A composition for use in the clinical diagnosis and treatment of hematological malignancies and uses thereof

Abstract

The application provides a composition for clinical diagnosis and treatment of hematological malignancies. The composition is obtained by database acquisition of TCR and BCR rearrangement specific primer fragments, and a primer set suitable for human immune library multiplex PCR is established, which can perform multiplex amplification on CDR3 sequences and incomplete rearrangement sequences. The multiple detection index primers include IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, TRD. Further research shows that the primer set can perform clinical multi-index detection in various hematological malignancies, can significantly improve the positive detection of clinical samples, can comprehensively detect the immune library of individual patients, realize high-sensitivity MRD tracking, and also can help obtain all abnormal clones of the body and accurately reflect the immune status of the body.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a composition for the clinical diagnosis and treatment of hematologic malignancies, which is applicable to the tracking and detection of minimal residual disease (MRD) in most hematologic malignancies and the dynamic monitoring of patients' immune systems. Background Technology

[0002] Hematologic malignancies are among the most common and fatal cancers. Most hematologic malignancies, such as acute lymphoblastic leukemia, have a particularly high incidence in children and are a leading cause of death in children. Studies show that B-cell lymphoma is currently the predominant type of lymphoma in my country. Clinical diagnosis mainly relies on histological morphology, which has certain limitations. Sometimes, routine pathological examinations are insufficient to differentiate between lymphomas, benign proliferations of lymphoid tissue, and dysplasia. Therefore, it is necessary to find more accurate detection methods and specific indicators to assist in diagnosis.

[0003] The sum of diversity of antigen-specific receptors (TCRs) or BCRs on the surface of functional T cells or B cells at any given time is called the immune repertoire (IR). With the rapid development of cytogenetics and molecular biology, the detection of V(D)J gene rearrangements in BCRs or TCRs is increasingly being applied clinically, such as in vaccine development and auxiliary clinical diagnosis. The immune repertoire status of patients with hematologic malignancies exhibits pathological changes compared to healthy individuals, potentially showing high-frequency subclones as tumor markers. Patients exhibit high clonal morphology and low diversity in their immune repertoire. Furthermore, previous studies have shown that almost all types of tumors exhibit high heterogeneity, with some tumors displaying differential clonal heterogeneity at different stages of origin and development. Therefore, identifying the clonal morphology of the immune repertoire in hematologic malignancies can help classify specific tumor subtypes, while monitoring minimal residual disease (MRD) may aid in assessing the stage of treatment efficacy and developing appropriate treatment strategies. Additionally, clonal evolution frequently occurs in hematologic malignancies and is currently considered to be associated with patient clinical prognosis. Monitoring major clones in hematologic malignancies (IR) in patients with hematologic malignancies and investigating whether clonal conversion and evolution occur can guide the development of clinical treatment plans for these patients.

[0004] Previously, studies on immune repertoire diversity were primarily conducted using multiparameter flow cytometry (FCM), CDR3 profiling, and PCR combined with Sanger sequencing. Multiparameter flow cytometry can differentiate tumor cells from normal cells based on their immunophenotypic characteristics; it is simple and rapid to perform and can target various tumor samples, including circulating tumor cells. However, flow cytometry has relatively low detection sensitivity, only around 10%. -3 -10 -4While multiparameter flow cytometry can classify and analyze the populations and expression patterns of different clones in an individual's immune repertoire at the protein level, it cannot obtain detailed information on different clonal subfamilies, making it unsuitable for tracking and monitoring MRD (Malignant Reproductive Disease) of precise malignant clonal sequences. CDR3 profile analysis can quantitatively compare the frequency changes of individual subfamilies of clones, but it can only detect CDR3 length polymorphisms and cannot analyze the specific CDR3 sequence. PCR combined with Sanger sequencing allows for detailed analysis of CDR3 length and sequence; however, due to limitations in sequencing throughput and efficiency, Sanger sequencing cannot comprehensively resolve an individual's immune repertoire information.

[0005] Although current medical technology has made significant progress in the treatment of hematological malignancies, with patients experiencing remission after chemotherapy, targeted therapy, and hematopoietic stem cell transplantation, progression-free survival cannot be guaranteed, and most patients face the risk of recurrence. Recent studies have shown a strong correlation between recurrence of hematological malignancies and tumor malignancy repercussions (MRD). The earlier the MRD appears and the higher the detection value, the faster the recurrence and the lower the three-year overall survival rate. Furthermore, previous research has indicated that the diversity and clonality of the tumor repercussion repercussions (TCRs) are being studied as indicators of patient prognosis and recurrence risk, suggesting that TCR diversity and clonality have the potential to serve as molecular markers for malignant tumors.

[0006] In current hematological malignancy diagnosis and treatment, MRD (metastatic leukemia) is not only a crucial prognostic indicator used to assess the effectiveness of medication and the likelihood of recurrence, but it also influences decisions regarding subsequent treatment plans. Therefore, monitoring post-treatment MRD levels and using NGS-based immune repertoire technology to track specific clones within the MRD, repeatedly detecting the expression levels of primary clones, and monitoring the occurrence of newly formed malignant clones, allows for early prediction of changes in the patient's bodily functions. This helps in tailoring precise medical strategies for each patient, achieving the most ideal treatment outcomes. Furthermore, direct observation of the patient's immune dynamics can assist in prospectively assessing patient prognosis, improving clinical treatment plans, and ultimately enhancing patient survival.

[0007] The detection of MRD presents significant challenges. Conventional methods such as morphological examination or immunohistochemistry often fail to accurately reflect the minute number of tumor cells in a patient's body, leading to false negatives. Determining whether target cells are lymphoid or myeloid through chemical staining relies heavily on the expertise and experience of the examining physician, making it highly subjective. While PCR detection of V(D)J gene rearrangements provides a more standardized identification method for MRD, it is still severely limited by factors such as high cost, complex operation, and low sensitivity. Furthermore, it cannot effectively obtain specific clonates for long-term tracking and accurate prognostic assessment. Therefore, a widely applicable, highly sensitive, and standardized product specifically designed for MRD detection has considerable application value and potential. Currently, domestically developed immune repertoire products for MRD detection are in the initial research and development stage. However, from both a market and technological perspective, current products cannot fully meet the needs of the number of patients and researchers in clinical practice. Similarly, technologies that directly reflect a patient's overall immune status in clinical testing are still lacking. The emergence and application of immune repertoire combined with NGS technology offer significant opportunities for clinical treatment and related research. Summary of the Invention

[0008] Based on the above description, this invention uses specific primer fragments obtained from databases for TCR and BCR rearrangements to design a primer set suitable for multiplex PCR of human immune repertoire. This set can amplify CDR3 sequences and incomplete rearranged sequences multiplex. The primers for various detection indicators include IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD. Further research shows that this primer set can be used for clinical multi-indicator detection in various hematological malignancies and can significantly improve the positive detection of clinical samples. It can comprehensively detect the individual immune repertoire of patients, obtain all abnormal clones, and accurately reflect the body's immune status.

[0009] One of the objectives of this invention is to protect a composition for clinical diagnosis and treatment of hematologic malignancies, namely the aforementioned primer set, which includes at least primer sets for amplifying IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD respectively.

[0010] In order to facilitate the detection of the success or failure of PCR amplification reaction and the calculation of PCR amplification fold, an internal reference set containing multiple sequences with a defined copy number is introduced as a quality control step. The structure of this internal reference set mainly simulates the natural V(D)J gene combination. In combination with this internal reference set, this invention can correct primer bias, calculate the actual amplification fold, and quantify the number of cells.

[0011] Furthermore, this invention can also introduce actin, a relatively conserved housekeeping gene in all species, as an internal control. This gene is stable and encoded by a cytoskeletal protein. By introducing actin, this invention can more accurately quantify the number of nucleated cells and abnormal cells, which is suitable for tracking and monitoring MRD.

[0012] In the above composition, the primers for amplifying IGH have the following sequences: the upstream primer sequence is shown in SEQ ID NO: 1-22, and the downstream primer sequence is shown in SEQ ID NO: 23-25; and / or

[0013] The primers for amplifying IGDH contain upstream primer sequences as shown in SEQ ID NO:26–33 and downstream primer sequences as shown in SEQ ID NO:34; and / or

[0014] The primers for amplifying IGK have the following sequences: upstream primer sequence as shown in SEQ ID NO:35–46, and downstream primer sequence as shown in SEQ ID NO:47–51; and / or

[0015] The primers for amplifying IGL contain upstream primer sequences as shown in SEQ ID NO:52–57 and downstream primer sequences as shown in SEQ ID NO:58; and / or

[0016] The primers for amplifying TRB-VJ contain upstream primer sequences as shown in SEQ ID NO: 59–96 and downstream primer sequences as shown in SEQ ID NO: 97–110; and / or

[0017] The primers for amplifying TRB-DJ have the following sequences: upstream primer sequence as shown in SEQ ID NO: 111–112, and downstream primer sequence as shown in SEQ ID NO: 97–110; and / or

[0018] The primers for amplifying TRD contain upstream primer sequences as shown in SEQ ID NO: 113–121 and downstream primer sequences as shown in SEQ ID NO: 122–127; and / or

[0019] The primers for amplifying TRG contain upstream primer sequences as shown in SEQ ID NO: 128–135 and downstream primer sequences as shown in SEQ ID NO: 136–139; and / or

[0020] The sequences of the artificial internal reference set are shown in SEQ ID NO:140~294.

[0021] Furthermore, the primer sequences for the housekeeping gene actin are shown in SEQ ID NO:295-296.

[0022] Furthermore, the composition also includes sequencing primers and tags, wherein the sequences of the upstream sequencing primers and tags are shown in SEQ ID NO:297-304, and the sequences of the downstream sequencing primers and tags are shown in SEQ ID NO:305-316.

[0023] A second objective of this invention is to protect a reagent kit for clinical diagnosis and treatment of hematologic malignancies, comprising the above-described composition.

[0024] Specifically, the kit includes reagent A and reagent B. Reagent A includes at least a primer set for amplifying IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD, and an artificial internal reference set simulating natural VJ and DJ sequences, respectively. Reagent B includes sequencing primers and a tag set.

[0025] Furthermore, reagent A also includes the housekeeping gene actin sequence.

[0026] The third objective of this invention is to protect the application of the above-mentioned composition or kit in the description of immune repertoires and / or MRD tracking and / or immune dynamic monitoring of patients with hematologic malignancies, and the above applications are all for non-disease diagnosis and treatment purposes, such as for scientific research, experiments and other studies.

[0027] Specifically, the procedure includes at least the following steps: obtaining genomic DNA, cDNA, or cfDNA from nucleated cells in the sample to be tested; adding the aforementioned artificial internal reference set with a defined copy number; performing multiplex PCR amplification using the aforementioned amplification primer set; performing secondary amplification on the multiplex PCR amplification products to add sequencing adapters; constructing a library; sequencing; performing quality control, preprocessing, and assembly on the resulting immune library file; comparing the assembled sequences with the IMGT database; performing clonal clustering based on the CDR3 sequence; generating files sorted by clonal abundance; calculating the MRD: MRD = (Rt*Cir / Rir) / TNk, where Rt is the number of tumor reads obtained from sequencing, Rir is the number of reads in the internal reference set obtained from sequencing, Cir is the copy number of each added internal reference set (quantified by digital PCR), TNk = Mk / A, where TNk is the total number of nucleated cells, Mk is the total amount of DNA from nucleated cells (which can be considered as the amount of DNA input in the experiment), and A is the average total amount of DNA in a single cell, i.e., 0.0064 ng; or

[0028] Genomic DNA, cDNA, or cfDNA from nucleated cells in the sample to be tested are obtained. An artificial internal reference set with a defined copy number and the aforementioned housekeeping gene actin sequence are added. Multiplex PCR amplification is performed using the aforementioned amplification primer set. The multiplex PCR amplification products are amplified a second time to add sequencing adapters. A library is constructed, and sequencing is performed. The resulting immune library file undergoes quality control, preprocessing, and assembly. The assembled sequences are compared with the IMGT database. Clones are clustered based on the CDR3 sequence to generate files sorted by clonal abundance. The MRD is calculated as: MRD = (Rt*Cir / Rir) / (RT*CH / RH), where RT is the number of housekeeping gene reads amplified from the clinical template, RH is the number of housekeeping gene plasmid reads obtained from the added specially labeled internal reference, and CH is the original copy number in the housekeeping gene plasmid internal reference.

[0029] Furthermore, the final concentrations of primers for multiplex PCR amplification were 0.29 μM for IGH, 0.2 μM for IGDH, 0.11 μM for IGK, and 0.09 μM for IGK. Simultaneously, the final concentrations of primers for multiplex PCR amplification were 0.5 μM for TRB-VJ, 1.45 μM for TRB-DJ, 2 μM for TRD, and 1.33 μM for TRG. By adjusting the appropriate primer concentrations and conducting multiple tests, the primer bias issue in multiplex PCR amplification was resolved.

[0030] The composition provided by this invention can be used to detect and assess various hematologic malignancies at the gene level, detect tumor cell clones in patients, calculate proportions, label and track related clones, and accurately monitor disease progression. Simultaneously, it can obtain the patient's immune status by detecting TCR immunological data, and assess the patient's prognosis. Through long-term real-time tracking of the patient's MRD, it can also promptly detect newly deteriorating tumor-related clones indicating disease progression, and simultaneously explore the dynamics of the patient's immune system. It mainly includes library construction and NGS bioinformatics analysis. The library construction mainly includes IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD, encompassing complete and incomplete rearrangements of BCR and TCR. It also includes a primer set covering the immune repertoire CDR3, an artificial sequence-internal reference sequence set simulating the natural V(D)J sequence, and a housekeeping gene internal reference sequence. A second round of amplification using Illumina index-related primers is used to add sequencing adapters to complete library construction. At the same time, the throughput and efficiency problems in sequencing are solved. Combining NGS with immune repertoire analysis allows for the acquisition of comprehensive immune repertoire information from different genetic materials such as DNA, RNA, and cfDNA. It also enables precise real-time monitoring of the frequency and diversity of clonal types within an individual's immune repertoire at different time points, providing reliable information for disease diagnosis and drug treatment evaluation. This method can target DNA, RNA, or cfDNA in the sample; when using RNA for detection, the RNA is first reverse transcribed.

[0031] In addition, data on clonal diversity, clonality, and amino acid polymorphism can be obtained from the TCR immune repertoire for analysis, monitoring the patient's clinical immune dynamics, and comprehensively assessing the patient's treatment prognosis. This includes indicators such as D50, Shannon's index, Gini coefficient, and Pielou's index. Specifically, D50 = the number of sequence types that first reach 50% / the total number of sequences; Shannon's entropy index = H′ = -ΣPi lnPi; Gini-Simpson Diversity index = D = 1 - ΣPi²; and Pielou's index = E = H / Hmax. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process for using immune repertoire technology to assist in the diagnosis of hematological malignancies and monitor MRD in Example 2;

[0033] Figure 2 This is a schematic diagram of the library construction process in Example 2;

[0034] Figure 3 This refers to the library quality control results of the bone marrow sample before treatment in Example 2, starting from DNA.

[0035] Figure 4 This is a schematic diagram of the MRD tracking calculation method in Example 2;

[0036] Figure 5 This is a schematic diagram of the bioinformatics analysis and processing workflow;

[0037] Figure 6 This is a display of the multi-indicator results of the immune repertoire in Example 3;

[0038] Figure 7 This is a report demonstration for Example 3. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0040] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0041] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0042] Example 1

[0043] This invention provides a composition for the clinical diagnosis and treatment of hematological malignancies, comprising at least a primer set for amplifying IGH, IGDH, IGK, IGL, TRVJ, TRDJ, TRG, and TRD respectively.

[0044] Among them, the upstream primer sequences for amplifying IGH are shown in SEQ ID NO:1 to 22, and the downstream primer sequences are shown in SEQ ID NO:23 to 25;

[0045] The primers for amplifying IGDH have the following sequences: upstream primer sequence as shown in SEQ ID NO:26-33, and downstream primer sequence as shown in SEQ ID NO:34.

[0046] The primer sequences for amplifying IGK are shown in SEQ ID NO:35-46, and the primer sequences are shown in SEQ ID NO:47-51.

[0047] The primers for amplifying IGL have the following sequences: upstream primer sequence as shown in SEQ ID NO:52-57, and downstream primer sequence as shown in SEQ ID NO:58.

[0048] The primers for amplifying TRB-VJ have the following sequences: upstream primer sequence as shown in SEQ ID NO: 59-96, and downstream primer sequence as shown in SEQ ID NO: 97-110.

[0049] The primers for amplifying TRB-DJ have the following sequences: upstream primer sequence as shown in SEQ ID NO: 111-112, and downstream primer sequence as shown in SEQ ID NO: 97-110.

[0050] The primers for amplifying TRD have the following sequences: upstream primer sequence as shown in SEQ ID NO: 113-121, and downstream primer sequence as shown in SEQ ID NO: 122-127.

[0051] The primers for amplifying TRG have the following sequences: upstream primer sequence as shown in SEQ ID NO: 128-135, and downstream primer sequence as shown in SEQ ID NO: 136-139.

[0052] Furthermore, it also includes an artificial set of internal references that simulates the natural VJ and DJ sequences, the sequences of which are shown in SEQ ID NO: 140–294.

[0053] Furthermore, it also includes the housekeeping gene actin sequence, as shown in SEQ ID NO:295-296.

[0054] Furthermore, it also includes sequencing primers and tags, wherein the sequences of the upstream sequencing primers and tags are shown in SEQ ID NO:297–304, and the sequences of the downstream sequencing primers and tags are shown in SEQ ID NO:305–316. Specific sequence information is as follows:

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[0062]

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[0067]

[0068] This invention also provides a kit for the clinical diagnosis and treatment of hematological malignancies, comprising the above-described composition.

[0069] Example 2

[0070] This invention provides a method for assisted diagnosis of hematological malignancies and detection of MRD based on immune repertoire technology, as detailed below:

[0071] (1) Sample acquisition:

[0072] Clinical samples obtained from the hospital will be categorized based on pre- and post-treatment conditions and sample specificity:

[0073] Human bone marrow samples before treatment, packaged in EDTA anticoagulant tubes;

[0074] Bone marrow samples from treated patients, packaged in EDTA anticoagulant tubes;

[0075] Peripheral blood samples from individuals prior to treatment, packaged in EDTA anticoagulant tubes;

[0076] Peripheral blood samples from patients after treatment, packaged in EDTA anticoagulant tubes.

[0077] (2) Construction of DNA-initiated multiplex PCR amplification libraries:

[0078] Red blood cells (Solarbio, R1010) were lysed from the sample, nucleated cells were isolated, and genomic DNA was extracted from the nucleated cells (MagCore Genomic DNA Whole Blood Kit).

[0079] The obtained genomic DNA was subjected to multiplex PCR reactions. Multiple primer sets for V gene fragments and J gene fragments were added to the multiplex PCR reaction system, along with the corresponding internal reference sets. Alternatively, the internal reference sets and the housekeeping gene actin were amplified simultaneously with the sample, and different calculation methods were used.

[0080] The primer sets for the V and J gene fragments include sequences as shown in SEQ ID NO:1–139; the internal reference set sequences are shown in SEQ ID NO:140–294; and the housekeeping gene action sequence is shown in SEQ ID NO:295–296.

[0081] The multiplex PCR reaction systems are shown in Table 1 below. Table 1 shows the 25 μL system. If you need to use a 50 μL or larger system, simply multiply the volume. The reaction program is shown in Table 2 below.

[0082] Table 1 Multiplex PCR reaction system

[0083]

[0084] Note: PCR Buffer II, MgCl2, and AmpliTaq Gold DNA polymerase used in the multiplex PCR reaction were from Thermo Fisher Scientific's "AmpliTaq Gold" kit. TM DNA Polymerase with Buffer II and MgCl2 (N8080259). The QC internal reference set is counted by copy number.

[0085] Table 2 Multiplex PCR reaction procedure

[0086]

[0087] (3) Construction of RNA-initiated multiplex PCR amplification libraries

[0088] RNA (PureLink) was extracted from the sample to be tested. TM RNA miniprep kit (Thermo Fisher Scientific, 12183018A, see kit instructions for details). After checking the quality of RNA product extraction by gel electrophoresis, the amount of RNA added was determined according to experimental requirements. Rapid and efficient RNA reverse transcription was performed using the Takara kit (RR037A), with details provided in the relevant instructions. The reverse transcription reaction system and procedure are shown in Tables 3 and 4, respectively. Subsequently, multiple primer sets for the V and J gene fragments were added to the multiplex PCR reaction system, along with corresponding internal controls, to amplify the reverse transcription products of RNA. The primer sets for the V and J gene fragments included sequences as shown in SEQ ID NO: 1–139; the internal control sequences were shown in SEQ ID NO: 140–294. The multiplex PCR reaction system and procedure are shown in Tables 1 and 2.

[0089] Table 3 Reverse transcription reaction system

[0090]

[0091] Table 4 Reverse transcription reaction procedure

[0092] temperature time 37℃ 15min 85℃ 5 seconds 4℃ ∞

[0093] (4) Sequencing and bioinformatics analysis

[0094] Using Novaseq from Illumina TM The Novaseq™ 6000 system was used to perform high-throughput sequencing on the PCR products in step (2) or (3). The operating principles and specific operating steps were all from the Illumina Novaseq™ 6000 system operating manual and were performed under the guidance of an engineer. The multiplex PCR amplification products in steps (2) and (3) were amplified a second time, introducing the adapters and tags specified by the Illumina sequencer. The reaction system for the second round of amplification is shown in Table 5 below, and the reaction procedure for the second amplification is shown in Table 6 below.

[0095] Table 5 Secondary amplification reaction system

[0096]

[0097]

[0098] Table 6 Secondary Amplification Reaction Procedure

[0099]

[0100] The primer sequences used for the secondary amplification are shown in SEQ ID NO:297~316.

[0101] The PCR products after secondary amplification were purified using magnetic beads, and the specific method is as follows:

[0102] a. Purify the amplification product with magnetic beads at a volume ratio of 1:1. Take a clean 1.5mL EP tube, mix XP magnetic beads with the amplification product at a volume ratio of 1:1, vortex centrifuge and incubate at room temperature for 5 minutes.

[0103] b. After incubation, place the EP tube on the magnetic rack and magnetically attract it for 5 minutes to ensure that the supernatant becomes clear and transparent;

[0104] c. Discard the supernatant and prepare 80% alcohol. Add 200 μL of 80% alcohol to each tube, let it stand for 30 seconds, then discard the supernatant. Repeat this process once more. Remove any remaining alcohol from the bottom of the tube and let it stand to air dry for 10 minutes.

[0105] d. When the magnetic beads are dry and no longer reflective, add low TE buffer to each tube as needed (at least 20 μL), remove the EP tube, vortex and centrifuge, and incubate at room temperature for 2 min;

[0106] e. Place the EP tube on a magnetic rack and magnetically attract it for 2 minutes until the solution is clear and transparent. Transfer the supernatant into a clean EP tube, which is the purified DNA.

[0107] The concentration of purified DNA was measured and recorded using a Qbit BR analyzer. Experiments could not proceed if the total concentration of the initial screening sample was below 1 μg or the total concentration of the MRD sample was below 10 μg. The purified, qualified samples underwent library quality control and sequencing.

[0108] (5) Bioinformatics analysis of sequencing data:

[0109] The resulting DNA library was analyzed using Illumina Novaseq. TM Sequencing was performed on a 6000 platform in PE150 mode. The library denaturation concentration was 2 nM, and the final concentration was 20 pM. The sequencing data was then processed using bioinformatics analysis. The FASTQ files of the immune repertoire obtained from NGS high-throughput sequencing underwent quality control, preprocessing, and R1 and R2 assembly. The assembled sequences were compared with the IMGT database, and clonal clustering was performed based on CDR3 sequences to generate files sorted by clonal abundance. The initial diagnostic sample result was calculated as: Clonal frequency = (clone reads / total reads) * 100%. A clonal frequency > 3% was considered positive.

[0110] The MRD value of the treated sample was calculated using two methods:

[0111] First, internal controls are used as the quantitative standard. MRD = (Rt / (Rir / Cir)) / TNk, where Rt is the number of reads obtained from sequencing of the tumor, Rir is the number of reads obtained from sequencing of the internal control set, Cir is the copy number of each set of internal controls added, obtained by digital PCR quantification, TNK = Mk / A, where TNK is the total number of nucleated cells, Mk is the total amount of DNA in nucleated cells, which can be regarded as the amount of DNA input in the experiment, and A is the average total amount of DNA in a single cell, i.e. 0.0064 ng.

[0112] Second, the internal control set and housekeeping gene sequence set are mixed and used simultaneously for the quantification of different components in the system. The housekeeping gene sequence set is used to quantify the total number of nucleated cells, while the internal control sequence set is used to quantify the number of tumor cells. MRD = (Rt*Cir / Rir) / (RT*CH / RH), where RT is the number of reads of the housekeeping gene sequence amplified from the clinical template, RH is the number of reads of the housekeeping gene plasmid obtained from the internal control sequence with added special marker (UMI), and CH is the original copy number of the housekeeping gene plasmid internal control.

[0113] Example 3

[0114] The composition from Example 1 was used to test 34 samples diagnosed with multiple myeloma by clinicopathology and imaging according to the method in Example 2. The results are shown in the table below:

[0115] Table 7. Positive detection rate results for multiple myeloma samples.

[0116] Sample type (number) IG rearrangement full indicator positive detection rate IG complete rearrangement positive detection rate Peripheral blood(20) 86.9％ 60.8％ Bone marrow (40) 95％ 77.5％

[0117] As can be seen from the above results, the composition provided by the present invention for the clinical diagnosis and treatment of hematological malignancies has a positive detection rate of 95% or higher for all IG rearrangement indicators (including incomplete rearrangements), and the detection results are basically consistent with the actual situation, demonstrating good reliability and repeatability.

[0118] Example 4: A Case Study of Clinical MRD Tracking

[0119] The patient was initially diagnosed with acute B-lymphoblastic leukemia (B-ALL) through other diagnostic methods.

[0120] First, a bone marrow sample is obtained from the patient before treatment, and nucleated cell gDNA is extracted. Pre-treatment diagnosis is then performed using high-throughput immune repertoire technology.

[0121] Sequencing was performed on the CDR3 and incomplete rearrangement regions of IGH, IGDH, IGK, IGK+, and IGL in the patient's B cells. Incomplete rearrangements were found in IGDH and IGK+, and three IGH clones were identified, with a frequency >3%. (See details in [link to relevant documentation]). Figure 7 .

[0122] Then, gDNA was extracted from bone marrow samples after treatment at different time intervals to detect and track the three IGH sequences identified as the master clone during the initial diagnosis. The operation procedure is as described in Example 2.

[0123] The results are as follows Figure 7After a period of treatment, the frequency of the three IGH master clones showed a downward trend, and after November 13, 2021, they all turned negative. At the same time, after July 27, 2021, the frequency of the IKG clones also dropped rapidly, and the third test turned negative. However, on April 18, 2022, the patient developed two new IGK master clones.

[0124] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition for clinical diagnosis and treatment of hematological malignancies, characterized in that, It should include at least a primer set that amplifies IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD respectively; The primers for amplifying IGH have the following sequences: upstream primer sequence as shown in SEQ ID NO: 1-22, and downstream primer sequence as shown in SEQ ID NO: 23-25. The upstream primer sequence is shown in SEQ ID NO:26-33, and the downstream primer sequence is shown in SEQ ID NO:

34. The primer sequences for amplifying IGK are shown in SEQ ID NO:35-46, and the sequences for the downstream primers are shown in SEQ ID NO:47-51. The primers for amplifying IGL have the following sequences: upstream primer sequence as shown in SEQ ID NO:52-57, and downstream primer sequence as shown in SEQ ID NO:

58. The primers for amplifying TRB-VJ have the following sequences: upstream primer sequence as shown in SEQ ID NO: 59-96, and downstream primer sequence as shown in SEQ ID NO: 97-110. The primers for amplifying TRB-DJ have the following sequences: upstream primer sequence as shown in SEQ ID NO: 111-112, and downstream primer sequence as shown in SEQ ID NO: 97-110. The primers for amplifying TRD have the following sequences: upstream primer sequence as shown in SEQ ID NO: 113-121, and downstream primer sequence as shown in SEQ ID NO: 122-127. The primers for amplifying TRG have the following sequences: upstream primer sequence as shown in SEQ ID NO: 128-135, and downstream primer sequence as shown in SEQ ID NO: 136-139.

2. The composition for clinical diagnosis and treatment of hematological malignancies according to claim 1, characterized in that, It also includes an artificial set of intrinsic parameters that simulates natural VJ and DJ sequences; The sequences of the artificial internal reference set are shown in SEQ ID NO:140~294.

3. The composition for clinical diagnosis and treatment of hematological malignancies according to claim 1, characterized in that, It also includes the housekeeping gene actin sequence; The sequence of the housekeeping gene actin is shown in SEQ ID NO:295~296.

4. The composition for clinical diagnosis and treatment of hematological malignancies according to any one of claims 1 to 3, characterized in that, It also includes sequencing primers and tags, wherein the sequences of the upstream sequencing primers and tags are shown in SEQ ID NO:297~304. The sequences of the downstream sequencing primers and tags are shown in SEQ ID NO: 305-316.

5. A reagent kit for clinical diagnosis and treatment of hematological malignancies, characterized in that, Includes the composition according to any one of claims 1 to 3.

6. The reagent kit for clinical diagnosis and treatment of hematological malignancies according to claim 5, characterized in that, It includes reagent A and reagent B, wherein reagent A includes at least a primer set for amplifying IGH, IGDH, IGK, IGL, TRB-VJ, TRB-DJ, TRG, and TRD, and an artificial internal reference set simulating natural VJ and DJ sequences, respectively; Reagent B includes sequencing primers and a tag set.

7. The reagent kit for clinical diagnosis and treatment of hematological malignancies according to claim 6, characterized in that, The reagent A also includes the housekeeping gene actin sequence.

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