A method and kit for prognosis evaluation of multiple myeloma

Abstract

The application belongs to the technical field of medical treatment, and particularly relates to a multiple myeloma prognosis evaluation method and kit, which comprises specific reagents for detecting mRNA or protein of at least one gene in BIRC6, DBR1 and MATR3. The specific steps of the multiple myeloma prognosis evaluation method are as follows: a. obtaining a biological sample of a to-be-detected MM patient; b. detecting the expression levels of BIRC6, DBR1 and MATR3 genes in the sample by using molecular biology technology; c. comparing the expression levels of one or more genes detected with preset reference thresholds, or calculating a comprehensive risk score according to the expression levels thereof; and d. judging the prognosis according to the reference threshold comparison or comprehensive risk score result. The expression levels of the hub members directly reflect the malignant degree of tumor cells, the genomic instability and the dependence degree on the DDR pathway, so as to become a powerful prognosis index.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to a method and kit for prognostic assessment of multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is a highly heterogeneous hematologic malignancy. Patients exhibit significant differences in clinical course and survival. Currently, the International Staging System (ISS) and the Revised International Staging System (R-ISS) are primarily used clinically, combined with high-risk cytogenetic abnormalities (such as del(17p), t(4;14), etc.) to stratify patients by risk and predict prognosis. However, even within the same risk stratification, patients' actual prognoses still vary significantly, indicating that the existing prognostic assessment system is not yet perfect.

[0003] Genomic instability of tumors is a key factor driving the progression and drug resistance of multiple myeloma (MM), while the DNA damage response (DDR) pathway, which maintains genomic stability, is abnormally active in MM cells. Therefore, identifying core molecular biomarkers that reflect the DDR "addiction" status and degree of genomic instability in MM cells is crucial for more accurate patient prognosis and guiding clinical decision-making.

[0004] Currently, the lack of corresponding solutions makes it difficult to accurately predict patient prognosis and guide clinical decision-making. Summary of the Invention

[0005] The purpose of this invention is to provide a method and kit for prognostic assessment of multiple myeloma, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a multiple myeloma prognostic assessment kit, comprising: Specific reagents for detecting the mRNA or protein of at least one of the genes BIRC6, DBR1, and MATR3.

[0007] Preferably, the specific reagent includes primer pairs, probes, or specific antibodies.

[0008] Preferably, the expression status of the BIRC6-DBR1-MATR3 axis is integrated into the R-ISS staging system to establish a multidimensional prognostic assessment model.

[0009] A prognostic assessment method for multiple myeloma, based on a multiple myeloma prognostic assessment kit, is described below: a. Obtain biological samples from the MM patient to be tested; b. Molecular biology techniques were used to detect the expression levels of BIRC6, DBR1, and MATR3 genes in the samples; c. Compare the expression levels of one or more detected genes with a preset reference threshold, or calculate a comprehensive risk score based on their expression levels; d. Determine the prognosis based on reference threshold comparisons or comprehensive risk scoring results: If the expression levels of one or more genes are significantly higher than the threshold, or the overall risk score is high, the patient is considered high-risk and has a poor prognosis. Conversely, if the expression levels of one or more genes are significantly lower than the threshold, or the overall risk score is low, the risk is considered low and the prognosis is good.

[0010] Preferably, in step a, the biological sample is tumor plasma cells isolated from bone marrow aspiration.

[0011] Preferably, in step b, the molecular biology techniques include RT-qPCR, RNA sequencing, gene chip, and immunohistochemistry.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention identifies the BIRC6-DBR1-MATR3 axis as a key regulatory unit for maintaining the genomic integrity of multifocal tumors (MM). It prevents excessive accumulation of DNA damage by analyzing the R-loop. The progressive activation of this axis is a core adaptive event in the evolution of MM from early to late stages and from indolent to aggressive. The expression levels of its members directly reflect the malignancy of tumor cells, genomic instability, and dependence on the DDR pathway, making it a powerful prognostic indicator. The prognostic predictive value and synergistic expression relationship of these three components were validated using the COMPASS MMRF database. Multivariate risk analysis confirmed that DBR1 is an independent prognostic predictor. Further analysis using datasets such as GSE6477, GSE161801, and GSE189460 revealed a synergistic and progressively increasing trend in DBR1 expression and DDR pathway activity from normal plasma cells to non-denial MM and then to recurrent MM, providing solid experimental data support for prognostic assessment. Furthermore, by integrating the expression status of this axis into R-ISS... The staging system establishes a multidimensional prognostic assessment model, using tumor plasma cells isolated from bone marrow aspirations as biological samples. It combines various mature molecular biology technologies such as RT-qPCR and RNA sequencing to determine prognosis through single-gene threshold comparison or multi-gene comprehensive risk scoring. This effectively solves the problem of large prognostic differences among patients within the same risk stratification in existing assessment systems. It can accurately identify newly diagnosed, relapsed / refractory MM patients and high-risk individuals hidden in traditional staging, providing a reliable basis for clinical treatment plan selection. It also provides potential expansion space for personalized treatment and dynamic monitoring of efficacy. Attached Figure Description

[0013] Figure 1 KM curves showing the prognostic value of DBR1, BIRC6, and MATR3 genes; Figure 2 To validate that DBR1 is an independent prognostic predictor in multivariate risk analysis; Figure 3 The graph shows a synergistic and progressively increasing trend in expression levels and DDR pathway activity. Figure 4 The graph shows a progressively increasing trend in pathway activity. Figure 5 To verify the co-expression of BIRC6 and DBR1, and the co-expression of MATR3 and DBR1. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example: Key findings and applications: The expression levels of the member genes of a functional molecular axis (BIRC6-DBR1-MATR3 axis), composed of the E3 ubiquitin ligase BIRC6, the RNA debranching enzyme DBR1, and the nuclear matrix protein MATR3, can serve as a novel combination of biomarkers for predicting the prognosis of multiple myeloma (MM) patients. Specifically, the higher the expression level of this axis member (one, two, or all three genes), the worse the patient's prognosis.

[0016] This invention is primarily aimed at all newly diagnosed or confirmed patients with multiple myeloma (MM), including but not limited to: Newly diagnosed multiple myeloma (NDMM) patients: Used for initial risk stratification before treatment initiation to guide the selection of the strength of the initial treatment regimen.

[0017] Patients with relapsed / refractory multiple myeloma (RRMM): Used to assess the aggressiveness of the disease, determine the urgency of subsequent treatment, and select appropriate treatment options.

[0018] For patients in the intermediate or low-risk categories in the traditional staging system: the method of this invention is used to perform secondary stratification to identify high-risk individuals with poor prognosis hidden within them.

[0019] Independent prognostic value evidence: In the large MMRFCoMMpass clinical cohort, Kaplan-Meier survival analysis clearly showed that patients with high BIRC6 expression (Figure 2D), high DBR1 expression (Figure 1A), and high MATR3 expression (Figure 5C) all had significantly shorter overall survival. Multivariate Cox regression analysis further confirmed that DBR1 expression was a prognostic predictor independent of other known clinical risk factors (Figure 1F), demonstrating its strong independent prognostic value.

[0020] Dynamic evidence for the co-activation of molecular axes: This dynamic evolution provides strong biological evidence for BIRC6-DBR1-MATR3 as a functional “axis” or “module”.

[0021] Evidence linking to disease progression: In the GSE6477 dataset, which includes samples from different disease stages, the expression level of DBR1 and the activity of the HR / ATM pathway increased significantly in a stepwise manner from normal plasma cells to NDMM and then to RRMM (Figure 1B). This directly links the activity of this molecular axis to the malignant progression stage of MM.

[0022] This invention provides a technical solution: A prognostic assessment kit for multiple myeloma, comprising: Specific reagents for detecting the mRNA or protein of at least one of the genes BIRC6, DBR1, and MATR3.

[0023] Based on the target molecule (mRNA / protein) and the technology platform, it is necessary to select the corresponding specific tool. The core is to ensure that the reagent can accurately identify the nucleic acid sequence or protein antigen epitope of the target gene.

[0024] Specific reagents for detecting mRNA: mRNA detection mainly relies on nucleic acid hybridization or amplification techniques, with the core reagents being specific probes or primers designed for the target gene sequence.

[0025] 1. Reagents for RT-PCR (Reverse Transcription Polymerase Chain Reaction) Specific primers: Designed based on the mRNA sequences of BIRC6 (gene ID: 10018), DBR1 (gene ID: 10400), and MATR3 (gene ID: 10752) (e.g., NCBIRefSeq sequences), primers must meet the following requirements: primer specificity (no cross-binding to other genes) and consistent amplification efficiency (e.g., similar Tm values). Example: BIRC6 primers can target conserved sequences in its coding region, avoiding non-specific amplification; cross-exon primers can be selected to exclude interference from genomic DNA contamination.

[0026] Fluorescent probes (for real-time quantitative RT-PCR): such as TaqMan probes, with a fluorescent group (such as FAM) labeled at the 5' end and a quenching group labeled at the 3' end. The sequence is complementary to the primer amplification region. Fluorescence is released only when the probe binds to the target mRNA amplification product, and the specificity is higher than that of ordinary RT-PCR.

[0027] RT-PCR kits: You can choose a universal reverse transcription-amplification kit (containing reverse transcriptase, DNA polymerase, dNTPs, etc.) and use it with the customized primers / probes mentioned above, or directly purchase a commercial RT-PCR detection kit for a single gene (some manufacturers offer pre-mixed primer and probe kits).

[0028] 2. RNA in situ hybridization (RNAISH) reagent Specific nucleic acid probes: These are divided into oligonucleotide probes (short chains, such as 20-30 nt) or complementary RNA probes (cRNA probes, long chains). Using fluorescent or chromogenic labeling (such as Digoxin, FITC), they can locate target mRNA in situ in tissues / cells. Characteristics: Probes must be designed specifically for the mRNA sequence of each gene, avoiding binding to homologous sequences. They are suitable for observing the expression distribution of target genes in tissues.

[0029] Specific reagents for protein detection: Protein detection relies on the specific binding of antigens and antibodies. The core reagents are specific antibodies against the target protein and the corresponding detection reagents.

[0030] 1. Reagents for Western Blot Primary antibody: An antibody that specifically recognizes BIRC6, DBR1, or MATR3 proteins. It must be a validated antibody (e.g., with Western blotting data). These are categorized as polyclonal antibodies (recognizing multiple epitopes with high affinity) and monoclonal antibodies (recognizing a single epitope with higher specificity). Example: BIRC6 protein has a large molecular weight (approximately 190 kDa), requiring an antibody targeting its specific domain (e.g., the BIR domain); DBR1 (approximately 38 kDa) and MATR3 (approximately 120 kDa) require antibodies with corresponding molecular weights.

[0031] Secondary antibody: An antibody against the host species of the primary antibody (e.g., rabbit anti-primary antibody needs to be combined with goat anti-rabbit secondary antibody), labeled with enzymes (e.g., HRP, AP) or fluorescent groups (e.g., IRDye800), used for signal amplification and detection.

[0032] Supporting reagents: protein extraction kit (such as RIPA lysis buffer), protein marker (for molecular weight control), developing substrate (such as ECL chemiluminescent substrate), etc.

[0033] 2. Immunohistochemistry (IHC) / immunofluorescence (IF) reagents Specific primary antibody: Antibodies suitable for IHC / IF should be selected (e.g., antibodies validated by paraffin or frozen sections) to avoid cross-reactivity. Example: MATR3 is expressed in the cell nucleus, so an antibody that can penetrate the cell membrane and specifically bind to nuclear MATR3 should be selected; DBR1 is expressed in the cytoplasm, so an antibody matching cytoplasmic localization should be selected.

[0034] Detection kits: such as the SP method kit for IHC (containing secondary antibody, enzyme-labeled streptavidin, and DAB chromogenic solution) and the fluorescent secondary antibody kit for IF (containing fluorescently labeled secondary antibody and anti-quenching agent), can be directly used with the primary antibody, simplifying the operation.

[0035] 3. Enzyme-linked immunosorbent assay (ELISA) reagents Commercial ELISA kits: Pre-coated kits targeting specific proteins, containing coating antibodies (capture antibodies), detection antibodies, enzyme-labeled secondary antibodies, substrates, etc., for the quantitative detection of target protein concentrations in samples (such as serum, cell supernatant). Features: Suitable for rapid quantification of large numbers of samples; kits with high sensitivity (e.g., detection limit <10 pg / mL) and good specificity (no cross-reactivity) should be selected.

[0036] The specific reagents include primer pairs, probes, or specific antibodies. The expression status of the BIRC6-DBR1-MATR3 axis is integrated into the existing R-ISS staging system to establish a multidimensional prognostic assessment model.

[0037] The R-ISS staging system (Revised International Staging System) is a core tool for assessing the prognosis of patients with multiple myeloma (MM). By integrating clinical indicators and molecular biological characteristics, it significantly improves the accuracy of risk stratification. The following is a detailed analysis of its core content and latest advancements: Stages and Survival Data R-ISS is stratified based on four key indicators: Serum β2-microglobulin (β2-MG): Reflects tumor burden and renal function and is an independent prognostic factor.

[0038] Serum albumin: reflects the patient's nutritional status and liver function.

[0039] Cytogenetic abnormalities: detected by fluorescence in situ hybridization (FISH), including del(17p), t(4;14), and t(14;16), which are closely associated with chemotherapy resistance and poor prognosis.

[0040] Lactate dehydrogenase (LDH): Elevated levels indicate high proliferative activity of tumor cells.

[0041] A prognostic assessment method for multiple myeloma, based on a multiple myeloma prognostic assessment kit, is described below: a. Obtain biological samples from the MM patient to be tested, preferably tumor plasma cells isolated from bone marrow aspiration; b. Molecular biology techniques were used to detect the expression levels of BIRC6, DBR1, and MATR3 genes in the samples. These techniques included RT-qPCR, RNA sequencing, gene chips, and immunohistochemistry. c. Compare the expression levels of one or more detected genes with a preset reference threshold, or calculate a comprehensive risk score based on their expression levels; d. Determine the prognosis based on reference threshold comparisons or comprehensive risk scoring results: If the expression levels of one or more genes are significantly higher than the threshold, or the overall risk score is high, the patient is considered high-risk and has a poor prognosis. Conversely, if the expression levels of one or more genes are significantly lower than the threshold, or the overall risk score is low, the risk is considered low and the prognosis is good.

[0042] Analysis of the above technical content: This solution mainly adopts the technical approach of "molecular marker detection + multidimensional model integration". By specifically detecting the mRNA or protein expression levels of BIRC6, DBR1 and MATR3 genes, and combining the molecular axis formed by them with the existing R-ISS staging system, it solves the technical pain point of large differences in prognosis among patients within the same risk stratum and difficulty in accurately identifying high-risk individuals in the traditional prognostic assessment system. This protocol uses tumor plasma cells isolated from bone marrow aspiration as the core biological sample, ensuring the purity of the detection target (tumor cells) while avoiding interference from normal cells in peripheral blood and other samples, laying the foundation for subsequent accurate detection. It utilizes mature molecular biology technologies such as RT-qPCR, RNA sequencing, gene chips, and immunohistochemistry to detect gene expression levels, balancing sensitivity (e.g., RNA sequencing can capture low-abundance mRNA), specificity (e.g., immunohistochemistry can target specific proteins with antibodies), and applicability (different technologies can be matched to different sample sizes and detection cost requirements). It innovatively introduces a dual-dimensional assessment of "single-gene threshold comparison + multi-gene comprehensive risk scoring," supporting rapid identification of high-risk patients through high expression of a single gene, while also improving the accuracy of prognostic assessment through multi-gene synergistic analysis, avoiding the limitations of single-marker detection. Finally, through a clear risk stratification standard (high expression / high score corresponds to poor prognosis, low expression / low score corresponds to good prognosis), it achieves accurate prognosis assessment for newly diagnosed, relapsed / relapsed patients. Accurate prognostic assessment of patients with refractory multiple myeloma can, in particular, uncover high-risk individuals hidden in traditional staging, providing a reliable basis for the selection of clinical treatment options (such as using more potent treatment options for high-risk patients).

[0043] From a technical perspective, this approach is deeply linked to the core pathological mechanism of multiple myeloma—genomic instability and abnormal DNA damage response (DDR) pathways. The BIRC6-DBR1-MATR3 molecular axis, as a key unit for maintaining the integrity of the tumor cell genome, directly reflects the malignancy of tumor cells (e.g., high expression suggests cells are more prone to gene mutations and uncontrolled proliferation), the degree of genomic instability (e.g., high expression means cells need to over-activate the DDR pathway through this axis to repair DNA damage, indirectly reflecting the accumulation of genomic damage), and the degree of dependence on the DDR pathway (e.g., patients with high expression may be more sensitive to DDR pathway inhibitors). This deep coupling of "pathological mechanism - molecular marker - prognostic assessment" makes this approach not only technically feasible at the detection level but also logically sound at the biological level. Compared to traditional staging systems that rely solely on clinical indicators (such as serum β2-microglobulin and albumin), it can capture the molecular essence of patient prognostic differences earlier and more accurately, filling the gap in the existing assessment system regarding the correlation between "molecular mechanism and clinical prognosis."

[0044] In terms of technological innovation, this solution also demonstrates significant clinical adaptability. For example, in the sample processing stage, it explicitly selects tumor plasma cells from bone marrow aspirations, rather than whole bone marrow samples. By enriching target cells, it reduces the dilution of test results by normal plasma cells and blood cells, making the test values ​​closer to the true expression level of tumor cells. This is especially beneficial for early-stage patients with a low proportion of bone marrow plasma cells, significantly improving test accuracy. Regarding the selection of testing technologies, it is not limited to a single method but offers multiple technical pathways. For instance, RT-qPCR is suitable for rapid clinical testing (results within hours for a single sample), meeting the rapid stratification needs of emergency and newly diagnosed patients. RNA sequencing is suitable for research and precision medicine scenarios that require simultaneous analysis of multiple gene expression profiles and exploration of potential synergistic biomarkers. Immunohistochemistry, combined with histological morphology observation, clarifies the localization of gene expression in tumor cells (e.g., the expression of MATR3 in the cell nucleus), providing dual evidence for pathological diagnosis and prognostic assessment. At the model integration level, it combines the molecular axis with the R-ISS staging system, rather than completely replacing traditional staging, utilizing the advantages of R-ISS. Based on its mature application in clinical practice (doctors have a broad understanding of this staging system), and supplemented by molecular markers, it achieves a multi-dimensional integration of "traditional clinical indicators + novel molecular markers", reducing the difficulty of clinical promotion and making it easier for medical institutions to accept and apply.

[0045] From a clinical perspective, this approach also offers potential for expanding personalized treatment and efficacy monitoring in multiple myeloma. On one hand, prognostic assessment results based on this molecular axis can further guide the selection of targeted therapy regimens. For example, patients with high expression of BIRC6-DBR1-MATR3, whose tumor cells rely on the DDR pathway for survival, could be considered for combined use of DDR pathway inhibitors (such as PARP inhibitors) to achieve a closed loop of "prognostic assessment - treatment regimen matching." On the other hand, the expression level of this molecular axis can serve as a dynamic indicator for efficacy monitoring. For instance, if the expression level of this axis gene significantly decreases after treatment, it may indicate a reduction in tumor cell malignancy and improved genomic instability, indirectly reflecting treatment effectiveness. This provides dynamic molecular-level evidence for clinical assessment of efficacy and adjustment of treatment strategies. Compared to traditional monitoring methods relying on imaging and serum M protein, it can reflect the impact of treatment on tumor cell molecular phenotypes earlier, allowing time for timely intervention (such as changing the regimen as early as possible if treatment is ineffective).

[0046] like Figure 1 As shown, sections A, B, and C of the graphs demonstrate the validation of the prognostic predictive value of DBR1, BIRC6, and MATR3 using bulk RNA sequencing data from the COMPASSMMRF database. These three graphs are prognostic KM curves, representing the prognostic value of the DBR1, BIRC6, and MATR3 genes, respectively.

[0047] like Figure 2 As shown in Figure D, multifactor risk analysis validates that DBR1 is an independent prognostic predictor.

[0048] This is a forest plot. After adjusting for age, sex, and ISS stage, DBR1 still has strong prognostic predictive value, with an HR of 1.4 and a significant P-value.

[0049] like Figure 3 As shown, in the E. GSE6477 dataset, from normal plasma cells to NDMM and then to RRMM, the expression level of DBR1 and the activity of the DDR pathway showed a synergistic and progressively increasing trend.

[0050] Using bulk RNA sequencing data from GSE6477, we found that the expression level of DBR1 and the activity of DNA damage repair pathways gradually increased during the progression of multiple myeloma (normal samples, MGUS, SMM, MM, RRMM).

[0051] like Figure 4 As shown, single-cell sequencing data of F. GSE161801 and GSE189460 clearly show that the activity of the DDR pathway progressively increases from normal plasma cells to NDMM and then to RRMM.

[0052] Using single-cell transcriptome data, we found that the activity of the DDR pathway progressively increases from normal plasma cells to NDMM and then to RRMM.

[0053] like Figure 5 As shown, G. used bulk RNA sequencing data from the COMPASSMMRF database to verify that BIRC6 and DBR1 are co-expressed, and MATR3 and DBR1 are co-expressed.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and therefore all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention.

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A prognostic assessment kit for multiple myeloma, characterized in that, include: Specific reagents for detecting the mRNA or protein of at least one of the genes BIRC6, DBR1, and MATR3.

2. The multiple myeloma prognostic assessment kit according to claim 1, characterized in that: The specific reagents include primer pairs, probes, or specific antibodies.

3. The multiple myeloma prognostic assessment kit according to claim 1, characterized in that: The expression status of the BIRC6-DBR1-MATR3 axis was integrated into the R-ISS staging system to establish a multidimensional prognostic assessment model.

4. A method for prognostic assessment of multiple myeloma, based on the multiple myeloma prognostic assessment kit according to any one of claims 1-3, characterized in that: The specific steps of this prognostic assessment method for multiple myeloma are as follows: a. Obtain biological samples from the MM patient to be tested; b. Molecular biology techniques were used to detect the expression levels of BIRC6, DBR1, and MATR3 genes in the samples; c. Compare the expression levels of one or more detected genes with a preset reference threshold, or calculate a comprehensive risk score based on their expression levels; d. Determine the prognosis based on reference threshold comparisons or comprehensive risk scoring results: If the expression levels of one or more genes are significantly higher than the threshold, or the overall risk score is high, the patient is considered high-risk and has a poor prognosis. Conversely, if the expression levels of one or more genes are significantly lower than the threshold, or the overall risk score is low, the risk is considered low and the prognosis is good.

5. The prognostic assessment method for multiple myeloma according to claim 4, characterized in that: In step a, the biological sample is tumor plasma cells isolated from bone marrow aspiration.

6. The prognostic assessment method for multiple myeloma according to claim 4, characterized in that: In step b, the molecular biology techniques include RT-qPCR, RNA sequencing, gene chips, and immunohistochemistry.

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