| BackgroundNK/T-cell lymphoma(NKTCL)is a subtype of non-Hodgkin’s lymphoma(NHL)with a relatively high incidence in East Asia,especially in China,which is highly malignant,aggressive,and has a poor overall prognosis.NKTCL mainly occurs in the nose and adjacent tissues,and a few involve the tonsils,gastrointestinal tract,skin,and testes.According to the different primary sites of NKTCL,it can be divided into a variety of types,such as nasal cavity and nasal type,nasal cavity and nasal appearance type,upper respiratory tract and non-upper respiratory tract type.In China,the nasal cavity and Wechsler’s ring are the most common sites of NKTCL,accounting for approximately 5%-15%of lymphomas.Epstein-Barr virus(EBV)is a common lymphophilic herpes virus in the population.Epidemiology has shown that a variety of tumors are associated with EBV infection,including NKTCL.The EBV positive rate of nasal type of NKTCL in Asian is almost 100%.Studies have confirmed that pre-treatment EBV DNA level can be used for tumor burden assessment,clinical staging,and treatment response evaluation in NKTCL.Post-treatment EBV level can be used to monitor NKTCL progression,and in addition,can be used as a valuable biomarker for minimal residual disease(MRD).There are two main forms of EBV in organisms:latent infection and lytic infection,of which latent infection is the most common.During latent infection,EBV encodes only some viral products in host cells,such as EBV latent membrane protein(LMP),EBV nuclear antigen(EBNA),terminal protein(TP)and EBV encoded small RNA(EBER).Studies have suggested that the above proteins and related pathways are the main mechanisms causing EBV-related tumorigenesis.However,it has also been partially shown that EBV DNA integration in the host genome leads to host genome instability,which results in abnormal gene expression has the potential to be one of the causes of disease.Although the pathogenesis of EBV-associated NKTCL has not been clarified,EBV key cellular events,including specific point mutations,large deletions,integration within the host genome,and abnormal activation of latent and lytic genes,are closely related to the development of NKTCL.Therefore,the study was mainly divided into two parts:First of all,the clinical data of 31 patients with NKTCL was retrospectively analyzed to assess the effect of EBV DNA replication on the survival;Secondly,whole genome sequencing and bioinformatics analysis were performed on 9 EBV(+)and EBV(-)NK/T-derived samples to preliminarily explore the integration of EBV DNA in the NKTCL genome and the possible target gene changes,providing new ideas for studying the role of EBV in the development of NKTCL.Objects1.A retrospective analysis of 31 NKTCL patients was performed to assess the effect of EBV DNA replication on clinical characteristics and survival,and to study the prognostic factors of NKTCL.2.Whole genome sequencing and bioinformatics analysis of 9 EBV(+)and EBV(-)NK/T derived samples were performed to determine whether EBV DNA was integrated in the NKTCL genome and to preliminarily explore its integration information.Materials and Methods1.The clinical data of 31 patients with refractory/relapsed NKTCL treated with the DDGP regimen from October 2014 to February 2019 in the Lymphoma Diagnosis and Treatment Center of the First Affiliated Hospital of Zhengzhou University were collected and retrospectively analyzed to assess the effect of EBV DNA replication on clinical characteristics and survival,and to study the prognostic factors of NKTCL.2.EBER expression was detected by in situ hybridization and EBV DNA was amplified by PCR to verify EBV infection in 9 NK/T derived samples;3.Whole genome sequencing and bioinformatics analysis were performed on 9 NK/T derived samples;4.The EBV DNA integrated sequences of samples were captured by whole genome sequence alignment;softclip reads were extracted by CREST software,paired reads were filtered and their number of distributions on chromosomes was counted;EBV fasta files of samples were aligned to the EBV fasta library by BLAST to confirm whether the captured sequences were viral sequences;5.IGV was used to display the distribution of reads on the chromosome of samples,PCR was used to amplify the high-frequency integration region of EBV DNA,and sanger sequencing was used to verify the EBV DNA integration sequences.Statistical MethodsSPSS 21.0 was applied for statistical analysis.The association between plasma EBV DNA replication levels and clinical characteristics of NKTCL patients was analyzed by Fisher’s exact test.Overall survival(OS)and progression-free survival(PFS)were calculated using the Kaplan-Meier method and compared with the Log-rank test.Cox proportional hazards regression models were used to determine prognostic factors for OS and PFS.P<0.05 was considered statistically significant.Results1.Patients with modified Ann Arbor stage Ⅲ-Ⅳ(P=0.002),elevated LDH levels(P=0.009),PINK-E scores 3-5(P<0.001),and ECOG PS scores 2-4(P=0.003)were more likely to have positive plasma EBV DNA.The 5-year PFS and 5-year OS of 31 NKTCL patients were 45.4%and 50.1%,respectively.Both pre-treatment EBV DNA negativity and post-treatment EBV DNA negativity were significantly associated with better PFS(Pre-treatment:P=0.005;post-treatment:P<0.001)and OS(Pre-treatment:P=0.006,post-treatment:P=0.002).In the subgroup analysis,patients whose EBV DNA turned negative after treatment had better PFS(P=0.005),but there was no significant difference in OS(P=0.225).Univariate analysis showed that modified Ann Arbor stage Ⅲ-Ⅳ,PINK-E score 3-5,ECOG PS score 2-4,pre-treatment EBV DNA positivity and post-treatment EBV DNA positivity were associated with poor PFS(P=0.041,0.028,0.009,0.005,<0.001,respectively);PINK-E score 3-5,ECOG PS score 2-4,pre-treatment EBV DNA positivity and post-treatment EBV DNA positivity were associated with poor OS(P=0.023,0.002,0.006,0.002,respectively).Multivariate analysis showed that EBV DNA status after treatment was an independent factor for PFS(P=0.015).2.EBV infection was present in 5 EBV(+)samples and absent in 4 EBV(-)samples.The coverage depth,sequencing depth,coverage rate and alignment rate of the samples all meet the requirements for subsequent studies.The capture sequence was confirmed as the viral sequence.EBV(+)nasal NTKCL tissues and EBV(+)NKTCL cell lines SNK and YTS had the highest number of reads,and showed non-random enrichment on chromosome 2.EBV DNA integration in the 400 bp region of chr2:30234084-30234483 caused insertion or deletion in the chr2p23.1 site.Conclusion1.EBV DNA replication is closely related to tumor burden and survival in NKTCL patients,post-treatment EBV DNA status is considered as an independent factor for PFS.2.EBV DNA is frequently integrated in the chr2p23.1 site of EBV(+)NKTCL cells and may affect target gene expression in this region. |
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