Immunobiomarkers for the auxiliary diagnosis of lymph node metastasis in esophageal cancer and their applications

Abstract

This invention provides immunobiomarkers for the auxiliary diagnosis of esophageal cancer lymph node metastasis and their applications. Specifically, this invention provides the use of immunobiomarkers for esophageal cancer lymph node metastasis and their detection reagents, for the preparation of diagnostic reagents or kits for assessing the risk of esophageal cancer lymph node metastasis. Studies have shown that the immunobiomarkers of this invention can serve as markers of esophageal cancer lymph node metastasis with high accuracy.

Description

Technical Field

[0001] This invention relates to the field of clinical laboratory diagnostics, specifically to a set of immunomarkers for the auxiliary diagnosis of lymph node metastasis in esophageal cancer and their applications. Background Technology

[0002] Esophageal cancer is a malignant tumor of the digestive tract that originates from the esophageal mucosal epithelium. Clinically, progressive dysphagia is the typical symptom of its advanced stage. Esophageal cancer has a persistently high mortality rate, seriously endangering the physical and mental health of the population. Lymph node metastasis is the most common mode of metastasis for esophageal squamous cell carcinoma, typically affecting the upper two-thirds of the esophagus. It is characterized by early onset, bidirectional spread, skip metastasis, and regional distribution, and is a key factor influencing the prognosis of esophageal cancer patients.

[0003] Effective lymph node dissection is the primary surgical approach for radical treatment of esophageal cancer. Early-stage esophageal cancer can be treated with minimally invasive endoscopic techniques, while suspected lymph node metastasis requires surgical intervention. Mid-to-late-stage esophageal cancer is primarily treated surgically, but when ≥2 lymph node metastases are found, combined radiotherapy and chemotherapy are necessary. Excessive lymph node dissection may also lead to more postoperative complications. Therefore, accurately identifying lymph node metastasis helps in developing a reasonable treatment plan and improving long-term patient prognosis. Currently, the clinical diagnosis of lymph node metastasis mainly relies on imaging examinations (enhanced CT, color Doppler ultrasound, or PET-CT) and physician experience, but its diagnostic value is controversial. It is evident that the above diagnostic methods have a certain rate of misdiagnosis for medical and laboratory personnel, lack precise quantitative biomarkers, make it difficult to accurately assess preoperative outcomes based on patient characteristics, and can easily lead to adverse effects on patient prognosis due to excessive dissection.

[0004] Therefore, timely and accurate preoperative diagnosis of esophageal cancer lymph node metastasis is an urgent clinical problem to be solved.

[0005] Therefore, there is an urgent need in this field to develop new methods for timely and accurate preoperative diagnosis of esophageal cancer lymph node metastasis. Summary of the Invention

[0006] This invention provides a novel method for timely and accurate preoperative diagnosis of lymph node metastasis in esophageal cancer.

[0007] In a first aspect of the present invention, the use of an immunobiomarker for esophageal cancer lymph node metastasis, or a detection reagent thereof, is provided for the preparation of a diagnostic reagent or diagnostic kit, said diagnostic reagent or diagnostic kit being used to determine the risk of esophageal cancer lymph node metastasis.

[0008] The immune biomarkers are selected from the following group:

[0009] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 +CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells;

[0010] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 + Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells;

[0011] (C) A combination of at least one marker selected from group A and at least one marker selected from group B.

[0012] In another preferred embodiment, the marker selected from group A, or a combination thereof, refers to the proportion of immune cells selected from any marker shown in A1 to A15 of group A, or a combination thereof, wherein the proportion of immune cells is the percentage of any marker shown in A1 to A15 of group A, or a combination thereof, relative to CD45. + Percentage of cells.

[0013] In another preferred embodiment, any marker selected from group A, or a combination thereof, refers to any cell or combination thereof shown in A1 to A15 accounting for CD45. + Percentage of cells.

[0014] In another preferred embodiment, the (B1)CD11c MdFI in myeloid dendritic cells is the median fluorescence intensity of CD11c in myeloid dendritic cells.

[0015] In another preferred embodiment, the (B2)CD11c MdFI in CD16 + Myeloid dendritic cells are CD11c on CD16 + Median fluorescence intensity of myeloid dendritic cells.

[0016] In another preferred embodiment, (B3)CD11c MdFI in intermediate monocytes is the median fluorescence intensity of CD11c in intermediate monocytes.

[0017] In another preferred embodiment, the (B4)CD38 MdFI in B cells is the median fluorescence intensity of CD38 in B cells.

[0018] In another preferred embodiment, the (B5)CD38 MdFI in CD27 + Memory B cells are CD38 at CD27 + Median fluorescence intensity of memory B cells.

[0019] In another preferred embodiment, the detection reagent or detection kit is used to detect the level of the marker in the sample to be tested.

[0020] In another preferred embodiment, the detection reagent or kit is also used to detect CD45 in the sample to be tested. + At the cellular level.

[0021] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A3)CD28 + CD3 + T cells; and (A11)CD16 + Myeloid dendritic cells.

[0022] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A1) Tfh cells; and (A13) HLADR. - CD38 - CD3 + T cells.

[0023] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A2) Tfh cells; and (A10) intermediate monocytes.

[0024] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A5) Tconv cells; and (A10) intermediate monocytes.

[0025] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A1) Tfh cells; and (A4) CD28. +CD8 + T cells.

[0026] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A1) Tfh cells; and (A7) central memory CD8. + T cells.

[0027] In another preferred embodiment, the immunobiomarker comprises a combination of the following biomarkers: (A1) Tfh cells; (A9) CXCR5. + CD8 + T cells.

[0028] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A4)CD28 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells.

[0029] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A2) Tfh1 cells; (A8) HLA-DR + CD38 + CD8 + T cells.

[0030] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A2) Tfh1 cells; (A5) Tconv cells.

[0031] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A7) central memory CD8 + T cells; (A11)CD16 + Myeloid dendritic cells; and (B4)CD38 MdFI in B cells.

[0032] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A1) Tfh cells; (A8) HLA-DR + CD38 + CD8 + T cells; and (A10) intermediate monocytes.

[0033] In another preferred embodiment, the immune biomarker comprises a combination of the following biomarkers: (A1) Tfh cells; (A7) central memory CD8. + T cells; and (A13)HLADR - CD38 - CD3 + T cells;

[0034] In another preferred embodiment, the sample to be tested is a blood sample or a whole blood sample.

[0035] In another preferred embodiment, the detection reagent or test kit is used to detect the level of the marker in a blood sample.

[0036] In another preferred embodiment, the immunobiomarker is selected from any one of the biomarkers A1-A15 and B1-B5 in Table A below, or a combination thereof:

[0037] Table A

[0038] code name The immune biomarkers of the present invention Upward / downward A1 Tfh cells Lower A2 Tfh1 cells Lower A3 <![CDATA[CD28 + CD3 + T cells Lower A4 <![CDATA[CD28 + CD8 + T cells Lower A5 Tconv cells Lower A6 CD4+ T cells Lower A7 <![CDATA[Central memory CD8 + T cell]]> Lower A8 <![CDATA[HLADR + CD38 + CD8 + T cells Lower A9 <![CDATA[CXCR5 + CD8 + T cells Lower A10 Intermediate monocytes Lower A11 <![CDATA[CD16 + Myeloid dendritic cells Lower A12 Th2 cells Lower A13 <![CDATA[HLADR - CD38 - CD3 + T cells Lower A14 <![CDATA[Naive CD8 + T cell]]> Lower A15 plasmacytoid dendritic cells Lower B1 CD11c MdFI in myeloid dendritic cells Lower B2 <![CDATA[CD11c MdFI in CD16 + Myeloid dendritic cells Lower B3 CD11c MdFI in intermediate monocytes Lower B4 CD38 MdFI in B cells Upward B5 <![CDATA[CD38 MdFI in CD27 + Memory B cells Upward

[0039] In a second aspect of the invention, a kit is provided, the kit containing a detection reagent for detecting immunobiomarkers of esophageal cancer lymph node metastasis in a sample to be tested;

[0040] The immunobiomarkers for esophageal cancer lymph node metastasis are selected from the following group:

[0041] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells;

[0042] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 +Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells;

[0043] (C) A combination of at least one marker selected from group A and at least one marker selected from group B.

[0044] In another preferred embodiment, the detection reagent is also used to detect CD45 in the sample to be tested. + cell.

[0045] In another preferred embodiment, the detection reagent is used to detect the level or quantity of the immunobiomarker of esophageal cancer lymph node metastasis in the test sample and / or CD45 in the test sample. + The level or number of cells.

[0046] In another preferred embodiment, the kit also includes a label or instructions indicating that the kit is used to assess the risk of esophageal cancer lymph node metastasis.

[0047] In another preferred embodiment, the detection of the immunobiomarker includes detection by a method selected from the group consisting of: immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunohistochemistry, dot blot, protein microarray, tissue microarray coupled with immunohistochemistry, or a combination thereof.

[0048] In another preferred embodiment, the detection reagent includes specific antibodies and specific binding molecules against immunobiomarkers of esophageal cancer lymph node metastasis.

[0049] In another preferred embodiment, the detection is performed on an ex vivo sample.

[0050] In another preferred embodiment, the ex vivo sample includes a blood sample or a whole blood sample.

[0051] In another preferred embodiment, the detection reagent is coupled with or carries a detectable marker.

[0052] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0053] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0054] In another preferred embodiment, the detection of the immunobiomarkers for esophageal cancer lymph node metastasis is quantitative.

[0055] In another preferred embodiment, the detection of immune biomarkers is to determine the proportion of immune cells or to determine the function of immune cells.

[0056] In another preferred embodiment, the proportion of immune cells refers to the percentage of immune cells in the peripheral blood.

[0057] In another preferred embodiment, the immune cell ratio refers to the proportion of immune cells in peripheral blood CD45. + Percentage of cells.

[0058] In another preferred embodiment, the immune cell ratio refers to the percentage of immune cells in the progenitor population or the percentage of immune cells in the parent population.

[0059] In another preferred embodiment, the kit also includes a positive control sample.

[0060] In another preferred embodiment, the sample is calibrated to represent the proportion and / or function of immune cells in patients or subjects with esophageal cancer lymph node metastasis.

[0061] In another preferred embodiment, the positive control sample is from one or more patients / subjects who are known to have esophageal cancer with lymph node metastasis.

[0062] In another preferred embodiment, the kit also includes a negative control sample.

[0063] In another preferred embodiment, the sample is calibrated to represent the proportion and / or function of immune cells in esophageal cancer patients or healthy subjects without lymph node metastasis.

[0064] In another preferred embodiment, the negative control sample is derived from one or more esophageal cancer patients or healthy subjects who are known not to have lymph node metastasis.

[0065] In a third aspect of the invention, a diagnostic device for esophageal cancer lymph node metastasis is provided, the device comprising:

[0066] (a) An input module, the input module being configured to input immunobiomarker data of esophageal cancer lymph node metastasis from a blood sample of the subject to be tested;

[0067] The immunobiomarkers for esophageal cancer lymph node metastasis are selected from the following group:

[0068] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 +T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells;

[0069] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 + Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells;

[0070] (C) A combination of at least one marker selected from group A and at least one marker selected from group B;

[0071] (b) A processing module configured to determine the following for input immunobiomarkers: when an immunobiomarker is upregulated, if its immune cell ratio is upregulated or its immune cell function value is higher than a reference value, it indicates a high risk of lymph node metastasis in the subject; when an immunobiomarker is downregulated, if its immune cell ratio is downregulated or its immune cell function value is lower than a reference value, it indicates a high risk of lymph node metastasis in the subject; conversely, it indicates a low risk of lymph node metastasis in the subject.

[0072] (c) Output module, which is configured to output the determination result.

[0073] In another preferred embodiment, when the immune biomarker is selected from group A, the immune cell ratio refers to the ratio of the biomarker level to CD45. + The ratio at the cellular level;

[0074] In another preferred embodiment, when the immunobiomarker is selected from group B, the immune cell function value refers to the median fluorescence intensity (MdFI) of CD11c or CD38 on the immune cells.

[0075] In another preferred embodiment, the output module includes any terminal, preferably a monitor, printer, tablet computer (PAD), or smartphone.

[0076] In another preferred embodiment, the modules are connected via wired or wireless means.

[0077] In another preferred embodiment, the device further includes (d) a detection module configured to detect the sample to be tested, thereby obtaining the levels of the immunobiomarkers and CD45 in the sample. + Cellular level.

[0078] In a fourth aspect of the invention, a set of immunobiomarkers for esophageal cancer lymph node metastasis is provided, the set of immunobiomarkers comprising biomarkers selected from the group consisting of:

[0079] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells; and / or

[0080] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 +Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells.

[0081] In another preferred embodiment, any marker selected from A1 to A15 in group A refers to any cell represented by A1 to A15 accounting for CD45. + Percentage of cells.

[0082] In another preferred embodiment, the (B1)CD11c MdFI in myeloid dendritic cells is the median fluorescence intensity of CD11c in myeloid dendritic cells.

[0083] In another preferred embodiment, the (B2)CD11c MdFI in CD16 + Myeloid dendritic cells are CD11c on CD16 + Median fluorescence intensity of myeloid dendritic cells.

[0084] In another preferred embodiment, (B3)CD11c MdFI in intermediate monocytes is the median fluorescence intensity of CD11c in intermediate monocytes.

[0085] In another preferred embodiment, the (B4)CD38 MdFI in B cells is the median fluorescence intensity of CD38 in B cells.

[0086] In another preferred embodiment, the (B5)CD38 MdFI in CD27 + Memory B cells are CD38 at CD27 + Median fluorescence intensity of memory B cells.

[0087] In a fifth aspect of the invention, a detection method is provided, comprising the steps of:

[0088] (a) Provide a test sample, said test sample being selected from a blood sample;

[0089] (b) Detect the level or intensity of immunobiomarkers for esophageal cancer lymph node metastasis in the test sample, denoted as C1, and detect CD45 in the test sample. + The level of cells is denoted as R1; and

[0090] (c) Compare the proportion of immune cells or the immune cell function value C1 of the immune biomarker with the control reference value C0;

[0091] The immunobiomarkers for esophageal cancer lymph node metastasis are selected from the following group:

[0092] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells;

[0093] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 + Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells;

[0094] (C) A combination of at least one marker selected from group A and at least one marker selected from group B.

[0095] If the immunobiomarkers for esophageal cancer lymph node metastasis in a subject meet the following criteria, it indicates a high risk of esophageal cancer lymph node metastasis in that subject:

[0096] (1) When a certain biomarker is an upregulated biomarker in Table A in the test subject, and the proportion of immune cells or the immune cell function value of the biomarker is higher than the reference value or standard value C0, the test subject has a high risk of lymph node metastasis.

[0097] (2) When a certain biomarker is a downregulated biomarker in Table A in the test subject, and the proportion of immune cells or the immune cell function value of the biomarker is lower than the reference value or standard value C0, the test subject has a high risk of lymph node metastasis.

[0098] In another preferred embodiment, when the immune biomarker is selected from group A, the immune cell proportion refers to the proportion of the biomarker level to CD45. + The proportion at the cellular level; when the immune biomarker is selected from group B, the immune cell function value refers to the median fluorescence intensity (MdFI) of CD11c or CD38 on immune cells.

[0099] In another preferred embodiment, the object of detection is a person.

[0100] In another preferred embodiment, the detection subjects include both men and women.

[0101] In another preferred embodiment, the detection subjects include infants, adolescents, or adults.

[0102] In another preferred embodiment, the subject of the test is an esophageal cancer patient.

[0103] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0104] In another preferred embodiment, the method is an in vitro method.

[0105] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0106] Figure 1 Figure AB shows the results of immune cell phenotypic measurements detected by flow cytometry in human samples from embodiments of the present invention, analyzed using Wilcoxon rank sum tests. LN represents esophageal squamous cell carcinoma patients without lymph node metastasis, and LNM represents esophageal squamous cell carcinoma patients with lymph node metastasis.

[0107] Figure 2 AB shows the ROC curve of a single indicator of the immune cell phenotypic marker of the present invention for diagnosing lymph node metastasis of esophageal cancer and assessing disease risk. Detailed Implementation

[0108] Through extensive and in-depth research and screening, the inventors have discovered for the first time a novel immunobiomarker for lymph node metastasis in esophageal cancer. This immunobiomarker includes markers A1-A15 and B1-B5. By detecting the proportion and / or functional values ​​of immune cells associated with these markers, the risk of lymph node metastasis in esophageal cancer patients can be effectively assessed. Based on this discovery, the present invention was completed.

[0109] Experiments of this invention show that the immunobiomarkers of this invention have good effects in judging lymph node metastasis of esophageal cancer, whether used alone or in combination.

[0110] the term

[0111] The terminology used in this invention has the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, some definitions and related terms are explained below. It should be noted that the explanations of the terminology provided herein are only to enable those skilled in the art to better understand this invention and are not intended to limit the invention.

[0112] As used in this article, the term "set of markers" refers to a collection of one, two, and / or more markers.

[0113] As used in this article, the term "individual" refers to an animal, particularly a mammal such as a primate, and preferably a human.

[0114] As used herein, terms such as “a,” “an,” and “this” refer not only to a singular number of individuals but also to a general class that can be used to describe a particular implementation.

[0115] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0116] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0117] As used herein, a “cell subpopulation” refers to a collection of arbitrary cells within a population of cells that possess various characteristics and share certain common features. This term may be used to refer to a specific cell subpopulation known in the art, or it may be used to refer to a specific cell subpopulation by describing any property (e.g., the expression of cell surface markers).

[0118] As used in this article, the term “relative quantity” for cells can be used interchangeably with “proportion”.

[0119] As used herein, the term "sample" or "sample" refers to material specifically associated with a subject from which specific information relating to the subject can be determined, calculated, or inferred. A sample may consist wholly or partially of biological material from the subject. A sample may also be material that has been in contact with the subject in a manner that allows testing of the sample to provide information relating to the subject. A sample may also be material that has been in contact with other materials, not belonging to the subject, but which enable subsequent testing of the first material to determine information relating to the subject; for example, a sample may be a cleaning solution for a probe or scalpel. A sample may be a source of biological material other than that in contact with the subject, as long as those skilled in the art can still determine information relating to the subject from the sample.

[0120] The sample is selected from biological fluids such as blood or whole blood, which are used interchangeably in this invention.

[0121] In all its embodiments, the method described above is applied to blood samples containing leukocytes (particularly at least mononuclear cells). The blood sample can be, for example, a sample of purified lymphocytes. It can also be a peripheral blood mononuclear cell (or PBMC) sample, which consists of lymphocytes (B cells, T cells, and NK cells), dendritic cells, and monocytes, and is typically obtained via the Ficoll method well known to those skilled in the art. However, to minimize manipulation of the sample and preserve physiological cell interactions between the different cell populations involved in the immune response, and to better reflect the complexity of the patient's / subject's innate and adaptive immune responses, it is preferable to use a whole blood sample (i.e., containing all leukocytes, erythrocytes, platelets, and plasma) collected directly via a venous route (e.g., using tubes containing an anticoagulant).

[0122] As used herein, the term "expression" refers to the final presentation of a cell surface marker (usually a protein) on the cell surface after RNA transcription, protein translation, and intracellular transport. The expression level or intensity of a cell surface marker can be measured using flow cytometry. Preferably, the expression level or intensity of a specific marker between two samples can be reflected by counting cells whose expression level or intensity reaches or exceeds a specified level, using the proportion of cells being "more" or "less". Preferably, the expression level of a specific marker between two samples can be reflected by the intensity of the fluorescence signal being "high" (Hi) or "low" (Low).

[0123] As used herein, the terms “reference value,” “reference quantity,” or “reference level” refer to the value (or quantity, or level) of a parameter or biomarker that indicates a subject’s status regarding a particular disease (or minor illness, or condition). An appropriate reference level for a parameter or biomarker can be quantified, determined, or measured by detecting the parameter / biomarker in several suitable reference subjects. Such reference levels can be adjusted for specific subject populations. Reference values ​​or reference levels can be absolute values; relative values; values ​​with upper or lower limits; a series of values; average values; medians, mean values, or values ​​compared to a specific control or baseline value. Reference values ​​can be based on values ​​from individual samples, such as values ​​obtained from samples from test subjects at an earlier time point. Reference levels can be based on large samples, such as a population of subjects from a fully age-matched group, or on a sample pool that includes or excludes the sample to be tested. Depending on the context, the reference level corresponds to the value of a parameter (or biomarker) that is quantified, determined, or measured on a sample from a healthy reference subject; or to the average (mean) of the values ​​of a parameter (or biomarker) that are quantified, determined, or measured on different samples from the same healthy reference subject (values ​​quantified / determined / measured on samples collected from the same healthy reference subject at various time intervals); or to the average (mean) of the values ​​of a parameter / biomarker determined / measured on the same sample from a healthy reference subject at various time intervals; or to the average (or mean) of the values ​​of a parameter / biomarker that are quantified / determined / measured on samples from several healthy reference subjects (at least two healthy reference subjects).

[0124] esophageal cancer

[0125] Esophageal cancer is a common malignant tumor of the digestive tract, mainly occurring in the epithelial cells or glands of the esophageal mucosa. Based on pathological type, esophageal cancer is mainly divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma. Clinical manifestations include dysphagia, chest pain, and weight loss.

[0126] Biomarkers of esophageal cancer metastasis

[0127] As used herein, the term "esophageal cancer metastasis biomarker of the present invention" refers to one or more of the biomarkers shown in Table A.

[0128] In this invention, the terms "esophageal cancer metastasis biomarker of the present invention", "esophageal cancer lymph node metastasis biomarker of the present invention", "esophageal cancer metastasis immune cell biomarker of the present invention", or "marker shown in Table A" are used interchangeably and all refer to having any one or more of the esophageal cancer lymph node metastasis biomarkers of the present invention.

[0129] The immunobiomarkers in this invention include one or more of the following immune cell phenotypes. By detecting the proportion or function of the above immunobiomarkers, they can be used for the diagnosis or risk assessment of esophageal cancer lymph node metastasis.

[0130] The immune cell phenotypes of group (A) in Table A of this invention are selected from CD16. + Myeloid dendritic cells, intermediate mononuclear cells, plasmacytoid dendritic cells, HLADR - CD38 - CD3 + T cells, CD28 + CD3 + T cells, CD4 + T cells, Tconv cells, Th2 cells, Tfh cells, Tfh1 cells, HLADR + CD38 + CD8 + T cells, central memory CD8 + T cells, CXCR5 + CD8 + T cells, CD28 + CD8 + T cells, immature CD8 + T cells.

[0131] CD16 as described in this invention + Myeloid dendritic cells (CD16) + Myeloid dendritic cells, CD16 + mDCs are a type of myeloid dendritic cell whose surface marker is CD3. - CD19 - CD56 - CD14 - HLADR + CD11c + CD123 - CD16 + T cells are a highly specialized class of antigen-presenting cells that are primarily responsible for capturing, processing, and presenting antigens to T cells, thereby initiating and regulating adaptive immune responses.

[0132] The intermediate monocyte described in this invention is a type of monocyte, and its surface marker expression characteristic is CD14. hi CD16 + It is responsible for the proliferation and stimulation of T cells, and plays a key role in antigen presentation and the secretion of cytokines or pro-inflammatory interleukins.

[0133] The plasmacytoid dendritic cells (pDCs) described in this invention are characterized by the expression of CD3 as a surface marker. - CD19 - CD56 - CD14 - HLADR + CD11c - CD123 + It can produce a large amount of type I interferon, which plays a vital role in antiviral immunity and is associated with the occurrence and development of many autoimmune and inflammatory diseases.

[0134] The HLADR described in this invention - CD38 - CD3 + T cells are characterized by the expression of CD3 as a surface marker. + HLADR - CD38 - T cells, whether inactive or dormant, possess the potential for an immune response and can be activated at the appropriate time. They play a crucial role in immune tolerance and surveillance.

[0135] CD28 as described in this invention + CD3 + T cells and CD28 + CD8 + T cells, whose surface marker expression characteristics are CD3 + CD28 + and CD3 + CD8 + CD4 - CD28 + CD28 is an important co-stimulatory molecule required for T cell activation, promoting T cell proliferation and enhanced function, and playing a key role in regulating immune responses, clearing infections and tumors.

[0136] The Tconv cells described in this invention are characterized by the expression of CD3 as a surface marker. + CD4 + CD8 - CD25 - CD127 - &CD3 + CD4 + CD8 - CD25 - CD127 + &CD3 + CD4 + CD8 -CD25 + CD127 + It is CD4 that has not differentiated into regulatory T cells. + T cell subsets are primarily responsible for recognizing foreign antigens and stimulating adaptive immune responses.

[0137] The Th2 cells described in this invention are characterized by the expression of CD3 as a surface marker. + CD4 + CD8 - CXCR3 - CCR4 + CCR6 - It mainly secretes IL-4, IL-5, and IL-13 to activate macrophages and enhance cell-mediated immune responses, participating in anti-parasitic immunity and allergic reactions.

[0138] The Tfh cells described in this invention are characterized by CD3 expression on their surface markers. + CD4 + CD8 - CXCR5 + It mainly secretes IL-21 and IL-4, and can be further divided into Tfh1 cells, Tfh2 cells, Tfh9 cells, Tfh17 cells and Tfh17 / Tfh1 cells. It can support the activation, differentiation and antibody production of B cells and play a key role in humoral immune response.

[0139] The Tfh1 cells described in this invention are characterized by the expression of CD3 as a surface marker. + CD4 + CD8 - CXCR5 + CXCR3 + CCR4 - CCR6 - It mainly secretes IF- - γ, IL-2, and TNF-α are involved in the regulation of Th1 immune responses, mainly helping B cells to carry out humoral immune responses in germinal centers.

[0140] The HLADR described in this invention + CD38 + CD8 + T cells are characterized by the expression of CD3 as a surface marker. + CD8 + CD4 - HLADR - CD38 - They are activated in response to infection or tumors, and can directly kill virus-infected cells and tumor cells by releasing effector molecules such as perforin and granzymes.

[0141] The central memory CD8 described in this invention + T cells are characterized by the expression of CD3 as a surface marker. + CD8 + CD4 - CD45RA - CCR7 + These are a type of long-lasting memory T cells that can rapidly proliferate and differentiate into effector CD8+ cells upon re-encountering the antigen. + T cells exert a powerful cytotoxic effect to eliminate virus-infected cells or tumor cells.

[0142] The CXCR5 mentioned in this invention + CD8 + T cells are characterized by the expression of CD3 as a surface marker. + CD8 + CD4 - CXCR5 + CXCR5 is a key chemokine receptor that enables these cells to localize to the germinal centers of lymph nodes and other specific lymphoid tissues. It plays a crucial role in promoting B cell activation and antibody production, regulating immune responses, and maintaining immune memory.

[0143] The juvenile CD8 mentioned in this invention + T cells are characterized by the expression of CD3 as a surface marker. + CD8 + CD4 - CD45RA + CCR7 + CD95 - It mainly resides in secondary lymphoid organs such as lymph nodes, and upon first encountering antigen presentation, it differentiates into effector CD8. + T cells or memory CD8 + T cells participate in specific immune responses.

[0144] Unless otherwise specified, any biomarker or combination thereof in Group A of Table A refers to the proportion of immune cells selected from any biomarker or combination thereof shown in A1 to A15 of Group A, wherein the proportion of immune cells selected from any biomarker or combination thereof shown in A1 to A15 of Group A is the percentage of CD45. + Percentage of cells.

[0145] The immune cell phenotypes of group (B) in Table A of this invention are composed of the median fluorescence intensity (MdFI) of the three CD molecules CD11c and CD38 on immune cell subsets. Median fluorescence intensity is used to quantify the expression level of molecular markers on the cell surface or inside the cell, and is usually used to measure the relative expression level of target molecules on cells.

[0146] CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16 + Myeloid dendritic cells and CD11c MdFI in intermediate monocytes represent the roles of CD11c in myeloid dendritic cells and CD16, respectively. + Median fluorescence intensity of myeloid dendritic cells and intermediate monocytes. CD11c is an integrin protein that is commonly used as a marker of myeloid cells, especially in dendritic cells and certain monocyte subsets, to mediate antigen capture, processing, and presentation, thereby activating T cells and initiating and regulating immune responses.

[0147] CD38 MdFI in B cells and CD38 MdFI in CD27 + Memory B cells are CD38 in B cells and CD27 in B cells. + Median fluorescence intensity of memory B cells. CD38 is a transmembrane protein with enzymatic activity, involved in NAD+ metabolism, signal transduction, and cell adhesion, and plays an important role in various immune cells. CD38 expression varies in B cells at different developmental stages, and is typically highly expressed in activated B cells and plasma cells. It is an important molecule regulating B cell maturation, antibody production, and immune memory. CD27 + CD38 expression in memory B cells may be associated with activation state and immune memory.

[0148] As used in this article, "median fluorescence intensity (MdFI)" is a quantitative indicator used in flow cytometry to measure the amount of fluorophore bound to cells or particles by monoclonal antibodies. Under appropriate conditions, fluorescence intensity is related to the number of binding sites of a specific fluorophore to a cell or particle. That is, the stronger the fluorophore binding to a cell or particle, the more target marker molecules are present, and vice versa. Stronger fluorescence intensity corresponds to a larger fluorescence signal channel value, and vice versa. Median fluorescence intensity represents the median of these fluorescence signal intensities, reflecting the expression level of the target molecule in the cell.

[0149] In this invention, “+” indicates that the marker expresses a positive result, “-” indicates that the marker expresses a negative result (no expression), “hi” indicates that the marker expresses a high level, and “Low” indicates that the marker expresses a low level. This is a marking method known to those skilled in the art.

[0150] The "proportion of immune cells" or "proportion of immune cell subsets" mentioned in this invention can be calculated in various different ways, and can be any of the following:

[0151] ① The percentage of immune cell subsets in peripheral blood;

[0152] ②The immune cell subsets mentioned account for a significant portion of peripheral blood CD45. + Percentage of cells;

[0153] ③ The percentage of the number of immune cell subsets relative to the number of progenitor immune cells;

[0154] ④ The percentage of the number of immune cell subsets relative to the number of immune cells in their parent population.

[0155] ⑤ The ratio between any two immune cell subsets.

[0156] In this invention, the "parent immune cells" are either the next higher level of immune cells based on the origin of immune cell differentiation, or the next layer of immune cells based on the logical order of flow cytometry gating.

[0157] In this invention, the "progenitor immune cells" refer to the upper two levels of immune cells based on the origin of immune cell differentiation, or the upper two layers of immune cells based on the logical order of flow cytometry gates.

[0158] The expression level of the immune functional molecules refers to the expression level of the immune functional molecules in the immune cell subset, which is referred to as "median fluorescence intensity" in this invention.

[0159] The immune functional molecules are selected from one or more of CD11c and CD38.

[0160] Using antibodies against the various cell surface antigen proteins / surface markers described above to detect the various immunobiomarkers described in this invention is a method well known to those skilled in the art.

[0161] Detection methods

[0162] Based on the differences in immunobiomarkers for esophageal cancer lymph node metastasis in blood samples, this invention provides a corresponding method for assessing the risk of esophageal cancer lymph node metastasis.

[0163] The method of this invention is an in vitro or ex vivo approach. For example, compared to etiological detection and SOFA scoring, this invention has the advantage of easily diagnosing esophageal cancer lymph node metastasis or assessing the risk of lymph node metastasis by providing directly measurable markers, particularly for patients preparing for radical esophagectomy or those diagnosed with esophageal squamous cell carcinoma, wherein the measurement can be performed in a laboratory near the patient or beside the patient. The measurement of the markers is fully adaptable to be performed by automated analytical machines or by testing methods known as rapid tests.

[0164] The sample for implementing the method described in this invention is also referred to as a test sample in this invention.

[0165] Test samples were taken from biological samples of patients / subjects from whom the risk of esophageal cancer lymph node metastasis was to be determined.

[0166] Specifically, the test sample is selected from biological fluids such as blood, whole blood, and preferably, in all embodiments, the method described above is applied to a blood sample containing leukocytes (particularly at least mononuclear cells). The blood sample can be, for example, a sample of purified lymphocytes. It can also be a peripheral blood mononuclear cell (or PBMC) sample, which consists of lymphocytes (B cells, T cells, and NK cells), dendritic cells, and monocytes, and is typically obtained using the Ficoll method well known to those skilled in the art. However, to minimize manipulation of the sample and preserve physiological cell interactions between different cell populations involved in the immune response, and to better reflect the complexity of the patient's / subject's innate and adaptive immune responses, it is preferable to use a whole blood sample (i.e., containing all leukocytes, erythrocytes, platelets, and plasma) collected directly via a venous route (e.g., using a tube containing an anticoagulant).

[0167] Any volume commonly used by those skilled in the art for hematological analysis will be convenient for this method. For example, the volume of biological samples can be 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, or 1000 μL (1 mL) and above.

[0168] The samples from which reference values ​​can be determined are also referred to as “control samples.” Specifically, to obtain reference values, these samples are preferably obtained from individuals with the same or most common characteristics, particularly those of the same sex and / or similar or the same age and / or ethnic origin, as subjects or patients / subjects whose risk of lymph node metastasis is to be determined. In this case, the reference sample can also be formed from any sample, whether or not it is a biological sample, that has been previously calibrated to contain mean immune cell proportions and / or immune function molecule expression levels corresponding to levels determined from a set of samples taken from patients / subjects who are known to have lymph node metastasis. In this case, and according to a particularly preferred variant, the reference sample is taken from one or more patients / subjects who are known to have lymph node metastasis.

[0169] In the context of this invention, the terms "detection," "measurement," or "determination" are used interchangeably and have the same meaning. These terms can refer to the detection and quantification of immune cell proportions or the detection and quantification of expression levels of immune functional molecules (the detection and quantification of these immune functional molecules at the protein level, referred to in this invention as "median fluorescence intensity"). Therefore, any detection and / or quantification method known to those skilled in the art can be used to carry out this invention.

[0170] In particular, the determination of the proportion of immune cells and / or the expression level of immune functional molecules (the detection and quantification of said functional molecules at the protein level) is performed using immune cell and / or immune functional molecule expression level specific tools or reagents that allow direct or indirect determination of their presence and / or quantification of their expression level.

[0171] Among these tools or reagents capable of detecting and / or quantifying the proportion of said immune cells and / or the expression level of immune functional molecules, specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, or fragments or derivatives thereof, may be mentioned.

[0172] In the method of the present invention, well-known analytical techniques can be used in particular to detect and / or quantify the proportion of immune cells and / or the expression level of immune functional molecules (immune cell function values), such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarrays, immunoprecipitation, immunohistochemistry, dot blots, protein microarrays, or tissue microarrays coupled with immunohistochemistry. Other suitable techniques include FRET or BRET, single-cell microscopy or histochemical methods using single or multiple excitation wavelengths and applying any suitable optical method, such as electrochemical methods (voltammetry and amperometric techniques), atomic force microscopy, and radio frequency methods, such as multipolar resonance spectroscopy, confocal and nonfocal methods, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsoidal polarization, resonant mirror methods, grating-coupled waveguide methods, or interferometry), cell ELISA, radioisotope analysis, magnetic resonance imaging, polyacrylamide gel electrophoresis (SDSPAGE), HPLC-mass spectrometry, and liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS). For example, when using flow cytometry, forward and side scattering information helps identify mononuclear cell populations among other blood cells. Preferably, flow cytometry is used to identify, select, sort, and quantify (and any combination thereof) the proportion of immune cells and / or the expression levels of immune functional molecules.

[0173] In this invention, the immune cell subsets and / or immune functional molecules (CD38, CD11c) are preferably measured by, for example, flow cytometry to determine whether the proportion of these immune cells and / or the expression level of these immune functional molecules are “high” or “low”.

[0174] Optionally, for the immune cell subsets and / or immune functional molecules (CD38, CD11c), the level of the immune cell subsets and / or immune functional molecules can be measured as "high" or "low" by measuring, for example, the amount of these immune cell subsets and / or immune functional molecules at high levels using flow cytometry. The exact amount of the surface immune cell subsets and / or immune functional molecules is not important; what is important is comparing the level of the immune cell subsets and / or immune functional molecules in the biological sample to be tested with the level of the immune cell subsets and / or immune functional molecules in a control sample. In other words, it is not necessary to measure the actual quantitative "level" of the immune cell subsets and / or immune functional molecules; it is preferable to measure the "high" or "low" level of the immune cell subsets and / or immune functional molecules and then compare the level of the immune cell subsets and / or immune functional molecules with the level of the immune cell subsets and / or immune functional molecules in a control sample.

[0175] All the instructions and preferences mentioned above concerning the measurement of the levels of the aforementioned immune cell subsets and / or immune function molecules are equally applicable to the measurement of levels in both the test sample and the reference sample.

[0176] The present invention also provides the use of measuring the proportion of immune cells and / or the expression level of immune functional molecules in biological samples in vitro or in vitro to distinguish whether esophageal cancer patients have lymph node metastasis in patients / subjects, wherein the patients / subjects are patients in hospitals, preferably patients diagnosed with esophageal cancer, more preferably patients diagnosed with esophageal squamous cell carcinoma, more preferably patients with enlarged lymph nodes, and even more preferably patients who are preparing to undergo radical esophagectomy.

[0177] Reagent test kit

[0178] Based on the correlation between immunobiomarkers of esophageal cancer lymph node metastasis and the risk of esophageal cancer lymph node metastasis, immunobiomarkers of esophageal cancer lymph node metastasis can be used as markers for assessing the risk of esophageal cancer lymph node metastasis.

[0179] This invention provides a kit for determining lymph node metastasis in esophageal cancer. The kit contains a detection reagent for detecting immunobiomarkers of lymph node metastasis in esophageal cancer. Preferably, the kit includes: binding molecules specific to each biomarker in the set, specific antibodies, specific amplification primers, specific probes or chips, isotopes, and enzymes. - The substrate complex, or combination thereof, may specifically be a specific polyclonal or monoclonal antibody, preferably a monoclonal antibody, or a fragment or derivative thereof. In a preferred embodiment, the biomarkers are detected or identified by one or more methods selected from the group consisting of: immunoprecipitation, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarrays, immunohistochemistry, dot blots, protein microarrays, tissue microarrays conjugated with immunohistochemistry, or other known routine immunological detection / analysis techniques.

[0180] In a preferred embodiment, the biomarkers are detected or identified using specific antibodies against the surface proteins of each biomarker.

[0181] In another preferred embodiment, the kit also includes a label or instructions.

[0182] In another preferred embodiment, the kit comprises:

[0183] - Specific tools or reagents for measuring the proportion of immune cells and / or the expression levels of immune functional molecules in the biological sample; and

[0184] -Auxiliary reagents for measuring the proportion of immune cells and / or the expression level of immune functional molecules in the biological sample; and / or

[0185] - Positive control samples, which are calibrated and represent the proportion of immune cells and / or the expression levels of immune function molecules in patients / subjects with esophageal squamous cell carcinoma and lymph node metastasis; and / or

[0186] - Negative control samples, which are calibrated and can represent the values ​​of immune cell subsets and / or expression levels of immune function molecules in patients with esophageal squamous cell carcinoma without lymph node metastasis / healthy subjects.

[0187] In particular, the kit of the present invention can be used to distinguish whether esophageal cancer patients have lymph node metastasis, wherein the patients / subjects are patients in hospitals, preferably patients diagnosed with esophageal squamous cell carcinoma, more preferably patients with enlarged lymph nodes, and even more preferably patients who are preparing to undergo radical esophagectomy.

[0188] Another positive control sample may also be a biological sample obtained from at least one patient / subject with esophageal squamous cell carcinoma known to have lymph node metastasis. Similarly, another negative control sample may also be a biological sample obtained from at least one patient or at least one healthy subject with esophageal squamous cell carcinoma known not to have lymph node metastasis.

[0189] Preferably, the kit contains a positive control sample and a negative control sample, and in particular, each sample is selected from calibration samples as defined above.

[0190] The main advantages of this invention include:

[0191] (a) This invention is the first discovery of a novel biomarker and its set, wherein the immune biomarker includes one or more of the following immune cell subsets:

[0192] (A) Any marker selected from group A, or a combination thereof: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A6) CD4 + T cells; (A7) central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 +T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells;

[0193] (B) Any marker selected from group B, or a combination thereof: (B1) CD11c MdFI in myeloid dendritic cells; (B2) CD11c MdFI in CD16 + Myeloid dendritic cells; (B3) CD11c MdFI in intermediate monocytes; (B4) CD38 MdFI in B cells; (B5) CD38 MdFI in CD27 + Memory B cells;

[0194] (C) A combination of at least one marker selected from group A and at least one marker selected from group B.

[0195] By detecting the proportion or function of the above-mentioned immunobiomarkers, they can be used for the diagnosis or risk assessment of esophageal cancer lymph node metastasis, and have very high predictive / assessment accuracy. Furthermore, the biomarker set of the present invention can also be used in combination with other indicators, and has a very good effect on the diagnosis or risk assessment of esophageal cancer lymph node metastasis.

[0196] (b) This invention can quickly and early determine the risk of lymph node metastasis in patients / subjects with esophageal squamous cell carcinoma and determine the appropriate surgical plan.

[0197] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0198] Detection methods

[0199] Unless otherwise specified, the cell subset ratios or cell proportions detected in the following examples refer to the corresponding cell subsets or the corresponding cells as a percentage of peripheral blood CD45. +Percentage of cell count. CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16 + Myeloid dendritic cells and CD11c MdFI in intermediate monocytes represent the roles of CD11c in myeloid dendritic cells and CD16, respectively. + Median fluorescence intensity (MdFI) of myeloid dendritic cells and intermediate monocytes. CD38 MdFI in B cells and CD38 MdFI in CD27 cells. + Memory B cells are CD38 in B cells and CD27 in B cells. + Median fluorescence intensity of memory B cells.

[0200] Example 1

[0201] 1.1 Sample Source

[0202] Peripheral blood samples were collected from the subjects, including 7 patients with esophageal squamous cell carcinoma without lymph node metastasis (recruited from the hospital's thoracic surgery department) and 16 patients with esophageal squamous cell carcinoma with lymph node metastasis (recruited from the hospital's thoracic surgery department). The patient samples were taken from the time the patients were diagnosed with esophageal squamous cell carcinoma and before undergoing radical esophagectomy.

[0203] 1.2 Detection Method

[0204] (1) Gently mix the peripheral blood whole blood sample and add 100 μL to the flow cytometer;

[0205] (2) Add fluorescently labeled anti-human CD45, CD3, CD14, CD15, CD16, CD11c, CD86, CD123, CD4, CD8, CD45RA, CCR7, HLADR, CD38, CD39, CXCR5, CD25, and CD127 antibodies to the flow cytometry tubes, respectively. The fluorescent labels are selected from Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, APC, APC / Cy7, APC / H7, Brilliant Violet 421, Brilliant Violet 510, Brilliant Blue 515, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 785, FITC, LEAF, and Pacific antibodies. One of Blue, PE, PE / Cy5, PE / Cy7, PE / Dazzle594, PerCP, and PerCP / Cy5.5. The fluorescence type can be freely combined according to the configuration of the laser and filters of the flow cytometer.

[0206] (3) Gently vortex the whole blood in the tube with the fluorescently labeled antibody and incubate at room temperature in the dark for 15 minutes.

[0207] (4) Add 2 mL of red blood cell lysis buffer to the flow cytometer, vortex to mix, and incubate at room temperature in the dark for 15 min.

[0208] (5) Centrifuge at 500g for 5 minutes at room temperature and discard the supernatant;

[0209] (6) Add 2 mL of PBS to the flow cytometer, vortex to mix, centrifuge at 500 g for 5 min at room temperature, and discard the supernatant;

[0210] (7) Add 250 μL of 1% PFA to the flow cytometer tube, vortex mix, and store at 4°C in the dark until detected by the flow cytometer.

[0211] 1.3 Results

[0212] As shown in Table 1 and Figure 1 As shown in AB. Table 1 presents the mean ± standard deviation (mean ± SD), fold change, and p-values ​​obtained by comparing various immune cell phenotypes that showed significant differences in different subjects (esophageal squamous cell carcinoma patients with or without lymph node metastasis) detected by flow cytometry, using Wilcoxon rank sum tests.- value result.

[0213] Figure 1 AB represents the box plot results obtained by comparing the immune cell phenotypes detected by the flow cytometer in different subjects (esophageal squamous cell carcinoma patients with or without lymph node metastasis) using Wilcoxon rank sum tests.

[0214] Among them, LN refers to esophageal squamous cell carcinoma patients without lymph node metastasis, while LMN refers to esophageal squamous cell carcinoma patients with lymph node metastasis.

[0215] Table 1

[0216]

[0217]

[0218] Note: Unless otherwise specified, the immune cells mentioned above refer to the corresponding cell subsets or the proportion of the corresponding cells in peripheral blood CD45. + Percentage of cells.

[0219] CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16+ myeloid dendritic cells, and CD11c MdFI in CD16+ intermediate monocytes are the median fluorescence intensities of CD11c in myeloid dendritic cells, CD16+ myeloid dendritic cells, and intermediate monocytes, respectively.

[0220] CD38 MdFI in B cells and CD38 MdFI in CD27 + Memory B cells are CD38 in B cells and CD27 in B cells. + Median fluorescence intensity of memory B cells.

[0221] This invention investigated the diagnostic ability of the immune cell phenotypes listed in Table 1 for esophageal cancer lymph node metastasis. The results are shown as AUC results for each indicator (see Table 2), and ROC curves are presented in the appendix. Figure 2 In AB.

[0222] Figure 2 AB showed that the immune cell phenotypes in Table 1 all had good diagnostic effects on lymph node metastasis, and could accurately distinguish between esophageal squamous cell carcinoma patients with lymph node metastasis and those without.

[0223] Among them, Tconv cells (i.e., Tconv cells account for 5% of peripheral blood CD45) + (percentage of cells), HLADR + CD38 + CD8 +T cells (HLADR) + CD38 + CD8 + T cells account for 45% of peripheral blood CD45 + Percentage of cells), intermediate monocytes (intermediate monocytes account for 5% of peripheral blood CD45) + The area under the ROC curve for single indicators such as the percentage of cells all exceeded 0.9.

[0224] Table 2 Diagnostic efficacy of individual indicators

[0225]

[0226]

[0227] Example 2

[0228] In Example 1, 7 patients with esophageal squamous cell carcinoma without lymph node metastasis and 16 patients with esophageal squamous cell carcinoma with lymph node metastasis were recruited from the thoracic surgery department of the hospital. The immune cell phenotypic markers from Table 2 were combined and a classification model was constructed using random forest. The predictive performance of the model was also evaluated using ROC.

[0229] The results showed that the above combination of immune cell phenotypes could demonstrate higher accuracy in distinguishing patients with lymph node metastasis from those without. The diagnostic efficacy of some indicator combinations is presented in Table 3, with the corresponding AUC results shown in parentheses, indicating the contribution of individual indicators during the random forest modeling process.

[0230] Table 3 Diagnostic efficacy of multi-indicator combinations

[0231]

[0232]

[0233] Example 3

[0234] To further demonstrate the effectiveness of the biomarker, three patients with esophageal squamous cell carcinoma and lymph node metastasis, and two patients with esophageal squamous cell carcinoma without lymph node metastasis (recruited from the hospital's thoracic surgery department) were collected separately as an external validation set for supplementary validation.

[0235] The diagnostic effectiveness of the immune cell phenotypes from Table 1 in the external validation set was evaluated based on the cutoff values ​​corresponding to each immune cell phenotype in Example 1 (see Table 2). The diagnostic effectiveness was assessed using accuracy, calculated using the following formula:

[0236] Accuracy=(TP+TN) / (TP+TN+FP+FN)

[0237] In the formula, TP represents the number of correctly classified positive examples (True Positives), TN represents the number of correctly classified negative examples (True Negatives), FP represents the number of incorrectly classified positive examples (False Positives), and FN represents the number of incorrectly classified negative examples (False Negatives).

[0238] The tests revealed that the aforementioned single immune cell subsets (or single immune cell markers) demonstrated very high accuracy in distinguishing patients with lymph node metastasis from those without in the external validation set, as shown in Table 4.

[0239] Except for childish CD8 + Apart from T cells, each individual indicator achieved an accuracy of 0.80 on the external validation set, which is sufficient for accurate diagnosis of patients with lymph node metastasis and those without.

[0240] Table 4. Diagnostic efficacy of individual indicators in the external validation set.

[0241] code name The immune biomarkers of the present invention Accuracy A1 Tfh cells 1.00 A2 Tfh1 cells 0.80 A3 <![CDATA[CD28 + CD3 + T cells 0.80 A4 <![CDATA[CD28 + CD8 + T cells 1.00 A5 Tconv cells 0.80 A6 CD4+ T cells 0.80 A7 <![CDATA[Central memory CD8 + T cells]]> 1.00 A8 <![CDATA[HLADR + CD38 + CD8 + T cells 0.80 A9 <![CDATA[CXCR5 + CD8 + T cells 0.80 A10 Intermediate monocytes 0.80 A11 <![CDATA[CD16 + Myeloid dendritic cells 0.80 A12 Th2 cells 0.80 A13 <![CDATA[HLADR - CD38 - CD3 + T cells 0.80 A14 <![CDATA[naive CD8 + T cell]]> 0.60 A15 plasmacytoid dendritic cells 0.80 B1 CD11c MdFI in myeloid dendritic cells 1.00 B2 <![CDATA[CD11c MdFI in CD16 + Myeloid dendritic cells 1.00 B3 CD11c MdFI in intermediate monocytes 1.00 B4 CD38 MdFI in B cells 0.80 B5 <![CDATA[CD38 MdFI in CD27 + Memory B cells 1.00

[0242] discuss

[0243] As can be seen from the AUC values ​​shown in the experimental results of the above embodiments, the biomarker of the present invention can effectively diagnose patients with esophageal squamous cell carcinoma and lymph node metastasis, and can effectively assess the risk of esophageal squamous cell carcinoma and lymph node metastasis, and has very good predictive / assessment accuracy.

[0244] It should be emphasized that, although the markers used in the above embodiments are immune cell subsets or the proportion of immune cells in peripheral blood CD45, + While cell proportions are used for computational modeling, it is generally understood by those skilled in the art that other cell types, such as the proportion of peripheral blood immune cells, or the proportion of their progenitor immune cells, or the proportion of their paternal immune cells, can also achieve the same or similar diagnostic / evaluation results.

[0245] The above demonstrates that peripheral blood immune biomarkers are significant in determining the risk of lymph node metastasis in esophageal squamous cell carcinoma, which is of great value for the clinical diagnosis and treatment of esophageal cancer.

[0246] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an immunobiomarker for esophageal cancer lymph node metastasis, or a reagent thereof, characterized in that, Used to prepare a diagnostic reagent or diagnostic kit for assessing the risk of esophageal cancer lymph node metastasis; The immunobiomarker is selected from any biomarker or combination thereof from group A; wherein group A consists of: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A7) Central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells; Wherein, the marker selected from group A, or any combination thereof, refers to the proportion of immune cells selected from any marker shown in A1-A5 and A7-A15 of group A, or any combination thereof, wherein the proportion of immune cells is the percentage of any marker selected from A1-A5 and A7-A15 of group A, or any combination thereof, on CD45. + Percentage of cells.

2. The use as described in claim 1, characterized in that, The detection reagent or test kit is used to detect the level of the marker in the sample to be tested.

3. The use as described in claim 2, characterized in that, The sample to be tested is a blood sample or a whole blood sample.

4. The use as described in claim 1, characterized in that, The detection reagent or kit is also used to detect CD45 in the sample to be tested. + At the cellular level.

5. The use as described in claim 1, characterized in that, The immune biomarkers include a combination of immune cell markers selected from the following group: (1)(A3)CD28 + CD3 + T cells and (A11)CD16 + Myeloid dendritic cells; (2)(A1)Tfh cells and (A13)HLADR - CD38 - CD3 + T cells; (3)(A2)Tfh cells and (A10) intermediate monocytes; (4)(A5) Tconv cells and (A10) intermediate monocytes; (5)(A1)Tfh cells and (A4)CD28 + CD8 + T cells; (6)(A1) Tfh cells and (A7) central memory CD8 + T cells; (7)(A1)Tfh cells and (A9)CXCR5 + CD8 + T cells; (8)(A4)CD28 + CD8 + T cells and (A9)CXCR5 + CD8 + T cells; (9)(A2)Tfh1 cells and (A8)HLADR + CD38 + CD8 + T cells; (10)(A2)Tfh1 cells and (A5)Tconv cells; (11)(A1)Tfh cells, (A8)HLADR + CD38 + CD8 + T cells and (A10) intermediate monocytes; (12)(A1) Tfh cells, (A7) central memory CD8 + T cells, and (A13)HLADR - CD38 - CD3 + T cells.

6. A reagent kit, characterized in that, The kit contains a detection reagent for detecting immunobiomarkers of esophageal cancer lymph node metastasis in the sample to be tested. The immunobiomarker for esophageal cancer lymph node metastasis is any biomarker selected from group A, or a combination thereof; wherein group A consists of the following: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28. + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A7) Central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells.

7. The kit according to claim 6, characterized in that, The detection reagent is also used to detect CD45 in the sample to be tested. + cell.

8. The kit according to claim 7, characterized in that, The detection reagent is used to detect the level or quantity of the immunobiomarkers for esophageal cancer lymph node metastasis in the test sample and / or the CD45 in the test sample. + The level or number of cells.

9. The reagent kit as described in claim 6, characterized in that, The kit also includes a label or instructions indicating that the kit is used to assess the risk of esophageal cancer lymph node metastasis.

10. The kit according to claim 6, characterized in that, The detection reagent is coupled with or carries a detectable marker, which is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

11. The kit as described in claim 6, characterized in that, The kit also includes a positive control sample.

12. The kit according to claim 6, characterized in that, The kit also includes a negative control sample.

13. A diagnostic device for esophageal cancer lymph node metastasis, characterized in that, The device includes: (a) An input module, the input module being configured to input immunobiomarker data of esophageal cancer lymph node metastasis from a blood sample of the subject to be tested; The immunobiomarker for esophageal cancer lymph node metastasis is any biomarker selected from group A, or a combination thereof; group A consists of the following: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28. + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A7) Central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells; (b) A processing module configured to determine the following for input immunobiomarkers: when an immunobiomarker is upregulated, an upregulated proportion of immune cells indicates a high risk of lymph node metastasis in the test subject; when an immunobiomarker is downregulated, a downregulated proportion of immune cells indicates a high risk of lymph node metastasis in the test subject; conversely, a downregulated proportion of immune cells indicates a low risk of lymph node metastasis in the test subject; and (c) An output module, the output module being configured to output the determination result; The proportion of immune cells refers to the relationship between marker levels and CD45. + The ratio at the cellular level.

14. A set of immunobiomarkers for esophageal cancer lymph node metastasis, characterized in that, The set of immunobiomarkers includes any biomarker selected from group A, or a combination thereof; wherein group A consists of: (A1) Tfh cells; (A2) Tfh1 cells; (A3) CD28 + CD3 + T cells; (A4)CD28 + CD8 + T cells; (A5) Tconv cells; (A7) Central memory CD8 + T cells; (A8)HLADR + CD38 + CD8 + T cells; (A9)CXCR5 + CD8 + T cells; (A10) intermediate monocytes; (A11) CD16 + Myeloid dendritic cells; (A12) Th2 cells; (A13) HLADR - CD38 - CD3 + T cells; (A14) immature CD8 + T cells; (A15) plasmacytoid dendritic cells.

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