Novel biomarker combinations for diagnosis, prognosis, stratification and / or therapy monitoring of cancer diseases

Abstract

The present invention relates to a novel method for the diagnosis, prognosis, stratification and / or therapy monitoring of cancer diseases in patients. The method is based on the determination of CD3+ tumor infiltrating lymphocyte levels and CD24 expression. The novel biomarker combination of the present invention allows the diagnosis, prognosis, stratification and / or therapy monitoring of various cancer diseases. Furthermore, diagnostic kits for performing the non-invasive method of the present invention are provided.

Description

Technical Field

[0001] This invention relates to a novel method for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer in patients. The method is based on the determination of CD3+ tumor-infiltrating lymphocyte levels and CD24 expression. The novel combination of biomarkers of this invention allows for the diagnosis, prognosis, stratification, and / or therapy monitoring of various cancers. Furthermore, diagnostic kits for performing the methods of this invention are also provided. Background Technology

[0002] Cancer remains a leading cause of morbidity and mortality worldwide, with millions of new cases diagnosed each year. According to the latest statistics, the global cancer incidence rate is estimated at over 18 million new cases annually. Among the various types of cancer, the most common include lung cancer, breast cancer, colorectal cancer, prostate cancer, and stomach cancer. Tumor staging systems, such as the TNM staging system, are widely used to assess the extent and progression of cancer. However, while classic tumor staging is extremely useful in assessing the extent and progression of cancer, it has limitations, such as limited accuracy in predicting patient outcomes and guiding personalized treatment strategies. Biomarkers play a crucial role in overcoming these limitations by providing more precise and personalized information about the biological behavior of tumors. They can help with early detection, monitoring treatment response, and identifying potential therapeutic targets, thereby enhancing the overall effectiveness of cancer management.

[0003] Therefore, the identification of specific and sensitive biomarkers suitable for effective diagnosis, prognosis, and treatment is highly relevant to many aspects of cancer-related research. Even though the number of potential biomarkers has increased significantly over the past few decades, only a small fraction have entered clinical validation, and even fewer have been used as reliable therapeutic targets or diagnostic markers.

[0004] Nasopharyngeal carcinoma (NPC) is a cancer originating in the nasopharynx. Although relatively uncommon, it still poses significant health problems, particularly in certain regions. NPC differs from other types of head and neck cancers in that EBV plays a crucial role in the disease's biology. NPC is more common in southeastern China, parts of the Middle East, and North Africa, with the majority of patients having locally advanced disease. Locally advanced nasopharyngeal carcinoma (LA-NPC), defined as stage III or IV NPC, is the most common form of NPC in Saudi Arabia. While most patients are successfully managed with concurrent radio-chemotherapy (CCRT), some relapse, and some eventually develop metastatic disease and die. Unfortunately, prognostic biomarkers are lacking to identify patients who may relapse, especially given the limited use of traditional TNM staging commonly used in cancer.

[0005] Therefore, there is a need for novel biomarkers that allow for reliable diagnosis, prognosis, stratification, and / or therapy monitoring of cancer. Furthermore, there is a need for new methods for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer in subjects. Additionally, there is a need for new methods for assessing treatment success in patients with cancer undergoing treatment. Furthermore, there is a need for a diagnostic kit for performing the aforementioned methods. Summary of the Invention

[0006] The elements of the invention will be described below. These elements are set forth along with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the invention to the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine two or more explicitly described embodiments or combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described in this application should be considered as disclosed in the description of this application.

[0007] In a first aspect, the present invention relates to a method for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer in subjects, comprising the following steps: (a) Providing biological samples from the subject; and (b) Determine the levels of at least two biomarkers in a biological sample, wherein at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; The levels of at least two biomarkers in biological samples from the subject that differ from those of healthy controls or reference values, as determined in step (b), indicate the presence of cancer in the subject.

[0008] In a preferred embodiment, the method is an ex vivo and / or in vitro and / or non-invasive and / or minimally invasive method.

[0009] In one embodiment, the biological sample is a sample of the subject, comprising tissue samples or body fluid samples, such as blood samples, serum samples, plasma samples, samples of a group of cells from a tumor, tumor tissue, urine samples, lymph samples, pleural fluid samples, or cerebrospinal fluid samples; wherein, preferably, the biological sample is a tissue sample.

[0010] In a preferred embodiment, the biological sample is a paraffin-embedded tissue sample, preferably a formalin-fixed paraffin-embedded tissue sample.

[0011] In one implementation, the levels of at least two biomarkers are assessed by their protein biomarkers, wherein determining the level of each of the at least two biomarkers involves determining the protein levels of the at least two biomarkers in a biological sample.

[0012] In one embodiment, the method further includes determining the level of one or more additional biomarkers in the biological sample, wherein the one or more additional biomarkers are selected from CD44 and markers for epithelial-mesenchymal transition, such as vimentin, E-cadherin, N-cadherin or β-catenin; preferably selected from CD44 and vimentin; even more preferably CD44.

[0013] In one implementation, the levels of one or more additional biomarkers in biological samples from the subject that differ from healthy controls or reference values ​​can further indicate the presence of invasive cancer in the subject.

[0014] In one implementation, if it is determined that the level in the biological sample leads to CD44+ high Phenotypic identification can further indicate aggressive cancer disease in subjects (i.e., cancer disease that does not respond to standard treatment and / or progresses faster than the typical progression of this type of disease).

[0015] In one implementation, if the level of vimentin in a biological sample is determined to cause... high Phenotypic identification can further indicate aggressive cancer disease in subjects (i.e., cancer disease that does not respond to standard treatment and / or progresses faster than the typical progression of this type of disease).

[0016] In one implementation, if the level of E-cadherin in the biological sample is determined to cause E-cadherin... low Phenotypic identification can further indicate aggressive cancer disease in subjects (i.e., cancer disease that does not respond to standard treatment and / or progresses faster than the typical progression of this type of disease).

[0017] In one implementation, if the determination of levels in a biological sample leads to the identification of a phenotype with upregulated β-catenin expression, that phenotype can further indicate an aggressive cancer disease in the subject (i.e., a cancer disease that does not respond to standard treatment and / or progresses faster than that type typically progresses).

[0018] In one implementation, if the determination of levels in a biological sample leads to the identification of a phenotype with upregulated N-cadherin expression, that phenotype can further indicate an aggressive cancer disease in the subject (i.e., a cancer disease that does not respond to standard treatment and / or progresses faster than that type typically progresses).

[0019] In one implementation, the method is a screening method for establishing a preliminary diagnosis of cancer in subjects.

[0020] In one implementation, if the levels of at least two biomarkers in a biological sample are determined to cause CD24+ high / CD3+TIL low The phenotype indicates that the subject is at high risk of disease progression.

[0021] In one implementation, when a subject is diagnosed as having a high risk of disease progression, the patient's treatment plan is modified; wherein, optionally, the modification of the treatment plan includes one or more of the following: changing the medication, optionally changing it to an investigational agent, and intensifying treatment, such as by increasing the treatment dose and / or adjusting the dosing regimen, to improve the patient's survival.

[0022] In one implementation, adjusting the dosing regimen includes one or more of the following: increasing the frequency of the drug, increasing the total dose of the drug, and / or increasing the duration of treatment.

[0023] In one implementation, modifying the drug includes one or more of the following: using a combination of one or more (other) therapeutic agents, using a more aggressive therapeutic agent, and / or using an investigational therapeutic agent.

[0024] In one embodiment, the cancer is selected from the group comprising nasopharyngeal carcinoma, head and neck cancer, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colorectal cancer, gastric cancer, lymphoma, including non-Hodgkin lymphoma such as Burkitt lymphoma, glioma, melanoma, small cell and non-small cell lung cancer, bile duct cancer, renal cell carcinoma, bladder cancer, uterine cancer, epithelial ovarian cancer, prostate cancer, and pancreatic cancer; wherein, preferably, the cancer is nasopharyngeal carcinoma, head and neck cancer, or hepatocellular carcinoma; even more preferably, the cancer is nasopharyngeal carcinoma; most preferably, the cancer is locally advanced nasopharyngeal carcinoma.

[0025] In one embodiment, one or more binding agents or antigen-binding peptides, such as antibodies, are used to detect biomarkers, wherein, preferably, the biomarkers are detected by immunohistochemistry, Western blotting, ELISA, proximity extension assay, or mass spectrometry; wherein, more preferably, the biomarkers are detected by immunohistochemistry.

[0026] In one implementation, immunohistochemistry is used to detect one or more of at least two biomarkers via a method comprising the following steps: (i) Provide formalin-fixed paraffin-embedded (FFPE) tissue samples derived from the subject; (ii) Dewaxing the FFPE tissue samples in xylene and rehydrating them using an ethanol / water gradient; (iii) Antigen retrieval was performed using an antigen retrieval solution in a Decloaking Chamber pressure cooker; (iv) Optionally, quench endogenous peroxidase and / or block endogenous biotin; (v) Incubate the sample with one or more primary antibodies that are specific to at least two biomarkers, preferably overnight at about 2-10°C, more preferably overnight at about 4°C, wherein the one or more primary antibodies are diluted in 1% BSA. (vi) Incubate the sample with one or more secondary antibodies that are specific to any one of the one or more primary antibodies, wherein the one or more secondary antibodies are conjugated to a reporter enzyme; wherein, optionally, if more than one secondary antibody is used, the incubation of the sample with one or more secondary antibodies may be performed simultaneously or sequentially. (vii) Detect the reporter enzyme conjugated with the secondary antibody, wherein, optionally, if more than one primary antibody and / or more than one secondary antibody are used, the detection of the secondary antibody may be performed simultaneously or sequentially.

[0027] In one embodiment, the method further includes one or more washing steps using a washing buffer such as TBST between steps (iv) and (v); and / or between steps (v) and (vi); and / or between steps (vi) and (vii); and / or between different secondary antibody detections in step (vii); and / or The antigen retrieval solution in step (iii) is Tris-EDTA with a pH of approximately 9; and / or The antigen retrieval in step (iii) is carried out at a temperature of about 90°C to about 150°C, preferably about 110°C to about 130°C, more preferably about 121°C, for about 1 min to about 15 min, preferably about 5 min to about 10 min, more preferably about 7 min.

[0028] In one implementation, the secondary antibody is diluted in 1% BSA.

[0029] In one embodiment, step (vi) is performed at room temperature for about 10 min to about 2 h, preferably about 15 min to about 45 min, more preferably about 20 min to about 30 min, and most preferably about 30 min.

[0030] In one embodiment, the conjugated reporter enzyme is selected from the group consisting of alkaline phosphatase (AP), horseradish peroxidase (HRP), β-galactosidase, glucose oxidase, and acid phosphatase; preferably, the conjugated reporter enzyme is alkaline phosphatase (AP) or horseradish peroxidase (HRP).

[0031] In a preferred embodiment, antigen expression is assessed using an H-score system, preferably CD24 expression.

[0032] In one embodiment, CD24 is detected using a binding agent or antigen-binding peptide that specifically binds to CD24, wherein CD24 contains at least 90% identity with the amino acids of SEQ ID NO:1, more preferably at least 95% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, optionally the same amino acid sequence or composed of therein; Preferably, the binder or antigen-binding peptide binds to the sequence of positions 46-48 according to SEQ ID NO:1.

[0033] In one embodiment, the binding agent or antigen-binding peptide that specifically binds to CD24 is selected from the group comprising anti-CD24 monoclonal antibody clone: ​​ML5, anti-CD24 monoclonal antibody clone: ​​M1 / 69, anti-CD24 monoclonal antibody clone: ​​SN3b, anti-CD24 monoclonal antibody clone: ​​SN3, anti-CD24 monoclonal antibody clone: ​​SWA11 and anti-CD24 monoclonal antibody clone: ​​ALB9; preferably selected from anti-CD24 monoclonal antibody clone: ​​ML5 and anti-CD24 monoclonal antibody clone: ​​M1 / 69; and even more preferably anti-CD24 monoclonal antibody clone: ​​ML5.

[0034] In one implementation, the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human, preferably a human.

[0035] In one aspect, the present invention relates to a method for evaluating the treatment success of a patient with cancer undergoing cancer treatment, comprising the following steps: (a) Providing biological samples from the subjects; (b) Determine the levels of at least two biomarkers in the biological sample, wherein the at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; and (c) Compare the levels of the at least two biomarkers identified in (b) with a reference sample or reference value. The decrease or increase in the levels of at least two biomarkers in the biological sample from the subject, compared to the reference sample or reference value, indicates the patient's response to the treatment.

[0036] In a preferred embodiment, the method is an ex vivo and / or in vitro and / or non-invasive and / or minimally invasive method.

[0037] In one implementation, the reference value corresponds to the levels of at least two biomarkers in a biological sample obtained from the patient prior to receiving the treatment.

[0038] In one implementation, high CD24 levels and / or low CD3+ TIL levels indicate an inadequate response to the treatment.

[0039] In one implementation, the patient is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human, preferably a human.

[0040] In one embodiment, the cancer is selected from the group consisting of nasopharyngeal carcinoma, head and neck cancer, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colorectal cancer, gastric cancer, lymphoma, including non-Hodgkin lymphoma such as Burkitt lymphoma, glioma, melanoma, small cell and non-small cell lung cancer, bile duct cancer, renal cell carcinoma, bladder cancer, uterine cancer, epithelial ovarian cancer, prostate cancer, and pancreatic cancer; wherein, preferably, the cancer is nasopharyngeal carcinoma, head and neck cancer, or hepatocellular carcinoma; even more preferably, the cancer is nasopharyngeal carcinoma; most preferably, the cancer is locally advanced nasopharyngeal carcinoma.

[0041] In one embodiment, one or more binding agents or antigen-binding peptides, such as antibodies, are used to detect biomarkers, preferably by immunohistochemistry, Western blotting, ELISA, ortho-extension assay, or mass spectrometry; more preferably, biomarkers are detected by immunohistochemistry.

[0042] In one implementation, immunohistochemistry is used to detect one or more of at least two biomarkers via a method comprising the following steps: (i) Provide formalin-fixed paraffin-embedded (FFPE) tissue samples derived from the subject; (ii) Dewaxing the FFPE tissue samples in xylene and rehydrating them using an ethanol / water gradient; (iii) Antigen retrieval was performed using an antigen retrieval solution in a Decloaking Chamber pressure cooker; (iv) Optionally, quench endogenous peroxidase and / or block endogenous biotin; (v) Incubate the sample with one or more primary antibodies that are specific to one or more of the at least two biomarkers, preferably overnight at about 2-10°C, more preferably overnight at about 4°C, wherein, more preferably, the one or more primary antibodies are diluted in 1% BSA; (vi) Incubate the sample with one or more secondary antibodies that are specific to any one of the one or more primary antibodies, wherein the one or more secondary antibodies are conjugated to a reporter enzyme; wherein, optionally, if more than one secondary antibody is used, the incubation of the sample with one or more secondary antibodies may be performed simultaneously or sequentially. (vii) Detect the reporter enzyme conjugated with the secondary antibody, wherein, optionally, if more than one primary antibody and / or more than one secondary antibody are used, the detection of the secondary antibody may be performed simultaneously or sequentially.

[0043] In one embodiment, the method further includes one or more washing steps using a washing buffer such as TBST between steps (iv) and (v); and / or between steps (v) and (vi); and / or between steps (vi) and (vii); and / or between different secondary antibody detections in step (vii); and / or The antigen retrieval solution in step (iii) is Tris-EDTA with a pH of approximately 9; and / or The antigen retrieval in step (iii) is carried out at a temperature of about 90°C to about 150°C, preferably about 110°C to about 130°C, more preferably about 121°C, for about 1 min to about 15 min, preferably about 5 min to about 10 min, more preferably about 7 min.

[0044] In one implementation, the secondary antibody is diluted in 1% BSA.

[0045] In one embodiment, step (vi) is performed at room temperature for about 10 min to about 2 h, preferably about 15 min to about 45 min, more preferably about 20 min to about 30 min, and most preferably about 30 min.

[0046] In one embodiment, the conjugated reporter enzyme is selected from the group consisting of alkaline phosphatase (AP), horseradish peroxidase (HRP), β-galactosidase, glucose oxidase, and acid phosphatase; preferably, the conjugated reporter enzyme is alkaline phosphatase (AP) or horseradish peroxidase (HRP).

[0047] In a preferred embodiment, antigen expression is assessed using an H-score system, preferably CD24 expression.

[0048] In one embodiment, CD24 is detected using a binding agent or antigen-binding peptide that specifically binds to CD24, wherein CD24 contains at least 90% identity with the amino acids of SEQ ID NO:1, more preferably at least 95% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, optionally the same amino acid sequence or composed of therein; Preferably, the binder or antigen-binding peptide binds to the sequence of positions 46-48 according to SEQ ID NO:1.

[0049] In one embodiment, the binding agent or antigen-binding peptide that specifically binds to CD24 is selected from the group comprising anti-CD24 monoclonal antibody clone: ​​ML5, anti-CD24 monoclonal antibody clone: ​​M1 / 69, anti-CD24 monoclonal antibody clone: ​​SN3b, anti-CD24 monoclonal antibody clone: ​​SN3, anti-CD24 monoclonal antibody clone: ​​SWA11 and anti-CD24 monoclonal antibody clone: ​​ALB9; preferably selected from anti-CD24 monoclonal antibody clone: ​​ML5 and anti-CD24 monoclonal antibody clone: ​​M1 / 69; and even more preferably anti-CD24 monoclonal antibody clone: ​​ML5.

[0050] In one implementation, the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human, preferably a human.

[0051] In one aspect, the present invention relates to a diagnostic kit for performing the methods as defined herein, the kit comprising a combination of at least two antibodies, derivatives thereof, or antigen fragments for detecting either CD24 or CD3.

[0052] In one embodiment, the kit further comprises additional components selected from the group consisting of: a visualization system; an blocking agent; a chromogenic solution; a washing buffer; instructions for appropriate operating parameters; a secondary antibody optionally conjugated to a reporter enzyme, wherein the secondary antibody is specific to at least one of the at least two antibodies; an antigen retrieval solution; an assay kit for detecting the conjugated reporter enzyme; and standard or control information such that the test sample can be compared with a standard control information.

[0053] In one aspect, the present invention relates to a method for the diagnosis, prognosis, stratification and / or therapy monitoring of cancer in a subject, comprising determining the levels of at least two biomarkers in a biological sample obtained from the subject, wherein the at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; The levels of at least two biomarkers in biological samples from subjects that differ from healthy controls or reference values ​​indicate the presence of cancer in the subjects.

[0054] In this respect, cancer, subjects, biomarkers, and the determination of said biomarkers are as defined herein.

[0055] In one aspect, the present invention relates to a method for evaluating the treatment success of a patient with cancer undergoing cancer treatment, comprising the following steps: (a) Determine the levels of at least two biomarkers in biological samples obtained from subjects, wherein at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; and (b) Compare the levels of the at least two biomarkers identified with a reference sample or reference value. The decrease or increase in the levels of at least two biomarkers in the biological sample from the subject, compared to the reference sample or reference value, indicates the patient's response to the treatment.

[0056] In this respect, cancer, subjects, biomarkers, and the determination of said biomarkers are as defined herein.

[0057] In one aspect, the present invention relates to CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells in methods for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer in subjects. Such uses typically involve determining the levels of at least CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells in a biological sample from a subject.

[0058] In this respect, cancer and subjects are as defined in this article.

[0059] In one aspect, the present invention relates to a method for detecting an antigen (preferably CD24) in a biological sample derived from a subject, the method comprising the following steps: (i) Provide formalin-fixed paraffin-embedded (FFPE) tissue samples derived from the subject; (ii) Dewaxing the FFPE tissue samples in xylene and rehydrating them using an ethanol / water gradient; (iii) Antigen retrieval was performed using an antigen retrieval solution in a Decloaking Chamber pressure cooker; (iv) Optionally, quench endogenous peroxidase and / or block endogenous biotin; (v) Incubate the sample with one or more primary antibodies that are specific to at least two biomarkers, preferably overnight at about 2-10°C, more preferably overnight at about 4°C, wherein the one or more primary antibodies are diluted in 1% BSA. (vi) Incubate the sample with one or more secondary antibodies that are specific to any one of the primary antibodies, wherein the one or more secondary antibodies are conjugated to a reporter enzyme; wherein, optionally, if more than one secondary antibody is used, the incubation of the sample with one or more secondary antibodies may be performed simultaneously or sequentially. (vii) Detect the reporter enzyme conjugated with the secondary antibody, wherein, optionally, if more than one primary antibody and / or more than one secondary antibody are used, the detection of the secondary antibody may be performed simultaneously or sequentially.

[0060] In one embodiment, the method further includes one or more washing steps using a washing buffer such as TBST between steps (iv) and (v); and / or between steps (v) and (vi); and / or between steps (vi) and (vii); and / or between different secondary antibody detections in step (vii); and / or The antigen retrieval solution in step (iii) is Tris-EDTA with a pH of approximately 9; and / or The antigen retrieval in step (iii) is carried out at a temperature of about 90°C to about 150°C, preferably about 110°C to about 130°C, more preferably about 121°C, for about 1 min to about 15 min, preferably about 5 min to about 10 min, more preferably about 7 min.

[0061] In one implementation, the secondary antibody is diluted in 1% BSA.

[0062] In one embodiment, step (vi) is performed at room temperature for about 10 min to about 2 h, preferably about 15 min to about 45 min, more preferably about 20 min to about 30 min, and most preferably about 30 min.

[0063] In one embodiment, the conjugated reporter enzyme is selected from the group consisting of alkaline phosphatase (AP), horseradish peroxidase (HRP), β-galactosidase, glucose oxidase, and acid phosphatase; preferably, the conjugated reporter enzyme is alkaline phosphatase (AP) or horseradish peroxidase (HRP).

[0064] In a preferred embodiment, antigen expression is assessed using an H-score system, preferably CD24 expression.

[0065] In one embodiment, CD24 is detected using a binding agent or antigen-binding peptide that specifically binds to CD24, wherein CD24 contains at least 90% identity with the amino acids of SEQ ID NO:1, more preferably at least 95% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, optionally the same amino acid sequence or composed of therein; Preferably, the binder or antigen-binding peptide binds to the sequence of positions 46-48 according to SEQ ID NO:1.

[0066] In one embodiment, the binding agent or antigen-binding peptide that specifically binds to CD24 is selected from the group comprising anti-CD24 monoclonal antibody clone: ​​ML5, anti-CD24 monoclonal antibody clone: ​​M1 / 69, anti-CD24 monoclonal antibody clone: ​​SN3b, anti-CD24 monoclonal antibody clone: ​​SN3, anti-CD24 monoclonal antibody clone: ​​SWA11 and anti-CD24 monoclonal antibody clone: ​​ALB9; preferably selected from anti-CD24 monoclonal antibody clone: ​​ML5 and anti-CD24 monoclonal antibody clone: ​​M1 / 69; and even more preferably anti-CD24 monoclonal antibody clone: ​​ML5. Attached Figure Description

[0067] The invention will now be further described with reference to the following figures.

[0068] All methods mentioned in the following figures are performed as described in detail in the embodiments.

[0069] Figure 1 ML5 anti-CD24 was validated using positive and negative controls.

[0070] (A) Representative images of formalin-fixed paraffin-embedded (FFPE) sections of breast cancer cell lines, (bottom) positive for CD24 and negative for CD44 (MCF-7 and SK-BR-3), or (top) negative for CD24 and positive for CD44 (MDA-MB-231 and Hs578T). Images were examined under an optical microscope at 400x magnification.

[0071] (B) Flow cytometry results using the same controls as in A for CD44 / CD24 staining, used for comparison between techniques.

[0072] (C) Representative images (400x) of CD24 immunohistochemistry using M1 / 69 antibody in formalin-fixed paraffin-embedded (FFPE) tissue sections of MCF-7 and SK-BR-3 cells (positive control) and MDA-MB-231 (negative control). Sections were counterstained with hematoxylin.

[0073] Figure 2 Immunohistochemistry was specifically validated using anti-CD24 ML5 antibody.

[0074] Representative images (400x) of CD24 immunohistochemistry using ML5 antibody in formalin-fixed paraffin-embedded (FFPE) tissue sections of SKBR3 cells after CD24 knockdown with specific siRNA (Si-CD24) are compared with scrambled control siRNA (Si-Neg) used to verify antibody specificity. Sections were counterstained with hematoxylin.

[0075] Figure 3 CD24 expression in breast tissue.

[0076] (A) CD24 expression is heterogeneous in breast cancer, exhibiting varying staining intensities. Representative images show high (+3), intermediate (+2), and low (+1) staining intensities of CD24.

[0077] (B) CD24 is expressed in the luminal epithelial cells of normal ducts / lobules and is overexpressed during ductal proliferation. Representative images of formalin-fixed paraffin-embedded (FFPE) sections of normal ducts / lobules immunostained against CD44 and CD24. Black arrows indicate CD24 staining in the apical side of luminal cells, while gray arrows indicate CD44 staining in myoepithelial / basal cells.

[0078] Figure 4 The impact of CD24 expression on the prognosis of breast cancer patients.

[0079] Kaplan-Meier survival curves show disease-free survival (DFS) in CD24-based breast cancer patients. Statistical significance was determined using the log-rank test.

[0080] Figure 5 Representative images of immunohistochemical sections.

[0081] Representative images of nuclear BMI1 (A), membrane CD44 (B), major cytoplasmic ALDH1 alone or with CD44 (C), and major cytoplasmic CD24 alone or with CD44 (membrane) (D). White arrows indicate cancer cells, while black arrows indicate immune-infiltrating cells.

[0082] Figure 6 The prognostic potential of CSC markers in LA-NPC.

[0083] Kaplan-Meier survival curves show disease-free survival (DFS) (A), metastasis-free survival (MFS) (B), or overall survival (OS) (C) in LA-NPC patients associated with CSC biomarkers. Statistical significance was calculated using the log-rank test.

[0084] Figure 7 In NPC, the expression of ALDH1 and CD24 is heterogeneous, with different staining intensities.

[0085] Representative images of high (+3), medium (+2), and low (+1) staining intensities of ALDH1 (A) or CD24 (B).

[0086] Figure 8Based on their H-scores, the prognostic potential of ALDH1 and CD24 CSC markers in LA-NPC.

[0087] Kaplan-Meier survival curves show disease-free survival (DFS), metastasis-free survival (MFS), or overall survival (OS) in LA-NPC patients associated with their H-score-based ALDH1 (bottom panel) or CD24 (top panel) CSC markers. Statistical significance was calculated using the log-rank test.

[0088] Figure 9 In LA-NPC, CD3+TIL provides a predictive ability.

[0089] (A) Representative images of high CD3+TIL (scores 4 and 3) and low CD3+TIL (scores 2 and 1). (B) Kaplan-Meier survival curves showing the effect of CD3+TIL on survival (DFS, MFS, and OS). Statistical significance was calculated using the log-rank test.

[0090] Figure 10 The prognostic potential of the combination of CD3+TIL and tumor CD24 expression in LA-NPC.

[0091] Kaplan-Meier survival curves show the impact of different combinations of CD3+ TILs and tumor CD24 (A) or tumor CD24 / CD44 (B) on disease-free survival (DFS), metastasis-free survival (MFS), or overall survival (OS). Statistical significance was calculated using the log-rank test. Detailed Implementation

[0092] As used herein, the term “subject” refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human, preferably a human.

[0093] As used herein, the term “patient” refers to mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, monkeys, or humans, preferably humans.

[0094] In a particularly preferred embodiment, the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human, preferably a human, such as a human patient, more preferably a human patient with a disease (such as cancer) and in need of treatment, even more preferably a human patient with nasopharyngeal carcinoma and in need of treatment, and most preferably a human patient with locally advanced nasopharyngeal carcinoma and in need of treatment.

[0095] As used herein, the terms “[this] invention”, “according to the present invention”, etc., are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0096] As used herein, the term “comprising” should be interpreted to encompass both “including” and “consisting of”, both meanings being specifically contemplated and therefore specific to the individual disclosures of the invention. When used herein, “and / or” should be considered a specific disclosure of each of the two specified features or components in the presence or absence of the other. For example, “A and / or B” would be considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were set forth separately herein. In the context of this invention, the terms “about” and “approximately” indicate, as those skilled in the art will understand, a range of precision in the technical effect of the features under discussion. This term typically indicates a deviation from the indicated value of ±20%, ±15%, ±10%, and, for example, ±5%. As will be understood by those skilled in the art, such a particular deviation for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can often have a larger such deviation than man-made or engineered effects. When referring to a singular noun (e.g., “one,” “a kind,” or “the”), the use of an indefinite or definite article includes the plural form of that noun, unless otherwise specified.

[0097] As used herein, the term "CD24" refers to a cell surface protein involved in cell adhesion and signal transduction. CD24 may also be referred to as "signal sensor CD24" or "thermally stable antigen CD24." CD24 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of various cell types, including hematopoietic cells, neurons, and epithelial cells. It plays a role in regulating immune responses and cell proliferation. As used herein, the term "CD24 positive" refers to cells that express the CD24 protein on their surface. These cells can be identified and characterized based on the presence of CD24, which can be detected using specific antibodies in techniques such as immunohistochemistry or flow cytometry. CD24 levels are considered high (also known as "CD24+") when CD24 is expressed in more than 10% of cells (such as cancer cells), preferably more than 20% of cells (such as cancer cells), and more preferably more than 30% of cells (such as cancer cells). high "or CD24+" hi CD24 is expressed in the membrane, cytoplasm, and / or nucleus of cells such as cancer cells.

[0098] In one implementation, the terms "CD24 level," "CD24+," etc., refer to the level of CD24 in the cell membrane.

[0099] In one implementation, the terms "CD24 level," "CD24+," etc., refer to the level of CD24 in the cytoplasm.

[0100] In one implementation, the terms "CD24 level," "CD24+," etc., refer to the level of CD24 in the cell nucleus.

[0101] As used herein, the term "CD3" refers to a protein complex and cell surface marker expressed on T cells. CD3 is composed of multiple subunits and is an essential component of the T cell receptor (TCR) complex, playing a crucial role in T cell activation, signal transduction, and immune responses. CD3 is commonly used as a marker to identify and study T cells in various immunoassays and studies. The terms "CD3+ cell" or "CD3-positive cell," etc., refer to cells that express the CD3 protein complex.

[0102] As used in this article, the term "tumor-infiltrating lymphocytes," also known as "TILs," refers to a class of immune cells, particularly lymphocytes, that have migrated from the bloodstream into a tumor. These cells are part of the body's immune response and can include various subgroups such as T cells, B cells, and natural killer (NK) cells. Tumor-infiltrating lymphocytes are typically analyzed to understand the immune environment within the tumor and their role in cancer progression and response to treatment.

[0103] As used herein, the term "CD3+ TIL" refers to tumor-infiltrating lymphocytes (TILs) that express the CD3 marker on their surface. CD3+ TILs are a subset of immune cells, particularly T cells, that have migrated into the tumor microenvironment and participate in the body's immune response against cancer cells. In this application, a CD3+ TIL score of 1 or 2 in a tumor section is considered to indicate a low level of CD3+ TILs (also referred to as "CD3+ TIL"). low The CD3+TIL score 1 is defined as CD3+TILs accounting for approximately 0% to approximately 10% of the tumor slice, and the CD3+TIL score 2 is defined as CD3+TILs accounting for more than approximately 10% to approximately 40% of the tumor slice.

[0104] As used herein, the terms “diagnosis” or “diagnostic” or similar expressions refer to the identification of the presence or absence of a pathological condition in a subject, or the identification of the nature of the pathological condition. The sensitivity and specificity of diagnostic methods differ. The “sensitivity” of a diagnostic assay is a measure of the percentage of diseased individuals who test positive (the “true positive” percentage). Diseased individuals who are not detected by the assay are “false negatives.” Subjects who are not diseased and test negative are called “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of non-diseased individuals who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0105] As used herein, the term "prognosis" refers to the prediction of the likely outcome of a disease, and the prospect of recovery, based on the nature of the case and symptom indications. Therefore, a negative or poor prognosis is defined by a lower duration of treatment-associated survival or survival rate. Conversely, a positive or good prognosis is defined by an increased duration of treatment-associated survival or survival rate. Typically, prognosis is provided in terms of progression-free survival, metastasis-free survival, and / or overall survival time.

[0106] As used herein, the term "stratification" refers to dividing a patient population into subgroups based on specified criteria. More specifically, it refers to dividing a group of subjects or patients into at least two groups based on specific criteria, which, in the context of this invention, include or constitute the levels of groups of biomarkers of the invention as determined. The methods according to the invention provide possible decisions regarding patient treatment and therapy, whether it be patient hospitalization, the use of one or more drugs, efficacy and / or dosage, treatment measures or monitoring of disease progression, and the duration or etiology or classification of disease, for example, into new or existing subtypes or distinctions between diseases and their patients. In particular, with respect to nasopharyngeal carcinoma, "stratification" herein means classifying nasopharyngeal carcinoma into early nasopharyngeal carcinoma, locally advanced nasopharyngeal carcinoma, or locally advanced nasopharyngeal carcinoma with a negative or poor prognosis.

[0107] As used in this article, the term "therapeutic monitoring" refers to observing the disease progression of a subject receiving a therapy (such as a cancer therapy). In other words, it involves regularly monitoring the effectiveness of the therapy administered to a subject during treatment. This allows healthcare practitioners to assess the effectiveness of the prescribed treatment early in the course of treatment and, consequently, continue, adjust, or terminate the treatment regimen.

[0108] As used in this article, the term “healthy control” refers to an individual or group of individuals who do not have the disease or condition being studied and is used as a baseline to compare with those individuals who do have the disease or condition.

[0109] As used herein, the term "reference value" refers to a value obtained by measuring a reference sample for comparative purposes in clinical and laboratory settings to interpret test results. In a preferred embodiment, the reference value should correspond to a sample obtained from a healthy individual or a sample obtained from adjacent tissue of a tumor in a subject with a tumor. In some embodiments, the reference value may also be derived from a sample obtained from the same subject at an earlier time point.

[0110] In a preferred embodiment, as used herein, the terms “increase” or “decrease” in biomarker levels, when compared to a control or reference value, refer to a significant increase or decrease, respectively.

[0111] In a preferred embodiment, the methods of the invention disclosed herein are non-invasive, minimally invasive, ex vivo, or in vitro methods. If the diagnostic methods described herein are non-invasive, the term "providing a biological" sample should preferably not be interpreted as including surgical procedures performed on a subject.

[0112] As used herein, the terms "cancer" and "cancer cell" refer to any cell exhibiting uncontrolled growth in the tissues or organs of a multicellular organism. In the context of this invention, particularly preferred cancers are selected from the group comprising nasopharyngeal carcinoma, head and neck cancer, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colorectal cancer, gastric cancer, lymphoma including non-Hodgkin lymphoma (e.g., Burkitt lymphoma), glioma, melanoma, small cell and non-small cell lung cancer, hepatocellular carcinoma, bile duct carcinoma, renal cell carcinoma, bladder cancer, uterine cancer, epithelial ovarian cancer, prostate cancer, and pancreatic cancer; wherein, preferably, the cancer is nasopharyngeal carcinoma, head and neck cancer, or hepatocellular carcinoma; even more preferably, the cancer is nasopharyngeal carcinoma; most preferably, the cancer is locally advanced nasopharyngeal carcinoma.

[0113] As used in this article, the term "nasopharyngeal cancer" (also known as NPC) refers to cancer originating in the nasopharynx. Although less common, it still presents significant health problems, particularly in certain regions. NPC differs from other types of head and neck cancer in that EBV (Epstein-Barr virus) plays a crucial role in the disease's biology, especially in undifferentiated NPC. NPC is more common in southeastern China, parts of the Middle East, and North Africa. As used in this article, the term "locally advanced nasopharyngeal cancer (LA-NPC)" refers to stage III or IV NPC and is the most common form of NPC in Saudi Arabia. While most patients are successfully managed with concurrent chemoradiotherapy (CCRT), some relapse, and some eventually develop metastatic disease and die.

[0114] As used herein, the term "invasive cancer" or "invasive form of cancer" refers to a class of cancers that do not respond to standard treatment and / or progress faster than is typically seen in such diseases. This includes cancers that exhibit rapid growth, a high propensity for metastasis, resistance to multiple lines of therapy, and a tendency to relapse rapidly after treatment. Furthermore, invasive cancers generally have a poor prognosis and may require stronger and more novel treatment options.

[0115] As used herein, the term "high risk of disease progression" refers to a clinical assessment indicating a significantly increased likelihood that an individual will experience disease deterioration or progression. This determination is based on a variety of factors, including but not limited to clinical symptoms, biomarker levels, genetic susceptibility, imaging findings, and other diagnostic criteria. Individuals identified as having a high risk of disease progression may require more intensive surveillance, treatment, or intervention to effectively manage their condition. Cancer subgroups at high risk of disease progression have been shown to be associated with reduced disease-free survival (DSF) and / or metastasis-free survival (MSF) and / or overall survival (OS).

[0116] As used herein, the term "biological sample" refers to a sample obtained and capable of measuring the expression of at least two biomarkers or their genes disclosed in this invention. Biological samples may include bodily fluid samples, such as blood samples, serum samples, plasma samples, samples of a group of cells from a tumor, tumor tissue, urine samples, lymph samples, pleural fluid samples, or cerebrospinal fluid samples, or tissue samples, such as samples from tissue biopsies. In some embodiments, the sample is a tissue sample, such as a tumor tissue sample, and may be fresh, frozen, or archived paraffin-embedded tissue. A tissue sample is preferred for the purposes of this invention.

[0117] As used herein, the term “biomarker” or “marker” refers to an organic biomolecule, particularly a polypeptide, or a cell expressing an organic biomolecule, particularly a polypeptide, that differs in its presence in a sample taken from a subject who has the specific condition, compared to a comparable sample taken from a subject who does not have the specific condition (e.g., a negative diagnosis, a normal or healthy subject, or a non-cancer patient, depending on whether the patient has undergone cancer testing or metastatic cancer testing).

[0118] As described herein, the term “determination of the level” of a biomarker in a sample, control, or reference refers to the quantification of the presence of the biomarker in the test sample. For example, the number or proportion of cells expressing a particular biomolecule can be assessed. Furthermore, the concentration of the biomarker in the sample can be directly quantified by measuring the amount of protein / peptide / polysaccharide present in the test sample. Additionally, the amount of a biomarker can be indirectly quantified by assessing the gene expression of the gene encoding the biomarker, for example, by quantifying the mRNA expression of the protein encoding the corresponding biomarker. This invention should not be limited to any particular method for determining the level of a given biomarker, but should encompass all means that allow for the direct or indirect quantification and / or estimation of the level of the biomarker. Therefore, as used herein, the term “level” is a parameter describing the absolute amount of a biomarker in a given sample; or alternatively, “level” refers to a relative amount, and preferably to the concentration of the biomarker in the test sample, such as mol / L, g / L, g / mol, etc.

[0119] As used herein, the term "paraffin-embedded tissue sample" refers to a biological tissue sample that has been preserved and embedded in paraffin to maintain its structural integrity for histological examination and analysis.

[0120] As used herein, the term "formalin-fixed paraffin-embedded tissue sample" refers to a biological tissue sample preserved by fixing it in formalin and subsequently embedding it in paraffin. This is a common method for preparing tissue samples to maintain their structural integrity for histological examination and analysis.

[0121] As used in this article, the term "CD44" refers to a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 is commonly used as a biomarker in cancer research and is associated with tumor progression and metastasis.

[0122] In this application, a CD44+ level is considered high (also referred to as "CD44+") when CD44 is expressed in ≥70% of the media of cancer cells in a tumor section. high (”).

[0123] As used herein, the term "vimentin" refers to a class of intermediate filament proteins expressed in mesenchymal cells. It is involved in maintaining cell integrity and providing resistance to stress. Vimentin serves as a marker in epithelial-mesenchymal transition (EMT), indicating the acquisition of mesenchymal, fibroblast-like properties by epithelial cells and exhibiting decreased intercellular adhesion and increased motility.

[0124] In this application, a high level of vimentin is defined as vimentin being upregulated in ≥10% of cancer cells in a tumor section (also known as "vimentin"). high (”).

[0125] As used herein, the term "E-cadherin" refers to a protein that, as a cell adhesion molecule, plays a crucial role in maintaining the integrity of the epithelial cell layer and tissue structure. It is a cadherin involved in cell-cell adhesion. E-cadherin serves as a marker in epithelial-mesenchymal transition (EMT), where its downregulation is typically associated with the loss of epithelial characteristics and the acquisition of mesenchymal traits, thereby contributing to increased cell motility and invasiveness.

[0126] In this application, a low level of E-cadherin (also known as "E-cadherin") is defined as the expression of E-cadherin in ≤70% of cancer cells in a tumor section. low (”).

[0127] As used in this article, the term "N-cadherin" refers to a type of cadherin, a type 1 transmembrane protein involved in cell-cell adhesion. N-cadherins are particularly well-known for their role in neural development and are also involved in cancer progression and metastasis.

[0128] As used herein, the term "β-catenin" refers to a protein that is part of the cadherin complex and plays a key role in regulating cell-cell adhesion and gene transcription. β-catenin is also a key component of the Wnt signaling pathway, which plays an important role in embryonic development and cancer.

[0129] As used herein, the term "investigational agent" refers to a substance or compound that is tested in a clinical trial to evaluate its safety, efficacy, or mechanism of action in treating a specific disease or condition. This term encompasses a broad range of potential therapeutic entities, including small molecules, biologics, vaccines, gene therapies, and other novel treatment options. Investigational agents are typically in the experimental stage and have not yet received approval from regulatory agencies such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA) for routine medical use. They undergo rigorous testing across multiple phases of preclinical research and clinical trials to determine their therapeutic potential, optimal dosage, side effect profile, and overall benefit-risk ratio.

[0130] As used herein, the term "enhanced treatment" refers to the process of increasing the intensity, dosage, frequency, or combination of therapeutic interventions to improve the efficacy of a treatment regimen. This may involve adjusting the current treatment plan to achieve better clinical outcomes, address suboptimal responses, or manage disease progression. Enhanced treatment may include adding new therapeutic agents, increasing the dosage of existing medications, shortening the intervals between treatments, or adopting more aggressive treatment strategies. The goal of enhanced treatment is to maximize therapeutic benefits while carefully monitoring and managing any potential increase in side effects or toxicity.

[0131] As used herein, the term "treatment plan" refers to a comprehensive and personalized strategy developed by healthcare professionals to manage a patient's medical condition. This plan outlines specific treatment options, including medications, therapies, lifestyle changes, and other interventions recommended to achieve desired health outcomes. Treatment plans are tailored to the patient's unique needs, taking into account their medical history, current health status, and personal preferences. It serves as a roadmap for both the patient and healthcare provider, detailing treatment goals, timelines for achieving those goals, and criteria for assessing progress. Treatment plans can be adjusted over time based on the patient's response to therapy and any changes in their condition.

[0132] As used herein, the term “TBST” refers to Tris buffered saline with a Tween (such as Tween 20), a type of buffer solution commonly used in biological research.

[0133] As used herein, the term "binding agent" refers to any molecule or molecular complex that has the ability to specifically recognize and bind to a target molecule. This includes, but is not limited to, antibodies, antibody fragments, antibody-like proteins, aptamers, peptides, and small molecules that can bind to antigens, receptors, or other specific targets.

[0134] As used herein, the term "antigen-binding peptide" refers to a peptide that specifically recognizes and binds to an antigen. These antigen-binding peptides can be engineered or naturally occurring and can be used in a variety of applications, including therapeutic agents, diagnostic assays, and research tools, to detect or neutralize specific antigens. The term includes antibodies, antibody fragments, Fab fragments, substantially complete antibodies, chimeric antibodies, bispecific antibodies, F(ab')2 fragments, single-chain Fv fragments, single-domain antibodies, and antibody-like proteins such as engineered ankyrin repeats (DARPins), affinity molecules, and other engineered binding proteins.

[0135] As used herein, the term "antibody" is used interchangeably with the term "immunoglobulin" and refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. The term also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, unglycosylated antibodies, and derivatives described below. Five different types of heavy chains exist, defined as antibody isotypes with different functional activities: IgM, IgD, IgG, IgA, and IgE. Each heavy chain contains a heavy chain variable region (abbreviated herein as V). H The light chain contains a variable region (V1) and a constant region for the heavy chain. Each light chain contains a variable region for the light chain (abbreviated as V1 in this paper). L (region) and light chain constant region. V H and V L The region can be further subdivided into hypervariable regions (called complement-determining regions (CDRs)) and more conserved regions scattered between them (called framework regions (FRs)). The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0136] As used herein, the term "primary antibody" refers to an antibody specifically designed to bind directly to an antigen of interest. These antibodies are used to detect and quantify the presence of specific proteins or other molecules in a sample.

[0137] As used herein, the term "secondary antibody" refers to an antibody bound to a primary antibody. Secondary antibodies are typically conjugated to a reporter molecule (such as an enzyme, fluorophore, or radioisotope) to facilitate the detection, quantification, or localization of the primary antibody-antigen complex. As used herein, the term "secondary antibody conjugated to a reporter enzyme" refers to a secondary antibody chemically linked to an enzyme capable of generating a detectable signal. Detection of the reporter enzyme is knowledgeable in those skilled in the art and includes methods such as colorimetric, chemiluminescent, or fluorescent assays.

[0138] As used herein, the term "antigen retrieval" refers to a set of techniques in immunohistochemistry (IHC) used to unmask or expose antigen sites in formalin-fixed paraffin-embedded tissue sections. This process enhances the binding of a primary antibody to its target antigen, which can be masked during fixation via cross-linking.

[0139] As used herein, the term "antigen retrieval solution" refers to a solution used in the antigen retrieval process in immunohistochemistry (IHC). These solutions typically contain buffers or chemicals, such as citrate buffer, EDTA, or Tris-EDTA, which help break down cross-links formed during tissue fixation, thereby exposing antigen sites for antibody binding.

[0140] As used herein, the term "binding" preferably refers to specific binding. In some embodiments, the term "binding" should be understood as "capable of binding"; therefore, the term "specific to" should be understood as "capable of binding specifically", and the term "specific binding" should be understood as "capable of binding specifically".

[0141] The terms "specific to" and "specific binding," used, for example, in the context of molecule A being specific to molecule B, or molecule A specifically binding to molecule B, or molecule A exhibiting specific binding to molecule B, refer to a situation where molecule A binds to molecule B but not to other unrelated molecules, or where the affinity is significantly reduced. This binding can be measured by conventional methods, such as by competitive ELISA or by measuring affinity (K0) via surface plasmon resonance. D Similarly, a molecule A that is specific to epitope C, or a molecule A that specifically binds to epitope C, or a molecule A that shows specific binding to epitope C, refers to a molecule A that binds to epitope C but does not bind to other unrelated epitopes, or has significantly reduced affinity.

[0142] As used herein, the term "H-score system" refers to a semi-quantitative method for evaluating the staining intensity and distribution of immunohistochemical markers in tissue sections. The H-score is calculated by assessing the percentage of cells stained at different intensity levels (e.g., 0, 1+, 2+, 3+) and then applying a formula to generate a score reflecting the degree and intensity of staining. The percentage of cells within the target region (…) is used as the basis for the H-score. P (0% to 100%) and sum them to calculate the H-score, where P 0 represents the proportion of negative (0) cells. P 1+ It represents the percentage of 1+ cells (weakly stained). P 2+ It represents the percentage of 2+ cells (moderately stained), and P 3+ This represents the percentage of 3+ cells (strongly stained). The H-score can be calculated using the following formula: H-score = (0 × P0) + (1 × P) 1+ )+ (2 × P 2+ ) + (3 × P 3+ ) This scoring system provides numerical values ​​that can be used to compare the expression levels of a specific antigen under different sample or experimental conditions. For biomolecule expression, 100 represents low / normal expression, while 300 is the maximum value. Cutoff values ​​can be used to bisect the data. Technicians will understand how to select cutoff values ​​based on biomolecules and biological samples.

[0143] For example, the H-score system can be used to score CD24 expression in cells such as cancer cells, with 100 representing low / normal expression and 300 representing the highest score. CD24 expression levels in normal cells serve as a baseline, with cells showing similar intensity (+1) and receiving a score of 100. The H-score is a useful tool in cases of expression heterogeneity. To divide the data, a cutoff value such as 140 can be used.

[0144] In a preferred embodiment, the binding agent or antigen-binding peptide that specifically binds to CD24 is an anti-CD24 monoclonal antibody clone: ​​ML5, also known as "ML5 anti-CD24 antibody". ML5 anti-CD24 antibody is an IgG1 antibody that recognizes the leucine-alanine-proline protein core of CD24.

[0145] In a preferred embodiment, the ML5 anti-CD24 antibody is used at a dilution factor (antibody: buffer) in the range of about 1:10 to about 1:1000, preferably in the range of about 1:25 to 1:500, more preferably in the range of about 1:50 to about 1:250, and even more preferably with a dilution factor of about 1:100; wherein, optionally, the buffer contains 1% BSA.

[0146] In one embodiment, the binding agent or antigen-binding peptide that specifically binds to CD3 is selected from the group comprising anti-CD3 monoclonal antibody 2GV6, anti-CD3 monoclonal antibody F7.2.38, anti-CD3 monoclonal antibody LN10 or anti-CD3 monoclonal antibody SP7, preferably anti-CD3 monoclonal antibody 2GV6, or polyclonal anti-CD3 antibodies.

[0147] In one embodiment, the method according to the invention can be used to identify cancer subgroups that are associated with short survival and / or require different therapeutic agents; wherein, preferably, the cancer is nasopharyngeal carcinoma, and even more preferably, locally advanced nasopharyngeal carcinoma.

[0148] In the following description, reference is made to the embodiments, which are intended to illustrate the invention and not to limit it.

[0149] Example The following examples demonstrate how the combination of newly identified low CD3+ TILs and CD24+ cells can be reliably used as an indicator for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer in subjects.

[0150] Example 1 Materials and methods patient This study was conducted under the Declaration of Helsinki and approved by the Research Advisory Council of King Faisal Specialist Hospital and Research Centre (KFSH&RC). Archived formalin-fixed paraffin-embedded (FFPE) tissue samples were obtained from a cohort of breast cancer patients diagnosed with invasive ductal carcinoma (IDC) who were treated between 2006 and 2008. Informed consent was provided by all patients.

[0151] Immunohistochemistry Sections (4 μm) of FFPE breast cancer tissue blocks were dewaxed in xylene and rehydrated using an ethanol / water gradient. Antigen retrieval was performed in a decloaking chamber pressure cooker, as detailed in Table 1. Endogenous peroxidase was quenched with 0.9% H2O2, and endogenous biotin was blocked with biotin and avidin, with three washes in between. Primary antibodies diluted in 1% bovine serum albumin were incubated overnight at 4°C. Two double staining methods were used, with CD44 as the rabbit antibody and CD24 as the mouse antibody. First, biotinylated anti-mouse secondary antibody was administered, followed by streptavidin alkaline phosphatase (both could be used directly), with a wash step in between. Then, Envision anti-rabbit horseradish peroxidase was used, followed by Solid Red (30 min) and 3,3'-diaminobenzidine (10 min). All washes were performed using Tris-buffered saline containing Tween.

[0152] The scoring was limited to IDC cells, ignoring the expression of tumor-associated immune cells, normal ducts / lobules, and ductal carcinoma in situ. Nuclear CD24 expression was not considered in this study.

[0153] Due to expression heterogeneity, the H-score system was used to score CD24 expression in cancer cells, with 100 representing low / normal expression and 300 representing the highest score. CD24 expression levels in normal luminal cells were used as a baseline, with cancer cells showing similar intensity (+1) and receiving a score of 100. Data were binary-coded using 140 as a cutoff value. Similarly, the H-score system was used to score Ep-CAM expression in cancer, with a cutoff value of 200 for Ep-CAM overexpression.

[0154] Table 1. Conditions for manual IHC in FFPE tissues

[0155] *The indicated temperature is SP1, while SP2 is always 95°C for 10 minutes.

[0156] **RTU, available immediately** # 4+ biotinylated goat anti-mouse from Biocare Medical, Cat# GM601H, followed by washing, and 4+ streptavidin-AP from Biocare Medical, Cat# AP605H. ⊥ Obtained from Dako / Agilent Flow cytometry In short, the tissue was digested using collagenase digestion medium. Cells were then washed with DNase, filtered through a 30-micron sieve, and stained with various antibodies, including Ep-CAM-APC, CD49f-PE, CD44 APC-Cy7, CD24 PE-Alexa610, and CD45 Alexa 430. Additionally, 4′,6-diamidinyl-2-phenylindole (DAPI) was added for viability assessment.

[0157] Cells were initially gated based on forward and side scattering to identify cell-like structures. Live cells (DAPI-negative) were then selected for single-cell gating only. Subsequently, CD45-positive (population 5 or p5) or CD45-negative (p4) cells were selected. Epithelial cells were further gated using Ep-CAM (p6) derived from the CD45-negative cell population.

[0158] CD24 hi Cells (MCF-7, SK-BR-3) and CD44 hi Cells (MDA-MB-231 and Hs578T) were also stained with CD44 APC-Cy7, CD24 PE-Alexa 610 or isotype controls.

[0159] result Multiple strategies were used to validate CD24 immunohistochemical staining for anti-CD24 (ML5). To optimize the CD44 / CD24 IHC double staining method in FFPE sections, various antigen retrieval solutions and temperatures were tested. Optimal results characterized by high signal and low background were achieved under the conditions outlined in Table 1.

[0160] To validate CD44 / CD24 dual staining, positive and negative controls were used. SK-BR-3 and MCF-7 breast cancer cell lines were used as CD24-positive and CD44-negative controls, respectively, while MDA-MB-231 and Hs578T breast cancer cell lines were used as CD24-negative / low and CD44-positive controls. Figure 1 A). IHC results ( Figure 1 A) Comparison with flow cytometry results showed high CD44 expression in MDA-MB231 and Hs578T and low expression in MCF-7 and SK-BR-3, while CD24 expression was high in MCF-7 and SK-BR-3 and low in MDA-MB-231 and Hs-578T. Figure 1 B).

[0161] In addition, two additional validation methods were employed in accordance with the recommendations of the Ad Hoc International Working Group for antibody validation. A separate antibody strategy was also used, in which another anti-CD24 antibody (clone M1 / 69) produced results similar to those of the ML5 anti-CD24 antibody in the cell line. Figure 1 C), and the gene strategy involves CD24 knockdown using specific siRNA, resulting in undetectability by ML5 anti-CD24 antibody (C). Figure 2 In summary, genetic and independent antibody strategies confirmed the specificity of ML5 anti-CD24 antibody IHC in FFPE sections.

[0162] Example 2 All methods mentioned in this embodiment are performed as described in Embodiment 1.

[0163] CD24 overexpression is associated with increased cell proliferation and shorter disease-free survival (DFS) in breast cancer tissues. In breast cancer patients, CD24 expression is mainly located in the cytoplasm, with different staining intensities ranging from mild (+) to strong (+++). Figure 3 A). Therefore, the H-score method was used to quantify staining intensity. Mild (+) CD24 intensity in cancer cells is similar to that observed in luminal cells of normal ducts. Figure 3 B). CD24 overexpression was observed in 33.8% of patients and was significantly associated with higher histological grade (p=0.013) and estrogen receptor negativity (p=0.010). Notably, CD24 overexpression was positively correlated with proliferation markers such as Ki-67 positivity (p=0.002) and SKP2 overexpression (p=0.009), and negatively correlated with the expression of the cell cycle inhibitor p21 (p=0.047). Furthermore, CD24 overexpression was found to be associated with shorter DFS (p=0.020). Figure 4 ).

[0164] Example 3 Materials and methods This study is a retrospective, single-center cohort study that tested the impact of different CSC biomarkers on the survival of LA-NPC patients.

[0165] This study was conducted in accordance with the Declaration of Helsinki and the Institutional Review Board (IRB) guidelines of the King Faisal Specialty Hospital and Research Centre (Research Ethics Committee REC and Basic Research Committee BRC), which approved the work under RAC# 2150-013. Informed consent was waived because the study was retrospective and the patients were not individually identifiable.

[0166] Patient selection NPC patients were from a previous trial that tested the effects of low-dose radiotherapy during induction neoadjuvant chemotherapy (INC) and before concurrent chemoradiotherapy. This trial terminated with negative results, indicating that low-dose INC had no significant impact on patient outcomes. Of the 108 enrolled patients, 83 had tissue blocks available for analysis (42 in the control arm and 41 in the experimental arm).

[0167] Immunohistochemistry (IHC) Formalin-fixed paraffin-embedded (FFPE) NPC tissue blocks obtained at the time of diagnosis were used for this study. Dewaxed and rehydrated 4 μm sections were immunostained for CD3+ and vimentin using the fully automated Ventana Benchmark Ultra system as described above, or manually stained for BMI1, CD24, ALDH1, and CD44.

[0168] BMI1 was stained with primary antibody mouse BMI1 (clone F6, 1:300 dilution) and then stained with Envision® anti-mouse secondary antibody.

[0169] CD24 / CD44 and ALDH1 / CD44 double staining Biotin / avidin was blocked from the sections, followed by protein blocking with 10% goat serum. The primary antibody used overnight at 4°C was either mouse anti-CD24 antibody (clone ML5) or mouse anti-ALDH1 antibody (clone 44 / ALDH, BD Biosciences, 1:200 dilution), mixed with rabbit anti-CD44 antibody (Cat# HPA005785, Sigma, St. Louis, USA, 1:500 dilution). The secondary antibody consisted of 4+ goat anti-mouse IgG, followed by 4+ streptavidin alkaline phosphatase (both from Biocaremedical, Cat# GM601H and AP605H, respectively), and then Envision anti-rabbit secondary antibody. A sequential protocol was used for signal visualization, first solid red, then DAB.

[0170] Pathological score CD3+ TILs were scored based on the percentage of tumor tissue area covered by CD3+ TILs. A score of 1 was used when CD3+ TILs comprised 0-10% of the tumor tissue area; a score of 2 was defined as CD3+ TILs comprising more than 10% but less than 40% of the tumor tissue area (both scores 1 and 2 were considered low CD3+ TILs). A score of 3 was used for cases where CD3+ TILs comprised >40% and ≤70% of the tumor tissue area, and a score of 4 was used for cases where CD3+ TILs comprised >70% of the tumor tissue area. (High CD3+ TILs = score 3 or 4). BMI1 intensity was given as a score of 0-3, where 0 was negative, 1 was mild, 2 was moderate, and 3 was strong. BMI1 staining intensity results were further dichotomized, with scores 0 and 1 considered negative, and scores 2 and 3 considered positive. ALDH1 and CD24 were scored using increments of 5-10%, with 10% and 30% used as cutoff values ​​for ALDH1 and CD24, respectively. ALDH1 and CD24 staining are heterogeneous; therefore, H-scores were used for different analyses, with a score of 90% as the cutoff value, as previously reported. Membrane CD44 was scored using 5-10% increments, with ≥70% as the cutoff value. Similarly, ALDH1 / CD44 was scored using 5-10% increments, with 10% as the cutoff value. Likewise, CD24 / CD44 double-positive cancer cells were scored using 5-10% increments, with 10% as the cutoff value.

[0171] Statistical analysis The association between CSC biomarkers and survival outcomes was assessed using a Cox proportional hazards model. Survival plots were generated using the Kaplan-Meier method, and the curves were compared using the log-rank test. Time statistics were performed for surviving or disease-free patients at the last follow-up. Categorical variables were compared using Fisher's exact test. A p-value of 0.05 was considered the significance threshold.

[0172] Example 4 All methods mentioned in this embodiment are performed as described in Embodiment 3.

[0173] Patient characteristics Most patients were male, with large tumors (T3 and T4) and advanced lymph node metastases (N), consistent with LA-NPC (Table 2). The tumors were predominantly non-keratinizing undifferentiated carcinoma, i.e., WHO type III histological subtype. The median follow-up time for LA-NPC patients was 8 years from the time of diagnosis, with 23 cases (28%) experiencing recurrence, including 6 cases of local or regional recurrence and another 17 cases (20%) of systemic recurrence. Of all LA-NPC patients, 12 (14%) eventually died from the disease.

[0174] Table 2. Patient Characteristics

[0175] Among NPC CSC markers, CD24 expression is significantly associated with survival.

[0176] BMI1 is nuclearly localized, and its intensity in tumor cells varies between "low" (i.e., negative / low (+) intensity) and "high" (i.e., intermediate (++) / high (+++) intensity) (white arrows). Figure 5 In contrast, BMI1 intensity remained relatively stable in immune cells and was therefore used as an internal control (black arrow). High BMI1 expression was present in 65% of cases, and it was not associated with DFS, MFS, or OS. Figure 6 ).

[0177] CD44 is membrane-localized and is overexpressed in cancer cells in 34% of cases. It is also overexpressed in immune-infiltrating cells. High tumor CD44 expression (>70% of cells) was significantly associated with disease-free survival (DFS) (p=0.007) and disease-free survival (MFS) (p=0.011). CD44 expression was not significantly associated with overall survival (OS).

[0178] ALDH1 is primarily localized in the cytoplasm and is overexpressed in tumor cells in 76% of cases. Figure 5 In addition, it is expressed by some histiocytes / macrophages (data not shown). There is no significant correlation between ALDH1 expression in tumor cells and patient survival. Figure 6 Some cases have cancer cells that are positive for both ALDH1 and CD44 (ALDH1 / CD44). Cancer cells with combined ALDH1 and CD44 expression are present in 50% of NPC cases. There is no correlation between ALDH1 / CD44 expression and survival.

[0179] ALDH1 staining intensity is heterogeneous. Figure 7 A). Therefore, the H-score was used for further scoring of ALDH1. ALDH1 staining assessed by the H-score was not correlated with DFS, MFS, or OS (A). Figure 8 ).

[0180] CD24 was overexpressed in 50% of NPC cases. Significant correlations were found between CD24 and DFS (p<0.001), MFS (p<0.001), and OS (p=0.005). Figure 6 Similarly, NPC cases with combined CD24 and CD44 expression accounted for 38% of NPC cases, and this CD24 / CD44 expression was significantly associated with DFS (p<0.001), MFS (p=0.005), and OS (p=0.029).

[0181] CD24 staining intensity is also heterogeneous. Figure 7 B). Therefore, the H-score was used to further score CD24. CD24 staining, as assessed by the H-score, was significantly associated with DFS (p<0.001), MFS (p=0.002), and OS (p=0.001). Figure 8 ).

[0182] In summary, among the CSC biomarkers tested, CD24 overexpression, alone or in combination with CD44, was significantly associated with shorter DFS, MFS, and OS, whether scored with 5-10% increments or using H-scores.

[0183] Example 5 All methods mentioned in this embodiment are performed as described in Embodiment 3.

[0184] The expression of EMT markers (vitrin) is associated with DFS.

[0185] Epithelial-mesenchymal transition (EMT) is associated with the development of cancer cells (CSCs). The prognostic potential of vimentin in LA-NPCs was analyzed. Vimentin was expressed in 31% of NPC cases (data not shown). Furthermore, vimentin was highly expressed in immune-infiltrating cells (data not shown). Vimentin expression in cancer cells was significantly associated with disease-free survival (DFS), but its association with disease-free survival (MFS) and overall survival (OS) was not statistically significant (data not shown).

[0186] Low CD3+ TILs are associated with shorter survival.

[0187] The prognostic potential of the immune prognostic marker CD3+ TIL was tested. Low CD3+ TIL (CD3+ TIL score 1 and 2) was compared with high CD3+ TIL (CD3+ TIL score 3 and 4). Figure 9 A) Significantly correlated with DFS (p<0.001), MFS (p=0.001), and OS (p=0.010) Figure 9 B).

[0188] Example 6 CD24 expression is an independent prognostic biomarker in LA-NPC. Cox regression analysis was used to examine whether the tested CSC biomarkers (including CD24) depended on other factors. Initially, univariate Cox regression analysis was applied to each CSC biomarker and vimentin. Among the tested CSC biomarkers, CD44, CD24, and the CD44 / CD24 combination were significantly associated with DFS (Table 3), while only CD24 and CD44 / CD24 were significantly associated with OS. Vimentin was significantly associated with DFS (p=0.021) but not with OS. As previously mentioned, low CD3+ TILs were significantly associated with shorter DFS and OS. As previously mentioned, the trial arm (with different targets unrelated to this study) had no significant effect on the prognosis of NPC patients.

[0189] Multivariate Cox regression analysis was performed to identify independent prognostic factors associated with survival in LA-NPC patients. In the multivariate analysis, only CD3+ TIL and CD24 were significantly associated with disease-free survival (DFS) (Table 4). CD24 was significantly associated with overall survival (OS), while CD3+ TIL was borderline. In conclusion, CD24 expression and CD3+ TIL were the only independent prognostic factors significantly associated with survival in the multivariate analysis.

[0190] Table 3. Univariate Cox proportional hazards regression analysis of different clinicopathological features and biomarkers with disease-free survival (DFS) and overall survival (OS) in 83 patients with LA-NPC.

[0191]

[0192] Table 4. Multivariate Cox proportional hazards regression analysis of different CSC markers with disease-free survival (DFS) and overall survival (OS) in 83 patients with LA-NPC.

[0193]

[0194] Since CD3+ TIL and CD24 tumor expression are the only two independent prognostic factors associated with DFS in multivariate analysis, these factors were combined and their combined correlation was tested. Low CD3+ TIL with high tumor CD24 expression occurred in the LA-NPC subgroup, which had the shortest DFS, MFS, and OS. Figure 10 A). This also applies to patients with low CD3+ TILs and high CD44 / CD24 expression, i.e., those with low CD3+ TILs and high CD44 / CD24 tumor expression, who have the worst DFS, MFS, and OS. Figure 10 B).

[0195] Example 7 In summary, the embodiments of this application convincingly demonstrate that the combination of low CD3+ TIL and CD24+ expression cells is a reliable prognostic factor in cancer.

[0196] In vivo chemoradiography (IHC) is considered the standard technique for studying tumor tissue because it preserves the in vivo tissue structure and avoids artifacts that sometimes occur in cell culture. However, this technique heavily relies on well-validated antibodies to obtain accurate results. Although well-characterized antibodies such as anti-CD44 antibodies can be used for selected biomarkers, identifying reliable anti-CD24 antibodies that are particularly suitable for IHC has remained challenging with current techniques.

[0197] In this application, the inventors have optimized a method for detecting CD24 expression in an IHC using anti-CD24 antibodies (ML5 and M1 / 69 clones). The specificity of these antibodies was rigorously validated in the FFPE IHC using multiple validation protocols established by an ad hoc international working group for antibody validation guidelines. Given the scarcity of well-validated anti-CD24 antibodies for the FFPE IHC and the importance of CD24 in tumor biology, this application provides a valuable tool for studying CD24 in archived clinical samples.

[0198] Nasopharyngeal carcinoma responds very well to concurrent chemoradiotherapy (CCRT). However, some patients relapse and have shorter survival. In certain geographic locations, including Saudi Arabia, the majority of patients have locally advanced nasopharyngeal carcinoma. By definition, all patients are TNM stage 3 or 4A according to the AJCC classification; therefore, prognosis using TNM staging is limited. In this application, multiple CSC markers were screened in parallel as prognostic factors in LA-NPC. CD24 expression in >30% of tumor cells was significantly associated with disease-free survival, metastasis-free survival, and overall survival. Unexpectedly, this application demonstrates for the first time that the combination of the cancer stem cell (CSC) marker CD24 and CD3+ TIL has significant prognostic value. Patients with LA-NPC tumors having low CD3+ TILs but rich in CD24+ CSC-like cells had significantly shorter DFS (median 2.9 years), MFS (median 3.9 years), and OS.

[0199] Therefore, this study breaks new ground in cancer prognosis by incorporating immune-related and cancer stem cell factors. The results of this embodiment demonstrate for the first time that analyzing the combination of the presence of CD24-positive cells and CD3+ TIL levels is highly suitable for methods of diagnosing, prognosticating, stratifying, and / or monitoring cancer in subjects.

[0200] In summary, this invention provides a novel method for the diagnosis, prognosis, stratification, and / or therapy monitoring of cancer. This method is based on the determination of CD3+ tumor-infiltrating lymphocyte levels and CD24 expression. The novel combination of biomarkers of this invention allows for the diagnosis, prognosis, stratification, and / or therapy monitoring of various cancers. Furthermore, this application provides a diagnostic kit for performing the method of this invention.

Claims

1. Use of reagents for determining the levels of at least two biomarkers in a biological sample in the preparation of a kit for the diagnosis, prognosis, stratification and / or therapy monitoring of cancer in a subject, wherein said at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; Preferably, the levels of the at least two biomarkers in the biological sample from the subject, which differ from those of a healthy control or reference value, indicate the presence of cancer in the subject.

2. The use according to claim 1, wherein the biological sample is a sample of a subject, and the biological sample comprises a tissue sample or a body fluid sample, such as a blood sample, serum sample, plasma sample, a sample of a group of cells from a tumor, tumor tissue, urine sample, lymph sample, pleural fluid sample, or cerebrospinal fluid sample; wherein, preferably, the biological sample is a tissue sample; even more preferably, a paraffin-embedded tissue sample; and most preferably, a formalin-fixed paraffin-embedded tissue sample.

3. The use according to any one of the preceding claims, wherein the levels of the at least two biomarkers are assessed by their protein biomarkers, and wherein determining the level of each of the at least two biomarkers involves determining the protein levels of the at least two biomarkers in the biological sample.

4. The use according to any one of the preceding claims, wherein the kit further comprises a reagent for determining the level of one or more additional biomarkers in the biological sample, wherein the one or more additional biomarkers are selected from CD44 and markers for epithelial-mesenchymal transition, such as vimentin, E-cadherin, N-cadherin or β-catenin; preferably selected from CD44 and vimentin; even more preferably CD44; in, More preferably, the levels of one or more additional biomarkers in the biological samples from the subject, which differ from healthy controls or reference values, can further indicate the presence of an aggressive form of cancer in the subject.

5. The use according to any one of the preceding claims, wherein the diagnosis is a screening method for establishing a preliminary diagnosis of cancer in the subject.

6. The use according to any one of the preceding claims, wherein determining the level of the at least two biomarkers in the biological sample results in CD24+ high / CD3+TIL low Phenotype indicates that the subject is at high risk of disease progression; in, Optionally, when the subject is diagnosed as having a high risk of disease progression, the patient's treatment plan is modified; wherein, further optionally, the modification of the treatment plan includes one or more of the following: changing the medication, optionally changing it to one or more investigational agents, and strengthening the treatment, such as by increasing the treatment dose and / or adjusting the dosing regimen.

7. Use of reagents for determining the levels of at least two biomarkers in a biological sample in the preparation of a kit for assessing treatment success in a patient with cancer undergoing cancer treatment, wherein said at least two biomarkers are CD3+ tumor-infiltrating lymphocytes (TILs) and CD24+ cells; Preferably, the levels of the at least two biomarkers are compared with a reference sample or reference value. A decrease or increase in the levels of at least two biomarkers in a biological sample from the subject, compared to the reference sample or reference value, indicates the patient's response to the treatment.

8. The use according to claim 7, wherein the reference value corresponds to the level of the at least two biomarkers in a biological sample obtained from the patient prior to receiving the treatment; and / or High CD24 levels and / or low CD3+ TIL levels indicate an inadequate response to the treatment.

9. The use according to any one of the preceding claims, wherein the cancerous disease is selected from the group comprising nasopharyngeal carcinoma, head and neck cancer, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colorectal cancer, gastric cancer, lymphoma, including non-Hodgkin lymphoma such as Burkitt lymphoma, glioma, melanoma, small cell and non-small cell lung cancer, bile duct cancer, renal cell carcinoma, bladder cancer, uterine cancer, epithelial ovarian cancer, prostate cancer, and pancreatic cancer; wherein, Preferably, the cancer is nasopharyngeal carcinoma, head and neck cancer, or hepatocellular carcinoma; even more preferably, the cancer is nasopharyngeal carcinoma; most preferably, the cancer is locally advanced nasopharyngeal carcinoma.

10. The use according to any one of the preceding claims, wherein one or more binding agents or antigen-binding peptides, such as antibodies, are used to detect the biomarker, wherein, Preferably, the biomarker is detected by immunohistochemistry, Western blotting, ELISA, adjacent extension assay, or mass spectrometry; more preferably, the biomarker is detected by immunohistochemistry.

11. The use according to any one of the preceding claims, wherein one or more of said at least two biomarkers are detected by immunohistochemistry using a method comprising the following steps: (i) Provide formalin-fixed paraffin-embedded (FFPE) tissue samples derived from the subject; (ii) Dewaxing the FFPE tissue samples in xylene and rehydrating them using an ethanol / water gradient; (iii) Antigen retrieval was performed using an antigen retrieval solution in a Decloaking Chamber pressure cooker; (iv) Optionally, quench endogenous peroxidase and / or block endogenous biotin; (v) Incubate the sample with one or more primary antibodies that are specific to one or more of the at least two biomarkers, preferably overnight at about 2-10°C, more preferably overnight at about 4°C, wherein, more preferably, the one or more primary antibodies are diluted in 1% BSA; (vi) Incubate the sample with one or more secondary antibodies specific to any one of the one or more primary antibodies, wherein the one or more secondary antibodies are conjugated to a reporter enzyme; wherein, Optionally, if more than one secondary antibody is used, the sample can be incubated with one or more secondary antibodies simultaneously or sequentially. (vii) Detect the reporter enzyme conjugated with the secondary antibody, wherein, optionally, if more than one primary antibody and / or more than one secondary antibody are used, the detection of the secondary antibody may be performed simultaneously or sequentially.

12. The use according to claim 11, wherein the method further comprises, between steps (iv) and (v); and / or between steps (v) and (vi); and / or between steps (vi) and (vii); and / or between different secondary antibody detections in step (vii), one or more washing steps using a washing buffer such as TBST; and / or The antigen retrieval solution in step (iii) is Tris-EDTA with a pH of approximately 9; and / or The antigen retrieval in step (iii) is carried out at a temperature of about 90°C to about 150°C, preferably about 110°C to about 130°C, more preferably about 121°C, for about 1 min to about 15 min, preferably about 5 min to about 10 min, more preferably about 7 min.

13. The use according to any one of the preceding claims, wherein CD24 is detected using a binding agent or antigen-binding peptide that specifically binds to CD24, wherein the CD24 contains at least 90% identity with the amino acids of SEQ ID NO:1, more preferably at least 95% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, optionally the same amino acid sequence or constituted therewith; in, Preferably, the binder or antigen-binding peptide binds to the sequence according to positions 46-48 of SEQ ID NO:1; and / or The specific binding agent or antigen-binding peptide for CD24 is selected from the group comprising anti-CD24 monoclonal antibody clone: ​​ML5, anti-CD24 monoclonal antibody clone: ​​M1 / 69, anti-CD24 monoclonal antibody clone: ​​SN3b, anti-CD24 monoclonal antibody clone: ​​SN3, anti-CD24 monoclonal antibody clone: ​​SWA11 and anti-CD24 monoclonal antibody clone: ​​ALB9; preferably selected from anti-CD24 monoclonal antibody clone: ​​ML5 and anti-CD24 monoclonal antibody clone: ​​M1 / 69; and even more preferably anti-CD24 monoclonal antibody clone: ​​ML5.

14. The use according to any one of the preceding claims, wherein the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey or human, preferably a human.

15. A diagnostic kit comprising a combination of at least two antibodies, their derivatives, or antigen fragments for detecting either CD24 or CD3; in, Optionally, the diagnostic kit further comprises one or more components selected from the group consisting of: a visualization system, an inhibitor, a chromogenic solution, a wash buffer, instructions for use with appropriate operating parameters, a secondary antibody optionally conjugated to a reporter enzyme, wherein the secondary antibody is specific to at least one of the at least two antibodies, an antigen retrieval solution, an assay kit for detecting the conjugated reporter enzyme, and standard or control information such that the test sample can be compared with a standard control information. Preferably, the diagnostic kit is a kit used according to any one of the preceding claims.

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