Treatments for oral cancer

Abstract

Provided herein is a method for treating oral cancer in an individual. According to an embodiment of the present disclosure, the method includes obtaining a first and a second biological sample from a lesion site and a non-lesion site of the individual, respectively; measuring the expression levels of CHEK1, PIK3CA or PIK3CD in the two biological samples by qRT-PCR, respectively, thereby obtaining a first and a second expression level; determining the ratio of the first expression level to the second expression level; and administering an effective amount of a checkpoint kinase 1 inhibitor to the individual when the ratio of CHEK1 is at least 1.7; or administering a phosphatidylinositol 3-kinase inhibitor when the ratio of PIK3CA is at least 2.4, or when the ratio of PIK3CD is at least 3.1.

Description

Technical Field

[0001] The present disclosure relates generally to the field of cancer treatment. More specifically, the present disclosure relates to methods for treating oral cancer in an individual in need thereof. Background Art

[0002] Cancer is a group of diseases characterized by abnormal cell growth that may invade or spread to other parts of the body, causing the death of the affected individual.

[0003] Oral cancer refers to cancer that occurs on the lining of the lips, mouth, or upper throat. Oral squamous cell carcinoma (OSCC) accounts for 90% of all oral malignancies. The main risk factors for oral cancer are smoking and heavy drinking, but reports indicate that betel quid chewing and human papillomavirus (HPV) infection are also one of the main predisposing factors. The current management of OSCC is still early surgical resection, followed by adjuvant therapy (e.g., radiotherapy), with or without chemotherapy. Among them, chemotherapy containing cisplatin or cetuximab is the current standard treatment for recurrent / metastatic OSCC. However, this treatment is not customized and often has serious adverse effects on patients, resulting in a poor quality of life, and rehabilitation is usually required to help them return to normal life.

[0004] In view of the above situation, the relevant field urgently needs a novel therapy that can accurately target oral cancer to improve the safety and effectiveness of treating oral cancer. Summary of the invention

[0005] A simplified summary of the present disclosure is presented below to provide the reader with a basic understanding. This summary is not an extensive overview of the present disclosure, nor does it identify the key / important elements of the present invention or describe the scope of the present invention. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as a preface to a more detailed description presented later.

[0006] As embodied and broadly described herein, one aspect of the present disclosure is a kit for detecting the sensitivity of an individual suffering from oral cancer to a kinase inhibitor, characterized in that it comprises a primer pair with sequence numbers: 1 and 2, sequence numbers: 3 and 4, or sequence numbers: 5 and 6; the primer pair with sequence numbers: 1 and 2 is used to detect the expression level of the CHEK1 gene of the individual; the primer pair with sequence numbers: 3 and 4 is used to detect the expression level of the PIK3CA gene of the individual; the primer pair with sequence numbers: 5 and 6 is used to detect the expression level of the PIK3CD gene of the individual; and the kinase inhibitor is selected from the group consisting of: checkpoint kinase 1 (CHK1) inhibitor, and phosphatidylinositol 3-kinase (PI3K) inhibitor.

[0007] According to certain embodiments of the present disclosure, the kit of the present invention is characterized in that it further comprises a primer pair with sequence numbers: 7 and 8, and the primer pair with sequence numbers: 7 and 8 is used to detect the expression level of the TBP gene of the individual.

[0008] According to certain embodiments of the present disclosure, the oral cancer is oral squamous cell carcinoma (OSCC).

[0009] Examples of CHK1 inhibitors suitable for use in the present disclosure may be

[0010] (S)-5-(3-Fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762);

[0011] 4-(((3S)-1-Azabicyclo(2.2.2)oct-3-yl)amino)-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR-124);

[0012] Rabusertib (LY2603618);

[0013] Prexasertib (LY2606368);

[0014] 4-(2,6-Dichlorophenyl)-9-hydroxy-6-(3-(methylamino)propyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione

[0015] (4-(2,6-dichlorophenyl)-9-hydroxy-6-(3-(methylamino)propyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione)(PD-321852);

[0016] (R)-2-amino-2-cyclohexyl-N-(2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-5,6-dihydro-1H-[1,2]diazepino[4,5,6-cd]indol-8-yl)acetamide (PF477736);

[0017] (R)-6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-3-yl)pyrazolo[1,5-a]pyrimidin-7-amine (SCH900776);

[0018] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinamide;

[0019] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-ylisobutyramide);

[0020] (R)-N-(5-bromo-4-(3-(methylamino)piperidin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinamide;

[0021] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-methylnicotinamide;

[0022] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)cyclopropanecarboxamide;

[0023] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-methylbutanamide; or

[0024] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyclopropylacetamide.

[0025] In some specific examples, the CHK1 inhibitor is (S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide;

[0026] Preseti; or (R)-2-amino-2-cyclohexyl-N-(2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-5,6-dihydro-1H-[1,2]diazepino[4,5,6-cd]indol-8-yl)acetamide.

[0027] Examples of PI3K inhibitors suitable for use in the present disclosure may be

[0028] (2S)-N1-[5-(2-tert-butyl-4-thiazolyl)-4-methyl-2-thiazolyl]pyrrolidine-1,2-dicarboxamide (A66);

[0029] (Z)-5-((5-(4-fluoro-2-hydroxyphenyl)furan-2-yl)methylene)thiazolidine-2,4-dione (AS-252424);

[0030] 5-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione (AS-604850);

[0031] (R)-2-(1-(7-methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethylamino)benzoic acid (AZD6482);

[0032] 2-amino-N-[7-methoxy-8-(3-morpholinopropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin (BAY 80-6945);

[0033] Copanlisib (BAY 80-6946);

[0034] 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one maleate (BGT226);

[0035] Buparlisib (BKM120);

[0036] Alpelisib (BYL719);

[0037] (5E)-5-{[5-(4-fluorophenyl)furan-2-yl]methylidene}-1,3-thiazolidine-2,4-dione (CAY10505);

[0038] 5-(2-Amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide (CZC24832);

[0039] Duvelisib (IPI-145);

[0040] 2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one (IC-87114);

[0041] Idelalisib (GS-1101, CAL-101);

[0042] Serabelisib(INK1117);

[0043] Taselisib (GDC-0032);

[0044] Pictilisib (GDC-0941);

[0045] Apitolisib (GDC-0980);

[0046] (Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene)thiazolidine-2,4-dione (GSK1059615);

[0047] Omipalisib (GSK2126458);

[0048] 2-methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6-morpholino-1H-benzo[d]imidazole-4-carboxylic acid (GSK2636771);

[0049] 6-[5-[(phenylsulfonyl)amino]-3-pyridinyl]-imidazo[1,2-a]pyridine-3-carboxylic acid, ethyl ester (HS-173);

[0050] 2-amino-N,N-dimethyl-5-(3-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine-5-yl)pyridine-3-sulfonamide (HS-527);

[0051] 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002);

[0052] Dactolisib (NVP-BEZ235);

[0053] Perifosine;

[0054] 2-Amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7-one (PF-04691502);

[0055] Gedatolisib (PF-05212384);

[0056] 3-(4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol (PI-103);

[0057] 2-Methyl-5-nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75);

[0058] N-(2,3-dihydro-7,8-dimethoxyimidazo[1,2-c]quinazolin-5-yl)-3-pyridinecarboxamide (PIK-90);

[0059] 2-[(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (PIK-293);

[0060] 2-[[4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (PIK-294);

[0061] 1-(4-(3-ethyl-7-morpholino-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl)-3-(4-(4-methylpiperazine-1-carbonyl)phenyl)urea)(PKI-402);

[0062] Sonolisib (PX-866);

[0063] (S)-2-(2-(2-Methylindolin-1-yl)-2-oxoethyl)-6-morpholinopyrimidin-4(3H)-one (SAR260301);

[0064] Pilaralisib (SAR245408);

[0065] (2S)-2-[[(2S)-3-Carboxy-2-[[2-[[(2S)-5-(diaminomethylideneamino)-2-[[4-oxo-4-[[4-(4-oxo-8-phenylchromen-2-yl)morpholin-4-ium-4-yl]methoxy]butanoyl]amino]pentanoyl]amino]acetyl]amino]propanoyl]amino]-3-hydroxypropanoate (SF1126);

[0066] 3-(2,4-Diaminopteridin-6-yl)phenol (TG100713);

[0067] Umbralisib (TGR-1202);

[0068] 5-(9-Isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584);

[0069] Voxtalisib (XL765); or

[0070] 4,4'-(6-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)di morpholine (ZSTK474).

[0071] In some specific embodiments, the PI3K inhibitor is apeliselice;

[0072] Pitriside; or (Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene)thiazolidine-2,4-dione.

[0073] According to some embodiments of the present disclosure, the individual is a human.

[0074]

[0011] Many of the attendant features and advantages of the present disclosure will be better understood with reference to the following detailed description considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings, in which:

[0076] Figures 1A-1D Depicted is the expression of CHEK1, PIK3CA, and PIK3CD in OSCC according to a working example of the present disclosure. Figures 1A-1C qRT-PCR analysis revealed that CHEK1 (Figure 1A), PIK3CA ( Figure 1B ) and PIK3CD( Figure 1C ) in 32 pairs of OSCC patients. The expression levels of these genes were measured by 2- ΔΔCt Methods: TATA-box binding protein (TBP) was used as an internal control. Figure 1D is a dot plot depicting the ratio of gene expression in tumor tissues of OSCC patients to that in adjacent normal tissues.

[0077] Figures 2A-2F The therapeutic effects of CHK1 inhibitors on OSCC are depicted. Figure 2A is a graph depicting the dose-dependent cytotoxic effects of various CHK1 inhibitors, including PF477736, AZD7762, and LY2606368, on OSCC cells SAS and OEC-M1, respectively, as determined by MTT assay. Figure 2B-2C The dose-dependent cytotoxic effect of PF477736 on OSCC cells was shown, as determined by colony-forming assay ( Figure 2B ) and determined by Annexin V staining ( Figure 2C ). Figure 2D The effects of CHK1 inhibitors on UV-induced CHK1 phosphorylation are illustrated. Figure 2EDepicts the dose-dependent cytotoxic effect of cisplatin on OSCC cells SAS, OC3 or OEC-M1 as determined by MTT assay. FIG. 2F is a bar graph depicting the effect of PF477736 and cisplatin on SAS or OC3 cells as determined by MTT assay. Data were repeated in three independent experiments. Results are expressed as mean ± SEM. Statistical analysis between the two groups was performed using Student's t-test. *p<0.05; **p<0.01; ***p<0.001.

[0078] Figures 3A-3G The therapeutic effects of PI3K inhibitors on OSCC are depicted. Figure 3A 3B-3C are bar graphs showing the dose-dependent cytotoxic effects of BYL719 on OSCC cells SAS or OEC-M1 as determined by colony formation assay ( Figure 3B ) and Annexin V staining ( Figure 3C ) determined. Figure 3D The effect of PI3K inhibitors on the phosphorylation of AKT is depicted. Figure 3E Depicted are the effects of BYL719 and PF477736 on SAS cells as determined by MTT assay. Figure 3F is depicted by Annexin V staining Figure 3F A bar graph of apoptotic cells in . Figure 3G Depicted are the effects of GDC-0941 and AZD7762 on SAS cells as determined by MTT assay. Data were repeated in three independent experiments. Results are expressed as mean ± SEM. Statistical analysis between two groups was performed using Student's t-test. *p<0.05; **p<0.01; ***p<0.001.

[0079] Figures 4A-4D Line graph depicting the effects of CHK1 inhibitor PF477736 and PI3K inhibitor BYL719 on OSCC xenografts. SAS cells were injected subcutaneously into NOD / SCID mice until tumors reached a volume of 300-500 mm 3 The mice were randomly divided into several groups. The transplanted mice were given vehicle control, 10 or 20 mg / kg PF477736 ( Figure 4A ), 25 or 50 mg / kg BYL719 ( Figure 4B), PF477736 (20 mg / kg) plus BYL719 (50 mg / kg) ( Figure 4C ) and 5mg / kg cisplatin ( Figure 4D ). Tumor volumes were measured and calculated at the indicated times. Data are expressed as mean ± SEM. Statistical analysis between the two groups was performed using Student's t-test. *p<0.05; **p<0.01; ***p<0.001.

[0080] Figures 5A-5E Shown are the effects of the CHK1 inhibitor PF477736 in combination with cisplatin or the PI3K inhibitor BYL719 on tumor growth in an OSCC patient-derived xenograft (PDX) model. Figure 5A Results are presented for three OSCC PDX mice (patient #1 PDX, patient #2 PDX, and patient #3 PDX) treated with vehicle control, cisplatin alone, PF477736 alone, and PF477736 plus cisplatin. Figure 5B Representative photographs of hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) staining (Ki-67; upper panels); line graphs depict corresponding quantitative results from patient #2 PDX model (lower panels). Figure 5C are line graphs depicting the respective changes in tumor volume for Patient #1 PDX and Patient #2 PDX models treated with vehicle control, BYL719 alone, PF477736 alone, or a combination of PF477736 and BYL719. Figure 5D Provided are the results of patient #2 PDX model determined by IHC staining (Ki-67; upper panel), and the corresponding quantification results (lower panel). Figure 5E is a line graph depicting the weight change of patient #2 PDX model after treatment. Tumor volume was measured twice a week and mice were monitored for 3 weeks. Data are presented as mean ± SEM. Statistical analysis between the two groups was performed using Student's t-test. *: p < 0.05; **: p < 0.01. DETAILED DESCRIPTION

[0081] The detailed description provided below in conjunction with the accompanying drawings is intended as a description of embodiments of the present invention and is not intended to represent the only form in which embodiments of the present invention may be constructed or utilized. The description sets forth the functions of the embodiments and the sequence of steps for constructing and operating the embodiments. However, the same or equivalent functions and sequences may be achieved by different embodiments.

[0082] I. Definitions

[0083] For convenience, certain terms used in the specification, embodiments and the appended claims are collected here. Unless otherwise defined herein, the scientific and technical terms used in this disclosure should have the meanings commonly understood and used by those of ordinary skill in the art. In addition, unless the context otherwise requires, it should be understood that singular terms should include the plural form of the thing, and plural terms should include the singular. Specifically, as used herein and in the claims, the singular forms of "a", "an" and "the" include plural references unless the context otherwise clearly indicates. Moreover, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three or more. Unless otherwise specified, the implementation of the present invention will adopt the conventional techniques of molecular biology, microbiology, recombinant DNA and immunology, which are all within the technical scope of the art. Such techniques are fully explained in the literature.

[0084] Although the numerical ranges and parameters describing the broad scope of the present invention are approximate, the numerical values ​​described in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors, which are necessarily caused by the standard deviation found in each test measurement. Similarly, as used herein, the term "about" generally refers to within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" refers to within the acceptable standard error of the mean value when considered by a person of ordinary skill in the art. Except for the content in the operating / working examples, or unless otherwise explicitly specified, all numerical ranges, quantities, values ​​and percentages disclosed herein, such as terms for material quantity, duration, temperature, operating conditions, quantity ratios, etc., should be understood as being modified by the term "about" in any case. Therefore, unless otherwise indicated, the numerical parameters described in the present disclosure and the attached claims are approximate values ​​that can be changed as needed. In any case, each numerical parameter should at least be understood as a numerical value obtained based on the reported significant digits and by applying ordinary rounding methods.

[0085] As used herein, the term "treatment" or "treating" may refer to a cure or palliative measure. In particular, the term "treatment" as used herein refers to the application of the method of the present invention to an individual suffering from cancer, symptoms associated with cancer, diseases or conditions secondary to cancer, with the purpose of partially or completely relieving, improving, alleviating, delaying the onset of cancer, inhibiting its progression, reducing its severity and / or reducing the incidence of one or more symptoms or characteristics thereof.

[0086] The terms "cancer" and "tumor" are used interchangeably in this disclosure and preferably refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer in this regard includes metastatic cancer and / or drug-resistant cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include bladder cancer, biliary tract cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, epidermal cancer, gastric cancer, gastrointestinal stromal tumor, glioblastoma, glioma, hepatocellular carcinoma, non-Hodgkin's lymphoma, Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma (e.g., oral squamous cell carcinoma, epithelial squamous cell carcinoma), thyroid cancer or thyroid follicular carcinoma. According to a specific working embodiment, the cancer is oral cancer; the cancer is oral cancer. More specifically, it is OSCC.

[0087] The term "subject" or "patient" refers to an animal, including human species, that can be treated with the methods of the present disclosure. Unless one gender is explicitly indicated, the term "subject" or "patient" means both male and female. Therefore, the term "subject" or "patient" includes any mammal that can benefit from cancer treatment. Examples of "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and fowl. In an exemplary embodiment, the subject is a mouse. In another exemplary embodiment, the subject is a human.

[0088] The term "lesion" refers to any pathological or traumatic discontinuity or partial loss of function of a tissue. For purposes described herein, focal (localized) lesions are the most concerning, that is, abnormalities that can be visually attributed to local areas, such as lesions located in the oral cavity. As described herein, lesions are associated with hyperproliferative cell divisions, which may be malignant, benign, or premalignant lesions between the two after biopsy analysis, wherein "premalignant" refers to benign lesions that are evolving into malignant lesions. Individuals with lesions may be susceptible or susceptible to diseases (e.g., cancer), and may not yet be diagnosed as having the disease. As described herein, the term "susceptible" refers to an individual's tendency to develop a certain state (e.g., a certain trait, phenotype, or disease), or a tendency to be less able to resist a certain state than an average individual.

[0089] As used herein, the term "biomarker" refers to a gene, the expression level of which is measured using a gene product.

[0090] As used herein, the term "at least" refers to a non-strict inequality that compares two numbers to be unequal. For example, the term "X is at least Y" means that X is greater than or equal to Y, or X is not less than Y.

[0091] The terms "administered," "administering," or "administration" are used interchangeably herein and refer to a means of administering an anti-cancer treatment as described in the present disclosure to a subject in need thereof.

[0092] As used herein, the term "effective amount" refers to an amount that can effectively achieve the desired therapeutic outcome for cancer treatment at a dosage and within the necessary time. For example, in the treatment of cancer, agents that reduce, prevent, delay, inhibit or prevent the expression of the CHEK1 gene or PIK3CA gene (i.e., checkpoint kinase 1 inhibitors or phosphatidylinositol 3-kinase inhibitors) can effectively prevent the spread and / or growth of cancer cells. The effective amount of the agent is not necessarily capable of curing the disease or condition, but will provide treatment for the disease or condition so that the onset of the disease or condition is delayed, hindered or prevented, or the symptoms of the disease or condition are improved. The specific effective amount or sufficient amount will vary with factors such as the following: the specific condition being treated, the patient's physical condition (e.g., the patient's weight, age or sex), the type of mammal or animal being treated, the duration of treatment, the nature of the concurrent therapy (if any), and the specific formulation used, etc. For example, the effective amount can be expressed as the total mass of the active agent (e.g., in grams, milligrams, or micrograms) or the ratio of the mass of the active agent to body weight, such as in milligrams per kilogram (mg / kg). The effective amount can be divided into one, two, or more doses in a suitable form to be administered once, twice, or more times throughout a specified period. Preferably, the effective amount refers to a human equivalent dose (HED), which is the maximum safe dose for a human subject. The HED can be calculated by estimating the maximum safe dose for a human subject in accordance with the industry guidance issued by the U.S. Food and Drug Administration (FDA) (titled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers").

[0093] The term "radiotherapy", also known as radiotherapy, refers to the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated ("target tissue") by damaging the cells' genetic material, thereby preventing these cells from continuing to grow. Radiation usually causes damage to both cancer cells and normal cells, with the latter being more able to resume normal functioning. In some embodiments, radiotherapy is used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, prostate, colon, uterus, and / or cervix. In some embodiments, radiotherapy is also used to treat leukemias and lymphomas.

[0094] As used herein, the term "chemotherapy" refers to the administration of one or more chemotherapeutic agents to a patient in need thereof, with the purpose of reducing, preventing, alleviating, limiting and / or delaying the growth or metastasis of a patient's tumor, or directly killing tumor cells by tumor necrosis or apoptosis or any other mechanism. As used herein, the term "chemotherapeutic agent" refers to any chemical substance known to clinical practitioners of ordinary skill in the art for the treatment or amelioration of cancer and / or as an inducer of apoptosis in patient cells.

[0095] II. Description of the Invention

[0096] The present disclosure is based at least in part on the discovery that genes CHEK1, PIK3CA and / or PIK3CD are differentially expressed between tumor tissue and matched adjacent normal tissue of OSCC patients. Therefore, administering a kinase inhibitor (e.g., a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor) to a patient based on the differentially expressed genes (i.e., CHEK1, PIK3CA and / or PIK3CD) in the patient will improve the therapeutic efficacy of the kinase inhibitor.

[0097] 1. Methods for treating cancer

[0098] Accordingly, the first aspect of the present disclosure is a method for treating cancer in an individual. The method comprises:

[0099] (a) obtaining first and second biological samples from a diseased site and a non-lesioned site of the individual, respectively;

[0100] (b) measuring the expression levels of the biomarkers in the first and second biological samples respectively by qRT-PCR to obtain a first expression level and a second expression level, wherein the biomarker is the CHEK1 gene, the PIK3CA gene or the PIK3CD gene;

[0101] (c) determining the ratio of the first expression level to the second expression level; and

[0102] (d) when the biomarker is the CHEK1 gene and the ratio determined in step (c) is at least 1.7; or when the biomarker is the PIK3CA gene and the ratio determined in step (c) is at least 2.4; or when the biomarker is the PIK3CD gene and the ratio determined in step (c) is at least 3.1, administering to the individual an effective amount of a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor.

[0103] According to preferred embodiments of the present disclosure, the methods of the present invention can be used to treat cancers as described above. Preferably, the methods of the present invention can be used to treat oral cancers, such as tumors of the floor of the mouth, gingival cancer, lip cancer, major salivary gland cancer, oropharyngeal cancer, OSCC, other oral cancers, jaw cancer, and tongue cancer. In some specific embodiments, the cancer treatable by the methods of the present invention is OSCC.

[0104] According to some embodiments of the present disclosure, the individual that can be treated by the method of the present invention is as described above. In some embodiments, the individual that will benefit from the method of the present invention is a mouse. In other embodiments, the individual that is suitable for treatment with the method of the present invention is a human.

[0105] First, paired samples (biological samples collected from the lesion site and non-lesion site of an individual, respectively) are referred to as the first and second biological samples (step (a)). Since the first biological sample is collected from the lesion site, the second biological sample is collected from normal tissue and may be any one of skin epithelium, nasal mucosa, oral mucosa, buccal epithelium, palate epithelium, sublingual epithelium, submucosal layer, rectal epithelium, vaginal epithelium, intrathecal tissue, intramuscular tissue, intravenous tissue, ligament or tendon. Preferably, the second biological sample is the oral mucosa adjacent to the lesion site. According to an alternative embodiment of the present disclosure, the first biological sample and the second biological sample may be from different individuals. For example, the first biological sample is from the lesion site of an individual associated with cancer, while the second biological sample is from the non-lesion site of a healthy donor.

[0106] Then, both the first and second biological samples are subjected to gene expression analysis for specific biomarkers (step (b)). The biomarker to be detected in the method of the present invention (i.e., in step (b)) can be a CHEK1 gene or a PIK3CA gene, or optionally, a PIK3CD. In some embodiments, the respective levels of the CHEK1 gene in the first and second biological samples are determined. In other embodiments, the respective levels of the PIK3CA gene in the first and second biological samples are determined. Alternatively, in other embodiments, the respective levels of the PIK3CD gene in the first and second biological samples are determined. The techniques suitable for evaluating gene expression are well known in the art, including but not limited to: whole genome expression profiles analyzed using expressed sequence tags (ESTs), serial analysis of gene expression (serial analysis of gene expression, SAGE), DNA microarrays, massively parallel signature sequencing (MPSS), RNA sequencing (RNA-seq), qRT-PCR, digital polymerase chain reaction (dPCR), two-dimensional gel electrophoresis (2-D electrophoresis), tissue arrays, immunohistochemistry (IHC) staining, etc. In some embodiments, gene expression is analyzed by RNA-seq. In other embodiments, gene expression is analyzed by qRT-PCR. In other embodiments, IHC staining is used. Preferably, in the methods of the present invention, gene expression is analyzed by qRT-PCR.

[0107] Alternatively or optionally, in order to minimize the individual differences from one individual to another, the level of the biomarker determined can be standardized. As used herein, the term "normalized" (normalization) refers to the standardized level of a gene product when applied to a gene, for example, a standardized value determined for the RNA expression level of a gene or the polypeptide expression level of a gene. Generally, standardization refers to dividing the value of a biomarker in a sample by the value of a housekeeping gene. "Biomarker" (biomarker) is defined as a laboratory measurement that reflects the activity of a disease process, in other words, a biomarker is quantitatively correlated (positive or negative) with the progression of the disease. "Housekeeping gene" (housekeeping gene) refers to a gene that is constitutively expressed at a relatively constant level under many or all known conditions, because it encodes a protein that a cell constantly needs, therefore, it is essential to the cell and is always present in any case. It is assumed that its expression is not affected by the experimental conditions. The protein it encodes is generally involved in the basic functions required for cell nutrition or maintenance. According to embodiments of the present disclosure, the housekeeping gene may be any one of β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hydroxymethylbinder synthase (HMBS), hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1), TATA box binding protein (TBP), or 18S ribosomal RNA (rRNA). In a specific embodiment, the housekeeping gene is TBP.

[0108] After determining the gene expression levels (e.g., CHEK1, PIK3CA or PIK3CD) in the first and second biological samples and optionally normalizing them, the ratio of the first expression level (i.e., the level of the biomarker in the first biological sample) to the second expression level (i.e., the level of the same biomarker in the second biological sample) is then calculated by dividing the first expression level by the second expression level, thereby determining the relative expression of the biomarker (e.g., CHEK1, PIK3CA, PIK3CD or a combination thereof) between the first biological sample and the second biological sample (i.e., the lesion and normal site) (i.e., step (c)).

[0109] Then, based on the respective ratios of the biomarkers determined in step (c), the individual with OSCC is treated. In the method of the present invention, when the ratio of a specific biomarker (e.g., CHEK1, PIK3CA or PIK3CD) is higher than a predetermined threshold, a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor is administered to the individual. For the biomarker CHEK1 gene, the predetermined threshold is 1.7; For the biomarker PIK3CA gene, the predetermined threshold is 2.4; For the biomarker PIK3CD gene, the predetermined threshold is 3.1. Therefore, if the ratio of CHEK1, PIK3CA and PIK3CD genes between the lesion site and the normal site is independently at least 1.7, 2.4 or 3.1, then a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor can be used to treat OSCC individuals.

[0110] Examples of CHK1 inhibitors suitable for use in the methods of the present invention include, but are not limited to:

[0111] (S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762);

[0112] 4-(((3S)-1-azabicyclo(2.2.2)oct-3-yl)amino)-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR-124);

[0113] Labuseti (LY2603618);

[0114] Presativa (LY2606368);

[0115] 4-(2,6-Dichlorophenyl)-9-hydroxy-6-(3-(methylamino)propyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (PD-321852);

[0116] (R)-2-amino-2-cyclohexyl-N-(2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-5,6-dihydro-1H-[1,2]diazepino[4,5,6-cd]indol-8-yl)acetamide (PF477736);

[0117] (R)-6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-3-yl)pyrazolo[1,5-a]pyrimidin-7-amine (SCH900776);

[0118] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinamide;

[0119] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-ylisobutyramide;

[0120] (R)-N-(5-bromo-4-(3-(methylamino)piperidin-1-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)nicotinamide;

[0121] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-methylnicotinamide;

[0122] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)cyclopropanecarboxamide;

[0123] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-methylbutanamide; or

[0124] (R)-N-(4-(3-aminopiperidin-1-yl)-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-cyclopropylacetamide.

[0125] In some embodiments, the CHK1 inhibitor used in the methods of the invention is (S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide;

[0126] Preseti; and (R)-2-amino-2-cyclohexyl-N-(2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-5,6-dihydro-1H-[1,2]diazepino[4,5,6-cd]indol-8-yl)acetamide.

[0127] Examples of PI3K inhibitors suitable for use in the methods of the present invention include, but are not limited to:

[0128] (2S)-N1-[5-(2-tert-butyl-4-thiazolyl)-4-methyl-2-thiazolyl]pyrrolidine-1,2-dicarboxamide (A66);

[0129] (Z)-5-((5-(4-fluoro-2-hydroxyphenyl)furan-2-yl)methylene)thiazolidine-2,4-dione (AS-252424);

[0130] 5-(2,2-Difluoro-benzo[1,3]dioxolan-5-ylmethylene)-thiazolidine-2,4-dione (AS-604850);

[0131] (R)-2-(1-(7-methyl-2-morpholinyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)ethylamino)benzoic acid (AZD6482);

[0132] 2-amino-N-[7-methoxy-8-(3-morpholinopropoxy)-2,3-dihydroimidazo[1,2-c]quinazoline (BAY80-6945);

[0133] Kupanisi (BAY 80-6946);

[0134] 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one maleate (BGT226);

[0135] Bupalise (BKM120);

[0136] Apelicet (BYL719);

[0137] (5E)-5-{[5-(4-fluorophenyl)furan-2-yl]methylene}-1,3-thiazolidine-2,4-dione (CAY10505);

[0138] 5-(2-amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide (CZC24832);

[0139] Duvelice (IPI-145);

[0140] 2-((6-amino-9H-purin-9-yl)methyl)-5-methyl-3-o-tolylquinazolin-4(3H)-one (IC-87114);

[0141] Idelarice (GS-1101, CAL-101);

[0142] Serelise (INK1117);

[0143] Tasilicept (GDC-0032);

[0144] Pitilicept (GDC-0941);

[0145] Apilicet (GDC-0980);

[0146] (Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene)thiazolidine-2,4-dione (GSK1059615);

[0147] Omibalisai (GSK2126458);

[0148] 2-Methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6-morpholinyl-1H-benzo[d]imidazole-4-carboxylic acid (GSK2636771);

[0149] 6-[5-[(Benzenesulfonyl)amino]-3-pyridinyl]-imidazo[1,2-a]pyridine-3-carboxylic acid, ethyl ester (HS-173);

[0150] 2-amino-N,N-dimethyl-5-(3-(2-methylpyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridine-3-sulfonamide (HS-527);

[0151] 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002);

[0152] Datolisei (NVP-BEZ235);

[0153] Perifosine;

[0154] 2-amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7-one (PF-04691502);

[0155] Gidalise (PF-05212384);

[0156] 3-(4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol (PI-103);

[0157] 2-Methyl-5-nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75);

[0158] N-(2,3-dihydro-7,8-dimethoxyimidazo[1,2-c]quinazolin-5-yl)-3-pyridinecarboxamide (PIK-90);

[0159] 2-[(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (PIK-293);

[0160] 2-[[4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (PIK-294);

[0161] 1-(4-(3-ethyl-7-morpholinyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl)-3-(4-(4-methylpiperazine-1-carbonyl)phenyl)urea (PKI-402);

[0162] Sonolis (PX-866);

[0163] (S)-2-(2-(2-methylindolin-1-yl)-2-oxoethyl)-6-morpholinopyrimidin-4(3H)-one (SAR260301);

[0164] Piralise (SAR245408);

[0165] (2S)-2-[[(2S)-3-carboxy-2-[[2-[[(2S)-5-(diaminomethyleneamino)-2-[[4-oxo-4-[[4-(4-oxo-8-phenylchromen-2-yl)morpholin-4-ium-4-yl]methoxy]butanoyl]amino]pentanoyl]amino]acetyl]amino]propanoyl]amino]-3-hydroxypropanoate (SF1126);

[0166] 3-(2,4-Diaminopteridin-6-yl)phenol (TG100713);

[0167] Upalis (TGR-1202);

[0168] 5-(9-isopropyl-8-methyl-2-morpholinyl-9H-purin-6-yl)pyrimidin-2-amine (VS-5584);

[0169] Vodalise (XL765); and

[0170] 4,4'-(6-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (ZSTK474).

[0171] In some embodiments, used in the methods of the present invention is apellisep;

[0172] Pitilicept; and

[0173] (Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene)thiazolidine-2,4-dione.

[0174] According to some embodiments of the present disclosure, when the ratio of the CHEK1 gene between the lesion and the normal part is at least 1.7, the OSCC individual is treated with a CHK1 inhibitor. In the case where the ratio of the CHEK1 gene is less than 1.7, the OSCC individual is treated with treatment other than the CHK1 inhibitor (e.g., surgery, radiotherapy, chemotherapy, immunotherapy, or these therapies). Alternatively or in addition, when the ratio of the PIK3CA gene or the PIK3CD gene between the lesion and the normal part is at least 2.4 or 3.1, the OSCC individual is treated with a PI3K inhibitor. In the case where the ratio of the PIK3CA gene or the PIK3CD gene is less than 2.4 or 3.1, the OSCC individual is treated with treatment other than the PI3K inhibitor. In some embodiments, the OSCC individual with a ratio of at least 1.7 of CHEK1 alone can show a good response to the combination therapy of CHK1 inhibitors (e.g., PF477736) plus chemotherapeutic agents (e.g., cisplatin). In other embodiments, OSCC subjects with a CHEK1 ratio of at least 1.7 and a PIK3CA and / or PIK3CD ratio of at least 2.4 and / or 3.1 may respond favorably to combination therapy with a CHK1 inhibitor (eg, PF477736 or AZD7762) plus a PI3K inhibitor (eg, BYL719 or GDC-0941).

[0175] Alternatively or optionally, the method of the present invention further comprises administering to the individual an additional anti-cancer therapy, such as surgery, radiotherapy, chemotherapy, immunotherapy, or a combination of these therapies, before, simultaneously or after the above-mentioned treatment step (i.e., step (d)). Combination therapy refers to a combination of at least two of the anti-cancer therapies listed above, such as a combination of surgery and radiotherapy, a combination of surgery and chemotherapy, etc.

[0176] The chemotherapy is carried out by administering a chemotherapeutic agent to a subject in need thereof to alleviate or improve cancer symptoms. Examples of chemotherapeutic agents suitable for use in the present method include, but are not limited to, alkylating agents, platinum drugs, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and / or enzyme inhibitors. Exemplary alkylating agents include, but are not limited to, Chlormethine, Ifosfamide, Trofosfamide, Melphalan, Prednimustine, Bendamustine, Uramustine, Carmustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, Mannosulfan, Treosulfan, Carboquone, Thiotepa, Triaziquone, and Triethylenemelamine. Examples of platinum drugs suitable for use in the methods of the present invention are Carboplatin, Cisplatin, Dicycloplatin, Nedaplatin, Oxaliplatin or Satraplatin.

[0177] In some embodiments, the chemotherapeutic agent used in the methods of the present invention is an antimetabolite, for example, Aminopterin, Hydroxyurea, Methotrexate, Pemetrexed, Pralatrexate, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Nelarabine, Tioguanine, Mercaptopurine, Fluorouracil, Capecitabine, Doxifluridine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, Azacitidine, Decitabine or Hydroxycarbamide. The antitumor antibiotic can be Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Actinomycin, Bleomycin, Dactinomycin, Mitomycin or Plicamycin. Likewise, the topoisomerase inhibitor can be Camptothecin, Cositecan, Belotecan, Gimatecan, Exatecan, Irinotecan, Lurtotecan, Silecan, Topotecan, Rubitecan, Etoposide or Teniposide.

[0178] Additionally, mitotic inhibitors suitable for use in the methods of the invention are Vinblastine, Vincristine, Vinflunine, Vindesine, Vinorelbine, Cabazitaxel, Docetaxel, Larotaxel, Ortataxel, Paclitaxel, Tesetaxel, or Ixabepilone. In addition, the enzyme inhibitor can be Tipifarnib, Abemaciclib, Alvocidib, Palbociclib, Ribociclib, Seliciclib, Bortezomib, Carfilzomib, Ixazomib, Anagrelide, Tiazofurin, Masoprocol, Niraparib, Olaparib, Rucaparib, Belinostat, Panobinostat, Romidepsin, Vorinostat or Idelagliside.

[0179] According to some embodiments of the present disclosure, the chemotherapeutic agent used herein may be bleomycin, carboplatin, capecitabine, cisplatin, docetaxel, 5-FU, hydroxyurea, methotrexate and paclitaxel. In a working example of the present disclosure, the chemotherapeutic agent is cisplatin.

[0180] Immunotherapy refers to killing cancer cells by one's own immune system. More specifically, by activating the patient's own immune system, it attacks the malignant cells that cause the disease. In some embodiments, Immunotherapy is related to the use of immune checkpoint inhibitors as immunotherapeutic agents to treat cancer. Exemplary immunotherapeutic agents include, but are not limited to, Pembrolizumab, Nivolumab, Cemiplimab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, Nivolumab, Atezolizumab, Avelumab, Durvalumab and Ipilimumab.

[0181] The effective amount of a therapeutic agent (e.g., a CHK1 inhibitor, a PI3K inhibitor, a chemotherapeutic agent, etc.) in the methods of the invention may vary depending on many factors, such as the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of the treatment, the nature of concurrent treatments (if any), the particular route of administration, and like factors that are within the knowledge and expertise of the health care practitioner.

[0182]

[00136] To induce a therapeutic effect in humans, the therapeutic agent (e.g., a CHK1 inhibitor, a PI3K inhibitor, a chemotherapeutic agent, etc.) in the methods of the invention is administered at a dosage of about 10 μg / kg to 50 mg / kg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 μg / kg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, Preferably, the therapeutic agent in the method of the present invention is administered to an individual at a dose of about 50 μg / kg to 10 mg / kg body weight; more preferably, the therapeutic agent in the method of the present invention is administered to an individual at a dose of about 0.5 mg / kg to 5 mg / kg body weight. According to one working embodiment, the therapeutic agent is the chemotherapeutic agent cisplatin, and the dose is about 0.5 mg / kg body weight to cause a therapeutic effect (e.g., reduce the volume of the tumor) in the individual. According to another working embodiment, the therapeutic agent is the CHK1 inhibitor PF477736, and the dose is about 1 or 2 mg / kg body weight to cause a therapeutic effect in the individual. According to another working example, the therapeutic agent is the PI3K inhibitor BYL719, and the dosage is about 2.5 or 5 mg / kg body weight to induce a therapeutic effect in the individual.

[0183] In the present method of the invention, an effective amount of a therapeutic agent (e.g., a CHK1 inhibitor, a PI3K inhibitor, a chemotherapeutic agent, etc.) can be administered as a single dose (e.g., a single intraperitoneal injection dose) or multiple doses (e.g., multiple intraperitoneal injection doses). In certain embodiments, when multiple doses are administered to an individual, the frequency of administering the multiple doses to the individual is three doses a day, two doses a day, one dose a day, five doses a week, four doses a week, three doses a week, two doses a week, one dose a week, every other Monday, one dose a month, or every other month. In certain embodiments, the frequency of administering multiple doses to an individual is five doses a week. In certain embodiments, the frequency of administering multiple doses to an individual is four doses a week. In certain embodiments, the frequency of administering multiple doses to an individual is two doses a week.

[0184] In certain embodiments, when multiple doses are administered to an individual, the duration between the first dose and the last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the individual. In certain embodiments, the duration between the first dose and the last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and the last dose of the multiple doses is the lifetime of the individual. In a specific embodiment, the duration between the first dose and the last dose of the multiple doses is four weeks.

[0185] In the methods of the invention, therapeutic agents suitable for use in the methods of the invention can be administered by routes known to those skilled in the art, including oral, intracranial, intraspinal, intrathecal, intramedullary, intracerebral, intracerebroventricular, intravenous, intraarterial, intracardiac, intradermal, subcutaneous, transdermal, intraperitoneal, and intramuscular routes.

[0186] It will be appreciated that the present disclosure also includes a method for predicting the likelihood that an individual with oral cancer will exhibit a beneficial response to a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor. The method comprises:

[0187] (a) measuring the expression level of a biomarker in a first and a second sample obtained from the individual by qRT-PCR, respectively, to obtain a first expression level and a second expression level of the biomarker, wherein the biomarker is a CHEK1 gene, a PIK3CA gene or a PIK3CD gene, and the first and the second samples are cancerous samples and non-cancerous samples, respectively;

[0188] (b) determining the ratio of the first expression level to the second expression level; and

[0189] (c) determining, based on the ratio determined in step (b), a likelihood that the individual will exhibit a beneficial response to a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor,

[0190] in:

[0191] If the biomarker is the CHEK1 gene and the ratio is at least 1.7, the individual may exhibit a beneficial response to a checkpoint kinase 1 inhibitor, or, if the biomarker is the PIK3CA gene and the ratio is at least 2.4; or if the biomarker is the PIK3CD gene and the ratio determined in step (c) is at least 3.1, the individual may exhibit a beneficial response to a phosphatidylinositol 3-kinase inhibitor.

[0192] As can be understood, the present disclosure also includes a kit for detecting that an individual with oral cancer is responsive to a kinase inhibitor. The kit comprises a primer pair of sequence numbers: 1 and 2, sequence numbers: 3 and 4, or sequence numbers: 5 and 6; wherein the primer pair of sequence numbers: 1 and 2 is used to detect the expression level of the CHEK1 gene; the primer pair of sequence numbers: 3 and 4 is used to detect the expression level of the PIK3CA gene; the primer pair of sequence numbers: 5 and 6 is used to detect the expression level of the PIK3CD gene; and the kinase inhibitor is selected from a group consisting of a checkpoint kinase 1 inhibitor or a phosphatidylinositol 3-kinase inhibitor. Alternatively, the kit further comprises a primer pair of sequence numbers: 7 and 8, wherein the primer pair of sequence numbers: 7 and 8 is used to detect the expression level of the TBP gene.

[0193] The following examples are provided to illustrate certain aspects of the present invention and to help those skilled in the art to implement the present invention. These examples are by no means to be considered as limiting the scope of the present invention in any way. Without further elaboration, it is believed that those skilled in the art can make the best use of the present invention according to the description herein. All publications cited herein are incorporated herein by reference in their entirety.

[0194] Example

[0195] Materials and methods

[0196] 1. Patients and Clinical Specimens

[0197] A group of patients was included in this study, and this study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Written informed consent was obtained from all participants before completing sample collection. The group included 32 patients with treatment-naive OSCC, and their clinical characteristics are listed in Table 1.

[0198] Table 1 Clinical characteristics of 32 OSCC patients

[0199]

[0200]

[0201] 2. Cell lines

[0202] In this study, OSCC cell lines including SAS ( 64403 TM), OEC-M1 (CVCL_6782) and OC3 (CVCL_D859). SAS cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 100U / ml penicillin and 100μg / ml streptomycin (Gibco, USA) and 10% fetal bovine serum (FBS; Gibco, USA). OEC-M1 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Thermo Fisher Scientific, USA), and OC3 cells were maintained in DMEM (Gibco, USA) containing keratinocyte serum-free medium (KSFM; Gibco, USA); both were supplemented with 10,000U / ml penicillin and 10,000μg / ml streptomycin (Gibco, USA) and 10% FBS (Gibco, USA). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0203] 3. Drugs, chemicals and antibodies

[0204] The CHK1 inhibitors PF477736 (Sigma-Aldrich, USA), AZD7762 (Selleck Chemicals, Canada), and LY2606368 (MedChem Express, USA); and the PI3K inhibitors GDC-0941 (Cayman Chemical, USA), GSK1059615 (Cayman Chemical, USA), and BYL719 (Novartis Pharma AG, Basel, Switzerland) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) and stored at -20°C. Cisplatin (Sigma-Aldrich, USA) was dissolved in 1× PBS and stored at 4°C. All antibodies used in immunoblotting experiments were: anti-CHK1, anti-p-CHK1-Ser296, anti-AKT, anti-p-AKT-Ser473, anti-Ki-67 (Cell Signaling Technology); anti-GAPDH (Bioworlde, USA); HRP-conjugated goat anti-rabbit IgG (PerkinElmer, USA); and HRP-conjugated goat anti-mouse IgG (PerkinElmer, USA).

[0205] 4. RNA Extraction and qRT-PCR

[0206] Total RNA from paired OSCC tumor tissues and surrounding normal tissues was extracted using TRIzol reagent (Gibco BRL), and the quality and quantity were confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Canada). For qRT-PCR, the first-strand complementary DNA was synthesized from 1 μg of total RNA using random hexamers (GeneDirex, Germany) and SuperScript III RT (Invitrogen, USA), and qRT-PCR analysis was performed using MicroAmp Fast 96-well reaction plates (0.1 mL) (Applied Biosystems, USA). The primers used in qRT-PCR are listed in Table 2. The average cycle threshold (Ct) of each replicate measurement was calculated. In order to determine the normalized value, the 2 -ΔCt Value. ΔCt=Ct Target –Ct TBP .

[0207] Table 2 qRT-PCR primers

[0208]

[0209]

[0210] 5. Cell Viability Assay

[0211] For cell viability assays, approximately 5 × 10 4 Cells were inoculated into each well of a 96-well plate, and treated with a specified concentration of vehicle control or inhibitor in complete medium for 48 hours, followed by MTT viability assay. The absorbance was read at 540nm in a multi-well spectrophotometer (SpectraMax M2; Molecular Devices, USA). All samples were repeated three times. For apoptosis assays, cells were plated in 6-well culture dishes and treated with vehicle control or various inhibitors at specified concentrations for 48 hours. The cells were then collected, washed with PBS, and stained with 2μl FITC-coupled annexin V antibody (Invitrogen, USA) and 50μg / ml propidium iodide (Sigma-Aldrich, USA), followed by flow cytometry analysis (Attune NxT; Invitrogen, USA) and data analysis using FlowJo software (Tree Star, Inc.).

[0212] 6. Immunoblotting

[0213] After treatment, the cells were lysed on ice for 15-30 minutes using RIPA lysis buffer (1% NP-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, 5 mM EDTA (pH 8.0), 10% glycerol, PMSF and 0.2% protease inhibitors), and then centrifuged at 15,000 × g for 20 minutes at 4°C. Equal amounts of protein from each sample were fractionated by SDS-PAGE and then transferred to PVDF membranes, which were incubated with appropriate primary antibodies. Proteins were detected using HRP-coupled secondary antibodies and ECL substrates (PerkinElmer, USA).

[0214] 7. Immunohistochemical Staining

[0215] Immunohistochemical staining was performed using an immunohistochemical stainer (Bond, Leica BioSystems). Tissue sections were recovered using a Bond-max automated immunostainer (Leica BioSystems) and stained with anti-Ki-67 antibody (mouse monoclonal antibody against Ki-67, Cell Signaling). A polymer detection system (Bond PolymerRefine Detection, Leia BioSystems) was used to reduce nonspecific staining. Tissue sections were stained with 3,3'-diaminobenzidine reagent as a chromogen, and hematoxylin was used as a counterstain.

[0216] 8. Animal Experimentation

[0217] NOD / SCID mice and NOD.Cg-Prkdcscid II2rgtm1Wjl / SzJ (NSG) mice were maintained in the animal facility of Chang Gung University. For the SAS cell line-derived xenograft (CDX) animal model, SAS cells (5 × 10 cells / mL) were prepared in serum-free DMEM. 6 100 μl of SAS cell suspension was injected subcutaneously into NOD / SCID mice (6-8 weeks) and tumors were established after 3-4 weeks. Once the xenografts reached 300-500 mm 3, mice were randomly divided into treatment or control groups. PF477736 was used at a dose of 10 or 20 mg / kg and administered intraperitoneally 4 days a week. BYL719 was used at a dose of 25 or 50 mg / kg and administered orally 5 days a week. Cisplatin was used at a dose of 5 mg / kg and administered intraperitoneally 2 days a week. During the treatment process (two to three weeks), tumor size was monitored twice a week and tumor volume was calculated.

[0218] For the OSCC PDX animal model, three newly diagnosed patients were recruited and written consent was obtained according to a protocol approved by the Institutional Review Board of Chang Gung Memorial Hospital. Patient tumor explants were prepared from surgical specimens and transplanted into NSG mice within 2 hours. Briefly, fresh tumor tissues were rinsed in PBS (Gibco, USA) containing antibiotic-antimycotic solution and then cut into small pieces (~1 mm 3 ). 50-100 mg of tumor was implanted subcutaneously in the left flank of each NSG mouse. When the tumor reached 400-500 mm 3 Animals were randomly divided into control and different treatment groups. The treatment methods for each drug were the same as those used in the SAS CDX model described above. For the combination treatment, in the cisplatin and PF477736 combination treatment part, cisplatin was used at a dose of 5 mg / kg, intraperitoneally administered 2 days a week for 1 week; PF477736 was used at a dose of 20 mg / kg, intraperitoneally administered 4 days a week for 1 week; and in the PF477736 and BYL719 combination treatment part, PF477736 was used at a dose of 20 mg / kg, intraperitoneally administered 4 days a week for 1 week; BYL719 was used at a dose of 50 mg / kg, orally administered 5 days a week for 2 weeks. Tumors were measured with calipers, and the volume was calculated as 1 / 2 × length × width 2 A total of three PDX models were evaluated for their responses to monotherapy or combination therapy. All animal experiments were approved by the Chang Gung University Animal Experimental Care Committee.

[0219] 9. Statistical Analysis

[0220] Patient characteristics were stratified by various clinicopathological factors and calculated by chi-square test. All statistical data are expressed as mean ± SEM. Intergroup comparisons were performed by Student's t test or Mann-Whitney U test for both groups. All statistical tests were performed using SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) or Prism. p values ​​< 0.05 were considered statistically significant.

[0221] Example 1 Differentially expressed genes in OSCC

[0222] To understand the genes differentially expressed in OSCC, a comprehensive transcriptome investigation approach, RNA-seq, was deployed to analyze matched tumor tissues and adjacent normal tissues from 32 OSCC patients (data not shown).

[0223] From the data obtained by the aforementioned method, three genes were found to be differentially expressed in OSCC: CHEK1, PIK3CA, and PIK3CD. The expression of these genes in 32 paired OSCC populations was further confirmed by qRT-PCR. Figure 1A As shown in Figure 2, CHEK1 expression was increased in OSCC compared with surrounding normal tissues. Similar results were found for PIK3CA and PIK3CD ( Figure 1B-1C The expression changes of genes in each paired OSCC sample were determined, and the thresholds for defining high expression were set, with the thresholds for CHEK1 being 1.7, PIK3CA being 2.4, and PIK3CD being 3.1 ( FIG1D ).

[0224] Taken together, these findings indicate that CHEK1, PIK3CA, and PIK3CD are significantly upregulated in OSCC patients.

[0225] Example 2 Effects of inhibiting CHK1 and PI3K on OSCC in vivo and in vitro

[0226] 2.1 Effect of CHK1 inhibition on OSCC in vitro

[0227] To evaluate the feasibility of inhibiting CHK1 and PI3K as an anti-tumor approach, several small molecule inhibitors of CHK1 and PI3K were tested in this example. Figure 2A Results are provided for the cell viability of specific CHK1 inhibitors (including PF477736, AZD7762, and LY2606368) on OSCC cell lines (including SAS and OEC-M1). All tested CHK1 inhibitors showed dose-dependent inhibitory effects on SAS and OEC-M1 cells ( Figure 2A The half maximal inhibitory concentration (IC 50 ) ranged from 120 to 400 nM, while the IC 50 The concentration was 10 μM ( Figure 2A In addition, the colony-forming ability of SAS and OEC-M1 cells was examined after treatment with PF477736, and the results showed that PF477736 significantly impaired the colony-forming ability of both cells ( Figure 2BIn addition, PF477736 significantly aggravated apoptosis in three OSCC cells including SAS, OC3 and OEC-M1 cells, as assessed by Annexin V and PI staining ( Figure 2C At the molecular level, the biochemical effects of three CHK1 inhibitors in SAS cells were characterized. DNA damage was first induced by UV irradiation, and then CHK1 inhibitors were applied to the damaged cells. Afterwards, phosphorylation of CHK1 at Ser296 and total CHK1 were analyzed by immunoblotting ( Figure 2D The results showed that CHK1 inhibitors (i.e., PF477736, AZD7762, and LY2606368) were able to specifically reduce the phosphorylation of CHK1 after UV irradiation, indicating that CHK1 activity was severely inhibited after CHK1 inhibitor treatment.

[0228] To evaluate the combination therapy with CHK1 inhibitors and current standard chemotherapy in the treatment of OSCC, PF477736 and cisplatin were selected as examples, as the CHK1 inhibitor part and the chemotherapy part of the combination therapy in this study, respectively. First, the IC of cisplatin for the treatment of three OSCC cells (SAS, OC3 and OEC-M1 cells) was determined. 50 , ranging from 2.5 to 5 μM ( Figure 2E ). Next, the efficacy of the combination therapy was evaluated by examining the ability to inhibit cell proliferation. Figure 2F As shown, the combination treatment of PF477736 and cisplatin synergistically impeded the proliferation of SAS and OC3 cells compared with control or single-agent treatment.

[0229] Collectively, these results demonstrate that CHK1 inhibitors, alone or in combination with chemotherapy (eg, cisplatin), effectively slowed cell proliferation, drove cell cycle arrest, and triggered cell death in OSCC.

[0230] 2.2 Effect of PI3K inhibition on OSCC in vitro

[0231] In this study, the efficacy of PI3K inhibitors (e.g., BYL719, GDC-0941, and GSK1059615) in eliminating OSCC cell lines (including SAS, OEC-M1, and OC3) was examined. First, cell proliferation assays after treatment with PI3K inhibitors (BYL719, GDC-0941, and GSK1059615) were performed, and the results are shown in Figure 3A According to the results, SAS and OEC-M1 cells treated with PI3K inhibitors showed a significant decrease in proliferation compared to those treated with vehicle control ( Figure 3A). IC of BYL719 in SAS, OC3 and OECM1 cell lines 50 The range is 8 to 20 μM ( Figure 3A ). Another indicator used to measure anticancer activity was to study the colony formation ability of SAS and OEC-M1 cells treated with BYL719. Figure 3B As shown in Figure 2, the colony-forming ability of the treated cells showed a dose-dependent impairment. Regarding apoptosis, OSCC cells treated with BYL719 showed a gradual increase in apoptosis with increasing BYL719 concentrations ( Figure 3C ). The effects of PI3K inhibitors on the PI3K / AKT / mTOR signaling pathway were also studied. Figure 3D , PI3K inhibitors (i.e., BYL719, GDC-0941, and GSK1059615) specifically inhibited the phosphorylation of AKT after UV irradiation, thereby attenuating the PI3K / AKT / mTOR signaling pathway after treatment.

[0232] In this study, the efficacy of a PI3K inhibitor combined with a CHK1 inhibitor as a combination therapy for treating cancer was evaluated. As an example, in the following experiments, PF477736 and BYL719 were used as a CHK1 inhibitor and a PI3K inhibitor, respectively. The results of cell proliferation after administration of the combination therapy are provided in Figure 3E In the study, it was shown that the combination treatment synergistically blocked proliferation in SAS cells compared with control or single-drug treatment. In addition, the combination treatment significantly increased the number of apoptotic cells in SAS cells ( Figure 3F Similar results were found in the use of combination therapy to inhibit cell proliferation, with the CHK1 inhibitor AZD7762 and the PI3K inhibitor GDC-0941 ( Figure 3G ).

[0233] Together, these findings suggest that PI3K inhibitors have promising anticancer activity and that combination therapy using PI3K and CHK1 inhibitors could help treat cancer.

[0234] 2.3 Effects of combination therapy on OSCC in vivo

[0235] The data described above have prompted the inventors to further investigate the antitumor activity of CHK1 and PI3K inhibitors alone or in combination in in vivo preclinical models of OSCC.

[0236] As a proof-of-concept purpose, SAS cells were used to prepare an in vivo model of OSCC (hereinafter referred to as OSCC CDX). The details of the preparation of OSCC CDX are described in “Materials and Methods”. Treatment with PF477736 at a concentration of 10 or 20 mg / kg barely inhibited tumor growth compared with the control group (Figure 4A, 10 mg / kg vs. control, p = 0.083; 20 mg / kg vs. control, p = 0.081). For BYL719, treatment at a dose of 50 mg / kg significantly inhibited tumor growth compared with the control group ( Figure 4B , 25 mg / kg vs. control, p=0.508; 50 mg / kg vs. control, p=0.011). In addition, co-treatment with PF477736 (20 mg / kg) and BYL719 (50 mg / kg) caused effective impairment of tumor growth ( Figure 4C , combination therapy vs. control, p<0.01). The data showed that the combination of PF477736 and BYL719 showed an enhanced therapeutic effect; the tumor volume in the combination treatment group was much smaller. Compared with the current standard chemotherapy cisplatin ( Figure 4D Combination treatment with PF477736 and BYL719 demonstrated superior efficacy in compromising tumor growth in the OSCC CDX model compared to 5 mg / kg vs. control, p=0.03).

[0237] Example 3 Effects of inhibiting CHK1 and PI3K on OSCC PDX model

[0238] In the more clinically relevant model OSCC PDX model, CHK1 and PI3K inhibitors were further evaluated as a means of controlling tumor growth. Tumor tissues from three stage IV OSCC patients (designated as patient #1, patient #2, and patient #3, respectively) were used to establish PDX models. Table 3 summarizes the clinical data of these three patients. The genetic characteristics of the three patients were characterized by RNA-seq and whole exome sequencing (data not shown), wherein patient #2 had TP53 and CDKN2A mutations, and patient #3 had TP53 mutations and PIK3CA mutations (data not shown). Similarly, it was noted that patient #2 and patient #3 had a significant increase in components in the PI3K / AKT / mTOR signaling pathway (data not shown). The gene expression of PIK3CA in the three patients was also confirmed by qRT-PCR, and the ratio of PIK3CA expression was higher than 2.4 in patient #2 and patient #3, respectively, but not in patient #1 (data not shown). After the OSCC PDX model was established, the mice were divided into a vehicle control group, a cisplatin group (5 mg / kg), a CHK1 inhibitor PF477736 group (20 mg / kg), and a cisplatin plus PF477736 group ( Figure 5A ). The results showed that either cisplatin or PF477736 monotherapy partially (but not significantly) inhibited the growth of PDX tumors. In contrast, the combination therapy showed synergistic inhibition of tumor growth in all PDX mouse models ( Figure 5A , combination treatment vs. control, p=0.0024 in patient #1 PDX, p=0.006 in patient #2 PDX, p=0.03 in patient #3 PDX).

[0239] Results of IHC staining of xenograft tumor sections for Ki-67, an indicator of cell proliferation, are provided in Figure 5B Only the combination therapy showed a significant decrease in the number of Ki-67 positive cells ( Figure 5B ).

[0240] Patient number #1 #2 #3 Age (years) 56 47 60 gender M M M T-stage 4A 4A 2 Nth phase 0 2B 2B pathology good medium medium Overall period IV IV IV Drinking Y Y Y Chewing betel nut Y Y Y smoke Y N Y Location Buccal mucosa tongue Buccal mucosa

[0241] The effect of combined treatment with CHK1 and PI3K inhibitors on the treatment of different PDX models was investigated as follows ( Figure 5C BYL719 (50 mg / kg) monotherapy and PF477736 (20 mg / kg) plus BYL719 (50 mg / kg) combination therapy failed to inhibit tumor growth in patient #1 PDX model ( Figure 5C, upper figure; BYL719 monotherapy vs. control p = 0.66; combination therapy vs. control p = 0.844). Meanwhile, BYL719 (50 mg / kg) monotherapy slightly inhibited tumor growth (50 mg / kg vs. control, p = 0.16), while BYL719 (50 mg / kg) plus PF477736 (20 mg / kg) combination therapy synergistically slowed tumor growth in patient #2 PDX ( Figure 5C , lower panel, combination therapy vs. control, p=0.036). PDXs of patient #2 with a specific genetic profile (higher CHEK1 and PIK3CA expression) were more responsive to the BYL719 plus PF477736 combination therapy. Figure 5D As shown in Figure 2, Ki-67 positive cells were significantly reduced in the BYL719 plus PF477736 combination treatment, indicating reduced cancer cell proliferation under this treatment. In addition, no significant weight loss was observed in any treatment group in the patient #2 PDX model ( Figure 5E ).

[0242] In summary, the data provided in this disclosure show that CHK1 inhibitors alone or in combination with chemotherapy (e.g., cisplatin) may be more effective in treating cancer patients with higher expression of CHEK1 alone. In addition, PI3K inhibitors plus CHK1 inhibitors can be used as potential anticancer treatment methods for treating cancer patients with high expression of CHEK1 and PIK3CA / PIK3CD. Patients with specific genetic characteristics as described herein can benefit particularly from personalized anticancer treatments as described in this disclosure.

[0243] It should be understood that the description of the above embodiments is given by way of example only, and various modifications may be made by those of ordinary skill in the art. The above description, examples and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present invention have been described above with a certain degree of particularity or with reference to one or more separate embodiments, those of ordinary skill in the art may make various changes to the disclosed embodiments without departing from the spirit or scope of the present invention. Sequence Listing <110> Chang Gung University Chang Gung Memorial Hospital Linkou <120> Treatments for oral cancer <130> P4100-CN <160> 8 <170> BiSSAP 1.3.6 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 1 tcggtataat aatcgtgagc g 21 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 2 acaggaccaa acatcaactg 20 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 3 acgatggaca actgtttca 19 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 4 gtctttgtgc attcttggg 19 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 5 gacatccagt atctcaagga c 21 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 6 agccagttca ctttggtt 18 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 7 tgctcacccc accaacaatt tag 23 <210> 8 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic sequence <400> 8 ctgggtttga tcattctgta gattaa 26

Claims

1. Use of a primer pair in the preparation of a kit for detecting the expression level of a biomarker in an individual suffering from oral squamous cell carcinoma, characterized in that: The kit is composed of primer pairs with sequence numbers: 1 and 2, sequence numbers: 3 and 4, and sequence numbers: 5 and 6; the primer pair with sequence numbers: 1 and 2 is used to detect the expression level of the CHEK1 gene of the individual; the primer pair with sequence numbers: 3 and 4 is used to detect the expression level of the PIK3CA gene of the individual; the primer pair with sequence numbers: 5 and 6 is used to detect the expression level of the PIK3CD gene of the individual.

2. The use according to claim 1, characterized in that The individual is a human.

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