Application of LMP1 gene copy number variation detection reagent in preparation of NK / T cell lymphoma prognosis kit
By detecting the copy number variation of LMP1 gene in the EB virus genome, the prognostic evaluation problem of NK/T cell lymphoma was solved, and accurate prediction of patients' survival status and improvement of treatment effect were achieved.
Patent Information
- Application Number
- CN202410023499.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-05
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art has failed to effectively explain the pathogenic mechanism and tumor progression mechanism of NK/T cell lymphoma associated with EB virus infection, and has not included it in the risk stratification and prognosis assessment of patients.
The LMP1 gene copy number variant detection reagent is provided, and the LMP1 gene copy number variant event in the EB virus gene is detected through whole genome sequencing. It is used to prepare an NK/T cell lymphoma prognosis kit to predict the overall survival and progression-free survival of patients.
As a new prognostic marker, LMP1 gene copy number variation can more accurately judge the prognosis of patients with NK/T cell lymphoma, improve treatment effects, and have good clinical promotion prospects.
Smart Images
Figure CN120272594A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of an LMP1 gene copy number variation detection reagent in the preparation of a prognosis kit for NK / T cell lymphoma. Background Art
[0002] Epstein-Barr virus is the first known virus associated with human cancer, discovered in Burkitt's Lymphoma in 1964. Subsequently, scientists found that the cancer types most closely associated with this virus are Natural-killer / T cell lymphoma and Nasopharyngeal carcinoma, and their tumor cells are generally infected with Epstein-Barr virus.
[0003] In recent years, a large number of studies have shown that gene mutations and chromosomal variations are closely related to the tumorigenesis and disease progression of NK / T cell lymphoma. These studies include: (1) Somatic mutations in NK / T cell lymphoma patients cause immune escape of tumor cells, changes in epigenetic modifications, and activation of multiple pro-cancer signaling pathways (such as JAK-STAT, NF-kB, and MAPK pathways); (2) Single nucleotide polymorphisms in the population genetic background can be widely used as a type of genetic marker, and genetic loci on genes such as HLA-DPB1, IL18RAP, and HLA-DRB1 have been identified as being related to the risk of NK / T cell lymphoma; (3) In addition, copy number variation events in the chromosomal 6q21 region have also been found in NK / T cell lymphoma, and these chromosomal structural variations can cause dysfunction of genes such as PRDM1, ATG5, and AIM1. The above studies have revealed the biological contributions of genetic variations to the occurrence and development of NK / T cell lymphoma from different levels, and a series of survival prognosis models and risk stratification strategies have also been proposed accordingly.
[0004] However, on the premise that Epstein-Barr virus is known to be an important pathogenic factor for NK / T cell lymphoma, these current findings are still not sufficient to effectively explain the pathogenic mechanism and tumor progression mechanism of NK / T cell lymphoma related to virus infection, and this important factor has not been incorporated into the risk stratification and prognosis assessment of NK / T cell lymphoma patients. Summary of the Invention
[0005] The purpose of the present invention is to provide a new NK / T cell lymphoma prognosis marker and its application in view of the deficiencies of the prior art, so as to solve the problems raised in the above background art.
[0006] To achieve the above object, the present invention provides the use of an LMP1 gene copy number variation detection reagent in the preparation of an NK / T cell lymphoma prognosis kit. The LMP1 gene is the LMP1 gene in the Epstein-Barr virus gene.
[0007] The LMP1 gene copy number variation detection reagent is used to detect copy number variation events of the LMP1 gene in the Epstein-Barr virus gene.
[0008] Preferably, the detection of the copy number variation event of the LMP1 gene includes, but is not limited to, using the method of whole genome sequencing on the tumor tissue of NK / T cell lymphoma patients.
[0009] Preferably, the copy number variation event of the LMP1 gene includes an increase in the copy number of the LMP1 gene.
[0010] Preferably, the prognosis of NK / T cell lymphoma includes the overall survival and progression-free survival of the patient.
[0011] Preferably, when the copy number of the LMP1 gene increases, the overall survival and progression-free survival of the patient are better.
[0012] Preferably, the LMP1 gene copy number variation detection reagent is a reagent for performing whole genome sequencing.
[0013] On the other hand, the present invention also provides a detection kit, which includes the LMP1 gene copy number variation detection reagent.
[0014] On the other hand, the present invention also provides the use of LMP1 gene copy number variation events in the prognosis of NK / T cell lymphoma.
[0015] On the other hand, the present invention also provides a method for the prognosis of NK / T cell lymphoma, which includes detecting whether the copy number variation event of the LMP1 gene increases.
[0016] According to the said method, it detects whether the copy number variation event of the LMP1 gene increases by whole genome sequencing. This method can be used for non-disease diagnosis purposes.
[0017] The present invention has the following beneficial effects:
[0018] Using the copy number variation event of the LMP1 gene in the Epstein-Barr virus genome as a novel prognostic marker has good predictive value for the overall survival and progression-free survival prognosis of NK / T cell lymphoma patients, thereby enabling more accurate and effective prognostic judgment for NK / T cell lymphoma patients, and targeted improvement of poor treatment prognosis, so it has good clinical promotion prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Samples of NK / T cell lymphoma patients with increased copy number of the LMP1 gene are shown.
[0020] Figure 2 The copy number variation map of the Epstein - Barr virus genome in NK / T cell lymphoma is shown.
[0021] Figure 3 The signaling pathways significantly up - regulated in mutant NK / T cell lymphoma patients with increased LMP1 copy number are shown.
[0022] Figure 4 The survival analysis results are shown. Detailed implementation manners
[0023] In order to more clearly understand the above - mentioned objects, features and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should know that these embodiments are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention; unless otherwise specified, the sequencing and analysis methods used in the following embodiments are all conventional methods.
[0024] As used herein, "prediction" or "prognosis" refers to the process or result of predicting a patient's condition, and does not mean a process or result that can predict the patient's condition with 100% accuracy. "Prediction" or "prognosis" refers to determining whether the likelihood of certain processes or results is increased, rather than determining the likelihood of certain processes or results by comparing with the situation where certain processes or results do not occur. In the context of the present invention, patients with increased LMP1 copy number are more likely to observe specific processes or results compared to those who do not exhibit this feature.
[0025] Example 1: Increased copy number of LMP1 in NK / T cell lymphoma patients
[0026] Tumor tissue biopsy samples of a total of 77 newly - diagnosed patients were collected from the Sun Yat - sen University Cancer Center and the Singapore General Hospital. According to the 2008 WHO diagnostic classification criteria for lymphoma, these patients were pathologically positive for CD3, EBERs, and cytotoxic molecules and were diagnosed with NK / T cell lymphoma.
[0027] Whole-genome sequencing was performed on the tumor tissues of these 77 NK / T cell lymphoma cases as follows: (1) First, DNA was extracted from these tumor samples using the QIAamp DNA Mini Kit (QIAGEN). This process required strict avoidance of exogenous DNA contamination and assessment of the quality and concentration of the extracted DNA for compliance; (2) Subsequently, the TruePre DNA Library Prep Kit V2 (Vazyme, catalog number TD501-02) was used to construct a whole-genome sequencing library from the above-extracted DNA. The concentration of the resulting library was detected using the high-precision dsDNA Quant Kit (Invitrogen, catalog number Q32854), and its quality was analyzed using the High Sensitivity NGS Fragment Analysis Kit (AATI, catalog number DNF-474) with the Fragment Analyzer instrument (AATI); (3) Finally, all samples were sequenced using the Illumina HiSeq X Ten sequencing platform to obtain paired-end sequence files with a length of 150 bp.
[0028] For the obtained whole-genome sequencing files, all sequence fragments were aligned to the Epstein-Barr virus reference genome using the BWA software. This Epstein-Barr virus reference genome sequence file was sourced from the NCBI GeneBank database (NCBI refseq ID: NC_007605.1). Then, the rmdup command of the samtools software was used to remove the duplicate fragments generated by PCR amplification during library construction, and the depth command was used to calculate the coverage and depth of alignment to the Epstein-Barr virus genome. Finally, the cnvPartition software was used to identify copy number variation events and calculate the copy number increase or decrease in all regions of the Epstein-Barr virus genome.
[0029] Through the above steps, a copy number variation map of the Epstein-Barr virus genome in NK / T cell lymphoma was constructed, and the position of the LMP1 gene was calibrated using the region fragment from 166483 to 169056 in the Epstein-Barr virus genome, thus discovering a high-frequency increase in the copy number of LMP1 (18 / 77, probability 23.4%) in NK / T cell lymphoma patients. Figure 1 It shows 18 samples with an increase in the copy number of this region among the samples of 77 NK / T cell lymphoma patients. Figure 2 It shows the copy number variation map of the Epstein-Barr virus genome in NK / T cell lymphoma, where the highest frequency of copy number increase of LMP1 exists, which is a landmark event for Epstein-Barr virus genetic variation in NK / T cell lymphoma.
[0030] Example 2: Copy number variation events of LMP1 lead to changes in functional pathways
[0031] Whole-transcriptome sequencing was performed on tumor samples from the same batch of NK / T cell lymphoma patients. Combining the event information of LMP1 copy number variation known from the above methods, transcriptome sequencing sequence files of mutant type (n = 9) with increased LMP1 copy number and wild type (n = 16) without increased copy number were obtained respectively.
[0032] Through conventional data processing steps such as quality control, alignment, and quantification of transcriptome sequencing data, transcriptome matrix files of mutant and wild type samples were obtained.
[0033] Furthermore, through analysis with the GSEA software in R language, significantly up-regulated functional pathways in the mutant type with increased LMP1 copy number compared to the wild type without increased copy number could be obtained.
[0034] Figure 3 The signal pathways significantly up-regulated in mutant NK / T cell lymphoma patients with increased LMP1 copy number are shown, including the P53 signal pathway, NFkB signal pathway, etc. with functional changes related to cancer development.
[0035] Example 3: Copy number variation events of LMP1 as a prognostic marker for NK / T cell lymphoma
[0036] Subsequently, clinical prognostic data of these NK / T cell lymphoma patients were collected, including their death events (n = 23) and disease progression events (n = 24), for subsequent survival analysis to determine whether the increased copy number of LMP1 can be used as a prognostic marker for NK / T cell lymphoma.
[0037] Survival analysis was performed on the copy number variation information and survival prognosis information paired for the above patients through the survival and survminer software in R language.
[0038] This survival analysis divided NK / T cell lymphoma patients into two groups according to whether there was an increase in the copy number of LMP1 in the Epstein-Barr virus genome - mutant type with increased LMP1 copy number (LMP-1 copy-gain ; n = 7) and wild type without increased copy number (LMP-1 wildtpe ; n = 16 or 17), and thus it was found that the increased copy number of LMP1 in tumor tissues was associated with a better prognosis of overall survival of patients.
[0039] Figure 4The survival analysis results are shown. As can be seen from the figure, for patients with an increased copy number of LMP1, the prognosis of overall survival (OS) and progression-free survival (PFS) is significantly better. This indicates that the copy number variation of LMP1 in the Epstein-Barr virus genome affects the survival of patients with NK / T cell lymphoma. Detecting the presence or absence of an increased copy number of LMP1 has good benefits for the risk stratification and prognosis assessment of patients.
Claims
1. Use of an LMP1 gene copy number variation detection reagent in the preparation of a prognostic kit for NK / T cell lymphoma.
2. The application according to claim 1, wherein The LMP1 gene copy number variation detection reagent is used to detect copy number variation events of the LMP1 gene in the Epstein-Barr virus gene.
3. The application according to claim 2, characterized in that, The detection of the copy number variation events of the LMP1 gene is performed by whole-genome sequencing of the tumor tissues of NK / T cell lymphoma patients.
4. The application according to claim 2, wherein The copy number variation events of the LMP1 gene include an increase in the copy number of the LMP1 gene.
5. The application according to claim 1, wherein The prognosis of NK / T cell lymphoma includes the overall survival and progression-free survival of the patients.
6. The application according to claim 1, characterized in that, When the copy number of the LMP1 gene increases, the overall survival and progression-free survival of the patients are better.
7. The application according to claim 1, characterized in that, The LMP1 gene copy number variation detection reagent is a reagent for performing whole-genome sequencing.
8. A detection kit, characterized in that It includes an LMP1 gene copy number variation detection reagent.
9. Use of LMP1 gene copy number variation events in the prognosis of NK / T cell lymphoma.
10. A prognostic method for NK / T cell lymphoma, characterized in that, The method includes detecting whether the LMP1 gene copy number variation events increase.