Early detection of nasopharyngeal carcinoma by plasma Epstein‐Barr virus DNA analysis in a surveillance program

BACKGROUND:

Nasopharyngeal carcinoma (NPC) is prevalent in Southeast Asia. Over the last decade, plasma Epstein‐Barr virus (EBV) DNA has been developed as a tumor marker for NPC. In this study, the authors investigated whether plasma EBV DNA analysis is useful for NPC surveillance.

METHODS:

In total, 1318 volunteers ages 40 to 60 years were prospectively recruited. Plasma EBV DNA and serology for viral capsid antigen immunoglobulin A (IgA) were measured. Participants who had detectable plasma EBV DNA or positive IgA serology underwent nasal endoscopic examination and a follow‐up plasma EBV DNA analysis in approximately 2 weeks. All participants were followed for 2 years to record the development of NPC.

RESULTS:

Three individuals with NPC were identified at enrolment. All of them were positive for EBV DNA and remained positive in follow‐up analysis. Only 1 of those patients was positive for EBV serology. In 1 patient who had NPC with a small tumor confined to the mucosa, the tumor was not detectable on endoscopic examination. Because of a 2‐fold increase in plasma EBV DNA on the follow‐up analysis, that patient underwent magnetic resonance imaging, which revealed the tumor. Among the participants who did not have NPC but had initially positive plasma EBV DNA results, approximately 66% had negative EBV DNA results after a median of 2 weeks.

CONCLUSIONS:

Plasma EBV DNA analysis proved useful for detecting early NPC in individuals without a clinical suspicion of NPC. Repeating the test in those who had initially positive results differentiated those with NPC from those who had false‐positive results. Cancer 2013. © 2013 American Cancer Society.     

Long‐term survival analysis of nasopharyngeal carcinoma by plasma Epstein‐Barr virus DNA levels

BACKGROUND: The objective of this study was to confirm the relation between plasma Epstein‐Barr virus (EBV) DNA (pEBV DNA) load and treatment outcomes after long‐term follow‐up in patients with nasopharyngeal carcinoma (NPC). METHODS: In total, 210 patients with NPC were enrolled, including 99 previously reported patients and 111 new patients. They prospectively received treatment with induction chemotherapy plus radiotherapy and were followed for at least 6 years. In these patients, pEBV DNA levels were measured before treatment and 1 week after treatment. The plasma viral load was correlated with treatment outcomes in the group of new patients and in the entire group. RESULTS : By using previously defined pEBV DNA cutoff values (1500 copies/mL pretreatment and 0 copies/mL post‐treatment), there was a significant correlation between the pEBV DNA value and relapse‐free survival, overall survival, and subsequent relapse rates in the new, independent patient cohort. Outcome analyses for the entire group revealed a higher relapse rate (45.6% vs 21.5% [P = .0037] or 76.7% vs 26.1% [P < .0001]), a worse relapse‐free survival rate (56.5% vs 79.3% [P < .0001] or 23.3% vs 75.6% [P < .0001]), and poorer overall survival (59.2% vs 86% [P = .0003] or 33.3% vs 79.4% [P < .0001]) in patients who had high pretreatment or persistently detectable post‐treatment pEBV DNA levels, respectively, versus their respective counterparts. Multivariate Cox analysis also confirmed these results. CONCLUSIONS : In this expanded study, the prognostic significance of pEBV DNA was confirmed using predefined cutoff values in an independent patient group, and pEBV DNA was identified as an independent prognostic marker for NPC. Cancer 2013. © 2012 American Cancer Society.     

Quantitative detection of Epstein-Barr virus DNA methylation in the diagnosis of nasopharyngeal carcinoma by blind brush sampling

Detecting EBV DNA load in nasopharyngeal (NP) brushing samples for the diagnosis of nasopharyngeal carcinoma (NPC) has attracted widespread attentions. Currently, NP brush sampling mostly relies on endoscopic guidance, and there are few reports on diagnostic markers suitable for nonguided conditions (blind brush sampling), which is of great significance for extending its application. One hundred seventy nasopharyngeal brushing samples were taken from 98 NPC patients and 72 non-NPC controls under the guidance of endoscope, and 305 blind brushing samples were taken without endoscopic guidance from 164 NPC patients and 141 non-NPC controls (divided into discovery and validation sets). Among these, 38 cases of NPC underwent both endoscopy-guided NP brushing and blind brushing. EBV DNA load targeting BamHI-W region and EBV DNA methylation targeting 11029 bp CpG site located at Cp-promoter region were detected by quantitative polymerase chain reaction (q-PCR). EBV DNA load showed good classification accuracy for NPC in endoscopy-guided brushing samples (AUC = 0.984). However, in blind bushing samples, the diagnostic performance was greatly reduced (AUC = 0.865). Unlike EBV DNA load, the accuracy of EBV DNA methylation was less affected by brush sampling methods, whether in endoscopy-guided brushing (AUC = 0.923) or blind brushing (AUC = 0.928 in discovery set and AUC = 0.902 in validation set). Importantly, EBV DNA methylation achieved a better diagnostic accuracy than EBV DNA load in blind brushing samples. Overall, detection of EBV DNA methylation with blind brush sampling shows great potential in the diagnosis of NPC and may facilitate its use in nonclinical screening of NPC.     

Quantification of Epstein–Barr virus DNA load in nasopharyngeal brushing samples in the diagnosis of nasopharyngeal carcinoma in southern China

Nasopharyngeal carcinoma (NPC) is highly incident in southern China, where 40% of world's new cases arise each year. Detection of Epstein–Barr virus (EBV) DNA load in nasopharyngeal (NP) brush/swab samples has gradually been established as a method for diagnosis of NPC. However, its applicable value in NPC diagnosis has never been investigated in southern China. It is important to explore whether such a test could be applicable to our local population. A total of 245 consecutive participants undergoing NP brushing examination were recruited to obtain the NP brushing samples in this study. Quantitative PCR assays were used to obtain the EBV DNA load. Mann–Whitney, ANOVA and receiver operating characteristic tests were used to analyze its diagnostic value. NP brushing samples from NPC patients showed extremely high levels of EBV DNA load (mean = 46360 copy/ng DNA) compared to its expression from non‐NPC control (mean = 28 copy/ng DNA) and high‐risk control (mean = 50 copy/ng DNA) groups. It produced 96% sensitivity and 97% specificity, at the COV = 225 copy/ng DNA. Furthermore, EBV DNA load could reflect disease progress. Our data showed a better performance of EBV DNA load in NP brushing samples compared with an initial biopsy, immunoglobulin A (IgA) antibody titers to viral capsid antigen in serum and EBV DNA load in plasma. Detection of EBV DNA load in NP brushing samples could be an effective supplement for NPC diagnosis. Being minimally invasive and low cost, NP brush sampling combined with EBV DNA detection demonstrates great potential for screening high‐risk populations for NPC.     

Epstein‐Barr virus strain variation and cancer

Epstein‐Barr virus (EBV) is a human tumor virus and is etiologically linked to various malignancies. Certain EBV‐associated diseases, such as Burkitt lymphomas and nasopharyngeal carcinomas, are endemic and exhibit biased geographic distribution worldwide. Recent advances in deep sequencing technology enabled high‐throughput sequencing of the EBV genome from clinical samples. Rapid cloning and sequencing of cancer‐derived EBV genomes, followed by reconstitution of infectious virus, have also become possible. These developments have revealed that various EBV strains are differentially distributed throughout the world, and that the behavior of cancer‐derived EBV strains is different from that of the prototype EBV strain of non‐cancerous origin. In this review, we summarize recent progress and future perspectives regarding the association between EBV strain variation and cancer.     

Modulation of the tumor microenvironment by Epstein‐Barr virus latent membrane protein 1 in nasopharyngeal carcinoma

Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein‐Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre‐invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1‐expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development.     

Prognostic value of serum Epstein–Barr virus antibodies in patients with nasopharyngeal carcinoma and undetectable pretreatment Epstein–Barr virus DNA

Epstein–Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). Serum IgA antibodies against early antigen (EA‐IgA) and viral capsid antigen (VCA‐IgA) are the most commonly used to screen for NPC in endemic areas. However, the prognostic value of serum EA‐IgA and VCA‐IgA in patients with NPC is less clear. We hypothesize that serum EA‐IgA and VCA‐IgA levels have prognostic impact for survival outcomes in NPC patients with undetectable pretreatment EBV (pEBV) DNA. In this series, 334 patients with non‐metastatic NPC and undetectable pEBV DNA were included. Serum EA‐IgA and VCA‐IgA were determined by ELISA. After analysis, serum EA‐IgA and VCA‐IgA loads correlated positively with T, N, and overall stage (all P < 0.05). Serum EA‐IgA was not associated with survival outcome in univariable analyses. But patients with serum VCA‐IgA >1:120 had significantly inferior 5‐year progression‐free survival (80.4% vs 89.6%, P = 0.025), distant metastasis‐free survival (88.4% vs 94.8%, P = 0.050), and locoregional relapse‐free survival (88.4% vs 95.6%, P = 0.023; log–rank test). Multivariable analyses revealed that N stage was the only independent prognostic factor (all P < 0.05), but the VCA‐IgA became insignificant. Further analyses revealed that serum VCA‐IgA was not an independent prognostic factor in early N (N0–1) or advanced N (N2–3) stage NPC. In summary, although both EA‐IgA and VCA‐IgA correlate strongly with TNM stage, our analyses do not suggest that these antibodies are prognostic biomarkers in patients with NPC and undetectable pEBV DNA.     

Genome‐wide identification of Epstein‐Barr virus–driven promoter methylation profiles of human genes in gastric cancer cells

BACKGROUND:

Aberrant methylation of tumor‐related genes has been reported in Epstein‐Barr virus (EBV)‐associated gastric cancers. This study sought to profile EBV‐driven hypermethylation in EBV‐infected cells.

METHODS:

The EBV‐positive AGS gastric cancer cell line (AGS‐EBV) and EBV‐negative AGS cells were used in this study. DNA methyltransferase‐3b (DNMT3b) activity was assessed by EpiQuick activity assay, and genome‐wide DNA methylation profiles were assessed by methyl‐DNA immunoprecipitation microarray assay.

RESULTS:

EBV infection was confirmed in AGS‐EBV cells by EBV‐encoded RNA in situ hybridization. Expression and activity of DNA methyltransferase‐3b (DNMT3b) was significantly increased in AGS‐EBV compared to AGS. Ectopic expression of LMP2A (latent membrane protein 2A) in AGS increased activity of DNMT3b. A total of 1065 genes were differentially methylated by EBV infection (fold‐changes ≥ 2, P < .05) in AGS‐EBV compared to AGS cells. The majority of the differentially methylated genes (83.2%, 886 of 1065 genes) had cytosine‐guanine dinucleotide (CpG) hypermethylation in AGS‐EBV (fold‐changes 2.43～65.2) versus that found in AGS cells. Gene ontology analysis revealed that hypermethylated genes were enriched in the important cancer pathways (≥ 10 genes each, P ≤ .05) including mitogen‐activated protein kinase signaling, cell adhesion molecules, wnt signaling pathway, and so forth. Six novel hypermethylated candidates (IL15RA, REC8, SSTR1, EPHB6, MDGA2, and SCARF2) were further validated. Higher levels of DNA methylation were confirmed for all these genes in AGS‐EBV cells by bisulfite genomic sequencing. Furthermore, these candidates were silenced or down‐regulated in AGS‐EBV cells, but can be restored by demethylation treatment.

CONCLUSIONS:

EBV infection in AGS cells induced aberrant CpG hypermethylation of 886 genes involving in important cancer‐related pathways. Induction of promoter methylation by EBV is regulated by up‐regulation of DNMT3b through LMP2A. Cancer 2013. © 2012 American Cancer Society.     

Frequency and clinical correlates of elevated plasma Epstein‐Barr virus DNA at diagnosis in peripheral T‐cell lymphomas

Epstein‐Barr virus (EBV)‐encoded RNAs (EBER) in tumor tissue and cell‐free plasma EBV‐DNA (pEBVd) are detected in EBV‐associated lymphomas. Studies have suggested that EBER+ peripheral T‐cell lymphomas (PTCL) have worse prognosis but the role of EBV in these neoplasms remains unclear. pEBVd is quantitative and more easily amenable to standardization than EBER, but frequency of pEBVd detection, clinical impact and agreement with EBER status in PTCL are unknown. We retrospectively assessed frequency of detectable pre‐treatment pEBVd, presence of EBER in tumor tissue, and outcomes in 61 of 135 EBV‐assessable PTCL patients. Fifteen of 61 patients (24.5%, 95% CI: 14–37%) were pre‐treatment pEBVd+, with no significant differences in baseline characteristics or treatment between pEBVd+ and pEBVd? patients. EBER‐ISH was performed on 10 pEBVd+ and 35 pEBVd? tumors. All 10 pEBVd+ patients were EBER+, but 9 pEBVd? patients were also EBER+. With median follow up of 24 months (range 1–96), overall survival (OS) was shorter in pEBVd+ compared to pEBVd? patients (13 vs . 72 months; p = 0.04). In our retrospective study, pre‐treatment pEBVd was elevated in 25% of PTCL patients, was highly specific for EBER+ tumors, and was associated with shorter survival. pEBVd should be further explored as a prognostic variable and tumor biomarker in PTCL.     

Epstein-Barr virus seroreactivity among unaffected individuals within high-risk nasopharyngeal carcinoma families in Taiwan

Most adults have been infected with EBV. Many studies have indicated that antibodies against specific EBV antigens, particularly IgA antibodies, can be predictive or prognostic of EBV-associated malignancies, such as NPC. We hypothesized that healthy individuals from families with a history of multiple members affected with NPC (who therefore might be genetically susceptible to NPC themselves) might have an EBV antibody profile that is distinct from that seen in healthy individuals from the community at large. To explore this possibility and examine determinants of anti-EBV antibody levels in healthy, high-risk individuals, we evaluated data from 2 parallel studies of NPC in Taiwan, which included 1,229 healthy members of families in which 2 or more individuals were affected with NPC and 320 controls from the community at large. Blood collected from participants was tested for IgA antibodies against EBV VCA and EBNA-1 and for neutralizing antibodies against EBV DNase using standard assays. We observed evidence of familial aggregation of EBV seroreactivity among individuals from high-risk, multiplex NPC families. Anti-VCA IgA and anti-EBNA-1 IgA antibody seroprevalence in unaffected family members of NPC cases was 5-6 times higher than in members of the community (p < 0.01). This elevated seroprevalence among unaffected individuals from high-risk families was observed regardless of the relationship of the unaffected individual to the closest affected relative (siblings, parents, children or spouses). No sociodemographic or environmental factors examined were found to strongly and consistently correlate with elevated seroprevalence, but patterns emerged of increasing seroprevalence among older individuals and among females. Unaffected individuals from high-risk NPC families have elevated anti-EBV IgA antibody titers. The etiologic and clinical implications of this finding remain to be established.     

Cancer stem cells in Epstein‐Barr virus‐associated gastric carcinoma

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein‐Barr virus (EBV)‐associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV‐infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9^+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV‐infected TMK1 cells and these cell populations showed high spheroid‐forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9^?/? cells. Sphere‐forming ability was dependent on the nuclear factor‐κB (NF‐κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11‐7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere‐forming ability of TMK1 cells, which was mediated through NF‐κB signaling. Together, these results indicate that CD44v6v9^+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF‐κB pathway. Future studies should investigate CD44v6/v9^+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.     

Plasma Epstein‐Barr virus DNA for pediatric Burkitt lymphoma diagnosis,prognosis and response assessment in Malawi

Point‐of‐care tools are needed in sub‐Saharan Africa (SSA) to improve pediatric Burkitt lymphoma (BL) diagnosis and treatment. We evaluated plasma Epstein–Barr virus (pEBV) DNA as a pediatric BL biomarker in Malawi. Prospectively enrolled children with BL were compared to classical Hodgkin lymphoma (cHL) and nonlymphoma diagnoses. Pediatric BL patients received standardized chemotherapy and supportive care. pEBV DNA was measured at baseline, mid‐treatment, and treatment completion. Of 121 assessed children, pEBV DNA was detected in 76/88 (86%) with BL, 16/17 (94%) with cHL, and 2/16 (12%) with nonlymphoma, with proportions higher in BL versus nonlymphoma (p < 0.001) and similar in BL versus cHL (p = 0.69). If detected, median pEBV DNA was 6.1 log₁₀copies/mL for BL, 4.8 log₁₀copies/mL for cHL, and 3.4 log₁₀copies/mL for nonlymphoma, with higher levels in BL versus cHL (p = 0.029), and a trend toward higher levels in BL versus nonlymphoma (p = 0.062). pEBV DNA declined during treatment in the cohort overall and increased in several children before clinical relapse. Twelve‐month overall survival was 40% in the cohort overall, and for children with baseline pEBV detected, survival was worse if baseline pEBV DNA was ≥6 log₁₀copies/mL versus <6 log₁₀copies/mL (p = 0.0002), and also if pEBV DNA was persistently detectable at mid‐treatment versus undetectable (p = 0.041). Among children with baseline pEBV DNA detected, viremia was the only significant risk factor for death by 12 months in multivariate analyses (adjusted hazard ratio 1.35 per log₁₀copies/mL, 95% CI 1.04–1.75, p = 0.023). Quantitative pEBV DNA has potential utility for diagnosis, prognosis, and response assessment for pediatric BL in SSA.     

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.     

Distribution of Epstein-Barr viral load in serum of individuals from nasopharyngeal carcinoma high-risk families in Taiwan

The utility of EBV load as a tumor marker in nasopharyngeal carcinoma (NPC) patients suggests that it might also serve as a screening test for individuals who are at high risk for developing NPC. We previously demonstrated that unaffected individuals from high-risk families had elevated anti-EBV antibody levels compared to community controls. In this study, we measured EBV load using 2 different real-time PCR assays (targeting BamH1W and polymerase gene sequences, respectively) carried out in 2 independent research labs in serum samples from 19 untreated NPC cases, 11 healthy community controls and 100 unaffected individuals from families in which 2 or more individuals were affected with NPC. EBV genomes were detectable in 68% of NPC cases by the EBV BamH1W assay and in 74% by the EBV polymerase assay (kappa = 0.64). Patients with stage III or IV disease had significantly higher EBV load compared to those with stage I or II disease (p = 0.008). EBV DNA was detected in a single community control sample by the EBV BamH1W assay and in none of the samples by the EBV polymerase assay. Only one of 100 unaffected family members tested positive by both assays. An additional 14 were positive by only one of the 2 EBV load assays used and usually in only one of the duplicate wells tested, all with very low viral loads (3-50 copies/ml). In addition, EBV load did not correlate with EBV serology results (anti-VCA, anti-DNase, anti-EBNA-1) among these unaffected family members. In conclusion, our study suggests limited clinical utility of the EBV load test, in its current configuration, to screen individuals from high-risk families. Should a more sensitive or specific molecular assay be developed that is capable of detecting and distinguishing tumor-derived EBV genomes or gene products from true negatives, it could be evaluated as a possible screening tool for asymptomatic and early-stage NPC.