IgD type multiple myeloma screening kit and screening system thereof

Abstract

The invention relates to an IgD type multiple myeloma screening kit and a screening system thereof, and finds that compared with non-IgD type multiple myeloma such as IgA, IgG, light chain type and non-secreting type MM, the expression quantity of ITM2C protein on a tumor cell membrane of an IgD type multiple myeloma patient is obviously reduced for the first time; the ITM2C protein can be used as a biomarker for screening the IgD type multiple myeloma, the reagent for detecting the expression quantity of the ITM2C protein is used for preparing the IgD type multiple myeloma screening kit, and the kit has high specificity and high sensitivity; the IgD type multiple myeloma can be accurately and effectively screened by detecting the expression quantity of the ITM2C protein on a tumor cell membrane.

Description

[0001] This application claims priority to the Chinese application number 202410643478X filed on May 22, 2024. Technical Field

[0002] The present invention relates to the field of biomedicine, and more specifically to an IgD multiple myeloma screening kit and a screening system thereof. Background Art

[0003] Multiple myeloma (MM) is the second most common blood cancer in the world. Its tumor cells originate from plasma cells in the bone marrow. It is characterized by abnormal proliferation of bone marrow plasma cells accompanied by excessive production of monoclonal immunoglobulins or light chains (M proteins), which in turn damage target organs, such as organ dysfunction, anemia, renal damage and bone damage. According to the difference in the secretion of monoclonal immunoglobulins, it can be divided into different subtypes such as IgG, IgA, IgM, IgD, IgE and light chain types. Among them, IgD type is a very rare special subtype. The treatment and prognosis of multiple myeloma are related to the subtype, and there is currently no separate targeted treatment for this subtype. Like other subtypes, the first-line treatment mostly adopts a proteasome inhibitor-based treatment plan, but the effect is not good. Therefore, accurate typing is crucial for the treatment or prognosis of multiple myeloma patients, especially the accurate distinction of IgD-type multiple myeloma, which is conducive to overcoming the technical difficulty that there is no specific treatment plan for IgD-type multiple myeloma.

[0004] However, it is difficult to diagnose IgD MM. First, IgD MM is a relatively rare MM subtype, and many clinicians or clinical workers are not familiar with it, which can easily lead to misdiagnosis and missed diagnosis. Secondly, IgD MM is usually very dangerous. When patients have atypical manifestations caused by extramedullary lesions, clinicians may misdiagnose patients as other serious diseases other than MM, thereby delaying the disease. In addition, clinical diagnosis of MM is generally carried out by serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE), and these mainstream diagnostic methods are prone to false negative results when detecting IgD protein due to low protein levels and its special electrophoretic migration performance. Moreover, the development of IgD IFE has not yet reached universalization, and many units can only detect common IgG, IgA and IgM. Therefore, some IgD patients will be missed or misdiagnosed as light chain or non-secretory MM. In summary, the complexity of the diagnosis of IgD MM is still a major problem that plagues clinicians, which fully demonstrates the urgent need to develop more sensitive, simple, fast and accurate new detection and screening technologies for the accurate diagnosis of IgD MM. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides an IgD multiple myeloma screening kit and a screening system thereof, the purpose of which is to discover that the expression level of ITM2C protein on the tumor cell membrane of IgD MM patients is significantly lower than that of non-IgD types such as IgA, IgG, light chain type, and non-secretory MM. Based on the ITM2C gene expression level, IgD MM and non-IgD MM can be well distinguished. The ITM2C protein expression level can be used as a biomarker to distinguish between IgD and non-IgD MM (including light chain type and non-secretory type) patients, thereby solving the technical problem that the prior art is difficult to accurately distinguish IgD from non-IgD types such as light chain type and non-secretory MM.

[0006] To achieve the above object, according to one aspect of the present invention, a use of an ITM2C protein detection reagent in the preparation of an IgD multiple myeloma screening kit is provided.

[0007] Preferably, the ITM2C protein detection reagent is used in the preparation of an IgD multiple myeloma screening kit, wherein the protein detection reagent is used for quantitatively detecting the expression amount of ITM2C protein on the cell membrane, and comprises an ITM2C monoclonal antibody or a polyclonal antibody.

[0008] Preferably, the ITM2C protein detection reagent is used in the preparation of an IgD multiple myeloma screening kit, wherein the protein detection reagent is used to quantitatively detect the expression amount of ITM2C protein on the tumor cell membrane.

[0009] According to another aspect of the present invention, a screening kit for IgD multiple myeloma is provided, wherein the screening kit comprises a reagent for detecting the expression level of ITM2C protein on the cell membrane.

[0010] Preferably, the IgD multiple myeloma screening kit comprises ITM2C monoclonal antibody or polyclonal antibody.

[0011] Preferably, the IgD multiple myeloma screening kit further comprises a secondary antibody, wherein the secondary antibody comprises a monoclonal or polyclonal antibody specifically binding to the ITM2C monoclonal antibody or polyclonal antibody and labeled with an enzyme or fluorescent dye, and is used to detect the binding amount of the ITM2C monoclonal antibody or polyclonal antibody to the ITM2C protein on the tumor cell membrane.

[0012] Preferably, the IgD multiple myeloma screening kit and the ITM2C monoclonal antibody thereof include proteintech.

[0013] Preferably, the IgD multiple myeloma screening kit further comprises a washing buffer.

[0014] According to another aspect of the present invention, there is also provided an IgD multiple myeloma screening system, which includes a data acquisition module, a judgment module and a result display module;

[0015] The data acquisition module is used to obtain the expression level of ITM2C protein on the tumor cell membrane of multiple myeloma patients and submit it to the judgment module;

[0016] The judgment module uses the obtained ITM2C protein expression level as the input of the classifier and judges according to the following method:

[0017] The lower the expression of ITM2C protein, the higher the risk of IgD multiple myeloma.

[0018] If the obtained ITM2C protein expression level is higher, the risk of IgD multiple myeloma is judged to be lower; and the judgment result is submitted to the result display module;

[0019] The result display module performs output display according to the judgment result of the judgment module.

[0020] Preferably, the judgment module of the IgD multiple myeloma screening system specifically performs judgment according to the following method:

[0021] If the expression level of ITM2C protein obtained is significantly lower than that of ITM2C protein in non-IgD MM, the sample is judged to be from an IgD MM patient;

[0022] If the expression level of ITM2C protein obtained is not significantly different from that of non-IgD MM, it is judged that the sample comes from a non-IgD MM patient;

[0023] The ITM2C protein expression level of the non-IgD MM is the average expression level of the ITM2C protein in one or more of the light chain type, non-secretory type, IgA type, and IgG type MM patients.

[0024] Preferably, in the IgD multiple myeloma screening system, the ITM2C protein expression level is obtained by flow cytometry.

[0025] In general, the above technical solution conceived by the present invention can achieve the following beneficial effects compared with the prior art, because it is found that the expression level of ITM2C protein can be used as a biomarker to distinguish IgD type and non-IgD type MM:

[0026] The present invention finds that the expression of ITM2C protein on the tumor cell membrane can be used as a biomarker to distinguish IgD-type and non-IgD-type MM. The ITM2C protein detection reagent can be used to prepare an IgD-type multiple myeloma screening kit. The screening kit has high specificity and high sensitivity, and can determine the possibility that the sample comes from an IgD-type multiple myeloma patient, that is, the risk level of IgD-type multiple myeloma, by detecting the expression of ITM2C protein on the tumor cell membrane of the multiple myeloma patient. The lower the amount, the greater the possibility that the sample is from an IgD multiple myeloma patient, indicating that the risk of suffering from IgD multiple myeloma is higher; the higher the ITM2C protein expression level, the smaller the possibility that the sample is from an IgD multiple myeloma patient, indicating that the risk of suffering from IgD multiple myeloma is lower. Based on the expression level of ITM2C, it is possible to accurately distinguish between IgD multiple myeloma and non-IgD multiple myeloma patients, thereby effectively screening IgD multiple myeloma from multiple myeloma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a differentially expressed gene in IgD and non-IgD malignant tumor cells;

[0028] Figure 2 It is a differentially expressed gene in IgD and non-IgD malignant tumor cells;

[0029] Figure 3 It is a differentially expressed gene in newly diagnosed IgD and non-IgD malignant tumor cells; Figure 3 A in the middle is the difference in activity levels of the 9 modules. Figure 3 Middle B shows the difference in activity of the protein processing (M2) module in IgD malignant cells compared to non-IgD cells;

[0030] Figure 4 This is the result of differential analysis of the protein expression of ITM2C on the cell membranes of IgD and non-IgD malignant tumors;

[0031] Figure 5 This is the result of differential analysis of BCL2 gene transcription levels in IgD and non-IgD malignant tumor cells;

[0032] Figure 6 The ROC curves of IgD MM and non-IgD MM (including IgG, IgA, light chain and non-secretory types) were drawn based on the expression of ITM2C protein on the tumor cell membrane;

[0033] Figure 7The ROC curves of IgD MM and non-IgD MM (including IgG, IgA, light chain and non-secretory types) were drawn based on the transcription level of BCL2 gene in tumor cells;

[0034] Figure 8 is the ROC curve drawn based on the transcription level of the ITM2C gene in Example 3;

[0035] Fig. 9 This is the result of differential analysis of the expression of ITM2C protein on tumor cell membranes between IgD MM patients and non-IgD MM patients in Example 3. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0037] Previous studies generally believed that ITM2C was overexpressed in multiple myeloma (MM) cells and was a potential marker gene for multiple myeloma. However, we found that the expression of ITM2C protein on the tumor cell membrane of IgD MM patients was significantly lower than that of non-IgD MM (including light chain, non-secretory, IgA, and IgG). Moreover, IgD and non-IgD MM could be well distinguished based on the expression level of ITM2C. The expression of ITM2C protein can be used as a biomarker for screening IgD multiple myeloma.

[0038] Based on this, the present invention provides an application of an ITM2C protein detection reagent in the preparation of an IgD multiple myeloma screening kit.

[0039] The protein detection reagent is used for quantitatively detecting the expression of ITM2C protein on the cell membrane, and preferably comprises ITM2C monoclonal antibody or polyclonal antibody. In some embodiments, it is used for quantitatively detecting the expression of ITM2C protein on the tumor cell membrane of multiple myeloma patients.

[0040] In addition, the present invention also provides an IgD multiple myeloma screening kit, which includes a reagent for detecting the expression level of ITM2C protein; preferably, the reagent for quantitatively detecting the expression amount of ITM2C protein on the cell membrane includes ITM2C monoclonal or polyclonal antibodies.

[0041] Furthermore, the screening kit also includes a secondary antibody, which refers to a monoclonal or polyclonal antibody that specifically binds to the primary antibody (ITM2C monoclonal antibody or polyclonal antibody) and is used to detect the binding of the ITM2C monoclonal antibody or polyclonal antibody to the ITM2C protein on the tumor cell membrane.

[0042] In some embodiments, the primary antibody is an ITM2C monoclonal antibody selected from the antibody of the brand proteintech (Cat. No.: 60095-1-Ig). The secondary antibody includes a monoclonal or polyclonal antibody labeled with an enzyme (such as horseradish peroxidase HRP or alkaline phosphatase AP) or a fluorescent dye, and the appropriate secondary antibody can be determined according to the panel self-selected fluorescence in actual application.

[0043] The screening kit of the present invention also includes auxiliary reagents for quantitatively detecting ITM2C protein on tumor cell membranes, such as washing buffer required for detecting the expression level of ITM2C protein on cell membranes by flow cytometry, wherein the washing buffer includes staining buffer or PBS buffer.

[0044] In addition, the present invention also provides an IgD multiple myeloma screening system, which includes a data acquisition module, a judgment module and a result display module;

[0045] The data acquisition module is used to obtain the ITM2C protein expression level on the surface of the tumor cell membrane of multiple myeloma patients and submit it to the judgment module; in some embodiments, the ITM2C protein expression level is obtained by flow cytometry, but the method for obtaining the ITM2C protein expression level on the cell membrane is not limited in the present invention.

[0046] The judgment module uses the obtained ITM2C protein expression level as the input of the classifier, and makes a judgment according to the fact that the lower the obtained ITM2C protein expression level, the higher the risk of suffering from IgD multiple myeloma; specifically as follows:

[0047] The lower the expression of ITM2C protein, the higher the risk of IgD multiple myeloma, that is, the greater the possibility that the sample comes from an IgD multiple myeloma patient;

[0048] If the obtained ITM2C protein expression level is higher, the risk of IgD multiple myeloma is judged to be lower, that is, the possibility that the sample comes from an IgD multiple myeloma patient is lower; and the judgment result is submitted to the result display module;

[0049] The result display module outputs and displays according to the judgment result of the judgment module, such as displaying that the sample comes from a person with high risk of IgD multiple myeloma or that the sample comes from a person with low risk of IgD multiple myeloma.

[0050] In some embodiments, the judgment module specifically performs judgment according to the following method:

[0051] If the expression level of ITM2C protein obtained is significantly lower than that of ITM2C protein in non-IgD MM, the sample is judged to be from an IgD MM patient;

[0052] If the expression level of ITM2C protein obtained is not significantly different from that of non-IgD MM, it is judged that the sample comes from a non-IgD MM patient;

[0053] The ITM2C protein expression level of the non-IgD MM is the average expression level of the ITM2C protein in one or more patients of light chain type, non-secretory type, IgA type, and IgG type MM.

[0054] In the present invention, the following method can also be used for determination:

[0055] If the obtained ITM2C protein expression level is greater than the preset value, the patient is judged to be non-IgD MM;

[0056] If the obtained ITM2C protein expression level is less than or equal to the preset value, the patient is judged to be IgD type MM; and the judgment result is submitted to the result display module.

[0057] The receiver operating characteristic curve (ROC curve for short) is a tool widely used to evaluate the performance of a binary classification model. In the medical field, ROC curves are commonly used to evaluate the performance of diagnostic tests, screening or prediction models. It is a curve drawn with the true positive rate (sensitivity) as the ordinate and the false positive rate (1-specificity) as the abscissa, and each point on the ROC curve corresponds to a different classification threshold. In some embodiments, the preset value can be determined by selecting the optimal threshold value by the ROC curve, such as selecting the classification threshold that maximizes the Youden index as the preset value, where the Youden index = sensitivity + specificity - 1.

[0058] The following are examples

[0059] The following IgD and non-IgD MM patients were included according to the following criteria:

[0060] ① Inclusion criteria: Patients with multiple myeloma admitted to the Department of Hematology of our hospital from 2018 to 2023 were collected, and the diagnosis met the domestic and international standards (References 1, 2): Chinese Medical Association Hematology Branch, Chinese Medical Association Hematology Branch, Chinese Multiple Myeloma Diagnosis and Treatment Guidelines (revised in 2024); Reference 1: Dimopoulos, M., Moreau, P., Terpos, E., Mateos, M., Zweegman, S., Cook, G., Delforge, M., Hájek, R., Schjesvold, F., and Cavo, M. (2022). Corrigendum to'Multiple myeloma: EHA-ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up': [Ann Oncol 2021; 32(3): 309-322]. Annals of Oncology 33, 117.

[0061] Document 2: Association, CH (2024). Guidelines for the diagnosis and management of multiple myeloma in China (2024revision). Zhonghua nei ke za zhi63,1186-1195.

[0062] According to the type of abnormal immunoglobulin, it is divided into IgG, IgA, IgD, light chain or non-secretory type.

[0063] ②Exclusion criteria: Patients with non-multiple myeloma or multiple myeloma accompanied by other tumor diseases were excluded.

[0064] All the following patients gave written informed consent.

[0065] Example 1 Analysis of differences in tumor cells between IgD and non-IgD MM patients at the transcriptome level, as follows:

[0066] Newly diagnosed (n=4) and relapsed (n=9) IgD MM patients in our hospital were randomly selected, including 2 t(11;14) positive cases and 11 t(11;14) negative cases, as well as newly diagnosed (n=17) and relapsed (n=7) non-IgD MM (IgG, IgA, light chain, non-secretory) patients. After written informed consent, all patients underwent bone marrow puncture biopsy, CD138 magnetic beads were used to sort the samples, and the retained CD138+ plasma cells were subjected to 10× single-cell RNA sequencing and VDJ sequencing. The sequencing system was Illumina NovoSeq 6000 with a sequencing depth of 2.5 million reads / cell. The query website of the original files (individual name D1-37) uploaded to the public database for single-cell sequencing and VDJ sequencing is https: / / ngdc.cncb.ac.cn / gsa-human / browse / HRA005760.

[0067] The sequencing data was then subjected to quality control, cell clustering, CNV analysis, gene regulatory network analysis, pseudo-time trajectory analysis, and cell interaction analysis. The specific analysis results are as follows:

[0068] (1) Single-cell sequencing data analysis

[0069] In this example, single-cell sequencing data analysis was used to compare the differences in transcriptome between IgD multiple myeloma (IgD / t(11;14)+, IgD / t(11;14)-) and non-IgD MM. By enriching pathways related to protein processing pathways (including endoplasmic reticulum stress, autophagy, and unfolded protein response), we found differentially expressed genes in IgD and non-IgD malignant tumor cells related to these pathways, including BCL2, ITM2C, etc. Some of the differentially expressed genes are as follows Figure 1 and Figure 2 shown.

[0070] In addition, the non-negative matrix factorization (cNMF) method was used to integrate the gene expression of individual cells into a set of interpretable biological gene expression programs (GEPs). 115 GEPs ( Figure 3 (A).

[0071] The inter-sample similarities between these GEPs were demonstrated by correlation clustering, which was further clustered into nine common modules, including conventional "basic" modules such as cell cycle, RNA / protein synthesis, and cell stress, as well as less understood immune-related, interferon-related, and extracellular matrix-related modules. For each module, we tested whether its expression level differed between IgD and non-IgD MMs, and found that the activity of the protein processing (M2) module in IgD malignant cells was significantly different from that in non-IgD cells, such as Figure 3 As shown in B.

[0072] ITM2C is a membrane protein, and membrane proteins are easier to detect in actual clinical tests, so in the following research, we focused on the differentially expressed membrane protein ITM2C in the M2 module. Previous studies generally believed that ITM2C was overexpressed in MM cells and was a potential marker gene for MM.

[0073] In this embodiment, the difference between ITM2C protein and non-IgD MM patients was analyzed by flow cytometry, and primary MM cells of IgD and non-IgD MM patients (IgG, IgA, light chain, non-secretory) were obtained. After washing 3 times with staining buffer or PBS buffer, the cells were resuspended in 50ul staining buffer or PBS buffer, and the CD138+ cells were stained with anti-ITM2C-APC surface antigen by adding antibodies. The primary antibody used was brand: proteintech, item number: 60095-1-Ig ITM2C monoclonal antibody, dosage 5ul / test, primary antibody was stained at 4°C for 20-30min, and 500ul PBS buffer was added after the staining was completed. After washing 2-3 times, the secondary antibody was added, wherein the secondary antibody was a monoclonal antibody specifically binding to the primary antibody and labeled with a fluorescent dye. In the present invention, the secondary antibody can be selected according to the actual application of the panel fluorescence, or a monoclonal or polyclonal antibody specifically binding to the primary antibody labeled with an enzyme.

[0074] The protein expression level of ITM2C on the cell membrane surface was expressed by mean fluorescence intensity (MFI). Figure 4 shown.

[0075] Depend on Figure 4However, we found that there was a significant difference in the expression level of ITM2C protein on the cell surface between patients with IgD multiple myeloma and patients with non-IgD multiple myeloma. Compared with non-IgD multiple myeloma, the expression level of ITM2C protein in patients with IgD multiple myeloma was significantly reduced. It can be seen that the results of flow cytometry analysis were consistent with single-cell sequencing, which once again demonstrated that IgD type MM has obvious heterogeneity relative to other subtypes. It is speculated that ITM2C protein on the cell membrane surface can be used as a potential biomarker for screening IgD type MM. By detecting the expression of ITM2C protein on the tumor cell membrane surface of patients with multiple myeloma, it can be used to screen IgD type MM from MM.

[0076] In addition, the above experimental results show that there are significant differences between IgD multiple myeloma and other subtypes of MM at the transcriptome level, and the differential genes in tumor cells are mainly related to protein processing pathways. Among them, the BCL-2 protein family acts as a core to integrate the stress signal network and regulate cell death, calcium homeostasis, endoplasmic reticulum stress and autophagy.

[0077] In this example, primary MM cells of IgD and non-IgD (IgG, IgA, light chain and non-secretory) MM patients were obtained, and the mRNA expression of BCL2 was detected. The qPCR experimental process is as follows:

[0078] CD138+ cells from IgD and non-IgD MM patients were collected, RNA was extracted and reverse transcribed, cDNA of primary specimens of patients was obtained, and qPCR mixture was prepared. The system is shown in the following table;

[0079] Table 1 RT-qPCR Gene Mix System (20 μL System)

[0080]

[0081] After the Gene mix is ​​prepared according to the above system, add 18 μL / well to the PCR well plate in turn under light-proof conditions, and pay attention to setting up 3 replicate wells; add 2 μL of the cDNA template of the corresponding treatment group to each well, stick the sealing film on the plate and prepare for the machine test. Perform the test according to the "two-step method" machine procedure. The specific procedure is shown in the table below;

[0082] Table 2 RT-qPCR amplification procedure

[0083]

[0084]

[0085] After obtaining the Ct value, calculate 2 -△△CtThe relative expression of the target gene was quantitatively analyzed. The results of the differential analysis of the BCL2 gene expression levels in IgD multiple myeloma and non-IgD malignant plasma cells are shown in Figure 5 shown.

[0086] Depend on Figure 5 It can be seen that the BCL2 expression level of IgD MM patients, whether t(11;14) positive or t(11;14) negative, was significantly higher than that of non-IgD MM patients, and there was no significant difference in BCL2 expression level between t(11;14) positive and t(11;14) negative IgD MM patients.

[0087] In addition, the results of the BCL2 IHC immunohistochemistry experiment showed that the percentage of BCL2-positive cells in t(11;14)-negative IgD-type multiple myeloma tissue samples was about 85-95%, indicating that t(11;14)-negative IgD-type multiple myeloma patients also generally overexpress BCL2. It can be seen that BCL2 protein may also be a potential biomarker for screening IgD-type MM.

[0088] Example 2 Screening of IgD MM based on ITM2C gene expression level

[0089] In order to verify the results of Example 1, another batch of IgD MM and non-IgD MM patients were selected for verification in this example. Although it is a basic operation in the art to use patients different from the screening set in Example 1 for verification in the verification experiment, it is still specially explained in the present invention: the IgD MM and non-IgD MM patients selected for verification in the present invention are not the same batch of patients as the IgD MM and non-IgD MM patients in Example 1, that is, the verification set and the screening set are different.

[0090] Considering that ITM2C is a membrane protein, it is easier to detect its gene expression product, ITM2C protein, in a clinical setting. This example uses flow cytometry to detect the expression of ITM2C protein on the cell membrane surface for verification, as follows:

[0091] Using flow cytometry, CD138+ cells of IgD MM and non-IgD MM patients were stained with anti-ITM2C-APC surface antigen and then loaded on the machine. The protein expression level of ITM2C on the cell surface was expressed by mean fluorescence intensity (MFI). The ROC curve was used to analyze the diagnostic performance of gene ITM2C for IgD MM. The ROC curves of IgD MM and non-IgD MM (including IgG, IgA, light chain and non-secretory types) are shown in the figure. Figure 6 shown.

[0092] Depend on Figure 6It can be seen that the ROC curve of IgD type and non-IgD type MM drawn based on the expression of ITM2C protein on the tumor cell membrane has an AUC area of ​​0.897, a specificity of 1, a sensitivity of 0.882, and a maximum equivalence index of 9.579. It can be seen that the detection of ITM2C protein expression can be used to distinguish IgD type and non-IgD type MM patients, especially IgD type and light chain type, non-secretory type, indicating that ITM2C protein can be used as a biomarker for screening IgD type MM, and the ITM2C protein expression on the tumor cell membrane can be used to distinguish IgD type and non-IgD type MM patients, thereby screening out IgD type MM from MM patients. In this embodiment, the patient can be judged as IgD type MM according to the classification threshold corresponding to the maximum equivalence index when the ITM2C protein expression is less than or equal to the maximum equivalence index, and the patient can be judged as non-IgD type MM when the ITM2C protein expression is greater than the classification threshold corresponding to the maximum equivalence index.

[0093] Comparative Example 1

[0094] A group of IgD and non-IgD MM patients different from the IgD and non-IgD MM patients in Example 1 were selected, and the BCL-2 transcription level (mRNA detection amount) of tumor cells of patients with IgD and non-IgD MM (including IgG, IgA, light chain type and non-secretory type) was determined by qPCR technology. The ROC curve between IgD and non-IgD MM was drawn based on the BCL-2 gene transcription level, and the diagnostic performance of BCL-2 gene expression level for IgD MM was determined and analyzed. The results are as follows Figure 7 shown.

[0095] Depend on Figure 7 It can be seen that the AUC area corresponding to the ROC curve for distinguishing IgD and non-IgD MM patients (including light chain type and non-secretory type) based on the expression level of gene BCL-2 is 0.691, which is lower than 0.7, and the specificity is 0.5, indicating that the expression level of gene BCL-2 cannot be used to distinguish IgD and non-IgD MM patients, especially IgD and light chain type, non-secretory type.

[0096] Example 3 Verification at the transcription and translation levels

[0097] There is no cell model or animal model for IgD MM at present. To ensure the credibility of the verification results of Example 2, we selected two groups of patients with IgD and non-IgD MM different from those in Example 1 and Example 2 for single cell sequencing and flow cytometry verification, as follows:

[0098] (1) Validation at the transcriptional level

[0099] Twenty newly diagnosed MM patients were included for single-cell sequencing. The specific information of these 20 newly diagnosed MM patients is shown in Table 3:

[0100] Table 3 Detailed information of 20 newly diagnosed MM patients

[0101]

[0102]

[0103] The collected samples were subjected to CD138 magnetic beads. The specific experimental process and experimental results are as follows:

[0104] 1. Sorting sample processing and single-cell sequencing

[0105] Bone marrow samples of the patients listed in Table 3 were collected and plasma cells were sorted using CD138 magnetic beads (MiltenyiBiotec GmbH, Germany) according to the instructions for use, as follows:

[0106] (1) Preparation of mononuclear cell suspension

[0107] About 20 ml of bone marrow was extracted, anticoagulated with sodium heparin, and centrifuged at 1500 rpm for 10 min. The supernatant plasma was transferred to a new centrifuge tube, centrifuged again at 3000 rpm for 10 min, and then placed in 1.5 ml centrifuge tubes (divided into 2 tubes) and frozen in a -20°C refrigerator.

[0108] Pour the remaining bone marrow blood into a 50ml centrifuge tube and dilute it to 40ml with PBS or saline. Slowly add the diluted sample to a 15ml lymph separation fluid centrifuge tube, add 6ml of lymph separation fluid to each tube, and spin at 2500 rpm for 30min (slow acceleration and slow deceleration).

[0109] After centrifugation, remove the misty cell layer (i.e., the mononuclear cell layer) and place it in another 15ml centrifuge tube (add 9ml of saline to each tube first), and centrifuge at 1000 rpm for 10min. Quickly pour off the supernatant, add 3ml of red blood cell lysis buffer to resuspend, lyse the red blood cells for 5min, add 10ml of saline to stop the lyse of the red blood cells, centrifuge at 1000 rpm for 10min, remove the supernatant (if there are many cells, the time can be left longer), and calculate the total number of mononuclear cells isolated. We calculated the appropriate volume of cell suspension to ensure that the number of cells in each sample is between 8,000 and 10,000, and prepare to add magnetic beads.

[0110] (2) CD138 magnetic bead sorting: 1×10 7 After thoroughly mixing, add 10 μL of cells + 10 μL of magnetic beads + 80 μL of buffer and incubate at 4-8°C for 20 min.

[0111] According to every 10 7Add 2 ml Buffer to each cell and rinse, centrifuge at 800 rpm for 5 min, remove the supernatant, and wash away the unbound magnetic beads. Place the LS type separation column on the separation device, first moisten the column with 1 ml Buffer, and set aside after completion.

[0112] Add the cell suspension to the sorting column and rinse twice with 3 ml buffer each time. Remove the sorting column from the sorter, then add 3 ml buffer and quickly push it out, repeat the previous steps with saline, spin at 1000 rpm for 10 min to collect CD138+ cells, count the cells, and freeze the cryopreservation solution for later use.

[0113] (3) Single-cell sequencing: Illumina NovoSeq 6000 of the 10x Genomics platform (Pleasanton, CA, USA) was used with a sequencing depth of 2.5 million reads / cell. Single-cell capture and library construction used the Chromium Next GEMSingle Cell 5' Kit v2 (10x Genomics) (PN-1000263) according to the manufacturer's instructions.

[0114] The cell suspension and magnetic beads are loaded onto the 10xGenomicsChromiumChipK to generate single-cell droplets (GEMs). Then, cDNA and cell barcodes are amplified by PCR, with the amplification program being 53°C for 45 min, 85°C for 5 min, and the final program being maintained at 4°C. The resulting sample can be stored at 4°C for a short period of time and the next experiment can be performed as soon as possible.

[0115] The scRNA-seq library was constructed using 5'LibraryKits (PN-1000190), and the scBCR-seq library was constructed using V(D)JEnrichmentKits, HumanB Cell (PN-1000252). The constructed library was sequenced on the Illumina NovaSeq6000 platform to generate 2×150-bp sequences.

[0116] Using GRCh38 as the reference library, CellRanger (v5.0.0) was used to process the original scRNA-seq data. The ROC curve analysis results based on the ITM2C expression level between IgD MM patients and non-IgD MM patients are shown in Figure 2 Figure 8 shown.

[0117] The receiver operating characteristic curve (ROC curve) is a tool widely used to evaluate the performance of binary classification models. In the medical field, ROC curves are often used to evaluate the performance of diagnostic tests, screening or prediction models. The larger the area under the curve (AUC), the higher the overall accuracy of the model. It is generally believed that when 0.90≤AUC<1.00, the model has excellent discrimination ability; 0.75≤AUC<0.90, the model has good discrimination ability; 0.60≤AUC<0.75, the model has certain discrimination ability, but it is not recommended; AUC<0.60, the model has poor discrimination ability.

[0118] According to the above Figure 8 The results show that the AUC corresponding to the ROC curve is 0.891>0.75, and the model has good discrimination ability, indicating that the expression level of ITM2C can be used to distinguish IgD MM patients from non-IgD MM patients, that is, gene ITM2C can be used as a biomarker for screening IgD MM patients.

[0119] (2) Verification at the translation level

[0120] Six different MM patients were included and flow cytometry was used to detect the amount of ITM2C protein on the tumor cell membrane for verification. The specific information of these six MM patients is shown in Table 4:

[0121] Table 46 Information of verified patients

[0122] Patient number gender age Subtype DS staging First visit time IgD-1 female 53 IgD IIIA 2023.07.24 IgD-2 male 57 IgD IIIA 2023.8.14 IgD-3 female 53 IgD IIIA 2019.10.08 Non-IgD-1 female 65 Light chain IIIA 2023.1.13 Non-IgD-2 female 59 Light chain IIIA 2023.12.19 Non-IgD-3 male 70 IgG IIIA 2023.6.7

[0123] Specific experimental process and experimental results:

[0124] Primary multiple myeloma cells of the patients verified in Table 4 were collected, washed 3 times with PBS buffer and then resuspended in 50ul PBS buffer. Antibodies were added to CD138+ cells for anti-ITM2C-APC surface antigen staining and then loaded onto the machine. The primary antibody was stained at 4°C for 20-30min, and 500ul PBS buffer was added after staining. After washing 2-3 times, the secondary antibody was added. Specific antibody information is shown in Table 5.

[0125] Table 5 Antibody information used in flow cytometry

[0126]

[0127] The protein expression level of ITM2C on the cell membrane surface was expressed by mean fluorescence intensity (MFI). Fig. 9 .

[0128] Depend on Fig. 9 As a result, compared with non-IgD MM patients, the amount of ITM2C protein in the tumor cell membrane of IgD MM patients is significantly reduced. Combined with the ROC curve analysis results of the single cell experiment, it can be seen that the gene ITM2C can be used as a biomarker for screening IgD MM patients, especially by detecting the expression of ITM2C protein in the tumor cell membrane, IgD MM can be screened out from MM patients. In this explanation, the number of IgD MM in the validation sample is small, mainly because IgD multiple myeloma patients are relatively more difficult to collect clinically. IgD multiple myeloma (Multiple Myeloma, MM) is a relatively rare subtype of multiple myeloma. Some foreign studies have reported that IgD MM accounts for 1.0% to 2.5% of all MM. Although the number of IgD multiple myeloma in the validation sample of this embodiment is small, it meets the general requirement of at least 3 cases, which can explain that the screening of IgD MM is achieved by detecting the ITM2C gene expression level of multiple myeloma patients, especially the detection of ITM2C protein expression on the tumor cell membrane.

[0129] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of an ITM2C protein detection reagent in the preparation of an IgD multiple myeloma screening kit.

2. Use of the ITM2C protein detection reagent according to claim 1 in the preparation of an IgD multiple myeloma screening kit, characterized in that: The protein detection reagent is used for quantitatively detecting the expression amount of ITM2C protein on the cell membrane, and includes ITM2C monoclonal antibody or polyclonal antibody.

3. A screening kit for IgD multiple myeloma, characterized in that: The screening kit includes reagents for detecting the expression level of ITM2C protein on the cell membrane.

4. The IgD multiple myeloma screening kit according to claim 3, characterized in that: The screening kit comprises ITM2C monoclonal antibody or polyclonal antibody.

5. The IgD multiple myeloma screening kit according to claim 4, characterized in that: The screening kit further comprises a secondary antibody, wherein the secondary antibody comprises a monoclonal or polyclonal antibody that is labeled with an enzyme or a fluorescent dye and specifically binds to the ITM2C monoclonal antibody or polyclonal antibody.

6. The IgD multiple myeloma screening kit according to claim 5, characterized in that: The ITM2C monoclonal antibody includes proteintech.

7. The IgD multiple myeloma screening kit according to claim 5 or 6, characterized in that: The screening kit also includes a wash buffer.

8. An IgD multiple myeloma screening system, characterized in that: It includes a data acquisition module, a judgment module and a result display module; The data acquisition module is used to obtain the expression level of ITM2C protein on the tumor cell membrane of multiple myeloma patients and submit it to the judgment module; The judgment module uses the obtained ITM2C protein expression level as the input of the classifier and judges according to the following method: The lower the expression of ITM2C protein, the higher the risk of IgD multiple myeloma. If the obtained ITM2C protein expression level is higher, the risk of IgD multiple myeloma is judged to be lower; and the judgment result is submitted to the result display module; The result display module performs output display according to the judgment result of the judgment module.

9. The IgD multiple myeloma screening system according to claim 8, characterized in that: The judgment module specifically performs judgment according to the following method: If the expression level of ITM2C protein obtained is significantly lower than that of ITM2C protein in non-IgD MM, the sample is judged to be from an IgD MM patient; If the expression level of ITM2C protein obtained is not significantly different from that of non-IgD MM, it is judged that the sample comes from a non-IgD MM patient; The ITM2C protein expression level of the non-IgD MM is the average expression level of the ITM2C protein in one or more of the light chain type, non-secretory type, IgA type, and IgG type MM patients.

10. The IgD multiple myeloma screening system according to claim 9, characterized in that: The ITM2C protein expression level was determined by flow cytometry.

Images