Epstein-barr Virus Related Biomarker In The Diagnosis Of Nasopharyngeal Carcinoma In Nasopharyngeal Brushing Samples

Objective:Nasopharyngeal carcinoma(NPC)is a highly invasive and metastatic cancer that is widely prevalent in southern China and Southeast Asia.Although the overall survival rate is approximately 90% in patients with early clinical stage after standard treatment,unfortunately,most patients are diagnosed with advanced-stage disease at their first visit,and the survival rate decreases to less than 50%.Developing a simple and reliable method facilitating NPC diagnosis and screening is of great significance to increase patient's survival in clinical practice.Previous studies have fully demonstrated that Epstein – barr virus infection play an important role in NPC occurrence.Although EBV is ubiquitous worldwide,it is so closely linked to NPC that nearly 100% of tumor lesions possess EBV genomes in undifferentiated type NPC(WHO type III),the predominant type of NPC in endemic areas.Moreover,squamous cell(WHO type I)and non-keratinizing(WHO type II)NPCs are also frequently associated with EBV in endemic areas.The infection of epithelial cells by EBV is a typical characteristic of NPC in high-risk areas.NP brush sampling combined with EBV related nucleic acid markers may provide some new strategies for NPC diagnosis and screening in high risk areas.The objective of our study contains the following three aspects:(1)Exploring whether detecting EBV DNA load in NP brushing samples is a good indicator for NPC diagnosis in our high-risk area.(2)Clarifying the molecular characteristic and the origin of EBV DNA load in NP brushing samples,and providing the theoretical basis for developing EBV related biomarkers.These contents includes revealing the EBV DNA is fragment or intact,and it is from the EBV infected tumor cells or extracellular EBV particle.It is not clear whetherEBV DNA load detection reflect the NPC development.(3)Developing more biomarkers for NPC diagnosis,especially the efficient biomarker in blind brushing(a sampling method that is independent on the clinical doctor and endoscopy),this is suitable for NPC screening in high risk areas.Methods:Our study focuses on NPC and we collaborate with the researchers from Sun Yat-Sen University Cancer Center,located at Guangdong province with a high incidence of NPC.(1)Firstly,a total of 303 NP brushing samples were collected from different participants,including 129 NPC patients,116 non-NPC controls and 58 NPC patients after the therapy.A stable NP brush sampling was established before conducting the following study.NP brushing samples was obtained from each participant,then DNA was extracted from the brushings samples and the EBV DNA load in NP brushing samples was quantified by Q-PCR.(2)Secondly,180 NP brushing samples including 104 NPC patients and 76 non-NC controls was used for further analysis.Both DNA and RNA were extracted from NP brushing samples.A comparative PCR was first conducted by detecting the 99 bp and 213 bp EBNA1 simultaneously in order to explore whether there was fragment EBV DNA.The EBV RNA profile including six EBV latent genes(including EBER1,BART,EBNA1,EBNA2,LMP1 and LMP2A)and five lytic gene(including Zebra,RTA,TK,PK and VCA-P18)transcripts were further measured.Besides,EBV miRNAs(including mir-bart1-5p,mir-bart5,mir-bart6-5p and mir-bart17-5p)were also quantified by Q-PCR.Finally,the methylated status(including methyalted degree and methylated type)of EBV DNA C-promoter was further revealed by methylation-specific PCR.(3)Thirdly,taking the EBV miRNA as an example,the diagnostic value was further validated in another independent cohort including 215 NPC patients and 209 controls.Besides,blind brushing from 38 NPC patients and 48 controls were conducted in parallel experiments.The diagnostic value of single EBV DNA load,EBV miRNA,EBV methylated degree and EBV methylated type or their combination was evulated no matter in NP brushing samples under the guide or without the guid of endosocopy.The ideal biomarker in blind brushing was expected to be discovered.Results:(1)All NP brushing samples from NPC patients were positive for EBV DNA,withextremely high DNA loads(mean=46 360 copies/ng DNA,range from 40 to 1 395 000copies/ng DNA).Although EBV DNA was detectable in 70.6% of the non-NPC control group(mean=28 copies/ng DNA,range from 0 to 158 copies/ng DNA),in 87.8% of the high risk control group(mean=50 copies/ng DNA,range from 0 to 469 copies/ng DNA)and in 63.8% of NPC patients after therapy(mean=27 copies/ng DNA,range from 0–417copies/ng DNA),the load value was very low.A significantly higher EBV DNA load was observed in NPC patients(P<0.0001),compared to their loads in controls.In contrast,no significant difference was observed between non-NPC control and high-risk control.The Cut off value(COV)was set by calculating the receiver operating characteristic curve(ROC)with the maximu area unde the curve(AUC).At the COV=225 copy?ng DNA,a high sensitivity(96%)and specificity(97%)was obtained.In parallel experiments,EBV DNA load in plasma showed 76% sensitivity and 87% specificity,and VCA-IgA titer exhibited 89% sensitivity and 77% specificity,both lower than the diagnostic value of EBV DNA load in NP brushing samples.Among the 129 biopsy-proven NPC patients,11patients(8.5% false negativity)needed to undergo repeated sampling before obtaining a correct pathological diagnosis.In contrast,the EBV DNA loads in the only NP brushing samples from these 11 patients were all above the COV,directly allowing for a correct diagnosis.(2)The results revealed that most of the EBV DNA in NP brushing samples from NPC patients was intact while higher levels of fragmented DNA indicated that an apoptotic origin was observed only in 12 samples.Qualitatively,the latency II transcription profile with high EBER1,BART,LMP1,LMP2 A,EBNA1 but not EBNA2 expression was observed in both NP brushing samples and tissues from NPC patients.Only EBER1 transcript(49.3%)with low expression was found in NP brushing samples and tissues from the control group.Besides the expression of latent genes,certain frequencies(31.4% to 93%)of lytic genes with high expression were also detected in samples from NPC patients.In contrast,low frequencies(0 to 15.5%)of lytic genes with low expression were found in samples from conrol group.There was a significant correlation between the EBV DNA load and frequencies of latent and lytic genes,but the the significant correlation between EBV DNA load and EBER1 expression was only observed in NPC patients.Besides,most of the NP brushing samples from NPC patients showed high expression of EBV mir-bar1-5p,mir-bart5,mir-bart6-5p and mir-bart17-5p(87.9% to 98.0%).In contrast,only a few NP brushing samples(5.4% to 17.6%)from control group showed low expression of EBV miRNAs.Significant correlation betweenEBV DNA load and mir-bart1-5p expression was also only observed in NPC patients.The methylation status of EBV DNA C-promoter was further examined in NP brushing samples from both NPC patients and controls.In NPC patients,the predominant status of EBV C promoter was methylated.There were 39 samples with only methylated bands(defined as M)and 65 samples with both methylated and unmethylated bands,but the methylated degree were significantly greater than the unmethylated degree(defined as M > U),according to the band brightness.In contrast,the predominant type of EBV DNA C promoter was unmethylated in the control group.There were 14 samples with only unmethylated bands(defined as U),9 samples with the band brightness U > M,6 samples with only methylated bands(M)and eight samples with the band brightness M > U.In addition,there were 39 samples with no PCR bands,probably due to the low EBV DNA loads.(3)Based on the difference of EBV DNA load,EBV miRNA and EBV DNA C-promoter methylation,further analysis showed they all had a high diagnostic accuracy in NPC diagnosis.Brush sampling both with and without the guide of endoscopy(named blind brushing)was conducted in 38 NPC patients.With the guide of endoscopy,94.7%(36/38)of brushing samples had EBV DNA load(from 94 to 40 095 copies)above the defined COV(225 copies),and the median load was 5144 copies.In contrast,only 55.3%(21/38)of NP samples via blind brushing had EBV DNA load(from 1 to 8744 copies)above the COV,and the median load decreased to 275 copies.Similarly,100% of brushing samples with the guide of endoscopy had EBV DNA methylated degree(from36.79 to 146.57)above the defined COV(33.48),and the median brightness of M band was 80.99.However,only 57.9%(22/38)of samples via blind brushing had EBV DNA methylated degree above the COV(from 0 to 90.36),and the median brightness decreased to 42.61.Different from the quantitative EBV DNA load and EBV DNA C-promoter methylated degree,up to 89.5%(34/38)of samples collected via blind brushing showed consistent results of qualitative methylated type with samples collected via endoscopy-guided brushing from NPC patients.All the samples were of methylated type with M or M > U.Conclusion:Our study first shows quantification of EBV DNA load,EBV miRNA,EBV methylated degree and EBV methylated type in NP brushing samples could be used as an effective supplement for NPC diagnosis in southern China.By the study of EBV RNAprofile,our results suggest that EBV genomic DNA from cells and free EBV virion both contribute to the EBV DNA load in the NP brushing samples.Subsequently,the methylation status of EBV DNA C-promoter in our study indicated that EBV DNA in NP brushing samples from NPC patients was mainly from EBV latent tumor cells.In contrast,unmethylation was predominant in most samples from the control group.It seemed that most of the EBV DNA in the samples was probably from free EBV virion.The methylation status of EBV DNA C-promoter in nasopharyngeal brushing samples was found to have a significant difference between NPC patients and the control group.This difference could be used to make a better diagnosis of NPC along with the EBV DNA load.More importantly,detection of EBV DNA methylation shows great potential in the diagnosis of NPC through blind brushing,a brush sampling technique that does not require clinical settings.Therefore,it has great potential to be applied for the screening of NPC in the future.