Immune biomarker for auxiliary diagnosis of esophageal cancer lymph node metastasis and application thereof

Abstract

The invention provides an immune biomarker for auxiliary diagnosis of esophageal cancer lymph node metastasis and application of the immune biomarker. Specifically, the invention provides an immune biomarker for esophageal cancer lymph node metastasis and application of a detection reagent of the immune biomarker for esophageal cancer lymph node metastasis, and the immune biomarker is used for preparing a diagnostic reagent or a diagnostic kit for judging the occurrence risk of esophageal cancer lymph node metastasis. Researches show that the immune biomarker can be used as a marker for esophageal cancer lymph node metastasis, and has high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of clinical testing and diagnosis, and in particular, to an immune marker set for auxiliary diagnosis of esophageal cancer lymph node metastasis and an application thereof. Background Art

[0002] Esophageal cancer is a malignant tumor of the digestive tract that originates from the esophageal mucosal epithelium. Clinically, progressive dysphagia is a typical symptom of the advanced stage. The mortality rate of esophageal cancer remains high, seriously endangering the physical and mental health of the people. Lymph node metastasis is the most important metastasis mode of esophageal squamous cell carcinoma, usually involving the upper two-thirds of the esophagus. It has the characteristics of early stage, bidirectionality, skipping and regionality, and is a key factor affecting the prognosis of esophageal cancer patients.

[0003] Effective clearance of metastatic lymph nodes is the main surgical option for radical treatment of esophageal cancer. Early esophageal cancer can be treated with minimally invasive endoscopy, and suspected lymph node metastasis requires surgical operation. Advanced esophageal cancer is mainly treated with surgical operation, but when ≥2 lymph node metastases are found, radiotherapy and chemotherapy are required. At the same time, too extensive lymph node clearance may cause more postoperative complications. Therefore, clarifying the presence or absence of lymph node metastasis is helpful to formulate a reasonable treatment plan and improve the long-term prognosis of patients. At present, the diagnosis of lymph node metastasis in clinical practice is mainly based on imaging examinations (enhanced CT, color ultrasound or PET-CT) and relies on the doctor's experience, but its diagnostic value is controversial. It can be seen that the above diagnostic methods have a certain misdiagnosis rate for medical staff and testing personnel, lack of accurate and quantitative biomarkers, and it is difficult to make accurate preoperative judgments based on the patient's own characteristics, which can easily affect the patient's prognosis due to excessive clearance.

[0004] Therefore, timely and accurate preoperative diagnosis of esophageal cancer lymph node metastasis is a clinical problem that needs to be solved urgently.

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

[0006] The present invention provides a new method for timely and accurate preoperative diagnosis of esophageal cancer lymph node metastasis.

[0007] In a first aspect of the present invention, there is provided an immune biomarker of esophageal cancer lymph node metastasis, or a use of a detection reagent thereof, for preparing a diagnostic reagent or a diagnostic kit, wherein the diagnostic reagent or the diagnostic kit is used to determine the risk of occurrence of esophageal cancer lymph node metastasis;

[0008] Wherein, the immune biomarker is 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) naive 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, any 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 in group A, or a combination thereof, wherein the proportion of immune cells is any marker selected from A1 to A15 in group A, or a combination thereof, accounting for CD45 + The percentage of cells.

[0013] In another preferred embodiment, any marker selected from group A, or a combination thereof, refers to any cell or a combination thereof shown in A1 to A15 accounting for CD45 + The 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 CD11cCD16 + Median fluorescence intensity of myeloid dendritic cells.

[0016] In another preferred embodiment, the (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 on 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 detection kit is also used to detect CD45 in the sample to be tested. + Cellular level.

[0021] In another preferred embodiment, the immune biomarker comprises a combination of the following markers: (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 markers: (A1) Tfh cells; and (A13) HLADR - CD38 - CD3 + T cells.

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

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

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

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

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

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

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

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

[0031] In another preferred embodiment, the immune biomarker comprises a combination of the following markers: (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 markers: (A1) Tfh cells; (A8) HLADR + CD38 + CD8 + T cells; and (A10) intermediate monocytes.

[0033] In another preferred embodiment, the immune biomarker comprises a combination of the following markers: (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 detection kit is used to detect the level of the marker in a blood sample.

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

[0037] Table A

[0038] Code Immune biomarkers of the present invention Adjust up / down A1 Tfh cells Downward adjustment A2 Tfh1 cells Downward adjustment A3 <![CDATA[CD28 + CD3 + T cells]]> Downward adjustment A4 <![CDATA[CD28 + CD8 + T cells]]> Downward adjustment A5 Tconv cells Downward adjustment A6 CD4+ T cells Downward adjustment A7 <![CDATA[Central memory CD8 + T cell]]> Downward adjustment A8 <![CDATA[HLADR + CD38 + CD8 + T cells]]> Downward adjustment A9 <![CDATA[CXCR5 + CD8 + T cells]]> Downward adjustment A10 Intermediate monocytes Downward adjustment A11 <![CDATA[CD16 + Myeloid Dendritic Cells]]> Downward adjustment A12 Th2 cells Downward adjustment A13 <![CDATA[HLADR - CD38 - CD3 + T cells]]> Downward adjustment A14 <![CDATA[Naive CD8 + T cell]]> Downward adjustment A15 Plasmacytoid dendritic cells Downward adjustment B1 CD11c MdFI in myeloid dendritic cells Downward adjustment B2 <![CDATA[CD11c MdFI in CD16 + Myeloid Dendritic Cells]]> Downward adjustment B3 CD11c MdFI in intermediate monocytes Downward adjustment B4 CD38 MdFI in B cells Increase B5 <![CDATA[CD38 MdFI in CD27 + Memory B cells]]> Increase

[0039] In a second aspect of the present invention, a kit is provided, the kit comprising a detection reagent, the detection reagent being used to detect immune biomarkers of esophageal cancer lymph node metastasis in a sample to be tested;

[0040] Wherein, the immune biomarker of esophageal cancer lymph node metastasis is 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) naive 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 immune biomarker of esophageal cancer lymph node metastasis in the sample to be tested and / or the CD45 + The level or number of cells.

[0046] In another preferred embodiment, the kit further comprises a label or instructions, wherein the label or instructions indicate that the kit is used to determine the risk of occurrence of lymph node metastasis of esophageal cancer.

[0047] In another preferred embodiment, the detection of the immune biomarker includes detection by a method selected from the following group: immunoprecipitation, flow cytometry, protein blotting, ELISA, ELISPOT, antibody microarray, immunohistology, 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 for the immune biomarkers of esophageal cancer lymph node metastasis.

[0049] In another preferred embodiment, the detection is a detection of an in vitro sample.

[0050] In another preferred embodiment, the in vitro 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 label.

[0052] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

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

[0054] In another preferred embodiment, the detection of the immune biomarker of esophageal cancer lymph node metastasis is quantitatively detectable.

[0055] In another preferred embodiment, detecting the immune biomarker is determining the proportion of immune cells or determining the function of immune cells.

[0056] In another preferred embodiment, the ratio 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 percentage of immune cells in peripheral blood CD45 + The percentage of cells.

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

[0059] In another preferred embodiment, the kit further comprises a positive control sample.

[0060] In another preferred embodiment, the sample is calibrated to represent the immune cell ratio value and / or immune cell function value of a patient or subject with esophageal cancer lymph node metastasis.

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

[0062] In another preferred embodiment, the kit further comprises a negative control sample.

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

[0064] In another preferred example, the negative control sample is from one or more esophageal cancer or healthy subjects known to have no lymph node metastasis.

[0065] In a third aspect of the present 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 immune biomarker data of esophageal cancer lymph node metastasis of a blood sample of a subject to be tested;

[0067] Wherein, the immune biomarker of esophageal cancer lymph node metastasis is 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) naive 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, wherein the processing module is configured to make the following determinations on the input immune biomarkers: when a certain immune biomarker is an up-regulated marker, when its immune cell ratio is up-regulated or its immune cell function value is higher than a reference value, it indicates that the subject to be tested has a high risk of lymph node metastasis; when a certain immune biomarker is a down-regulated marker, when its immune cell ratio is down-regulated or its immune cell function value is lower than a reference value, it indicates that the subject to be tested has a high risk of lymph node metastasis; otherwise, it indicates that the subject to be tested has a low risk of lymph node metastasis; and

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

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

[0074] In another preferred example, when the immune biomarker is a marker 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 display, a printer, a tablet computer (PAD), or a smart phone.

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

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

[0078] In a fourth aspect of the present invention, a set of immune biomarkers for esophageal cancer lymph node metastasis is provided, wherein the immune biomarker set comprises markers selected from the following group:

[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) naive 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 one of the markers A1 to A15 in group A refers to any one of the cells shown in A1 to A15 accounting for CD45 + The 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 CD11cCD16 + Median fluorescence intensity of myeloid dendritic cells.

[0084] In another preferred embodiment, the (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 on CD27 + Median fluorescence intensity of memory B cells.

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

[0088] (a) providing a test sample, wherein the test sample is selected from a blood sample;

[0089] (b) detecting the level or intensity of immune biomarkers of esophageal cancer lymph node metastasis in the test sample, denoted as C1, detecting the level or intensity of CD45 + The level of cells is denoted as R1; and

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

[0091] Wherein, the immune biomarker of esophageal cancer lymph node metastasis is 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) naive 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 immune biomarkers of esophageal cancer lymph node metastasis of the detected subject meet the following conditions, it indicates that the subject has a high risk of esophageal cancer lymph node metastasis:

[0096] (1) When a certain marker is an upregulated marker in Table A in the test subject, and the immune cell ratio or immune cell function value of the marker 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 marker is a down-regulated marker in Table A in the test subject, and the immune cell ratio or immune cell function value of the marker 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 a marker selected from group A, the immune cell ratio refers to the marker level accounting for CD45 + When the immune biomarker is a marker 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 detection subject is a human.

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

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

[0102] In another preferred embodiment, the detection subject 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 the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 AB shows the results of the immune cell phenotype measurement detected by flow cytometry in the human samples in the embodiment of the present invention, and the results obtained after analysis by Wilcoxon rank sum tests, where LN is a patient with esophageal squamous cell carcinoma without lymph node metastasis, and LNM is a patient with esophageal squamous cell carcinoma with lymph node metastasis.

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

[0108] After extensive and in-depth research and a large number of screenings, the inventors have discovered for the first time a new immune biomarker for esophageal cancer lymph node metastasis. The immune biomarker includes markers A1 to A15 and B1 to B5. By detecting the immune cell ratio and / or immune cell function value of the marker, the risk of lymph node metastasis in esophageal cancer patients can be effectively determined. On this basis, the present invention was completed.

[0109] The experiments of the present invention show that the immune biomarkers of the present invention have a good effect in judging esophageal cancer lymph node metastasis when used alone or in combination.

[0110] the term

[0111] The terms used in the present invention have the meanings commonly understood by those of ordinary skill in the relevant art. However, in order to better understand the present invention, some definitions and explanations of related terms are as follows. It should be noted that the explanations of the terms are provided here only to enable those skilled in the art to better understand the present invention and are not intended to limit the present invention.

[0112] As used herein, the term "marker panel" refers to a collection of one, two, and / or more than two markers.

[0113] As used herein, the term "subject" refers to an animal, particularly a mammal, such as a primate, preferably a human.

[0114] As used herein, terms such as "a," "an," and "the" refer not only to the singular individual, but include the general class that may be used to describe a particular embodiment.

[0115] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. 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 term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0117] As used herein, "cell subset" refers to a collection of any cells in a cell population containing multiple characteristics and having certain common characteristics. With respect to cell subsets known by specific names in the art, the term can be used to refer to the specific cell subset, and any property (e.g., expression of cell surface markers) can also be recorded to refer to the specific cell subset.

[0118] As used herein, the term "relative amount" with respect to cells can be used interchangeably with "ratio".

[0119] As used herein, the term "sample" or "specimen" refers to a material that is specifically associated with a subject, from which specific information about the subject can be determined, calculated, or inferred. A sample can be composed in whole or in part of biological material from the subject. A sample can also be a material that has been in contact with the subject in a manner that allows testing of the sample to provide information about the subject. A sample can also be a material that has been in contact with other materials that are not from the subject, but that enable the first material to be subsequently tested to determine information about the subject, such as a cleaning fluid from a probe or scalpel. A sample can be a source of biological material other than that in contact with the subject, as long as a person skilled in the art can still determine information about the subject from the sample.

[0120] The sample is selected from a biological fluid such as blood or whole blood, "blood" or "whole blood" being used interchangeably in the present invention.

[0121] In all its embodiments, the method as described above is applied to a blood sample containing leukocytes (particularly at least containing 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 is composed of lymphocytes (B cells, T cells and NK cells), dendritic cells and monocytes, and is usually obtained by the Ficoll method well known to those skilled in the art. However, in order to minimize the operation of the sample and to maintain the physiological cell interactions between the different cell populations involved in the immune response, and to better reflect the complexity of the innate and adaptive immune responses of the patient / subject, it is preferred to directly use a whole blood sample (i.e., comprising all leukocytes, erythrocytes, platelets and plasma) collected by an intravenous route (e.g., by using a tube containing an anticoagulant).

[0122] As used herein, the term "expression" means that a cell surface marker (usually a protein) is finally presented on the cell surface after RNA transcription, protein translation and intracellular transport. The expression level or expression intensity of a cell surface marker can be measured by flow cytometry. Preferably, the cells whose expression level or expression intensity reaches or exceeds a specified level can be counted, and the "high" or "low" expression amount of a specific marker between two samples can be reflected by the "more" or "less" ratio of the number of cells. Preferably, the "high" or "low" expression amount of a specific marker between two samples is reflected by the "high" (Hi) or "low" (Low) intensity of the fluorescent signal.

[0123] As used herein, the term "reference value" or "reference amount" or "reference level" refers to the value (or amount, or level) of a parameter or biomarker that indicates the state of a subject with respect to a particular disease (or ailment, or condition). Suitable reference levels of parameters or biomarkers can be quantified, determined or measured by detecting the parameter / biomarker in several suitable reference subjects. Such reference levels can be adjusted according to a specific subject population. Reference values ​​or reference levels can be absolute values; relative values; values ​​with upper or lower limits; a range of values; average values; median values, mean values ​​or values ​​compared to a specific control or baseline value. Reference values ​​can be based on values ​​of individual samples, such as values ​​obtained from a sample from a tested subject, but at an earlier time point. Reference levels can be based on a large number of samples, such as a subject population from a chronological age-matched group, or based on a sample pool that includes or does not include the sample to be tested. Depending on the context, the reference level corresponds to the value of a parameter (or biomarker) quantified, or determined, or measured on a sample from a healthy reference subject; or to the average (mean) of the values ​​of a parameter (or biomarker) quantified, or 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 but at various time intervals; or to the average (or mean) of the values ​​of a parameter / biomarker 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, which mainly occurs in the epithelial cells or glands of the esophageal mucosa. According to the pathological type, esophageal cancer is mainly divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma. Clinical manifestations include dysphagia, chest pain, weight loss, etc.

[0126] Biomarkers of esophageal cancer metastasis

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

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

[0129] The immune biomarkers in the present invention include one or more of the following immune cell phenotypes. By detecting the ratio or function of the above immune biomarkers, it is used for the diagnosis or risk assessment of esophageal cancer lymph node metastasis.

[0130] The immune cell phenotype of group (A) in Table A of the present invention is selected from CD16 + Myeloid dendritic cells, intermediate monocytes, 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, naive CD8 + T cells.

[0131] The CD16 described in the present invention + Myeloid dendritic cells (CD16 + Myeloid dendritic cells, CD16 + mDCs are a type of myeloid dendritic cells, and their surface marker is CD3 - CD19 - CD56 - CD14 - HLADR + CD11c + CD123 - CD16 + , are a type of highly specialized 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 monocytes described in the present invention are a type of monocytes, and their surface marker expression characteristics are CD14 hi CD16 + , 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 the present invention have a surface marker expression characteristic of CD3 - CD19 - CD56 - CD14 - HLADR + CD11c - CD123 + , can produce large amounts 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 the present invention - CD38 - CD3 + T cells, whose surface marker expression characteristic is CD3 + HLADR - CD38 - T cells are in an unactivated or dormant state, but have potential immune response capabilities and can be activated at the right time. They play an important role in immune tolerance and surveillance.

[0135] The CD28 + 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 and clearing infections and tumors.

[0136] The Tconv cells described in the present invention have a surface marker expression characteristic of CD3 + CD4 + CD8 - CD25 - CD127 - &CD3 + CD4 + CD8 - CD25 - CD127 + &CD3 + CD4 + CD8 -CD25 + CD127 + , which are CD4 T cells that have not differentiated into regulatory T cells + T cell subsets are mainly responsible for recognizing foreign antigens and stimulating adaptive immune responses.

[0137] The Th2 cells described in the present invention have a surface marker expression characteristic of CD3 + CD4 + CD8 - CXCR3 - CCR4 + CCR6 - , mainly secreting 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 the present invention have a surface marker expression characteristic of CD3 + CD4 + CD8 - CXCR5 + , which mainly secrete IL-21 and IL-4, can be further divided into Tfh1 cells, Tfh2 cells, Tfh9 cells, Tfh17 cells and Tfh17 / Tfh1 cells. They can support the activation and differentiation of B cells and the production of antibodies, and play a key role in humoral immune response.

[0139] The Tfh1 cells described in the present invention have a surface marker expression characteristic of CD3 + CD4 + CD8 - CXCR5 + CXCR3 + CCR4 - CCR6 - , which 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 the germinal center.

[0140] The HLADR described in the present invention + CD38 + CD8 + T cells, whose surface marker expression characteristic is CD3 + 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 perforins and granzymes.

[0141] The central memory CD8 + T cells, whose surface marker expression characteristic is CD3 + CD8 + CD4 - CD45RA - CCR7 + , is a type of long-lived memory T cell that can rapidly proliferate and differentiate into effector CD8 T cells when encountering antigens again. + T cells exert potent cytotoxic effects to eliminate virus-infected cells or tumor cells.

[0142] The CXCR5 + CD8 + T cells, whose surface marker expression characteristic is CD3 + 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 is very important in promoting B cell activation and antibody production, regulating immune responses and maintaining immune memory.

[0143] The naive CD8 + T cells, whose surface marker expression characteristic is CD3 + CD8 + CD4 - CD45RA + CCR7 + CD95 - , mainly reside in secondary lymphoid organs such as lymph nodes, and when they encounter antigen presentation for the first time, they differentiate into effector CD8 + T cells or memory CD8 + T cells are involved in specific immune responses.

[0144] Unless otherwise specified, any marker of group A in Table A, or a combination thereof, refers to: the proportion of immune cells selected from any marker shown in A1 to A15 in group A, or a combination thereof, wherein the proportion of immune cells is any marker selected from any marker shown in A1 to A15 in group A, or a combination thereof, accounting for CD45 + The percentage of cells.

[0145] The immune cell phenotype of group (B) in Table A of the present invention is composed of the median fluorescence intensity (MdFI) of three types of CD molecules, CD11c and CD38, on immune cell subsets. The 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 of the target molecule on the cell.

[0146] CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16 + Myeloid dendritic cells and CD11c MdFI in intermediate monocytes are CD11c in myeloid dendritic cells, CD16 + Median fluorescence intensity of myeloid dendritic cells and intermediate monocytes. CD11c is an integrin protein that is commonly used as a marker for 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 on B cells and CD27 + Median fluorescence intensity of memory B cells. CD38 is a transmembrane protein with enzymatic activity that participates in NAD+ metabolism, signal transduction, and cell adhesion, and plays an important role in a variety of immune cells. CD38 is expressed differently in B cells at different developmental stages and is usually highly expressed in activated B cells and plasma cells. It is an important molecule that regulates B cell maturation, antibody production, and immune memory. CD27 + The expression of CD38 in memory B cells may be associated with activation status and immune memory.

[0148] As used herein, "Median fluorescence intensity (MdFI)" is a quantitative indicator of the amount of monoclonal antibody fluorescein bound to cells or particles detected by flow cytometry. Under appropriate conditions, fluorescence intensity is related to the number of sites where a cell or particle binds to a specific fluorescein. That is, the stronger the fluorescein bound to the cell or particle, the more target marker molecules there are, and vice versa. The stronger the fluorescence intensity, the larger the channel value of the fluorescence signal, and vice versa. The median fluorescence intensity represents the median of these fluorescence signal intensities, reflecting the expression level of the target molecule in the cell.

[0149] The "+" marked in the present invention indicates that the marker is positively expressed, "-" indicates that the marker is negatively expressed (not expressed), "hi" indicates that the marker is expressed and the expression is high, and "Low" indicates that the marker is expressed but the expression is low. These are labeling methods known to conventional technicians in the field.

[0150] The "immune cell ratio" or "immune cell subset ratio" mentioned in the present invention can be calculated in a variety of different forms, which can be any of the following:

[0151] ① The percentage of the immune cell subpopulation in the number of immune cells in peripheral blood;

[0152] ②The immune cell subsets account for CD45 + The percentage of cells;

[0153] ③The percentage of the immune cell subpopulation in the number of its progenitor immune cells;

[0154] ④The percentage of the immune cell subpopulation to the immune cells of its parent group,

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

[0156] In the present invention, the "parent group immune cells" are the upper level immune cells according to the source of immune cell differentiation, or the upper layer immune cells according to the logical order of flow cytometry gates.

[0157] In the present invention, the "progenitor immune cells" are the upper two levels of immune cells based on the source of immune cell differentiation, or the upper two layers of immune cells based on the logical order of flow cytometry gates.

[0158] The immune function molecule expression level is the expression level of the immune function molecule in the immune cell subpopulation, which is referred to as "median fluorescence intensity" in the present invention.

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

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

[0161] Detection Methods

[0162] Based on the differences in immune biomarkers of esophageal cancer lymph node metastasis in blood samples, the present invention provides a corresponding method for determining the risk of esophageal cancer lymph node metastasis.

[0163] The method of the present invention is an in vitro or ex vivo method. For example, compared with etiological detection and SOFA score, the present invention has the advantage of being able to easily diagnose esophageal cancer lymph node metastasis or assess the risk of lymph node metastasis by providing a directly measurable marker, especially the risk of patients who are ready for radical esophagectomy or diagnosed with esophageal squamous cell carcinoma, wherein the measurement can be performed in a laboratory near the patient or at the patient's side. The measurement of the marker is fully suitable for being performed by an automated analysis machine or by a test method called a rapid test.

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

[0165] The test sample is obtained from a biological sample of a patient / subject for whom it is desired to determine the risk of developing lymph node metastasis of esophageal cancer.

[0166] In particular, the test sample is selected from biological fluids such as blood, whole blood, preferably, in all its embodiments, the method as described above is applied to a blood sample containing leukocytes (especially 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 usually obtained by the Ficoll method well known to those skilled in the art. However, in order to minimize the manipulation of the sample and maintain the physiological cell interactions between the different cell populations involved in the immune response, and to better reflect the complexity of the innate and adaptive immune responses of the patient / subject, it is preferred to directly use a whole blood sample (i.e., containing all white blood cells, red blood cells, platelets and plasma) collected by the intravenous route (e.g., by using a tube containing an anticoagulant).

[0167] Any volume commonly used by those skilled in the art for hematological analysis will be convenient for the present method. For example, the volume of the biological sample 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 sample from which reference value can be determined is also referred to as "control sample", and in particular, in order to obtain reference value, these samples are preferably obtained from people with identical features or most common features, especially the same sex and / or similar or identical age and / or ethnic origin as the experimenter or patient / experimenter of the risk of lymph node metastasis determined by expectation. In this case, reference sample can also be formed by any sample, and no matter whether it is a biological sample, the sample was calibrated to contain average immune cell ratio value and / or immune function molecule expression level value before, which corresponds to the level measured in the set of the sample taken from the following patient / experimenter, and the patient / experimenter is a patient known to have suffered from lymph node metastasis. In this case and according to a particularly preferred variant, the reference sample is taken from one or more patients / experiments known to have suffered from lymph node metastasis.

[0169] In the context of the present invention, the terms "detection" or "measurement" or "determination" are used interchangeably and have the same meaning. These terms can represent the detection and quantification of the immunocyte ratio value, and can also represent the detection and quantification of the immune function molecule expression level value (the detection and quantification of the immune function molecule at the protein level are referred to as "median fluorescence intensity" in the present invention). For this reason, any detection and / or quantitative method known to those skilled in the art can be adopted for implementation of the present invention.

[0170] In particular, the determination of immune cell ratio values ​​and / or immune function molecule expression level values ​​(detection and quantification of the functional molecules at the protein level) is carried out using immune cell and / or immune function molecule expression level-specific tools or reagents that allow direct or indirect determination of their presence and / or quantification of their expression levels.

[0171] Among the tools or reagents capable of detecting and / or quantifying the immune cell ratio value and / or immune function molecule expression level value, specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, or fragments or derivatives thereof may be mentioned.

[0172] In the method of the present invention, in particular, well-known analytical techniques can be used to detect and / or quantify the immune cell ratio value and / or the expression level of immune function molecules (immune cell function value), such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, flow cytometry, Western blotting, ELISA, ELISPOT, antibody microarray, immunoprecipitation, immunohistology, dot blot, protein microarray, or tissue microarray 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 amperometry techniques), atomic force microscopy and radio frequency methods, such as multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectivity, transmittance and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, resonant mirror method, grating coupled waveguide method or interference method), cell ELISA, radioisotope, magnetic resonance imaging, polyacrylamide gel electrophoresis (SDSPAGE) analysis; HPLC-mass spectrometry; liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS). For example, when flow cytometry is used, forward scatter and side scatter information help identify mononuclear cell populations in other blood cells. Preferably, immune cell ratio values ​​and / or immune function molecule expression levels are identified, selected, sorted, and quantified (and any combination thereof) by flow cytometry.

[0173] In the present invention, the immune cell subpopulations and / or immune function molecules (CD38, CD11c) are preferably measured by, for example, flow cytometry to determine whether the ratio of these immune cells and / or the expression level of these immune function molecules is "high" or "low".

[0174] Alternatively, for the immune cell subpopulations and / or immune function molecules (CD38, CD11c), the "high" or "low" amount of these immune cell subpopulations and / or immune function molecules at high levels can be measured by, for example, flow cytometry to measure the "high" or "low" amount of these immune cell subpopulations and / or immune function molecules at high levels, to measure the "high" or "low" level of the immune cell subpopulations and / or immune function molecules. The exact amount of the surface immune cell subpopulations and / or immune function molecules is not important, what is important is to compare the level of the immune cell subpopulations and / or immune function molecules in the biological sample to be detected with the level of the immune cell subpopulations and / or immune function molecules in the control sample. In other words, it is not necessary to measure the actual quantitative "level" of the immune cell subpopulations and / or immune function molecules, and it is preferred to measure the "high" or "low" level of the immune cell subpopulations and / or immune function molecules, and then compare the level of the immune cell subpopulations and / or immune function molecules with the level of the immune cell subpopulations and / or immune function molecules in the control sample.

[0175] All indications and preferences above concerning measuring the levels of said immune cell subpopulations and / or immune function molecules apply equally to measuring the levels in a test sample and in a reference sample.

[0176] The present invention also provides the use of measuring the ratio of immune cells and / or the expression level of immune function molecules in a biological sample in vitro or ex vivo to distinguish whether or not esophageal cancer patients have lymph node metastasis in patients / subjects. The patients / subjects are patients in a hospital, preferably patients diagnosed with esophageal cancer, preferably patients diagnosed with esophageal squamous cell carcinoma, more preferably patients with enlarged lymph nodes, and more preferably patients who are preparing to undergo radical surgery for esophageal cancer.

[0177] Reagent test kit

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

[0179] The present invention provides a kit for determining esophageal cancer lymph node metastasis, the kit comprising a detection reagent, the detection reagent is used to detect immune biomarkers of esophageal cancer lymph node metastasis. Preferably, the kit includes: binding molecules, specific antibodies, specific amplification primers, specific probes or chips, isotopes, enzymes, etc. that are specific to each biomarker in the set. - Substrate complex, or a combination thereof, specifically, specific polyclonal or monoclonal antibodies, preferably monoclonal antibodies, or fragments or derivatives thereof can be mentioned. In a preferred embodiment, each biomarker is detected or identified by one or more methods selected from the group consisting of immunoprecipitation, flow cytometry, Western blot, ELISA, ELISPOT, antibody microarray, immunohistology, dot blot, protein microarray, tissue microarray coupled with immunohistochemistry, or other well-known immunological conventional detection / analysis techniques.

[0180] In a preferred embodiment, each biomarker is detected or identified by specific antibodies against its surface protein.

[0181] In another preferred embodiment, the kit further comprises 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 level of immune function molecules in the biological sample; and

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

[0185] - A positive control sample, which is calibrated and can represent the immune cell ratio value and / or immune function molecule expression level value of an esophageal squamous cell carcinoma patient / subject with lymph node metastasis; and / or

[0186] - Negative control sample, which is calibrated and can represent the immune cell subset values ​​and / or immune function molecule expression level values ​​of esophageal squamous cell carcinoma patients / healthy subjects without lymph node metastasis.

[0187] In particular, the kit of the present invention can be used to distinguish whether esophageal cancer patients have lymph node metastasis or not. The patients / subjects are patients in the hospital, preferably patients diagnosed with esophageal squamous cell carcinoma, more preferably patients with enlarged lymph nodes, and more preferably patients who are preparing to undergo radical surgery for esophageal cancer.

[0188] Another positive control sample can also be the biological sample obtained from at least one patient / subject of esophageal cancer squamous cell carcinoma known to have lymph node metastasis.Similarly, another negative control sample can also be the biological sample obtained from at least one patient of esophageal cancer squamous cell carcinoma known to have no lymph node metastasis or at least one healthy subject.

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

[0190] The main advantages of the present invention include:

[0191] (a) The present invention discovers for the first time a new biomarker and a set thereof, 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) naive 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 immune biomarkers, it can be used for the diagnosis or risk assessment of esophageal cancer lymph node metastasis with very high prediction / assessment accuracy. The biomarker set of the present invention can also be used in combination with other indicators, which has a very good effect on the diagnosis or risk assessment of esophageal cancer lymph node metastasis.

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

[0197] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0198] Detection Methods

[0199] The cell subpopulation ratio or cell ratio detected in the following examples, unless otherwise specified, refers to the corresponding cell subpopulation or corresponding cell ratio in peripheral blood CD45 +The percentage of cells. CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16 + Myeloid dendritic cells and CD11c MdFI in intermediate monocytes are CD11c in myeloid dendritic cells, CD16 + Median fluorescence intensity (MdFI) of myeloid dendritic cells and intermediate monocytes. CD38 MdFI in B cells and CD38 MdFI in CD27 + Memory B cells are CD38 on B cells and CD27 + Median fluorescence intensity of memory B cells.

[0200] Example 1

[0201] 1.1 Sample Source

[0202] Peripheral blood samples were collected from subjects, including 7 patients with esophageal squamous cell carcinoma without lymph node metastasis (recruited from the Department of Thoracic Surgery of the Hospital) and 16 patients with esophageal squamous cell carcinoma with lymph node metastasis (recruited from the Department of Thoracic Surgery of the Hospital). The patient samples were taken from patients before radical esophagectomy after diagnosis of esophageal squamous cell carcinoma.

[0203] 1.2 Detection Method

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

[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 tube, respectively. The fluorescent marker is 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, BrilliantViolet 711, Brilliant Violet 785, FITC, LEAF, Pacific One of Blue, PE, PE / Cy5, PE / Cy7, PE / Dazzle594, PerCP and PerCP / Cy5.5. The fluorescent species can be freely combined according to the configuration of the laser and filter of the flow cytometer.

[0206] (3) Gently vortex the whole blood and fluorescent-labeled antibody in the tube and incubate at room temperature in the dark for 15 min;

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

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

[0209] (6) Add 2 mL of PBS to the flow tube, 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 cytometry tube, vortex to mix, and store at 4°C in the dark until detected by flow cytometry.

[0211] 1.3 Results

[0212] As shown in Table 1 and Figure 1 Table 1 shows the mean ± standard deviation (mean ± SD), fold change of various immune cell phenotypes detected by flow cytometry that are significantly different in different subjects (ESCC patients with or without lymph node metastasis), and P values ​​obtained by comparison using Wilcoxon rank sum tests.- value result.

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

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

[0215] Table 1

[0216]

[0217]

[0218] Note: Unless otherwise specified, the above immune cells refer to the corresponding cell subsets or the corresponding cells accounting for the percentage of peripheral blood CD45 + The percentage of cells.

[0219] CD11c MdFI in myeloid dendritic cells, CD11c MdFI in CD16+ myeloid dendritic cells and CD11cMdFI 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 on B cells and CD27 + Median fluorescence intensity of memory B cells.

[0221] The present invention studies the diagnostic ability of the immune cell phenotype in Table 1 for esophageal cancer lymph node metastasis. The results are shown in the form of AUC results of each indicator (see Table 2), and ROC curves are shown in the attached figure. Figure 2 In AB.

[0222] Figure 2 AB showed that the immune cell phenotypes in Table 1 all had good diagnostic effects for lymph node metastasis and could accurately distinguish ESCC patients with lymph node metastasis from those without lymph node metastasis.

[0223] Among them, Tconv cells (i.e., Tconv cells account for the proportion of peripheral blood CD45 + percentage of cells), HLADR + CD38 + CD8 +T cells (HLADR + CD38 + CD8 + T cells account for CD45 + cells), intermediate monocytes (intermediate monocytes accounted for the percentage of peripheral blood CD45 + The areas under the ROC curves of single indicators such as the percentage of cells were all over 0.9.

[0224] Table 2 Diagnostic effect 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 Department of Thoracic Surgery of a hospital. The immune cell phenotype markers from Table 2 were combined and a classification model was constructed using random forests. ROC was also used to evaluate the prediction effect of the model.

[0229] The results showed that the above immune cell phenotypes can be combined to show higher accuracy in distinguishing patients with lymph node metastasis from those without lymph node metastasis. The diagnostic effects of some indicator combinations are randomly displayed, and the corresponding AUC results are shown in Table 3. The contribution of a single indicator in the random forest modeling process is shown in brackets.

[0230] Table 3 Diagnostic effect of multiple index combinations

[0231]

[0232]

[0233] Example 3

[0234] To further prove the effectiveness of the marker, three patients with esophageal squamous cell carcinoma with 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 as external validation sets for supplementary proof.

[0235] In the external validation set, the diagnostic effect of the immune cell phenotypes in Table 1 in the external validation set was diagnosed based on the Cutoff corresponding to each immune cell phenotype in Example 1 (see Table 2). The diagnostic effect was judged by accuracy, and the specific calculation formula was:

[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 test found that the above-mentioned single immune cell subsets (or single immune cell markers) can show very high accuracy in the external validation set to distinguish patients with lymph node metastasis from patients without lymph node metastasis. The results are shown in Table 4.

[0239] Removal of naive CD8 + Except for T cells, the accuracy of each single indicator on the external validation set reached 0.80, which can almost accurately diagnose patients with and without lymph node metastasis.

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

[0241] Code 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] From the AUC values ​​shown in the experimental results of the above embodiments, it can be seen that the markers of the present invention can effectively diagnose patients with esophageal squamous cell carcinoma lymph node metastasis, and can effectively assess the risk of esophageal squamous cell carcinoma lymph node metastasis, and have very good prediction / assessment accuracy.

[0244] It should be emphasized that although the markers used in the above embodiments are immune cell subsets or immune cells accounting for peripheral blood CD45 + However, according to the common knowledge of technicians in this field, the proportion of other types of cells, such as the proportion of peripheral blood immune cells, the proportion of their progenitor immune cells, or the proportion of their parent immune cells, can also achieve the same or similar diagnostic / evaluation effects.

[0245] The above shows that peripheral blood immune biomarkers are meaningful 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 the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An immune biomarker for esophageal cancer lymph node metastasis, or use of a detection reagent thereof, characterized in that: Used for preparing a diagnostic reagent or a diagnostic kit, wherein the diagnostic reagent or the diagnostic kit is used for judging the risk of occurrence of lymph node metastasis of esophageal cancer; Wherein, the immune biomarker is selected from the following group: (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) naive CD8 + T cells; (A15) plasmacytoid dendritic cells; (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; (C) A combination of at least one marker selected from Group A and at least one marker selected from Group B.

2. The use according to claim 1, characterized in that The any 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 in group A, or a combination thereof, wherein the proportion of immune cells is any marker selected from A1 to A15 in group A, or a combination thereof, accounting for CD45 + The percentage of cells.

3. The use according to claim 1, characterized in that The immune biomarkers include a combination of immune cell markers selected from the group consisting of: (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; (A9) CXCR5 + CD8 + T cells; (8)(A4)CD28 + CD8 + T cells; (A9) CXCR5 + CD8 + T cells; (9) (A2) Tfh1 cells; (A8) HLADR + CD38 + CD8 + T cells; (10) (A2) Tfh1 cells; (A5) Tconv cells; (11)(A7) Central Memory CD8 + T cells; (A11) CD16 + myeloid dendritic cells; and (B4) CD38 MdFI in B cells; (12) (A1) Tfh cells; (A8) HLADR + CD38 + CD8 + T cells; and (A10) intermediate monocytes; (13) (A1) Tfh cells; (A7) central memory CD8 + T cells; and (A13) HLADR - CD38 - CD3 + T cells.

4. A kit, characterized in that: The kit contains a detection reagent, which is used to detect immune biomarkers of esophageal cancer lymph node metastasis in the sample to be tested; Wherein, the immune biomarker of esophageal cancer lymph node metastasis is selected from the following group: (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) naive CD8 + T cells; (A15) plasmacytoid dendritic cells; (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; (C) A combination of at least one marker selected from Group A and at least one marker selected from Group B.

5. The kit according to claim 4, characterized in that The detection reagent is also used to detect CD45 in the sample to be tested + cell.

6. The kit according to claim 4, characterized in that The kit also contains a label or instructions, which indicates that the kit is used to determine the risk of esophageal cancer lymph node metastasis.

7. The kit according to claim 4, characterized in that The detection reagent is coupled with or carries a detectable label, and the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

8. A diagnostic device for esophageal cancer lymph node metastasis, characterized in that: The device comprises: (a) an input module, the input module being configured to input immune biomarker data of esophageal cancer lymph node metastasis of a blood sample of a subject to be tested; Wherein, the immune biomarker of esophageal cancer lymph node metastasis is selected from the following group: (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) naive CD8 + T cells; (A15) plasmacytoid dendritic cells; (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; (C) a combination of at least one marker selected from Group A and at least one marker selected from Group B; (b) a processing module, wherein the processing module is configured to make the following determinations on the input immune biomarkers: when a certain immune biomarker is an up-regulated marker, when its immune cell ratio is up-regulated or its immune cell function value is higher than a reference value, it indicates that the subject to be tested has a high risk of lymph node metastasis; when a certain immune biomarker is a down-regulated marker, when its immune cell ratio is down-regulated or its immune cell function value is lower than a reference value, it indicates that the subject to be tested has a high risk of lymph node metastasis; otherwise, it indicates that the subject to be tested has a low risk of lymph node metastasis; and (c) an output module, wherein the output module is configured to output the determination result.

9. The device according to claim 8, characterized in that When the immune biomarker is a marker selected from Group A, the immune cell ratio refers to the marker level and CD45 + When the immune biomarker is a marker selected from group B, the immune cell function value refers to the median fluorescence intensity (MdFI) of CD11c or CD38 on immune cells.

10. An immune biomarker set for esophageal cancer lymph node metastasis, characterized in that: The immune biomarker panel includes markers selected from the group consisting of: (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) naive CD8 + T cells; (A15) plasmacytoid dendritic cells; and / or (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.

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