Application of chromosome instability score detection reagent in lymphoma prognosis evaluation and detection device

Abstract

The invention relates to the technical field of molecular medicine, in particular to a detection device for prognosis evaluation of diffuse large B-cell lymphoma based on a chromosome instability score, and the chromosome instability score is proved to be a biomarker for independent prediction prognosis of DLBCL in multi-factor Cox analysis. It is shown that chromosome instability scoring can provide additional prognosis information besides traditional clinical prognosis factors (such as IPI scoring, age, staging and the like), chromosome instability scoring provides a quantitative method for evaluating chromosome instability, and by quantifying chromosome instability, the evaluation accuracy of the chromosome instability is improved. The chromosome instability score can more accurately predict the prognosis of the patient, and the score can be used for guiding the prediction of the postoperative recurrence risk of the diffuse large B-cell lymphoma patient, so that the subsequent treatment of the diffuse large B-cell lymphoma patient can be prompted, the subjectivity in prognosis evaluation can be reduced, and the consistency and repeatability of evaluation can be improved.

Description

Technical Field The present invention relates to the technical field of molecular medicine, and in particular to a detection technology for prognosis assessment of diffuse large B-cell lymphoma based on chromosome instability scoring. Background Art Diffuse Large B-Cell Lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) in adults, accounting for approximately 25% to 50% of NHL[1]. More than half of patients are over 70 years old when diagnosed, and the survival rate decreases with age[2,3]. The prognosis of DLBCL is affected by many factors, including age, stage, double hit / double expression, and TP53 mutation, which are all related to prognosis [3-6]. At present, the clinical prognostic evaluation of DLBCL mainly relies on the International Prognostic Index (IPI) [7] and Gene Expression Profiling (GEP) classification [8]. IPI is based on the patient's age, LDH level, physical status, disease stage, and number of extra-lymph node affected sites, while GEP divides DLBCL into different subtypes based on the gene expression pattern of tumor cells [7,8]. Although these indicators are helpful in predicting the prognosis of patients to a certain extent, they also have limitations in some cases. For example, IPI may have poor prognostic evaluation effect in young patients [9], and GEP requires the detection of sample RNA, but RNA is easily degraded. Therefore, it is crucial to study new prognostic evaluation indicators to meet clinical needs and improve the accuracy of prognosis prediction. Given the limitations of existing prognostic indicators, researchers have been seeking new indicators that can provide more accurate prognostic information. Non-patent literature:

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[0006] Xu-Monette Z Y,Wu L,Visco C,et al.Mutational profile and prognosticsignificance of TP53 in diffuse large B-cell lymphoma patients treated withR-CHOP:report from an International DLBCL Rituximab-CHOP Consortium ProgramStudy[J].Blood,The Journal of the American Society of Hematology,2012,120(19):3986-3996.

[0007] International Non-Hodgkin's Lymphoma Prognostic FactorsProject.Apredictive model for aggressive non-Hodgkin's lymphoma[J].NewEngland Journal of Medicine,1993,329(14):987-994.

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[0009] Hamlin PA, Zelenetz AD, Kewalramani T, et al. Age-adjusted International Prognostic Index predicts autologous stem cell transplantation outcome for patients with relapsed or primary refractory diffuse large B-cell lymphoma[J]. Blood, 2003, 102(6):1989-1996. Summary of the invention The present invention can screen key markers for the prognosis assessment of diffuse large B-cell lymphoma, obtain gene variation results and chromosomal instability scores, and indicate the risk of postoperative recurrence in patients through different scoring results. To achieve the above object, the present invention provides the following technical solutions: Use of a reagent for detecting chromosomal instability in the preparation of a prognostic assessment reagent for diffuse large B-cell lymphoma. The reagent for detecting chromosome instability is a reagent for obtaining chromosome instability information by detecting gene copy number. A detection device for prognosis assessment of diffuse large B-cell lymphoma, comprising: The tissue sample preparation library module extracts DNA from the patient's tumor tissue sample and prepares the library after ultrasonic fragmentation; A sequencing module is used to perform high-throughput sequencing on the library and obtain the copy number of different fragments on the genome; The data analysis module divides the total length of the fragments with abnormal copy numbers obtained in the sequencing module by the full length of all the detection fragments to obtain the CIS value; In the judgment module, if the CIS value is greater than the set threshold, it is judged as chromosome instability and the sample has a poor prognosis. The fragments with abnormal copy number are determined by calculating the log2 ratio value of the number of machine-readable segments of each fragment. If Log2ratio≥0.2 or Log2 ratio≤-0.2, the fragments with abnormal copy number are determined. The threshold range is 0.35-0.40. A computer readable medium having recorded thereon a computer program code capable of executing a method for evaluating the prognosis of diffuse large B-cell lymphoma: Step 1, obtaining the copy number of different fragments on the genome by high-throughput sequencing of the library; the library is obtained by extracting DNA from a patient's tumor tissue sample, and preparing the library after ultrasonic fragmentation; Step 2, dividing the total length of the obtained fragments with abnormal copy number by the full length of all the detected fragments to obtain the CIS value; Step 3: If the CIS value is greater than the set threshold, it is judged as chromosomal instability and the sample has a poor prognosis. Compared with the prior art, the present invention has the following beneficial effects: The chromosomal instability score in the present invention was confirmed to be a biomarker for independent prognosis prediction of DLBCL in multivariate Cox analysis, indicating that the chromosomal instability score can provide additional prognostic information in addition to traditional clinical prognostic factors (such as IPI score, age, stage, etc.), and the chromosomal instability score provides a quantitative method to evaluate chromosomal instability. By quantifying chromosomal instability, the chromosomal instability score can more accurately predict the prognosis of patients. The application of this score can be used to guide the prediction of the risk of postoperative recurrence in patients with diffuse large B-cell lymphoma, thereby providing a prompt for their subsequent treatment, and can reduce the subjectivity in prognostic evaluation and improve the consistency and repeatability of the evaluation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The data analysis flow chart of the present invention is Figure 2 The total population in the present invention discovery group ( Figure 2 A) Ann Arbor Stage Figure 2 B) IPI score ( Figure 2 C) and immunohistochemical typing ( Figure 2 D) Kaplan-Meier survival curve of overall survival Figure 3 The distribution of chromosome instability scores and threshold selection in the present invention discovery group Figure 4 Kaplan-Meier survival curves of the overall survival of the two groups with high and low chromosomal instability scores in the discovery group (4A) and the validation group (4B) of the present invention Figure 5 The ROC curve of the chromosome instability score, IPI score, immunohistochemistry typing and ECOG physical performance score in the present invention group for predicting overall survival DETAILED DESCRIPTION The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The purpose of the present invention is to screen key markers for the prognosis evaluation of diffuse large B-cell lymphoma, obtain gene variation results and chromosomal instability scores, and indicate the risk of postoperative recurrence in patients through different scoring results. The present invention is based on the analysis of clinical data, prognostic data and sample sequencing data of 41 Chinese patients with diffuse large B-cell lymphoma (DLBCL), exploring the clinical and molecular characteristics that predict prognosis. The sequencing data are primary lesion tissue samples of patients. After DNA is extracted, it is tested by next-generation sequencing technology (NGS) to obtain the gene variation results and chromosome instability scores (CIS) of patient samples. The new prognostic assessment molecular feature CIS discovered in the study was verified using an independent external validation set. See the data analysis flow chart. Figure 1 . The definition of "gene variation" in the present invention is: Gene variation: including gene mutation, copy number variation, and gene fusion. Gene mutation includes single nucleotide mutation and gene insertion / deletion mutation; copy number variation includes gene amplification and gene deletion; gene fusion is mainly caused by chromosome translocation, deletion or inversion, resulting in the fusion of partial gene sequences of two genes. The "chromosome instability score" described in the present invention is defined as: Chromosomal instability score (CIS): For the region where targeted sequencing is performed, the specific target genes detected are shown in Table 1. The exon region of the target gene is divided into segments using the segment module of CNVkit (PMID: 27100738), and the copy number changes of each segment can be obtained; further, the total length of the fragments with copy number abnormalities is counted, and the ratio of the total length of the abnormal length to the total length of the fragments is obtained; in the specific calculation process, the log2 ratio value of each segment is calculated, and the total length of the segment region with Log2 ratio ≥ 0.2 or Log2 ratio ≤ -0.2 is divided by the total length of all segments. The definition of "CIS-high" in the present invention is: CIS-high: Chromosome instability score CIS ≥ 0.366. The definition of "CIS-low" in the present invention is: CIS-low: Chromosome instability score CIS < 0.366. Discovery group patient information and sample testing: The discovery group consisted of 41 patients diagnosed with DLBCL from November 2013 to November 2017 and with sufficient tumor tissue samples for NGS sequencing. Males accounted for 54%, with a relatively balanced gender ratio; patients aged >60 accounted for 27%; patients with stage III & IV accounted for 76%; patients with LDH>250 accounted for 56%; patients with GCB accounted for 27%; patients with ≥2 extranodal lesions accounted for 46%; patients with ECOG score >1 accounted for 22%; IPI scores of 0-5 accounted for 12%, 15%, 22%, 37%, 10%, and 5%, respectively. DNA was extracted from the patient's tumor tissue samples, and after ultrasonic disruption, a commercial kit was used to prepare the library. The targeted enrichment library was constructed using the Hemosheng targeted sequencing kit produced by Nanjing Shihe Gene Biotechnology Co., Ltd., and then the enrichment library was sequenced. Finally, the offline data was analyzed using bioinformatics methods. The Hemosin test kit used in the present invention can be used to detect 475 genes. Discovery group patient information and sample testing: The discovery group consisted of 41 patients diagnosed with DLBCL from November 2013 to November 2017 and with sufficient tumor tissue samples for NGS sequencing. Males accounted for 54%, with a relatively balanced gender ratio; patients aged >60 accounted for 27%; patients with stage III & IV accounted for 76%; patients with LDH>250 accounted for 56%; patients with GCB accounted for 27%; patients with ≥2 extranodal lesions accounted for 46%; patients with ECOG score >1 accounted for 22%; IPI scores of 0-5 accounted for 12%, 15%, 22%, 37%, 10%, and 5%, respectively. DNA was extracted from the patient's tumor tissue samples, and after ultrasonic disruption, a commercial kit was used to prepare the library. The targeted enrichment library was constructed using the Hemosheng targeted sequencing kit produced by Nanjing Shihe Gene Biotechnology Co., Ltd., and then the enrichment library was sequenced. Finally, the offline data was analyzed using bioinformatics methods. The detection panel used in the present invention refers to the CN109880910A patent, which can be used to detect 425 genes. The genes involved are shown in Table 1: Correlation analysis between clinical features or immunohistochemical features and prognosis of DLBCL in the training group The overall survival (OS) of patients was calculated from the date of pathological diagnosis of DLBCL to the date of death or the last follow-up. The Kaplan-Meier curve of the overall survival of 41 patients in the discovery group is shown in Figure 2 A, mOS has not yet been reached, the 3-year survival rate is 72.5%, and the 5-year survival rate is 67.2%. The survival curves related to IPI, Ann Arbor stage, and GCB classification are shown in Figure 2 There was no significant difference in prognosis between patients with B-2D, III & IV stages (P>0.05), and there was no significant difference in prognosis between patients with IPI scores of 1-4 points (P>0.05). The prognosis of GCB type in this dataset was worse, which was contrary to the better prognosis of GCB type reported in some studies (PMID: 26857366). Based on the above research results, it shows that the prediction performance of the existing prognostic evaluation indicators used in clinical practice still has room for improvement. Cox was used to analyze the correlation between clinical and immunohistochemical features and overall survival in the training group. Based on the above clinical issues, the Ann Arbor stage was divided into groups according to stage I-II vs. stage III-IV; the IPI score was divided into groups according to 0-1 vs.>1; other groups are shown in Table 2. There was no significant difference in OS between the male and female groups, between different ages, between high and low LDH (lactate dehydrogenase) groups, and between the two groups with 0-1 vs.>1 extranodal lesions. However, patients with high IPI scores (≥4) had a worse prognosis (P=0.034); patients with GCB classification had a worse prognosis (P=0.028); patients with high ECOG scores (>1) also had a worse prognosis (P=0.019), which can be considered as predictive markers for OS. Table 2 Clinical factors related to the prognosis of cholangiocarcinoma in the univariate Cox screening training group *P values ​​were calculated based on Cox regression analysis. Correlation analysis between molecular features and prognosis of DLBCL in the training group First, genes with somatic mutation frequency ≥ 10% in the discovery group were screened as candidates. High-frequency mutation genes included KMT2D (34.1%), PIM1 (34.1%), MYD88 (31.7%), TP53 (26.8%), CD70 (26.8%), B2M (24.4%), etc. In the discovery group, gene markers that may be associated with the prognosis of DLBCL were screened by Cox analysis and Log-rank test (P < 0.050). The screening results are shown in Table 2. Table 3 Cox analysis and Log-rank test to screen DLBCL prognosis-related gene markers (n = 23) As can be seen from the table above, the frequencies of the 23 gene changes ranged from 12.2% to 34.1%. No significant difference in prognosis was found between the above gene variants and non-variants by either Cox analysis or Log-rank test (P>0.05). In addition to detecting mutations, copy number variations, and gene fusions in 475 genes, the Hemosin panel also detects the chromosome instability score CIS. The optimal threshold analysis was performed using the surv_cutpoint function in the survminer package in R (version 4.1.0), and the optimal threshold was determined to be 0.366, see Figure 3 When CIS ≥ 0.366, it is CIS-high, and when CIS < 0.366, it is CIS-low. The survival curve was drawn and it was found that there was a significant difference in the overall survival between the CIS-low and CIS-high groups (P < 0.001). Figure 4 A. The significant features in the univariate analysis were further combined for multivariate Cox analysis, and it was found that CIS was an independent biomarker for predicting the prognosis of DLBCL. Table 4 Multivariate Cox analysis of biomarkers that independently predict prognosis in DLBCL *P values ​​were calculated based on Cox regression analysis. Verification of chromosomal instability as an independent predictor of prognosis in DLBCL The validation group of patients came from the cBioPortal database: (https: / / www.cbioportal.org / study / summary?id=mbn_msk_2024), the patient screening process can be found in Figure 1 A total of 363 patients with DLBCL underwent NGS sequencing at Memorial Sloan Kettering Cancer Center (MSK). After excluding patients with unknown (NA) or 0 overall survival and patients whose CIS could not be evaluated, 343 patients were included in the final analysis. Males accounted for 58%; patients aged > 60 years accounted for 74%; 14 patients had unknown stage, and among the remaining patients, stage IV patients accounted for 51%. All 343 patients were tested using the IMPACT-HEME-400 kit, which covers 1.0837Mb of the human genome [PMID: 37898613]. Considering that CIS is data that is corrected based on the genome size covered by the kit, the same threshold of 0.366 was used for verification. When CIS ≥ 0.366, it was CIS-high, and when CIS < 0.366, it was CIS-low. The survival differences between the CIS-high group and the CIS-low group were compared and analyzed, and the Kaplan-Meier method was used to draw the survival curve, see Figure 4 B, Patients with high chromosomal instability have a worse prognosis, further validating the discovery set results. Comparison of the prognostic performance of chromosomal instability with other clinical features: CIS and other single-factor significant clinical characteristics predicted overall survival respectively. The results showed that CIS had the largest UC, which was higher than other clinical characteristics, indicating that CIS was better than IPI score, immunohistochemical typing and ECOG performance score in predicting the prognosis of DLBCL. The results are shown in Figure 5 . It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.

Claims

1. Use of a reagent for detecting chromosomal instability in the preparation of a reagent for prognosis assessment of diffuse large B-cell lymphoma.

2. The use according to claim 1, characterized in that: The reagent for detecting chromosome instability is a reagent for obtaining chromosome instability information by detecting gene copy number.

3. A detection device for prognosis assessment of diffuse large B-cell lymphoma, characterized in that: include: The tissue sample preparation library module extracts DNA from the patient's tumor tissue sample and prepares the library after ultrasonic fragmentation; A sequencing module is used to perform high-throughput sequencing on the library and obtain the copy number of different fragments on the genome; The data analysis module divides the total length of the fragments with abnormal copy numbers obtained in the sequencing module by the full length of all the detected fragments to obtain the CIS value; In the judgment module, if the CIS value is greater than the set threshold, it is judged as chromosome instability and the sample has a poor prognosis.

4. The detection device according to claim 3, characterized in that: The fragments with abnormal copy number are determined by calculating the log2 ratio value of the number of machine-readable segments of each fragment. If Log2 ratio ≥ 0.2 or Log2 ratio ≤ -0.2, the copy number is determined to be abnormal.

5. The detection device according to claim 3, characterized in that: The threshold range is 0.35-0.

40.

6. A computer-readable medium, characterized in that: It describes a computer program that can perform a prognostic assessment method for diffuse large B-cell lymphoma: Step 1, obtaining the copy number of different fragments on the genome by high-throughput sequencing of the library; the library is obtained by extracting DNA from a patient's tumor tissue sample, and preparing the library after ultrasonic fragmentation; Step 2, dividing the total length of the obtained fragments with abnormal copy number by the full length of all the detected fragments to obtain the CIS value; Step 3: If the CIS value is greater than the set threshold, it is judged as chromosomal instability and the sample has a poor prognosis.

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