Molecular markers and their application in the detection of angioimmunoblastic T-cell lymphoma

Abstract

The present invention discloses a molecular marker and its application in detecting angioimmunoblastic T-cell lymphoma, belonging to the technical field related to blood disease detection. It includes a combination of multiple markers designed by the present invention, which is applied to flow cytometry detection of angioimmunoblastic T-cell lymphoma and / or monitoring of micro-residual lesions. The molecular markers designed by the present invention can be widely used in patients' bone marrow, peripheral blood, lymph nodes, punctures, skin tissues, pleural effusions, peritoneal effusions and other living cell samples. By adopting a combination of multiple markers for effective gating, abnormal cells of AITL can be quickly screened out at an early stage, with a sensitivity and specificity of 100% for tissue samples, and a small amount of cells are used, and the operation is fast and simple. At the same time, for patients undergoing chimeric antigen receptor T cell therapy, the composition of the present invention can also evaluate the immunofluorescence intensity of antigen targets, which is an indispensable part of cell therapy, and the subsequent residual monitoring is not affected by the treatment.

Description

Technical Field

[0001] The present invention relates to a useful marker for disease diagnosis, belonging to the technical field of blood disease detection, and specifically to a molecular marker and its application in detecting angioimmunoblastic T-cell lymphoma. Background Art

[0002] Angioimmunoblastic T-cell lymphoma (AITL) is a type of peripheral T-cell lymphoma, accounting for about 20% of peripheral T-cell lymphomas. Its long-term prognosis is poor, with a 5-year survival rate of about 30-40%. Pathologically, the most typical changes are follicular structure destruction, high endothelial venous hyperplasia and dendritic cell hyperplasia. However, since the abnormal cells in AITL are often very low in proportion and the background cells are rich, mostly a large number of normal T lymphocytes, B lymphocytes, immunoblasts, eosinophils or scattered RS cells, it is sometimes difficult to distinguish AITL from reactive hyperplasia, Hodgkin lymphoma, diffuse large B-cell lymphoma, etc. only through pathology and immunohistochemistry. Therefore, in the early stages of the disease or when typical morphological features are lacking, multiple pathological biopsies are often required for the final diagnosis. In addition, a small number of patients cannot undergo lymph node examination due to immune cytopenia. All of the above make it difficult to diagnose the disease in the early stage.

[0003] Both the WHO and NCCN guidelines recommend the CHOP regimen as the first-line treatment for AITL, but this regimen still has a high recurrence rate. At present, a new treatment regimen is urgently needed to improve the efficacy. With the continuous emergence of new therapies such as targeted therapy and immunotherapy, cell immunotherapy has become a new research direction. Chimeric antigen receptor T cells (Chimeric-Antigen-Receptors-T-cell, CART) achieve specific recognition of tumor-associated antigens through genetically modified T cells, so that effector T cells can fully exert their anti-tumor effects. It is the focus of current cell immunotherapy and was first used in the treatment of acute B lymphocytic leukemia. With the further development of CART therapy, it was found that anti-CD5 CART cells were also used in relapsed and refractory T-cell lymphoma. In fact, before CART treatment, the antigen expression density of the targeted antigen molecules on the surface of abnormal cells needs to be evaluated. If the antigen density is too low, the target cells, i.e., tumor cells, cannot be effectively killed, and it may cause off-target, so the detection of antigen expression density is very important. Using normal CD4+T lymphocytes of AITL patients as internal controls and comparing them with the average fluorescence intensity of target antigens on the surface of tumor cells can reflect the expression of target antigens in tumor cells to a certain extent. This is a relative quantitative test, which is simple and fast. Combined with the PEPhycoerythrin Fluorescence Quantitation Kit, the antigen expression density of tumor cells can be absolutely quantitatively detected, further reducing the impact of instrument status, cell size, reagents and operations, etc. However, the market price of this kit is not cheap.

[0004] Accurately finding tumor cells and detecting the average fluorescence intensity and antigen expression density of tumor cell target antigens can currently only be done through flow cytometry.

[0005] Multiparameter flow cytometry uses fluorescent antibodies to label cells, which can achieve quantitative analysis of single cells. It has high sensitivity and is one of the most advanced cell quantitative analysis methods. It has been widely used in the diagnosis and follow-up of leukemia and lymphoma. However, multiparameter flow cytometry currently only uses pan-T markers for the initial screening of angioimmunoblastic T-cell lymphoma. Pan-T markers include cell membrane CD2, CD3, CD5, CD7, CD4, CD8, etc. Typical CD3-CD4+T lymphocytes are easily detected, but there are also a few patients with normal or slightly low CD3 intensity, which often leads to missed diagnosis. For bone marrow samples, the expression of CD7 on T lymphocytes is heterogeneous, and reactive T lymphocytes can also show the loss of CD7, so it is impossible to distinguish tumor cells from polyclonal reactive T lymphocytes. Lymphocyte changes caused by increased eosinophils can also result in the appearance of CD3-CD4+ cells.

[0006] In summary, the existing multi-parameter flow cytometry has the following problems: 1. It is not suitable for patients without pan-T marker expression deficiency, and such patients often cannot find abnormal cells through conventional gating methods. 2. If the abnormal cells only express membrane CD3 with a slightly weaker expression intensity and CD7 expression is normal, it is often easy for inexperienced personnel to miss the diagnosis. 3. The current initial screening methods cannot determine the clonality of T lymphocytes and cannot identify benign and malignant CD4+CD7- cell populations. 4. The use of TCR Vβ for T cell clonality identification requires a large amount of samples, is costly, and is time-consuming and laborious to operate. 5. For punctures or bone marrow samples with a small amount of cells, the type of tumor cells cannot be determined at one time. 6. If abnormal cells need to use multiple pan-T markers for gating, the cost of subsequent additions will be high. 7. If tumor cells cannot be targeted, the average fluorescence intensity of the antigen cannot be evaluated, which will affect subsequent treatment. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a molecular marker and its application in detecting angioimmunoblastic T-cell lymphoma. The molecular marker can not only realize the early screening of AITL, but also has the advantages of fast, simple and economical operation, and can also evaluate the immunofluorescence intensity of the target antigen, thereby guiding the treatment.

[0008] To achieve the above-mentioned purpose, the present invention discloses a molecular marker, which includes CD4 antibody, CD7 antibody, CD45 antibody, TRBC 1 antibody, CD5 antibody, CD3 antibody, CD10 antibody, PD1 antibody and CD8 antibody. All antibodies designed and adopted in the present invention are cell membrane antibodies, and the sample loss is small. At the same time, due to the use of a combination of multiple markers to set the gate, compared with the traditional antibody combination, it can avoid missed diagnosis caused by normal or slightly weak CD3 expression intensity, which helps to improve the sensitivity of diagnosis; and the molecular marker is mainly used to detect angioimmunoblastic T-cell lymphoma and / or bone marrow micro-residual lesions and / or T-cell lymphoma with pan-T antigen loss.

[0009] Furthermore, each of the antibodies is a monoclonal antibody.

[0010] Furthermore, each of the antibodies is a fluorescein-labeled antibody, and the CD4 antibody is labeled with BV605, the CD7 antibody is labeled with V450, the CD45 antibody is labeled with V500, the TRBC 1 antibody is labeled with FITC, the CD5 antibody is labeled with PE, the CD3 antibody is labeled with PerCP-cy5.5, the CD10 antibody is labeled with PE-Cy7, the PD1 antibody is labeled with APC, and the CD8 antibody is labeled with APC-Cy7.

[0011] Furthermore, the molecular marker is a mixture of CD4 antibody, CD7 antibody, CD45 antibody, TRBC1 antibody, CD5 antibody and CD3 antibody, CD10 antibody, PD1 antibody and CD8 antibody in a volume ratio of 2:2:3:5:5:6:2:3:2.

[0012] In addition, the present invention also discloses a kit, which includes the above-mentioned molecular marker and a storage component for storing the molecular marker. The storage component here is a conventional storage component. And the kit is mainly used to detect angioimmunoblastic T-cell lymphoma and / or bone marrow micro-residual lesions and / or T-cell lymphoma with pan-T antigen loss.

[0013] Furthermore, the kit also includes hemolysin, buffer, and flow tubes used in conjunction with a flow cytometer. The buffer may be PBS buffer or other buffer. The kit is mainly used to detect angioimmunoblastic T-cell lymphoma and / or bone marrow micro-residual lesions and / or T-cell lymphoma with pan-T antigen loss.

[0014] At the same time, the present invention also discloses the use of the above molecular markers in preparing a kit for detecting angioimmunoblastic T-cell lymphoma and / or bone marrow micro-residual lesions.

[0015] In addition, the above molecular markers can also be used in the preparation of a kit for detecting T-cell lymphoma with pan-T antigen loss.

[0016] At the same time, the present invention also discloses a molecular marker for detecting angioimmunoblastic T-cell lymphoma and / or bone marrow minimal residual lesions and / or pan-T antigen-losing T-cell lymphoma, which is a mixture of CD4 antibody, CD7 antibody, CD45 antibody, TRBC 1 antibody, CD5 antibody and CD3 antibody, CD10 antibody, PD1 antibody and CD8 antibody in a volume ratio of 2:2:3:5:5:6:2:3:2.

[0017] Furthermore, the process of detecting angioimmunoblastic T-cell lymphoma and / or bone marrow minimal residual lesions comprises the following steps:

[0018] 1) Add the sample to be tested into the flow cytometry tube to make the single cells in suspension state and ensure that the cell volume is 1×10 5 / tube~1×10 7 / tube; if the amount of cells is very small, add all the cells; the sample volume of each tube should not exceed 150uL. If the volume exceeds 150uL, 1× hemolysin without fixative can be used for cell enrichment first;

[0019] 2) mixing the sample obtained in step 1) with the molecular marker, and incubating at room temperature in the dark for 10 to 20 minutes;

[0020] 3) Add 1× hemolysin to the sample obtained in step 2) and incubate at room temperature in the dark for 5 to 15 minutes;

[0021] 4) centrifuging the sample obtained in step 3) to remove the supernatant;

[0022] 5) adding PBS buffer to the sample obtained in step 4) and mixing, centrifuging to remove the supernatant, and then resuspending the cells with PBS buffer;

[0023] 6) Sending the sample obtained in step 5) to a flow cytometer for on-machine detection and analyzing the detection results.

[0024] Furthermore, in step 6), the on-machine detection includes adopting a fixed gate setting and a multi-flag combination gate setting method, which is specifically as follows:

[0025] Fixed gate setting: remove the adhesion cell gate, live cell gate and blood cell gate, wherein the blood cell gate is set by CD45 / SSC;

[0026] Multi-marker combination gating: Synchronously with the CD45 / SSC gating, use CD3 / SSC to set the mature T lymphocyte gate and check the expression of CD4+T lymphocytes, the expression of CD8+T lymphocytes, and the expression of CD3+T lymphocytes TRBC1.

[0027] Further, in step 6), the analysis and detection process is as follows:

[0028] Set up two-dimensional dot plots of CD4 / CD7, CD3 / CD4, CD3 / CD5, CD5 / CD7, TRBC1 / CD3, and CD10 / PD1, and circle cells that meet any of the following phenotypes as suspicious cells: CD3-CD4+, CD3-CD5+, CD4+CD10+, CD4+PD1 bri +、CD10+PD1 bri +, and further clarify whether the suspicious cells are abnormal phenotypes and abnormal clones. The specific judgment criteria are as follows:

[0029] 1) Abnormal phenotype: changes in the expression intensity of pan-T markers, such as the absence or weakening of CD3 expression, and in rare cases, enhanced expression; and / or enhanced, weakened or absent expression of CD7; and / or both CD4 and CD5 are positive, with or without intensity changes; and if PD1 is strongly positive and CD10 is positive, it can be judged as AITL; if PD1 is strongly positive and CD10 is negative, it is highly suspected to be AITL;

[0030] 2) Abnormal clone: ​​If the TRBC1 positivity rate is greater than 85%, it can be judged as monoclonal expression and determined as an abnormal clone; if the TRBC1 positivity rate is less than 15%, it can be suspected to be an abnormal clone; if the TRBC1 positivity rate is 15-85%, it can be judged as polyclonal expression and is a normal polyclonal T lymphocyte. Furthermore, in step 1), the sample to be tested includes any one of the living cell samples of bone marrow, peripheral blood, lymph nodes, punctures, skin tissue, pleural effusion, and peritoneal effusion. Therefore, the present invention is not affected by the sample type. For patients with critical conditions, immune thrombocytopenia, or inability to obtain sufficient tissue samples, it is applied to bone marrow, peripheral blood or body fluid samples, which is conducive to early diagnosis; and micro-residue detection can be performed.

[0031] The beneficial effects of the present invention are mainly reflected in the following aspects:

[0032] 1. The molecular markers and related kits designed by the present invention are a combination of multiple antibodies, which can not only clarify the expression of multiple pan-T markers, but also use TRBC 1 to clarify the clonality of cells, and can simultaneously identify immunophenotypes and clonality, which is conducive to improving accuracy.

[0033] 2. The molecular markers designed by the present invention can also accurately classify T-cell lymphoma through the strong expression of PD1, with or without the expression of CD10, which is beneficial to improve the specificity of diagnosis. For details, please refer to the auxiliary explanations of Documents 1 and 2.

[0034] 3. The molecular markers and related kits designed in the present invention save more samples and costs than traditional screening methods. The type of tumor cells can still be determined at one time for fine needle aspiration, cerebrospinal fluid and bone marrow samples, thus achieving trace detection.

[0035] 4. The molecular markers designed in the present invention are all cell membrane antibodies, and their detection is not limited by sample type. They are applicable to all living cell samples and have a wide range of applications, especially for patients who cannot undergo histological examination, and can be used for diagnosis.

[0036] 5. The molecular markers and related kits designed by the present invention are simple to use, easy to operate, and save time and effort.

[0037] 6. The molecular markers designed by the present invention are not limited to angioimmunomaternal T-cell lymphoma, but can be used to screen T-cell lymphoma with pan-T antigen loss with high sensitivity.

[0038] 7. The molecular marker designed by the present invention uses PE fluorescein to measure the fluorescence intensity of the antigen expression of CD5 molecules on the surface of abnormal cells to achieve relative quantitative detection. If combined with a polyethylene phycoerythrin fluorescence quantitative kit, absolute quantitative detection can be performed to accurately reflect the antigen expression density of the molecule on the surface of abnormal cells, providing support for cellular immunotherapy.

[0039] 8. The molecular markers designed by the present invention can be used to monitor tiny residual lesions of AITL and are not affected by immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Gating and analyzing flow cytometry results for normal bone marrow samples;

[0041] Figure 2 Gating and analyzing flow cytometry results for normal bone marrow samples;

[0042] Figure 3 Gating and analysis of flow cytometry results for bone marrow samples from HES patients;

[0043] Figure 4 Flow cytometry analysis of bone marrow samples from CML patients with a low number of CD3-CD4+ T lymphocytes was performed by gating;

[0044] Figure 5 , Figure 6 , Figure 7 , Figure 8 and Fig. 9 Gating and analysis of flow cytometry results of bone marrow, lymph node, and peripheral blood samples from AITL patients;

[0045] Fig.10 These are statistical graphs of the mean fluorescence intensity of CD5 and PD1 in AITL patients. DETAILED DESCRIPTION

[0046] English abbreviations

[0047] AITL Angioimmunoblastic T-cell lymphoma

[0048] HES Hypereosinophilic syndrome

[0049] CML Chronic myeloid leukemia

[0050] T-NHL T-cell lymphoma

[0051] CD4 Human leukocyte differentiation antigen 4

[0052] CD7 Human leukocyte differentiation antigen 7

[0053] CD45 Human leukocyte differentiation antigen 45

[0054] TRBC1 T cell receptor β chain constant region 1

[0055] CD5 Human leukocyte differentiation antigen 5

[0056] CD3 Human leukocyte differentiation antigen 3

[0057] CD10 Human leukocyte differentiation antigen 10

[0058] PD1 Programmed death receptor-1

[0059] BD BD Company of America

[0060] The fluorescence intensity of each antibody expresses the meaning

[0061] + Positive

[0062] bri + Strong positive

[0063] bri strong

[0064] P + Partially positive

[0065] Part P

[0066] - Negative

[0067] dim + Weak positive

[0068] Cited literature 1: Yabe M, Gao Q, Ozkaya N, et al. Bright PD-1 expression by flowcytometry is a powerful tool for diagnosis and monitoring of angioimmunoblastic T-cell lymphoma. Blood Cancer J. 2020; 10(3):32. Published2020Mar 6.doi:10.1038 / s41408-020-0301-x).

[0069] Cited literature 2: Attygalle A, Al-Jehani R, Diss TC, et al. Neoplastic T cells in angioimmunoblastic T-cell lymphoma express CD10. Blood. 2002; 99(2): 627-633. doi: 10.1182 / blood.v99.2.627.

[0070] 1. Collect 208 patients clinically diagnosed with AITL in Wuhan from January 2013 to July 2021, and live cell samples of the 208 AITL patients, including 98 lymph node samples, 10 puncture tissue samples, 1 skin tissue sample, 201 bone marrow samples, 10 peripheral blood samples, 6 ascites samples, and 12 pleural effusion samples. Among them, the clinical data of AITL patients showed that among the 208 patients, there were 142 males and 66 females, with a male-to-female ratio of 2.15:1, with an age range of 19-82 years old and an average age of 58 years old. 99 cases were >60 years old and 109 cases were ≤60 years old. Summarizing all phenotypes of AITL through the above cases is the basis for the design of this antibody complex. There are 30 patients who actually used this antibody complex, and 44 specimens, including 7 peripheral blood samples, 12 tissue (including puncture and lymph node) samples, and 25 bone marrow samples.

[0071] 2. Detect the expression of each antibody in AITL patients by flow cytometry, as described in the examples.

[0072] 3. Main experimental reagents and instruments

[0073] 3.1. Main experimental reagents

[0074] The types, dosages, and manufacturer models of antibodies and the types and manufacturer models of fluorescein are shown in Table 1 ;

[0075] Table 1 List of molecular markers

[0076]

[0077]

[0078] 3.2. Main experimental instruments

[0079] 3-laser 10-color FACS Canto II flow cytometer: Becton Dickinson

[0080] 4. The present invention also discloses the use of the molecular markers shown in Table 1 above to detect angioimmunoblastic T-cell lymphoma and / or bone marrow micro-residual lesions. The specific process is as follows:

[0081] 1) Add the sample to be tested into the flow tube to make the single cell in suspension state; the number of cells in each flow tube is 1×10 6 ~1×10 7 If the number of cells is very small, such as puncture tissue and cerebrospinal fluid samples, all cells are added; the sample volume of each tube usually does not exceed 150uL. If the volume exceeds 150uL, 1× hemolysin without fixative can be used for cell enrichment first.

[0082] 2) mixing the sample obtained in step 1) with the molecular marker, and incubating at room temperature in the dark for 10 to 20 minutes;

[0083] 3) Add 1× hemolysin to the sample obtained in step 2) and incubate at room temperature in the dark for 5 to 15 minutes;

[0084] 4) Centrifuging the sample obtained in step 3) to remove the supernatant; wherein the centrifugal force is 300-450g and the centrifugation is performed for 5 minutes;

[0085] 5) adding PBS buffer to the sample obtained in step 4) and mixing, centrifuging to remove the supernatant, and then resuspending the cells with PBS buffer; wherein the amount of PBS buffer added is about 500uL;

[0086] 6) Sending the sample obtained in step 5) to a flow cytometer to complete the detection and analyze the detection results, specifically using diva 8.0 software to analyze the data.

[0087] 5. The present invention also discloses a gating process of samples in a flow cytometer, which is as follows:

[0088] (1) Fixed gates, including sequentially removing the adhesion cell gate, live cell gate, and blood cell gate;

[0089] Removing the adherent cell gate: Adherent cells can be removed by FSC-area (Area, A) / height (Height, H). The principle is that cells are spherical and A is positively correlated with H.

[0090] Live cell gate: The commonly used method for clinical specimen testing is FSC / SSC. The principle is that the size and granularity of live cells are nearly normally distributed, clustering around a center, and have a clear boundary with dead cells, apoptotic cells, debris and background noise.

[0091] Blood cell gate: The blood cell gate includes lymphocytes (Lym), monocytes (Mono), neutrophils (Gran), eosinophils (Eo) and nucleated red blood cells (P6).

[0092] The CD45 / SSC ratio is used to roughly distinguish the blood cells of different groups. The principle is that the fluorescence intensity of CD45 expression of hematopoietic cells is different, among which mature lymphocytes>monocytes>neutrophils>nucleated red blood cells, and the SSC size difference is different, such as Figure 1 As shown, neutrophils > monocytes > mature lymphocytes > nucleated erythrocytes. The purpose is to prevent the omission of large tumor cell populations after misdiagnosis or tumor evolution, and to set internal controls.

[0093] (2) Multi-marker combination gating is performed simultaneously with CD45 / SSC gating, starting with the lymphocyte (Lym) gate.

[0094] In the lymphocyte gate, use CD3 / SSC to set the mature T lymphocyte gate, and perform the following tests in the CD3 / SSC gate: expression of CD4+T lymphocytes and CD8+T lymphocytes; expression of TRBC-1 in CD3+T lymphocytes.

[0095] Accurately lock the target cells based on the combination of observed markers: In the lymphocyte gate, use a multi-marker combination to set the gate and look for tumor cells. In most cases, tumor cells will have weakened or missing CD3 expression. Therefore, when setting the gate with a multi-marker combination, all live cell gates should be displayed first, and then all lymphocytes should be displayed last to avoid missing tumor cells with special phenotypes. For example, when setting a two-dimensional dot plot of CD4 / CD7, CD3 / CD4, CD3 / CD5, CD5 / CD7, TRBC1 / CD3, and CD10 / PD1, you need to circle cells that meet any of the following phenotypes in the lymphocyte gate: CD3-CD4+, CD3-CD5+, CD4+CD10+, CD4+PD1 bri +、CD10+PD1 bri +, these cells are suspicious cells. Display the expression of each antibody in this antibody complex in suspicious cells to clarify whether abnormal cells have abnormal phenotypes and abnormal clones.

[0096] Abnormal cell judgment criteria: 1. Abnormal phenotype: changes in the expression intensity of pan-T markers, the most common change is the loss or weakening of CD3 expression; CD7 expression is variable, it can be enhanced, weakened or missing, but the most common is missing; CD4 and CD5 are positive; if PD1 is strongly positive and CD10 is positive, it can be judged as AITL, if CD10 is negative and PD1 is strongly positive, it is highly suspected. 2. Abnormal clone: ​​If the suspicious cell TRBC1 is monoclonally expressed, it is an abnormal clone. If TRBC1 is not expressed, it should be carefully judged in combination with the immune phenotype. If TRBC1 is polyclonally expressed, it is further excluded.

[0097] Specifically, if the CD3 intensity is not obvious, circle the CD4+ / CD7- or CD5+CD7- cells, and clarify the expression of CD10, PD1, TRBC1, and CD3. Since only some cases of AITL express TCR ab, if the suspicious cells are polyclonal in the TRBC 1 / CD3 combination, their abnormality can be ruled out, but their non-expression cannot be used as a basis for monoclonality. At this time, the immune phenotype should be combined to see the degree of loss of pan-T markers. If TRBC 1 is expressed and its positivity rate is 85% to 100%, excluding 85%, it can be determined to be a monoclonal cell and determined to be an abnormal clone; if the TRBC1 positivity rate is less than 15%, it can be suspected to be an abnormal clone; if the TRBC1 positivity rate is 15 to 85%, it can be judged as polyclonal expression and is a normal polyclonal T lymphocyte. Since there are varying proportions of CD3+CD4+CD7-T lymphocytes in normal bone marrow, the proportion of these reactively proliferating T lymphocytes will increase significantly after being stimulated by other factors, interfering with the judgment, for example Figure 1 to Figure 2 As shown, the proportion of CD4+CD7- cells increased significantly. The marker combination designed by the present invention can accurately lock the target cells and exclude reactive polyclonal T lymphocytes by using TRBC1.

[0098] In reactive cases, such as lymphocyte changes caused by increased eosinophils, abnormal phenotype T lymphocytes of CD3-CD4+ may appear during pan-T marker screening, which can be easily mistaken for tumor cells. In unfamiliar situations, it is very easy to misdiagnose as T-cell lymphoma (T-NHL). Figure 3 The abnormal phenotype of CD3-CD4+ is actually a change in lymphocytes caused by an increase in eosinophils. Figure 4 The abnormal phenotype of CD3-CD4+ shown is actually a very small number of T lymphocytes of unknown significance that appear in chronic myeloid leukemia, and is not a tumor T lymphocyte.

[0099] In addition, since the proportion of AITL tumor cells is low and the number of reactive cells is much higher than that of tumor cells, when the tumor cells are less than 0.01%, the number of tumor cells obtained should be greater than 50. Otherwise, the clinic needs to be informed because the proportion is extremely low or the number of cells is extremely small. The results are for reference only and follow-up is recommended.

[0100] At the same time, AITL is not 100% bone marrow invasion, and the result analysis is affected by channels, cell numbers, antigen molecules, experience, etc. Therefore, when the bone marrow is negative, AITL cannot be directly ruled out.

[0101] In order to better explain the present invention, the main contents of the present invention are further explained below in conjunction with specific embodiments, but the contents of the present invention are not limited to the following embodiments.

[0102] This example provides 2 normal samples, 2 reactive samples, and 5 AITL cases as examples. Figure 1 to Figure 2 For normal specimens, in addition to routine gating, the CD4 / CD7 marker combination was used to circle out CD4+CD7- cells for immunophenotyping and clonality identification. Figure 3 to Figure 4 Reactive CD3-CD4+ T lymphocytes were present in all cases, and the suspicious cells did not express CD10 and PD1. bri . Figures 5 to 9 For AITL cases, the average fluorescence intensity of the abnormal cell CD5 antigen can be reported at the same time for AITL cases with positive results, and the following CD3 is defaulted to cell membrane CD3; Fig.10 These are the statistics of the mean fluorescence intensity of CD5 and PD1 in AITL patients.

[0103] Specifically, combined Figure 1 , Figure 2 It can be seen that for normal bone marrow specimens. Set in sequence: FSC-A / H sets Total as the de-adhesion cell gate, and obtains a single cell in Total; displays FSC / SSC in Total and sets P1 as the live cell gate, and obtains a single live cell in P1; uses CD45 / SSC and CD3 / SSC to set gates in the P1 gate. Among them, CD45 / SSC sets the blood cell gate, and obtains lymphocyte (Lym), granulocyte (Gran), monocyte (Mono), and eosinophil (Eo) cell gates respectively; CD3 / SSC sets the gate to view the expression of CD4+ and CD8+; uses CD4 / CD7 to set the target cell gate (P3) to obtain the target cell population. Display the commonly used combinations of lymphocytes CD3 / CD4, CD3 / CD5, CD5 / CD7, TRBC1 / CD3 (target cells), TRBC1 / CD3 (normal control cells), and PD1 / CD10, and place the target cells on the top layer for display. The target cells (CD4+CD7-) showed no loss of expression of other pan-T antigens except CD7 expression, and TRBC1 was polyclonal, and PD1 and CD10 were not positive. Therefore, even if CD7 expression was lost in this specimen, it cannot be determined that it is a tumor cell.

[0104] Specifically, if Figure 3In the bone marrow sample shown, CD45 / SSC and CD3 / SSC were used to set gates in the P1 gate. Among them, CD45 / SSC was used to set the blood cell gate, and lymphocytes (Lym), granulocytes (Gran), monocytes (Mono), and eosinophils (Eo) were obtained respectively; the cell membrane CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+; CD3-CD4+T lymphocytes appeared in the lymphocyte gate, but PD1 and CD10 expressions were negative, excluding AITL. However, the proportion of eosinophils was high, and the clinical history did not have fever, lymphadenopathy, and hepatosplenomegaly, so it was judged to be HES. At the same time, the skin pathology results reported reactive hyperplasia in the T zone, which was consistent with the above judgment results.

[0105] Specifically, if Figure 4 In the bone marrow sample shown, CD45 / SSC and membrane CD3 / SSC were used to set gates in the P1 gate. Among them, CD45 / SSC was used to set the blood cell gate, and lymphocytes (Lym), granulocytes (Gran), monocytes (Mono), and eosinophils (Eo) were obtained respectively; the membrane CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+; CD4+CD7- and CD3-CD4+T lymphocytes were gated respectively for phenotype and clonality identification, and no expression of CD10 and strong expression of PD1 were found. At the same time, CD4+CD7- cells showed polyclonal expression of TRBC1. The clinical history showed no fever or lymphadenopathy, and genetic and molecular examinations showed that Philadelphia chromosome P210 was positive, which did not support lymphoma.

[0106] Specifically, if Figure 5 In the bone marrow sample shown, CD45 / SSC and membrane CD3 / SSC were used to set gates in P1. CD45 / SSC was used to set the blood cell gate to obtain lymphocytes (Lym), granulocytes (Gran), and monocytes (Mono). The membrane CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+. dim +CD4+T lymphocytes for phenotypic and clonality identification. Abnormal cells express CD3 dim 、CD4 dim The expression of CD5, CD7, and PD1 was uniformly enhanced, CD10 was not expressed, and TRBC1 did not show polyclonal expression. Although CD10 expression was negative, it still indicated that it was an abnormally mature T lymphocyte and was highly suspected of AITL. This group of cells still existed in the lymph node samples, and the pathological report was AITL.

[0107] Specifically, if Figure 6In the bone marrow sample shown, the patient has been diagnosed with AITL by histopathology. Further, CD45 / SSC and CD3 / SSC were used to set gates in the P1 gate. CD45 / SSC was used to set the blood cell gate to obtain lymphocytes (Lym), granulocytes (Gran), and monocytes (Mono), respectively; the cell membrane CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+; CD4+CD7-T lymphocytes were circled in the lymphocyte gate, showing that two groups of cells appeared in the CD3 / CD5 marker combination, and CD3+CD5 was circled in the CD4+CD7-T lymphocytes. bri +Cell population, displayed in other combined gates, abnormal cells express CD3, CD4, CD5 bri 、CD10、PD1 bri , CD7 was not expressed. This group of cells showed monoclonal expression in TRBC1, which was judged as bone marrow involvement. The bone marrow T cell receptor gene rearrangement was positive, which was consistent with our results, further verifying the correctness of our results.

[0108] Specifically, if Figure 7 In the lymph node sample shown, the nucleated cells are mainly lymphocytes. CD45 / SSC and CD3 / SSC are used to set gates in the P1 gate. CD45 / SSC is used to set the blood cell gate to obtain lymphocytes (Lym); CD3 / SSC is used to set the gate to check the expression of CD4+ and CD8+; in the lymphocyte population, CD10+PD1 ... bri + Lymphocytes, displayed in other combination gates, abnormal cells expressing CD3, CD4, CD5, CD10, PD1 bri Most of them expressed CD7 and were diagnosed as AITL. Lymph node biopsy showed AITL, which was consistent with our results and further verified the correctness of our results.

[0109] Specifically, if Figure 8 In the bone marrow sample shown, CD45 / SSC and CD3 / SSC were used to set gates in the P1 gate. CD45 / SSC was used to set the blood cell gate to obtain lymphocytes (Lym); CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+; in the lymphocyte population, CD3-CD4+T lymphocytes appeared in the CD3 / CD4 combination gate, and abnormal cells expressed CD4, CD5, and PD1 in other combination gates. bri 、CD10 p + phenotype, no expression of CD3 and CD7, and monoclonal expression of TRBC1, which was judged to be AITL bone marrow involvement. Lymph node biopsy showed AITL and positive bone marrow receptor gene rearrangement, which was consistent with our results and further verified the correctness of our results.

[0110] Specifically, combined Fig. 9 In the peripheral blood sample shown, CD45 / SSC and CD3 / SSC were used to set gates in P1. CD45 / SSC was used to set the blood cell gate to obtain lymphocytes (Lym); CD3 / SSC was used to set the gate to check the expression of CD4+ and CD8+; in the lymphocyte population, CD3+ appeared in the CD3 / CD4 combined gate. dim +CD4+ T lymphocytes, shown in other combination gates, abnormal cells expressing CD3 dim , CD4, CD5, PD1 bri , CD10 and CD7 were not expressed, and TRBC1 was expressed monoclonally, which was judged as residual positive. The morphology and receptor gene rearrangement were positive, which was consistent with our results, further verifying the correctness of our results.

[0111] Specifically, combined Fig.10 It can be seen that the abnormal cells of AITL patients express PD1 strongly, which can be used to diagnose AITL. Abnormal cells also express CD5, and the average fluorescence intensity of CD5 is not statistically different from that of normal T lymphocytes. Therefore, CD5 is a suitable target for cellular immunotherapy.

[0112] Combined with the above Figures 5 to 9 It can be seen that the molecular markers designed by the present invention can not only clarify the expression of multiple pan-T markers, but also use TRBC 1 to clarify the clonality of cells, and identify cell phenotypes and cell clonality at the same time; on the other hand, the strong expression of PD1 with or without the expression of CD10 can be used to accurately type T-cell lymphoma, which is beneficial to improve the sensitivity and specificity of diagnosis; in addition, the type of tumor cells can still be determined at one time for fine needle aspiration, cerebrospinal fluid and bone marrow samples, that is, trace detection, cheap monitoring, and is not affected by immunotherapy. It can be used for diagnosis, especially for patients who cannot undergo histological examination; at the same time, if Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 Abnormal cell CD5 expression in Fig.10 It can be seen that the molecular marker designed by the present invention uses PE fluorescein to measure the fluorescence intensity of antigen expression of CD5 molecules on the surface of abnormal cells, and can perform relative and absolute quantitative detection of antigen expression through the average fluorescence intensity and antigen expression density, thereby providing support for CART cell immunotherapy.

[0113] The above embodiments are only the best examples, and are not intended to limit the implementation of the present invention. In addition to the above embodiments, the present invention has other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A molecular marker, characterized in that It comprises CD4 antibody, CD7 antibody, CD45 antibody, TRBC 1 antibody, CD5 antibody, CD3 antibody, CD10 antibody, PD1 antibody and CD8 antibody; each of the antibodies is a monoclonal antibody.

2. The molecular marker according to claim 1, characterized in that Each of the antibodies is a fluorescein-labeled antibody, and the CD4 antibody is labeled with BV605, the CD7 antibody is labeled with V450, the CD45 antibody is labeled with V500, the TRBC 1 antibody is labeled with FITC, the CD5 antibody is labeled with PE, the CD3 antibody is labeled with PerCP-cy5.5, the CD10 antibody is labeled with PE-Cy7, the PD1 antibody is labeled with APC, and the CD8 antibody is labeled with APC-Cy7.

3. The molecular marker according to claim 1 or 2, characterized in that The molecular marker is a mixture of CD4 antibody, CD7 antibody, CD45 antibody, TRBC 1 antibody, CD5 antibody, CD3 antibody, CD10 antibody, PD1 antibody and CD8 antibody in a volume ratio of 2:2:3:5:5:6:2:3:

2.

4. A kit, characterized in that: It comprises the molecular marker described in any one of claims 1 to 3 and a storage component for placing the molecular marker.

5. The kit according to claim 4, characterized in that It also includes red blood cell lysis solution, buffer, and flow tubes for use with flow cytometers.

6. Use of the molecular marker according to claim 1, 2 or 3 or the kit according to claim 4 or 5 in the preparation of a kit for detecting angioimmunoblastic T-cell lymphoma and / or minimal residual lesions in the bone marrow.

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