Use of LMP1 gene copy number variation detection reagent in preparation of NK / t cell lymphoma prognosis kit

Abstract

Disclosed in the present invention is a use of an MP1 gene copy number variation detection reagent in preparation of an NK / T cell lymphoma prognosis kit. The present invention further provides a detection kit comprising the LMP1 gene copy number variation detection reagent, a use thereof, and an NK / T cell lymphoma prognosis method. In the present invention, a copy number variation event of LMP1 gene in an EB virus genome is used as a novel prognosis marker, which has a good prediction value for the overall survival and progression-free survival prognosis of an NK / T cell lymphoma patient, and can realize more accurate and effective prognostic assessment for the NK / T cell lymphoma patient.

Description

Application of LMP1 gene copy number variation detection reagent in the preparation of NK / T cell lymphoma prognosis kit Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of an LMP1 gene copy number variation detection reagent in the preparation of a NK / T cell lymphoma prognosis kit. Background Art

[0002] Epstein-Barr virus (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 the virus are natural-killer / T cell lymphoma and nasopharyngeal carcinoma, whose tumor cells are commonly infected by 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 cancer-promoting signaling pathways (JAK-STAT, NF-kB, and MAPK pathways); (2) Single nucleotide polymorphisms in the genetic background of the population can be widely used as a type of genetic marker. Genetic loci on genes such as HLA-DPB1, IL18RAP, and HLA-DRB1 have been identified as being associated with the risk of NK / T cell lymphoma; (3) In addition, copy number variation events in the chromosome 6q21 region have been found in NK / T cell lymphoma. These chromosomal structural variations can cause functional disorders of genes such as PRDM1, ATG5, and AIM1. The above studies have revealed the biological contribution of genetic variation to the occurrence and development of NK / T cell lymphoma from different levels, and have also proposed a series of survival prognostic models and risk stratification strategies based on this.

[0004] However, given that Epstein-Barr virus is an important pathogenic factor in NK / T-cell lymphoma, these findings are still insufficient to effectively explain the pathogenic mechanism and tumor progression mechanism of NK / T-cell lymphoma associated with viral infection, and this important factor has not been incorporated into the risk stratification and prognostic assessment of NK / T-cell lymphoma patients.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art and provide a new NK / T cell lymphoma prognostic marker and its application to solve the problems raised in the above background technology.

[0007] To achieve the above objectives, the present invention provides the use of an LMP1 gene copy number variation detection reagent in the preparation of a NK / T cell lymphoma prognosis kit. The LMP1 gene is the LMP1 gene in the Epstein-Barr virus gene.

[0008] The LMP1 gene copy number variation detection reagent is used to detect the copy number variation event of the LMP1 gene in the EB virus gene.

[0009] Preferably, the detection of copy number variation events of the LMP1 gene includes, but is not limited to, whole genome sequencing of tumor tissues from NK / T cell lymphoma patients.

[0010] Preferably, the copy number variation event of the LMPl gene includes an increase in the copy number of the LMPl gene.

[0011] Preferably, NK / T cell lymphoma prognosis includes the patient's overall survival and progression-free survival.

[0012] Preferably, when the LMPl gene copy number is increased, the patient's overall survival and progression-free survival are better.

[0013] Preferably, the LMP1 gene copy number variation detection reagent is a reagent for whole genome sequencing.

[0014] On the other hand, the present invention also provides a detection kit, which includes the LMP1 gene copy number variation detection reagent.

[0015] On the other hand, the present invention also provides the application of LMP1 gene copy number variation events in the prognosis of NK / T cell lymphoma.

[0016] On the other hand, the present invention also provides a method for prognosis of NK / T cell lymphoma, which comprises detecting whether the LMP1 gene copy number variation event is increased.

[0017] According to the method, whole genome sequencing is used to detect whether the LMP1 gene copy number variation event is increased. This method can be used for non-disease diagnosis purposes.

[0018] The present invention has the following beneficial effects:

[0019] Using the copy number variation events of the LMP1 gene in the Epstein-Barr virus genome as a new prognostic marker has good predictive value for the overall survival and progression-free survival prognosis of NK / T cell lymphoma patients, thereby achieving more accurate and effective prognostic judgment for NK / T cell lymphoma patients and targetedly improving poor treatment prognosis, and therefore has good prospects for clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows NK / T cell lymphoma patient samples with increased LMP1 gene copy number.

[0021] FIG2 shows a map of EBV genome copy number variation in NK / T cell lymphoma.

[0022] FIG3 shows signaling pathways that are significantly upregulated in patients with mutant NK / T cell lymphoma with increased LMP1 copy number.

[0023] FIG4 shows the results of survival analysis. DETAILED DESCRIPTION

[0024] To more clearly understand the above-mentioned objects, features, and advantages of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific examples. Those skilled in the art should be aware that these examples are merely intended to facilitate understanding of the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the sequencing and analysis methods used in the following examples are conventional methods.

[0025] As used herein, "predict" or "prognosis" refers to predicting the course or outcome of a patient's condition and does not imply predicting the course or outcome of a patient's condition with 100% accuracy. "Predict" or "prognosis" refers to determining whether the likelihood of a certain course or outcome is increased and does not imply determining the likelihood of a certain course or outcome by comparing it to a situation where the course or outcome does not occur. For purposes of the present invention, patients with an increased number of LMP1 copies are more likely to observe a particular course or outcome than those who do not exhibit this characteristic.

[0026] Example 1: LMP1 copy number increase in NK / T cell lymphoma patients

[0027] A total of 77 tumor biopsy specimens were collected from patients with previously untreated disease at the Sun Yat-sen University Cancer Center and Singapore General Hospital. Based on 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.

[0028] Whole-genome sequencing was performed on the 77 NK / T cell lymphoma tissues as follows: (1) DNA was extracted from the tumor samples using the QIAamp DNA Mini Kit (QIAGEN). This process required strict avoidance of contamination with exogenous DNA, and the quality and concentration of the extracted DNA were evaluated. (2) The extracted DNA was then used to construct a whole-genome sequencing library using the TruePre DNA Library Prep Kit V2 (Vazyme, Catalog No. TD501-02). The concentration of the resulting library was determined using the High Precision dsDNA Quant Kit (Invitrogen, Catalog No. Q32854), and its quality was analyzed using the High Sensitivity NGS Fragment Analysis Kit (AATI, Catalog No. DNF-474) using a Fragment Analyzer (AATI). (3) All samples were sequenced using the Illumina HiSeq X Ten sequencing platform to obtain 150 bp paired-end sequence files.

[0029] For the resulting whole-genome sequencing files, all sequence fragments were aligned to the Epstein-Barr virus reference genome using BWA software. The Epstein-Barr virus reference genome sequence file was obtained from the NCBI GeneBank database (NCBI refseq ID: NC_007605.1). The rmdup command of samtools software was then used to remove duplicate fragments generated by PCR amplification during library construction, and the depth command was used to calculate the coverage and depth of the alignment to the Epstein-Barr virus genome. Finally, cnvPartition software was used to identify copy number variation events and calculate copy number gains or losses for all regions in the Epstein-Barr virus genome.

[0030] Through the above steps, a copy number variation map of the EBV genome in NK / T cell lymphoma was constructed, and the location of the LMP1 gene was mapped using the region fragment 166483 to 169056 in the EBV genome, thereby discovering a high frequency of LMP1 copy number increase in NK / T cell lymphoma patients (18 / 77, with a probability of 23.4%). Figure 1 shows 18 samples with increased copy number in this region among 77 samples from NK / T cell lymphoma patients. Figure 2 shows a copy number variation map of the EBV genome in NK / T cell lymphoma, among which LMP1 has the highest frequency of copy number increase, which is a landmark event of EBV genetic variation in NK / T cell lymphoma.

[0031] Example 2: LMP1 copy number variation events cause changes in functional pathways

[0032] Whole-transcriptome sequencing was performed on tumor samples from the same cohort of NK / T cell lymphoma patients. Combined with the LMP1 copy number variation event information known from the above method, transcriptome sequencing sequence files were obtained for mutants with LMP1 copy number gain (n=9) and wild-types without copy number gain (n=16).

[0033] Through routine data processing steps such as quality control, alignment, and quantification of transcriptome sequencing data, the transcriptome matrix files of mutant and wild-type samples were obtained.

[0034] Further analysis using GSEA software in R language revealed that the functional pathways that were significantly upregulated in the mutant type with increased LMP1 copy number compared to the wild type without increased copy number were compared.

[0035] FIG3 shows signaling pathways that are significantly upregulated in patients with mutant NK / T cell lymphoma with increased LMP1 copy number, including the P53 signaling pathway and NFkB signaling pathway, which are functionally altered and are related to the occurrence and development of cancer.

[0036] Example 3: LMP1 copy number variation events as a prognostic marker for NK / T cell lymphoma

[0037] Subsequently, the clinical prognostic data of these NK / T cell lymphoma patients, including their death events (n=23) and disease progression events (n=24), were collected for subsequent survival analysis to determine whether increased copy number of LMP1 could serve as a prognostic marker for NK / T cell lymphoma.

[0038] Survival analysis was performed on the copy number variation information and survival prognosis information of the above-mentioned patient pairs using the survival and survminer software in R language.

[0039] This survival analysis divided NK / T cell lymphoma patients into two groups based on the presence or absence of increased copy number of LMP1 in the EB virus genome: patients with LMP1 copy number increased mutant (LMP-1 copy-gain ; n=7) and wild type without copy number gain (LMP-1 wildtpe ; n = 16 or 17), and found that increased LMP1 copy number in tumor tissues was associated with a better prognosis for overall survival of patients.

[0040] Figure 4 shows the results of the survival analysis. As can be seen, patients with increased LMP1 copy number had significantly better overall survival (OS) and progression-free survival (PFS) prognoses. This suggests that LMP1 copy number variation in the EBV genome affects the survival of patients with NK / T cell lymphoma, and that detecting the presence of increased LMP1 copy number is beneficial for risk stratification and prognostic assessment.

Claims

Use of an LMP1 gene copy number variation detection reagent in the preparation of a prognosis kit for NK / T cell lymphoma.

2. The application according to claim 1, characterized in that, 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 tissue 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, characterized in that The prognosis of NK / T cell lymphoma includes the overall survival and progression-free survival of patients.

6. The application according to claim 1, wherein When the copy number of the LMP1 gene increases, the overall survival and progression-free survival of patients are better.

7. The application according to claim 1, wherein 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.

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