An igd type multiple myeloma screening kit and screening system thereof

Abstract

The present application relates to a kind of IgD type multiple myeloma screening kit and screening system thereof, the present application is first discovered compared with non-IgD type multiple myeloma, such as IgA, IgG, light chain type, non-secretory MM, the expression amount of ITM2C protein on the tumor cell membrane of IgD type multiple myeloma patient is significantly reduced, and ITM2C protein can be used as a biomarker for screening IgD type multiple myeloma, the reagent for detecting the expression amount of ITM2C protein is used to prepare IgD type multiple myeloma screening kit, the kit has high specificity and high sensitivity, can realize accurate and effective screening IgD type multiple myeloma by detecting the expression amount of ITM2C protein on the tumor cell membrane.

Description

[0001] This application claims priority to Chinese application No. 202410643478X filed on May 22, 2024. TECHNICAL FIELD

[0002] The present application relates to the field of biomedicine, and more particularly to an IgD type multiple myeloma screening kit and a screening system thereof. BACKGROUND

[0003] Multiple myeloma (MM) is the second most common hematological malignancy worldwide, and its tumor cells originate from plasma cells in the bone marrow. Its characteristics are abnormal proliferation of bone marrow plasma cells accompanied by overproduction of monoclonal immunoglobulin or light chain (M protein), which in turn damages target organs, such as organ dysfunction, anemia, kidney damage, and bone damage. According to the different secreted monoclonal immunoglobulin, it can be divided into IgG, IgA, IgM, IgD, IgE and light chain type, etc. Among them, IgD type is a rare special subtype. The treatment plan and prognosis of multiple myeloma are related to the subtype, and at present there is no separate targeted treatment plan for this subtype. Like other subtypes, the first-line treatment is mainly based on proteasome inhibitors, but the effect is not good. Therefore, accurate typing is crucial for the treatment or prognosis of multiple myeloma patients, especially accurate differentiation of IgD type multiple myeloma, which is beneficial to overcome the technical difficulties of IgD type multiple myeloma without specific treatment plan.

[0004] However, there are certain difficulties in diagnosing IgD type MM at present. First of all, as a relatively rare subtype of MM, many clinicians or clinical workers are not familiar with it, which can easily lead to misdiagnosis and missed diagnosis. Secondly, IgD type MM is usually very dangerous, and when patients develop extramedullary lesions and cause atypical manifestations, it may lead clinicians to misjudge the patients as other non-MM severe diseases, thereby causing delay in the disease. In addition, serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) are generally used in clinics to diagnose MM, but these mainstream diagnostic methods are prone to false negatives in detecting IgD protein due to low protein levels and special electrophoretic migration performance. Moreover, IFE of IgD has not yet been popularized, and many units can only detect common IgG, IgA and IgM. Therefore, a part of IgD patients may be missed or misdiagnosed as light chain type or non-secretory MM. In summary, the complexity of IgD type MM diagnosis is still a big problem for clinicians, fully demonstrating the urgent need to develop more sensitive, simple, fast and accurate new detection screening technologies for accurate diagnosis of IgD type MM. SUMMARY

[0005] In order to solve the above defects or improvement needs of the prior art, the present application provides an IgD type multiple myeloma screening kit and a screening system thereof, which aims to find that the expression amount of ITM2C protein on the tumor cell membrane of IgD type MM patients is significantly lower than that of non-IgD type MM such as IgA, IgG, light chain type and non-secretion type, that the IgD type MM and the non-IgD type MM can be well distinguished based on the expression level of ITM2C gene, and that the expression amount of ITM2C protein can be used as a biomarker for distinguishing IgD type and non-IgD type MM (including light chain type and non-secretion type) patients, thereby solving the technical problem that the prior art cannot accurately distinguish IgD type and non-IgD type MM such as light chain type and non-secretion type.

[0006] In order to achieve the above-mentioned purpose, according to one aspect of the present application, there is provided an application of an ITM2C protein detection reagent in the preparation of an IgD type multiple myeloma screening kit.

[0007] Preferably, the application of the ITM2C protein detection reagent in the preparation of the IgD type multiple myeloma screening kit uses the protein detection reagent for quantitatively detecting the expression amount of ITM2C protein on the cell membrane, which includes an ITM2C monoclonal antibody or a polyclonal antibody.

[0008] Preferably, the application of the ITM2C protein detection reagent in the preparation of the IgD type multiple myeloma screening kit uses the protein detection reagent for quantitatively detecting the expression amount of ITM2C protein on the tumor cell membrane.

[0009] According to another aspect of the present application, there is also provided an IgD type multiple myeloma screening kit, which includes a reagent for detecting the expression level of ITM2C protein on the cell membrane.

[0010] Preferably, the IgD type multiple myeloma screening kit includes an ITM2C monoclonal antibody or a polyclonal antibody.

[0011] Preferably, the IgD type multiple myeloma screening kit further includes a secondary antibody, which includes a monoclonal or polyclonal antibody labeled with an enzyme or a fluorescent dye and specifically combined with the ITM2C monoclonal antibody or the polyclonal antibody, for detecting the binding amount of the ITM2C monoclonal antibody or the polyclonal antibody to the ITM2C protein on the tumor cell membrane.

[0012] Preferably, the IgD type multiple myeloma screening kit includes an ITM2C monoclonal antibody, which includes proteintech.

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

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

[0015] The data acquisition module is configured to acquire the expression level of ITM2C protein on the tumor cell membrane of a multiple myeloma patient and submit the expression level to the judgment module.

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

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

[0018] The higher the acquired expression level of ITM2C protein, the lower the risk of IgD type multiple myeloma.

[0019] The result display module displays the judgment result of the judgment module.

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

[0021] If the acquired expression level of ITM2C protein is significantly lower than the expression level of ITM2C protein of non-IgD type MM, it is judged that the sample is from an IgD type MM patient.

[0022] If the acquired expression level of ITM2C protein has no significant difference from the expression level of ITM2C protein of non-IgD type MM, it is judged that the sample is from a non-IgD type MM patient.

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

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

[0025] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects 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 application finds that the expression amount of ITM2C protein on the tumor cell membrane can be used as a biomarker for distinguishing IgD type and non-IgD type MM, and the ITM2C protein detection reagent can be applied to the preparation of an IgD type multiple myeloma screening kit. The screening kit has high specificity and high sensitivity, and can determine the possibility of the sample from an IgD type multiple myeloma patient, that is, the risk degree of suffering from IgD type multiple myeloma, by detecting the expression amount of ITM2C protein on the tumor cell membrane of the multiple myeloma patient. The lower the expression amount of ITM2C protein, the greater the possibility that the sample is from an IgD type multiple myeloma patient, and the higher the risk of suffering from IgD type multiple myeloma. The higher the expression amount of ITM2C protein, the smaller the possibility that the sample is from an IgD type multiple myeloma patient, and the lower the risk of suffering from IgD type multiple myeloma. Based on the expression level of ITM2C, IgD type multiple myeloma and non-IgD type multiple myeloma patients can be accurately distinguished, and IgD type multiple myeloma can be effectively screened from multiple myeloma patients. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0029] Figure 3 is a differentially expressed gene in newly diagnosed IgD type and non-IgD malignant tumor cells; Figure 3 A in the formula is the difference in activity level of 9 modules, Figure 3 B in the formula is the difference in activity of the protein processing (M2) module in IgD malignant cells and non-IgD cells;

[0030] Figure 4 is a differential analysis result of the protein expression amount of ITM2C on the membrane of IgD type and non-IgD malignant tumor cells;

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

[0032] Figure 6 is a ROC curve of IgD type MM and non-IgD type MM (including IgG, IgA, light chain type and non-secretion type) based on the protein expression amount of ITM2C on the tumor cell membrane;

[0033] Figure 7is the ROC curve of IgD type MM and non-IgD type MM (including IgG, IgA, light chain type and non-secretion type) based on the transcription level of BCL2 gene in tumor cells;

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

[0035] Figure 9 is the difference analysis result of the expression amount of ITM2C protein on the tumor cell membrane of IgD type MM patients and non-IgD type MM patients in Example 3. DETAILED DESCRIPTION

[0036] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects according to the present application will be described in detail below in combination with the drawings and preferred embodiments.

[0037] It is generally believed in previous studies that ITM2C is overexpressed in multiple myeloma (MM) cells and is a potential marker gene of multiple myeloma. However, we found that the expression amount of ITM2C protein on the tumor cell membrane of IgD type MM patients is significantly lower than that of non-IgD type MM (including light chain type, non-secretion type, IgA and IgG), and the expression level of ITM2C can well distinguish IgD type and non-IgD type MM, and the expression amount of ITM2C protein can be used as a biomarker for screening IgD type multiple myeloma.

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

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

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

[0041] Further, the screening kit further includes a secondary antibody, which is a monoclonal or polyclonal antibody specifically combined with the primary antibody (ITM2C monoclonal antibody or polyclonal antibody), for detecting the binding of ITM2C monoclonal antibody or polyclonal antibody to ITM2C protein on the tumor cell membrane.

[0042] In some embodiments, the primary antibody is an ITM2C monoclonal antibody selected from the antibody with brand proteintech (item number: 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. The appropriate secondary antibody can be determined according to the fluorescence of the panel actually used.

[0043] In the present application, the screening kit also includes auxiliary reagents for quantitatively detecting ITM2C protein on the membrane of tumor cells, such as the washing buffer required for detecting the expression level of ITM2C protein on the cell membrane by flow cytometry, which includes a staining buffer or a PBS buffer.

[0044] In addition, the present application also provides an IgD type 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 acquire the expression amount of ITM2C protein on the membrane surface of tumor cells of a multiple myeloma patient and submit it to the judgment module. In some embodiments, the expression amount of ITM2C protein is obtained by flow cytometry, but the method for acquiring the expression amount of ITM2C protein on the cell membrane is not limited in the present application.

[0046] The judgment module takes the acquired expression amount of ITM2C protein as the input of the classifier and judges according to the lower the acquired expression amount of ITM2C protein, the higher the risk of IgD type multiple myeloma. Specifically as follows:

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

[0048] The higher the acquired expression amount of ITM2C protein, the lower the risk of IgD type multiple myeloma, that is, the smaller the possibility that the sample comes from an IgD type multiple myeloma patient; and the judgment result is submitted to the result display module.

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

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

[0051] If the acquired expression amount of ITM2C protein is significantly lower than the expression amount of ITM2C protein of non-IgD type MM, it is judged that the sample comes from an IgD type MM patient.

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

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

[0054] In the present application, the determination can also be performed according to the following method:

[0055] If the acquired ITM2C protein expression level is greater than the preset value, the patient is determined to be a non-IgD type MM patient.

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

[0057] The receiver operating characteristic curve (ROC curve) is a tool widely used to evaluate the performance of a binary classification model. In the medical field, the ROC curve is often used to evaluate the performance of diagnostic tests, screening or prediction models. It is a curve with true positive rate (sensitivity) as the vertical coordinate and false positive rate (1-specificity) as the horizontal coordinate. 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 on the ROC curve, such as selecting the classification threshold value that maximizes the Youden index as the preset value, where the Youden index = sensitivity + specificity - 1.

[0058] The following is an example

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

[0060] ①Inclusion criteria: Collect multiple myeloma patients admitted to the Department of Hematology of our hospital from 2018 to 2023, the diagnosis meets the domestic and foreign standards (Literature 1, Literature 2): Chinese Medical Association Hematology Branch, Chinese Medical Association Hematology Branch, China Multiple Myeloma Diagnosis and Treatment Guidelines (2024 Revision); Literature 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] Literature 2: Association, C.H. (2024). Guidelines for the diagnosis and management of multiple myeloma in China (2024 revision). Zhonghua nei ke za zhi 63, 1186-1195.

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

[0063] ②Exclusion criteria: Exclude multiple myeloma patients who are not multiple myeloma or have other tumor diseases.

[0064] All the following patients have given written informed consent.

[0065] Example 1 Difference analysis of tumor cells between IgD type and non-IgD type MM patients at the transcriptome level, as follows:

[0066] Randomly selected IgD MM patients newly diagnosed (n=4) and relapsed (n=9) in our hospital, of which 2 cases were t(11;14) positive and 11 cases were t(11;14) negative, and non-IgD MM (IgG, IgA, light chain type, non-secretory type) patients newly diagnosed (n=17) and relapsed (n=7), after written informed consent, all patients in this batch were subjected to bone marrow biopsy, and the sample was sorted by CD138 magnetic beads, and the CD138+ plasma cells were retained for 10x single cell RNA sequencing and VDJ sequencing. The sequencing system is Illumina NovoSeq 6000, and the sequencing depth is 2.5 million reads / cell. The query website of the original file (individual name D1-37) uploaded on the public database of single cell sequencing and VDJ sequencing is https: / / ngdc.cncb.ac.cn / gsa-human / browse / HRA005760.

[0067] After the sequencing data is quality controlled, cell clustering, CNV analysis, gene regulation 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] This example uses single cell sequencing data analysis to compare the differences between IgD multiple myeloma (IgD / t(11;14)+, IgD / t(11;14)-) and non-IgD MM at the transcriptome level. Through enrichment analysis of protein processing pathway-related pathways (including endoplasmic reticulum stress, autophagy, and unfolded protein response), we found differentially expressed genes related to these pathways in IgD and non-IgD malignant tumor cells, including BCL2, ITM2C, etc., some of which are shown in Figure 1 and Figure 2 .

[0070] In addition, the gene expression of single cells is integrated into a combination of a set of interpretable biological gene expression programs (GEPs) using the non-negative matrix factorization (cNMF) method. From 21 newly diagnosed samples (4 of which are IgD MM and 17 of which are non-IgD MM), 115 GEPs (A) were resolved. Figure 3

[0071] ​The sample-sample similarity among these GEPs is demonstrated by correlation clustering, which further clusters into nine common modules, including regular "housekeeping" modules such as cell cycle, RNA / protein synthesis and cell stress, and less understood immune-related, interferon-related and extracellular matrix-related modules. For each module, we test whether its expression level is different between IgD and non-IgD MM, and find that especially the protein processing (M2) module is significantly different in activity between IgD and non-IgD cells, as shown in Figure 3 FIG. 6B.

[0072] ITM2C is a membrane protein, and membrane proteins are more likely to be detected in clinical practice. Therefore, we focused on the differentially expressed membrane protein ITM2C in the M2 module in the following study. Previous studies generally believe that ITM2C is overexpressed in MM cells and is a potential marker gene for MM.

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

[0074] The protein expression level of cell membrane surface ITM2C was represented by mean fluorescence intensity (MFI), and the results are shown in Figure 4

[0075] As shown in Figure 4 ​We found that there was a significant difference in the protein expression level of cell surface ITM2C between IgD type multiple myeloma patients and non-IgD type multiple myeloma patients, and the protein expression level of ITM2C in IgD type multiple myeloma patients was significantly lower than that in non-IgD type multiple myeloma. It can be seen that the flow cytometry analysis results are consistent with single cell sequencing, which again shows that IgD type MM has obvious heterogeneity compared with other subtypes of MM. It is speculated that the cell membrane surface ITM2C protein can be used as a potential biomarker for screening IgD type MM, and the detection of the expression level of ITM2C protein on the cell membrane surface of multiple myeloma tumor cells can be used to screen IgD type MM from MM.

[0076] In addition, from the above experimental results, it can be seen that IgD type multiple myeloma and other subtypes of MM have significant differences 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, regulate cell death, calcium homeostasis, endoplasmic reticulum stress and autophagy.

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

[0078] CD138+ cells of IgD type and non-IgD type MM patients were collected respectively, RNA extraction and reverse transcription were performed, patient primary specimen cDNA was obtained, and qPCR mixed solution was prepared. The system is shown in the following table;

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

[0080]

[0081] After preparing the Gene mix according to the above system, add 18 μL / well to the PCR well plate under light-proof conditions, and note to set 3 replicate wells; add 2 μL of cDNA template corresponding to the treatment group to each well, and prepare for machine detection after sticking the sealing film. The machine detection is carried out according to the "two-step method", and the specific program is shown in the following table;

[0082] Table 2 RT-qPCR amplification program

[0083]

[0084]

[0085] After obtaining the Ct value, calculate 2 -△△CtThe relative expression amount of the target gene was quantitatively analyzed, and the difference in the expression level of BCL2 gene between the IgD type multiple myeloma and the non-IgD type malignant plasma cell was analyzed, as shown in Table 2. Figure 5

[0086] As shown in Table 2, the expression amount of BCL2 in the IgD type MM patients was significantly higher than that in the non-IgD type MM patients, and there was no significant difference in the expression amount of BCL2 between the t(11;14) positive IgD type MM patients and the t(11;14) negative IgD type MM patients. Figure 5

[0087] In addition, the IHC experiment immunohistochemical result of BCL2 showed that the percentage of BCL2 positive cells in the t(11;14) negative IgD type multiple myeloma tissue sample was about 85-95%, which indicated that the t(11;14) negative IgD type multiple myeloma patients also universally overexpressed BCL2. It can be seen that the BCL2 protein can also be a potential biomarker for screening IgD type MM.

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

[0089] To verify the result of Example 1, another batch of IgD type MM and non-IgD type patients were selected for verification. Although it is a basic operation in the art to use different patients from the screening set in Example 1 for verification, it is still particularly mentioned herein that the IgD type MM and non-IgD type MM patients selected for verification in the present application are not the same batch of patients as the IgD type MM and non-IgD type MM patients in Example 1, i.e., the verification set and the screening set are not the same.

[0090] Considering that ITM2C is a membrane protein, it is easier to detect the expression product of its gene, ITM2C protein, in a clinical environment. In this embodiment, flow cytometry was used to detect the expression amount of ITM2C protein on the cell membrane surface for verification, as follows:

[0091] Flow cytometry was used to perform anti-ITM2C-APC surface antigen staining on CD138+ cells of IgD type MM and non-IgD type MM patients, and the expression level of ITM2C protein on the cell surface was represented by the mean fluorescence intensity (MFI). The diagnostic performance of the gene ITM2C for IgD type MM was analyzed by ROC curve, and the ROC curve of IgD type MM and non-IgD type MM (including IgG, IgA, light chain type and non-secretion type) is shown in Figure 2. Figure 6

[0092] As shown in Table 2, the expression amount of BCL2 in the IgD type MM patients was significantly higher than that in the non-IgD type MM patients, and there was no significant difference in the expression amount of BCL2 between the t(11;14) positive IgD type MM patients and the t(11;14) negative IgD type MM patients. Figure 6 ​​​It can be seen that the AUC area corresponding to the ROC curve of IgD type and non-IgD type MM based on the expression amount of ITM2C protein on the membrane of tumor cells is 0.897, the specificity is 1, the sensitivity is 0.882, and the maximum approximate index is 9.579. It can be seen that detecting the expression amount of ITM2C protein can be used to distinguish IgD type and non-IgD type MM patients, especially to distinguish IgD type and light chain type, non-secretion type, which shows that ITM2C protein can be used as a biomarker for screening IgD type MM. By detecting the expression amount of ITM2C protein on the membrane of tumor cells, IgD type and non-IgD type MM patients can be distinguished, and IgD type MM can be screened from MM patients. In this embodiment, if the expression amount of ITM2C protein is less than or equal to the classification threshold value corresponding to the maximum approximate index, the patient is judged to be IgD type MM, and if the expression amount of ITM2C protein is greater than the classification threshold value corresponding to the maximum approximate index, the patient is judged to be non-IgD type MM.

[0093] Comparative Example 1

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

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

[0096] Example 3 verifies at transcriptional and translational levels

[0097] At present, there is no cell model or animal model of IgD type MM. In order to ensure the credibility of the verification results of Example 2, we selected two batches of IgD type and non-IgD type MM patients different from those in Example 1 and Example 2 for single cell sequencing and flow cytometry detection verification, as follows:

[0098] (1) Verification at the transcriptional level

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

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

[0101]

[0102]

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

[0104] 1. Sample processing and single-cell sequencing

[0105] Bone marrow samples of patients in Table 3 were collected, and CD138 magnetic beads (Miltenyi Biotec GmbH, Germany) were used for plasma cell sorting according to the instructions, as follows:

[0106] (1) Preparation of single nuclear cell suspension

[0107] About 20 ml of bone marrow was drawn and anticoagulated with sodium heparin. Centrifugation was performed at 1500 rpm for 10 min, and the supernatant plasma was transferred to a new centrifuge tube. After centrifugation at 3000 rpm for 10 min, it was stored in a 1.5 ml centrifuge tube (2 tubes) and frozen in a -20°C refrigerator.

[0108] The remaining bone marrow blood was poured into a 50 ml centrifuge tube and diluted with PBS or physiological saline to 40 ml. The diluted sample was slowly attached to a 15 ml lymphocyte separation medium centrifuge tube, and 6 ml of lymphocyte separation medium was added to each tube. Centrifugation was performed at 2500 rpm for 30 min (slow acceleration and slow deceleration).

[0109] After centrifugation, the misty cell layer (i.e., single nuclear cell layer) was removed and placed in another 15 ml centrifuge tube (9 ml of physiological saline was added to each tube), and centrifugation was performed at 1000 rpm for 10 min. The supernatant was quickly poured, 3 ml of red blood cell lysis solution was added for resuspension, and red blood cell lysis was performed for 5 min. Physiological saline 10 ml was added to terminate red blood cell lysis, and centrifugation was performed at 1000 rpm for 10 min. The supernatant was removed (if the cells are more, the time can be placed for a long time), and the total number of separated single nuclear cells was calculated. The appropriate cell suspension volume was calculated to ensure that the number of cells in each sample was between 8,000 and 10,000, and the magnetic beads were prepared.

[0110] (2) CD138 magnetic bead sorting: 1 x 10 7 After mixing well, incubate at 4-8°C for 20 min.

[0111] 10 7Cells were washed with 2 ml Buffer, centrifuged at 800 rpm for 5 min, and the supernatant was removed to remove unbound magnetic beads. The LS type sorting column was placed on the sorter, and 1 ml Buffer was used to rinse the column. After completion, it was ready for use.

[0112] The cell suspension was added to the sorting column, and 3 ml Buffer was used to rinse twice. The sorting column was removed from the sorter, and 3 ml Buffer was immediately added to push it out quickly. The previous action was repeated with normal saline. CD138+ cells were collected at 1000 rpm for 10 min, counted, and stored in a frozen solution for storage.

[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. Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics) (PN-1000263) was used for single-cell capture and library construction, and the operation was performed according to the manufacturer's instructions.

[0114] The cell suspension and magnetic beads were loaded onto the 10x Genomics Chromium Chip K to generate single-cell microdroplets (GEMs). Then, the cDNA and cell barcodes were amplified by PCR with a 53°C 45 min, 85°C 5 min amplification procedure, and finally maintained at 4°C. The resulting sample can be stored at 4°C for a short time, and the next step should be performed as soon as possible.

[0115] scRNA-seq libraries were constructed using 5' Library Kits (PN-1000190), and scBCR-seq libraries were constructed using V(D)J Enrichment Kits, Human B Cell (PN-1000252). The constructed libraries were sequenced on the Illumina NovaSeq 6000 platform to generate 2x150-bp sequences.

[0116] The raw scRNA-seq data was processed using CellRanger (v5.0.0) with GRCh38 as the reference library. The ROC curve analysis results based on the expression levels of ITM2C between IgD-type MM patients and non-IgD-type MM patients are shown in Figure 8 .

[0117] Receiver Operating Characteristic Curve (ROC curve) is a tool widely used to evaluate the performance of a binary classification model. In the medical field, ROC curve is often used to evaluate the performance of diagnostic tests, screening or prediction models. The larger the Area Under Receiver Operating Characteristic Curve (AUC), the higher the overall accuracy of the model. Generally, 0.90≤AUC<1.00 is considered as excellent model discrimination ability; 0.75≤AUC<0.90 is considered as good model discrimination ability; 0.60≤AUC<0.75 is considered as model has certain discrimination ability, but not recommended for use; AUC<0.60 is considered as poor model discrimination ability.

[0118] According to the above Figure 8 The results show that the AUC of the ROC curve is 0.891, which is greater than 0.75, indicating that the model has good discrimination ability. This means that the expression level of ITM2C based on ITM2C can be used to distinguish IgD type MM patients from non-IgD type MM patients, i.e. the gene ITM2C can be used as a biomarker for screening IgD type MM patients.

[0119] (2) Verification at the translation level

[0120] Six different MM patients were included in the study to verify the amount of ITM2C protein on the tumor cell membrane using flow cytometry. The specific information of these six MM patients is shown in Table 4:

[0121] Table 4 Information of 6 verification patients

[0122] Patient No. Gender Age Subtype DS Stage Time of Initial Diagnosis 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 Type IIIA 2023.1.13 Non-IgD-2 Female 59 Light Chain Type IIIA 2023.12.19 Non-IgD-3 Male 70 IgG IIIA 2023.6.7

[0123] Specific experimental procedures and results:

[0124] Primary multiple myeloma cells from the patients in Table 4 were collected and washed with PBS buffer for 3 times, then resuspended in 50ul PBS buffer. After staining the CD138+ cells with anti-ITM2C-APC surface antigen, the sample was loaded into the machine. The primary antibody was incubated at 4℃ for 20-30min, then 500ul PBS buffer was added and washed for 2-3 times before adding the secondary antibody. The 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 represented by the mean fluorescence intensity (MFI), and the results are shown in Figure 9 .

[0128] By Figure 9 It can be seen from the results that the amount of tumor cell membrane ITM2C protein in IgD type MM patients is significantly lower than that in non-IgD type MM patients. Combined with the ROC curve analysis results of single cell experiments, it can be seen that the gene ITM2C can be used as a biomarker for screening IgD type MM patients, especially by detecting the expression amount of tumor cell membrane ITM2C protein to screen IgD type MM from MM patients. It is explained below that the number of IgD type MM in the verification sample is relatively small, mainly because it is relatively difficult to collect IgD type multiple myeloma patients in clinical practice. IgD type multiple myeloma (Multiple Myeloma, MM) is a relatively rare subtype of multiple myeloma. Some foreign studies report that IgD type MM accounts for 1.0% to 2.5% of all MM. Although the number of IgD type multiple myeloma in the verification sample of the present embodiment is relatively small, it meets the general requirement of at least 3 cases, and it can be shown that IgD type MM can be screened by detecting the expression level of ITM2C gene in multiple myeloma patients, especially by detecting the expression amount of ITM2C protein on tumor cell membrane.

[0129] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to obtain equivalent embodiments with equivalent changes, without departing from the scope of the technical solution of the present application. Any modification, equivalent change and modification of the above embodiments made in accordance with the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. Use of an ITM2C protein detection reagent in the manufacture of a screening kit for IgD type multiple myeloma, characterized in that, The ITM2C protein detection reagent is used for quantitatively detecting the expression amount of ITM2C protein on the tumor cell membrane of multiple myeloma.

2. The use of the ITM2C protein detection reagent according to claim 1 in the preparation of an IgD type multiple myeloma screening kit, characterized in that, The protein detection reagent comprises an ITM2C monoclonal antibody or a polyclonal antibody.

3. An IgD type multiple myeloma screening system, characterized by, The method comprises a data acquisition module, a judgment module and a result display module. The data acquisition module is used for acquiring the expression amount of ITM2C protein on the tumor cell membrane of a multiple myeloma patient sample and submitting the expression amount to the judgment module. The judgment module takes the acquired expression amount of ITM2C protein as the input of a classifier and judges according to the following method: The lower the acquired expression amount of ITM2C protein, the higher the risk of IgD-type multiple myeloma. The higher the acquired expression amount of ITM2C protein, the lower the risk of IgD-type multiple myeloma. The judgment result is submitted to the result display module.

4. The IgD-type multiple myeloma screening system of claim 3, wherein, The result display module displays the judgment result of the judgment module. The judgment module judges according to the following method: If the acquired expression amount of ITM2C protein is obviously lower than the expression amount of ITM2C protein of non-IgD-type multiple myeloma, it is judged that the sample is from an IgD-type multiple myeloma patient. If the acquired expression amount of ITM2C protein has no obvious difference from the expression amount of ITM2C protein of non-IgD-type multiple myeloma, it is judged that the sample is from a non-IgD-type multiple myeloma patient.

5. The IgD-type multiple myeloma screening system of claim 4, wherein, The expression amount of ITM2C protein of non-IgD-type multiple myeloma is the average expression amount of ITM2C protein of one or more patients of light chain type, non-secretion type, IgA and IgG type multiple myeloma. The expression amount of ITM2C protein is obtained by flow cytometry.

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