least 6,7, 8, 10, 12, 15 or 20 consecutive amino acids of the marker polypeptide. A change in the expression level of a protein is at least about 1.1, 1.25, 1.5, 2, 3, 4,5, 6,7, 8, 9, or 10-fold or more of 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 of the expression level of a control or standard as compared to 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 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 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 to a carrier or support material. For the method or kit of the inventionThe oligonucleotide of (a) may have 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)CDKN2A/2BProtein or protein fragment encoded by a gene) in the opposite direction of the sense polynucleotide).

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 virtue of 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 detectingCDKN2A/2BReagents 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 can be prepared conventionally 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)CDKN2A/2B) 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 easily 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; 546), antibody heavy chains, intrabodies, humanized antibodies, human antibodies, antibody light chains, single chain fv (scFv) (e.g., including monospecific, bispecific, etc.), anti-idiotypic (ant-Id) antibodies, proteins comprising an antibody portion, chimeric antibodies (e.g., an antibody comprising the binding specificity of a murine antibody 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 modification of an immunoglobulin molecule comprising an antigen recognition site of the desired specificity 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., Abnova (e.g., cat # PAB0823, H00001030-A01), Abcam (e.g., cat # ab54210, ab150916), Invitrogen (e.g., cat # MA1-16664, MA5-14260, MA1-12294, PA5-99491, PA5-49749), 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-; 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 detectionCDKN2A/2BGenes or their mrnas or their encoded proteins or protein fragments). 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 together or separately to a given subjectThe recipient of (2). As an example, the kit may contain a reagent for detectionCDKN2A/2BReagents, buffers and instructions for use of the genes or their mrnas or their encoded proteins or protein fragments. The kit may further contain polymerase, dNTP, etc. 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 greater than 1000/cm². Microarray technology is disclosed, for example, in the following references: a Practical Approach (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 disclosesCDKN2A/2BThe 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 uses and techniques without departing from the spirit, scope and spirit 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 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.

1 method of experiment

(1) Construction of esophageal squamous carcinoma PDC

The 123 ESCC patient pathology information used to construct the PDC (i.e., the patient-derived cancer cell line) was confirmed by a qualified pathologist. Fresh ESCC tumor tissue was washed 3 times with PBS and cut into 0.5-1 mm pieces³And (4) fragmenting. Then, the cells were transferred to a cell culture dish and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1X non-essential amino acids, 50U/mL penicillin and 50. mu.g/mL. The culture conditions in the incubator are 37 ℃ and 5% CO₂. After 5 to 7 days, the cells are digested and gradient adherent culture is carried out to purify the tumor cells, the adherent cells are marked as 0 generation, and STR identification is carried out when the purified cells are passed to the fifth generation. After the identification is completed, the cells are subjected to amplification culture to obtain enough cells, and then the related test is carried out. Excess cell cryo-preservation was performed in liquid nitrogen.

(2) In vitro cell viability assay

Cells in good growth status were digested, resuspended, counted, and plated in 96-well plates at 4000 cell densities in quadruplicate at standard medium culture conditions per well. After the cells were adherent, an appropriate concentration of CDK4/6 inhibitor (palbociclib and ribociclib) was 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 was calculated by four parameter curve analysis (IC 50). IC50 values and standard deviations of CDK4/6 inhibitors were calculated from four independent experiments to provide data references for further in vitro validation.

(3) In vitro plate clone proliferation assay

Tumor cells were digested, resuspended and counted and plated at a density of 2000 cells per well into 6-well plates. After the cells are attached well, CDK4/6 inhibitor with proper concentration (different concentration gradients, namely 0, 0.5 and 1 mu M) is added, the cells are cultured for 2 weeks under standard conditions in a cell culture box, the growth state of the cells is observed under a microscope at regular intervals, and a cell culture solution with proper concentration is replaced at regular intervals. After 2 weeks, the cells were washed twice with PBS, and then methanol was added to fix the cells, for 10 minutes at room temperature. The cell fixing solution was removed, and then a staining solution containing 0.1% crystal violet was added to the 6-well plate, and the plate was left at room temperature for 15 minutes. After the cell staining was observed and confirmed to be satisfactory, the crystal violet stain was removed and washed with PBS. After drying, counting, photographing and statistical analysis are performed under a microscope.

(4) Establishment of PDCX model and CDK4/6 inhibitor in-vivo growth inhibition experiment

Will be 1x10⁷The primary cells were inoculated into hind legs of 4-week-old male BALB/c nude mice. Tumor size was measured every three days, while nude mice body weight and tumor growth curves were recorded. When the volume of the transplanted tumor is close to 1000 mm³When large, mice were sacrificed. Rapidly and uniformly cutting the tissue into 1-2 mm³Size. Tissue fragments were implanted in the left axilla of 4-week-old male BALB/c nude mice. After two weeks, when the size of the transplanted tumor of 40 nude mice reaches 100-³In time, three groups were randomized and received control (saline), 75 mg/kg palbociclib, and 150 mg/kg palbociclib, respectively. After 28 days or the volume of the tumor tissue reaches 1500 mm³At this time, the experiment was terminated, the nude mice were sacrificed, and tumor tissue and blood samples were taken and stored.

(5) High throughput sequencing and gene copy number variation analysis

High-throughput sequencing and analysis of tumor tissues of 161 cases of esophageal squamous carcinoma patients, PDCX samples from fresh tumor patients and blood samples from corresponding patients are completed by Shanghai thought Didi medical inspection institute Co., Ltd, and the company has NGS solid tumor tissue gene mutation detection capability 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 tumor tissues contained in the FFPE glass slide, and meanwhile, a PDCX sample from a fresh tumor patient and a blood sample from a corresponding patient are also obtained. IDTX gen hybridization buffer was used to prepare and prepare the library required for analysis, Illumina NextSeq 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.

2 results and analysis of the experiments

(1) According to the statistics of the gene map analysis of the tumor tissue of 161 cases of esophageal squamous carcinoma patients, in the population of esophageal carcinoma patients,CDKN2A/2Bthe proportion of no copy number change was 73%,CDKN2A/2Bthe deletion ratio was 27% (FIG. 1).

(2) By using 4 primary cells derived from tumor tissues of patients with esophageal squamous carcinomaCDKN2AAndCDKN2Bthe Copy Number Variation (CNV) condition is detected and classified asCDKN2A/2BMissing groups (numbered ZEC145 and ZEC 127) and no CNV groups (numbered ZEC166 and ZEC 118). Analysis of drug sensitivity data for targeted CDK4/6 inhibitors showed relativeCDKN2A/2BIn the deletion group, the primary cell response CDK4/6 inhibitor palbociclib (figure 2) and the Ribociclib (figure 3) of tumor tissue-derived primary cells of patients without CNV group esophageal squamous carcinoma have obviously increased IC50 concentration. The above results show that it is possible to obtain,CDKN2A/2Bdeficient esophageal squamous carcinoma patient tumor tissue-derived primary cells as compared toCDKN2A/2BPrimary cells without copy number variation, inhibited CDK4/6The agent is more sensitive.

(3) For further verificationCDKN2A/2BThe absence of a biomarker that could be responsive to CDK4/6 inhibitor sensitivity in esophageal squamous carcinoma was further confirmed using an in vitro plate clone proliferation assay. The plate clone experiment selects primary cells ZEC127 and ZEC118 from tumor tissues of esophageal squamous carcinoma patients, wherein ZEC127 isCDKN2A/2BDeletion type, ZEC118 isCDKN2A/2BWild type (A)CDKN2A/2BNo copy number change). Experimental data analysis results show that both the CDK4/6 inhibitors palbociclib and ribociclib can significantly inhibit the ZEC127 (fig. 4 and 6) clonogenic capacity, and the inhibitory effect gradually increases with increasing CDK4/6 inhibitor concentration; and isCDKN2A/2BDeleted primary cell ZEC127 compared toCDKN2A/2BPrimary cells ZEC118 without copy number changes were more sensitive to CDK4/6 inhibitor under equivalent conditions, in particular as treatment with equivalent palbociclib and rebbociclib drug concentration conditions resulted in a lower number of primary cell clones (fig. 4-fig. 7). The results of the above data show that,CDKN2A/2Bthe deletion has the potential to become a biomarker for predicting the sensitivity of the esophageal squamous carcinoma to the CDK4/6 inhibitor.

(4) For further comprehensive evaluationCDKN2A/2BThe application of the deletion in the evaluation of the sensitivity of esophageal squamous cell carcinoma drugs and targeted therapy is deeply verified by adopting a primary cell xenograft (PDCX) model. The primary esophageal squamous carcinoma cells selected in the in vivo xenograft experiment are ZEC145 and ZEC166, wherein ZEC145 is ZEC145CDKN2A/2BDeletion type, ZEC166 isCDKN2A/2BThe wild type. The results of data analysis of the effect of CDK4/6 inhibitor on the PDCX model show that,CDKN2A/2Bthe deleted primary cell ZEC145 was more sensitive to the CDK4/6 inhibitor palbociclib, which is shown in that two doses (75 and 150 mg/kg) of palbociclib have obvious tumor formation inhibiting effect, and the effect of the high dose treatment group is more obvious (FIG. 8A, FIG. 8B). Both fig. 8A and fig. 9A were at day 0 of the transplantable tumor, with no significant difference between groups; fig. 8B and 9B are day 28 and day 17, respectively, after the transplantation of the tumor.CDKN2A/2BZEC166 wild-type primary cells failed to respond effectively to the CDK4/6 inhibitor palbociclib action and was characterized by somaticThe growth of the internal transplantable tumor was not significantly different from the control group even at the highest dose (150 mg/kg) of palbociclib (fig. 9A, fig. 9B). The above results show that it is possible to obtain,CDKN2A/2Bthe deleted esophageal squamous carcinoma PDCX responded to CDK4/6 inhibitor more strongly, and the tumor volume was significantly reduced. This further verifies in vivoCDKN2A/ 2BThe absence can be used as a biomarker for predicting CDK4/6 inhibitor sensitivity.

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

1. For detectingCDKN2AGenes and/orCDKN2BUse of genes or their mrnas or their encoded proteins or protein fragments 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 a CDK4/6 inhibitor, preferably as compared to a control,CDKN2Agenes and/orCDKN2BA low expression level and/or a loss of copy number of the gene indicates that the cancer cell or patient is sensitive to a CDK4/6 inhibitor, anCDKN2A/2BA high expression level and/or an unchanged copy number of the gene indicates that the cancer cell or patient is not sensitive to the CDK4/6 inhibitor.

2. Use according to claim 1, characterized in that the CDK4/6 inhibitor is selected from pabociclib, rebusciclib, abercinib mesylate, Trilaciclib, G1T38, SHR-6390, FLX-925, alvocidib, voruciclib, AT-7519, INOC-005, BPI-1178, G1T30-1, GZ-38-1, P-276-00, R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996 and PD-171851.

3. Use according to claim 1, characterized in that it is for detectingCDKN2AGenes and/orCDKN2BAgents for genes or their mrnas or their encoded proteins or protein fragments include:

(i) andCDKN2Agenes and/orCDKN2BSubstances hybridizing with the genes or their mRNAs, or

(ii) Amplification ofCDKN2AGenes and/orCDKN2BThe gene or mRNA thereof, or

(iii) AndCDKN2Agenes and/orCDKN2BA binding agent to which a gene-encoded protein or protein fragment binds.

4. Use according to claim 3, characterized in that said andCDKN2Agenes and/orCDKN2BThe binding agent to which the gene-encoded protein or protein fragment binds is an antibody against CDKN2A and/or CDKN 2B.

5. Use according to claim 3, characterized in that said andCDKN2Agenes and/orCDKN2BThe substance hybridizing with the genes or their mRNAs is an oligonucleotide primer or probe, and/or the amplificationCDKN2AGenes and/orCDKN2BThe substance of the genes or their mrnas 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 a CDK4/6 inhibitor, characterized in that said kit or microarray comprises means for detectingCDKN2AGenes and/orCDKN2BGenes or their mrnas or their encoded proteins or protein fragments.

7. The kit or microarray of claim 6, wherein the microarray comprises, in comparison to a control,CDKN2Agenes and/orCDKN2BA low expression level and/or a loss of copy number of the gene indicates that the cancer cell or patient is sensitive to a CDK4/6 inhibitor, anCDKN2AGenes and/orCDKN2BA high expression level and/or an unchanged copy number of the gene indicates that the cancer cell or patient is not sensitive to the CDK4/6 inhibitor; preferably said CDK4/6 inhibitsThe agent is selected from the group consisting of palbociclib, rebusciclib, abbesinib mesylate, Trilaciclib, G1T38, SHR-6390, FLX-925, alvocidib, voruciclib, AT-7519, INOC-005, BPI-1178, G1T30-1, GZ-38-1, P-276-00, R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996, and PD-171851.

8. Kit or microarray according to claim 6, characterized in that it is used for detectionCDKN2AGenes and/orCDKN2BAgents for genes or their mrnas or their encoded proteins or protein fragments include:

(ii) Amplification ofCDKN2AGenes and/orCDKN2BThe gene or mRNA thereof, or

(iii) And withCDKN2AGenes and/orCDKN2BA binding agent to which a gene-encoded protein or protein fragment binds.

9. The kit or microarray of claim 8, wherein the hybridization is performed withCDKN2AGenes and/orCDKN2BThe binding agent to which the gene-encoded protein or protein fragment binds is an antibody against CDKN2A and/or CDKN2B, and/or

The said andCDKN2Agenes and/orCDKN2BThe substance to which the genes or their mRNAs hybridize is an oligonucleotide primer or probe, and/or

Said amplificationCDKN2AGenes and/orCDKN2BThe substance of the genes or their mrnas is an oligonucleotide primer or probe.

10. Use of a CDK4/6 inhibitor for the manufacture of a medicament for the treatment of oesophageal cancer, such as oesophageal squamous carcinoma, wherein said oesophageal cancer is for example oesophageal squamous carcinomaCDKN2AGenes and/orCDKN2BLow expression level and/or copy number deletion of the gene; preferably the CDK4/6 inhibitor is selected from the group consisting of Pabociclib, Ribociclib, Abelinib mesylate, Trilaciclib, G1T38, SHR-6390, FLX-925, alvocidib, voruciclib, AT-7519, INOC-005, BPI-1178, G1T30-1, GZ-38-1, P-276-00, R-547, PAN-1215, PD-0183812, AG-024322, NSC-625987, CGP-82996 and PD-171851; more preferably, the CDK4/6 inhibitor is in an effective amount.