Nasopharyngeal Carcinoma DNA Research Articles

Identifying patients at high risk of recurrence by rebound of plasma Epstein-Barr virus DNA in nasopharyngeal carcinoma.

6030 Background: About 10-30% of patients with nasopharyngeal carcinoma (NPC) would develop disease recurrence following treatment. The optimal method to screen patients needing early medical intervention remains to be studied. We investigated the potential cut-off values of plasma EBV DNA in identifying patients at high risk of recurrence. Methods: From 2012 to 2020, 950 patients with detectable pre-treatment plasma EBV DNA and undetectable EBV DNA within 3 months after treatment were retrospectively reviewed. Survival outcomes of patients with different plasma EBV DNA rebound patterns were analyzed. The diagnostic performance of different cut-off values was evaluated in patients with ≥ 2 positive tests. Results: The number of patients with continuous negative plasma EBV DNA, only 1 positive test, and ≥ 2 positive tests during post-treatment surveillance were 747, 89, and 114, respectively. Patients with ≥ 2 positive plasma EBV DNA tests had worse PFS, LRRFS, and DMFS when compared with the ones with negative tests ( P < 0.001, P < 0.001, and P < 0.001) and only 1 positive test ( P < 0.001, P < 0.001, and P < 0.001). Superior PFS was observed for patients with negative tests compared to patients with only 1 positive test ( P = 0.006). Of 56 patients with more than 50 copies/ml plasma EBV DNA in both tests, 48 developed recurrences within 4 years. The sensitivity, specificity, PPV, and NPV of this cut-off for recurrence prediction were 35.8%, 99.0%, 85.7%, and 90.4, respectively. Then, the whole population was divided into the low-risk group (≤ 1 positive test), the intermediate-risk group (≥ 2 positive tests and at least one test had < 50 copies/ml) and the high-risk group (≥ 2 positive tests and both tests had ≥ 50 copies/ml). The PFS of the high-risk group was worse than that of the low-risk group ( P < 0.001) and the intermediate-risk group ( P < 0.001). The PFS of the high-risk patients with and without capecitabine or tegafur/gimeracil/oteracil before clinical confirmed relapse were 59.3% and 15.0% ( P = 0.004). Conclusions: NPC patients with ≥ 2 positive tests and ≥ 50 copies/ml in both tests during follow-up were at high risk of relapse. Early medical intervention could bring survival benefits to this cohort.

Recent Advances in the Development of Biomarkers and Chemoradiotherapeutic Approaches for Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) is endemic in Southern China, and the prognosis of this cancer has improved in part due to advances in radiotherapy (RT) techniques, broadened therapeutic options, and more precise prognostic stratification of patients. RT is the primary curative treatment of NPC, and the incorporation of chemotherapy (induction, concurrent, adjuvant) to RT has contributed to improved survival in patients with locoregionally advanced NPC. Concurrent chemoradiotherapy (CCRT) in combination with adjuvant or induction chemotherapy is now the standard treatment of locoregionally advanced NPC, but the ideal CCRT therapeutic strategy for NPC remains controversial. Plasma EBV DNA is the archetypal tumor-derived DNA in NPC, and three generations of studies have gradually expanded its clinical applications. Recently, the advent of whole exome/genome sequencing of NPC and the promising clinical activity of immune checkpoint inhibitors have also spurred interest in the development of newer biomarkers. This review will focus on two clinical advances in NPC research that have made substantial impact on the contemporary management of NPC: (1) The integration of plasma EBV DNA in an expanding spectrum of clinical indications, and the development of promising immune-related biomarkers; (2) the current development of CCRT with special emphasis on the use of induction and adjuvant chemotherapy, as well as the potential applications of metronomic chemotherapy and immune checkpoint inhibitors in the treatment of locoregionally advanced NPC.

Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma: A Brief Review.

Epstein-Barr virus (EBV) is a B-lymphocytes herpes virus and can transform B lymphocytes to malignant tumor cells if infected. Nasopharyngeal carcinoma (NPC) is strongly associated with EBV. Circulating EBV DNA in plasma has been recognized as an important biomarker of NPC. Much work has been done to validate the ability of circulating EBV DNA for screening, diagnosis, risk stratification, monitoring, and predicting prognosis. This chapter reviews the clinical progress of circulating cell-free EBV DNA in NPC.

Clinical value of a plasma Epstein-Barr virus DNA assay in the diagnosis of recurrent or metastatic nasopharyngeal carcinoma: a meta-analysis.

Background: To evaluate the diagnostic value of Epstein–Barr virus (EBV) DNA in nasopharyngeal carcinoma (NPC) patients with locoregional or distant recurrence.Methods: Articles related to the diagnosis of recurrent or metastatic NPC by the detection of EBV DNA in plasma or serum were retrieved from different databases. Sensitivity, specificity, summary receiver operating characteristic (SROC) curves, and likelihood ratios were pooled to assess the diagnostic value of individual diagnostic tests.Results: This meta-analysis pooled 25 eligible studies including 2496 patients with NPC. The sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (−LR) of EBV DNA in the diagnosis of NPC were 0.858 (95% confidence interval (CI): 0.801–0.901), 0.890 (95% CI: 0.866–0.909), 7.782 (95% CI: 6.423–9.429) and 0.159 (95% CI: 0.112–0.226), respectively. The diagnostic odds ratio (DOR) was 48.865 (95% CI: 31.903–74.845). The SROC for EBV DNA detection was 0.93 (95% CI: 0.90–0.95).Conclusion: The detection of EBV DNA for the diagnosis of recurrent or metastatic NPC has good sensitivity and specificity and might be helpful in monitoring recurrent or metastatic NPC.

Molecular Prognostic Value of Circulating Epstein-Barr Viral DNA in Nasopharyngeal Carcinoma: A Meta-Analysis of 27,235 Cases in the Endemic Area of Southeast Asia.

Aims: This meta-analysis evaluated the value of using Epstein-Barr virus (EBV) DNA titers as a predictive factor in assessing the clinical course of nasopharyngeal carcinoma (NPC) patients in Southeast Asia. Methods: A systematic search was performed using PubMed, the Cochrane Library, and Embase online databases. Eligible studies with complete baseline information and extractable hazard ratios (HRs), 95% confidence intervals (CIs), and other details were included in this analysis. All pooled statistics were calculated using RevMan 5.3 software with inverse variance methods, forest plots, or funnel plots directly; the results were analyzed both in toto and by subgroups using meta-regression and sensitivity analyses. Results: Forty studies involving 27,235 subjects were included in this meta-analysis. When the recommended EBV DNA viral cutoff values of 2,000, 0, and 0 copies/mL were used for the pretreatment, mid treatment, and post treatment groups, respectively, all values above these cutoffs were associated with at least a 2.5-fold increased risk of death compared with patients with lower levels of EBV DNA (HRs and 95% CIs were 2.47 [2.10-2.89], 2.67 [1.50-4.75], and 5.25 [3.58-7.71], respectively, p < 0.05). This finding could also explain heterogeneities among the studies. Conclusion: Higher pretreatment, mid-treatment, and post-treatment EBV DNA levels were all significantly correlated with poor outcomes for patients afflicted with NPCs. Further investigations of EBV DNA levels, combined with addressing tumor size, lymph nodes, metastases stages, are needed to determine whether they can be used as clinical guidelines for diagnosis and treatment.

Interplay of Tumor Spread, Volume and Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma: Feasibility of An Integrative Risk Stratification Scheme.

Purpose: To investigate the inter-correlation of tumor spread, volume and quantitative plasma Epstein-Barr virus DNA level (pEBV DNA), and to further assess the prognostic efficacy of a novel risk stratification combining anatomic, volumetric and biological features in nasopharyngeal carcinoma (NPC).Methods and Materials: One hundred and twelve patients with non-metastatic NPC were prospectively enrolled. Correlation of pEBV DNA with tumor invasiveness, lymph node (LN) metastasis, tumor volume and classification was tested by univariate and multivariate analyses. 5-year distant metastasis free survival (DMFS) was evaluated using Kaplan-Meier method and Cox proportional hazards model.Results: Tumor volume, TNM stage and pEBV DNA were strongly inter-correlated to each other. Nodal volume, skull base invasion and LN metastasis to supraclavicular fossa were determined to be independent predictors for pEBV DNA level. To exclude collinearity, a risk stratification based on combination of EBV DNA, nodal volume and anatomic features was established, offering significant distinguishing ability in 5-year DMFS. Further multivariate Cox regression analysis found the novel stratification to be independent predictor of DMFS.Conclusions: Both anatomic spread and tumor volume contribute to pEBV DNA level, leading to strong inter-correlation between NPC stage, volume and EBV DNA. The proposed risk stratification combining anatomic, volumetric and biological features showed potential in refining DMFS prediction.

Plasma Epstein–Barr viral DNA load at midpoint of radiotherapy course predicts outcome in advanced-stage nasopharyngeal carcinoma

To test the hypothesis that prognostication of treatment outcome is feasible by biomarker response at midcourse of chemoradiotherapy (CRT)/radiotherapy (RT), with respect to the plasma load of Epstein-Barr viral (EBV) DNA in nasopharyngeal carcinoma (NPC). One hundred seven patients with stage IIB-IV NPC were prospectively studied. Plasma EBV DNA load was measured by quantitative PCR before therapy (pre-DNA), at completion of 4 weeks of CRT/RT (mid-DNA), and within 3 months of completion of therapy (post-DNA). The end points are post-DNA load, a recognized surrogate of survival, and clinical outcome. Ninety-three percent of patients had detectable EBV DNA before therapy (median load = 972 copies/ml). EBV DNA became undetectable in 55 (51%) patients at the end of week 4 of therapy. Detectable mid-DNA was associated with worse clinical outcome (median follow-up time, 6.2 years), for distant failure [hazard ratio (HR) 12.02, 95% confidence interval (CI) 2.78-51.93; P < 0.0001], progression-free survival (PFS; HR 4.05, 95% CI 1.89-8.67, P < 0.0001), and overall survival (OS; HR 3.29, 95% CI 1.37-7.90, P = 0.0077). Seventy-four percent of all failures were associated with detectable mid-DNA, whereas 34% of all failures were associated with detectable post-DNA. Stratification by tumor stage (IIB, III, IV) has no significant prognostic effect. Unfavorable EBV DNA response at midcourse of RT/CRT is an adverse prognosticator for treatment outcome, is linked to majority of all failures, and discriminates outcome better than tumor stage. The data could provide a basis for trial design that addresses alteration of therapy intensity during the latter phase of CRT, and adjuvant therapy. Validation studies are awaited.

Comparison of two laboratory extraction techniques for the detection of Epstein–Barr virus in the saliva of nasopharyngeal carcinoma patients

The aim of the present study was to compare the effectiveness of DNA extraction using an extraction kit against the standard boiling technique for the detection of Epstein-Barr virus (EBV) DNA in nasopharyngeal carcinoma (NPC) patients. Stimulated whole saliva samples from newly-diagnosed NPC patients were collected. EBV DNA was extracted by both techniques (n=23) followed by quantitative real-time polymerase chain reaction (PCR) using the primer/probe set for BALF5. The results of the quantitative real-time PCR were reproducible in both groups. The two techniques were moderately correlated (r=0.67, P<0.05), and the degree of agreement was good. However, the mean EBV DNA level in the boiling group (3.02±8.67×10(6) copies/μL) was significantly higher than the extraction kit group (1.15±2.66×10(6) copies/μL) (P<0.05). The EBV DNA level was higher in patients at an advanced overall stage (P=0.05). The results of the present study showed that the performance of the extraction kit was not superior to the simple boiling technique for the detection of salivary EBV DNA in NPC patients using real-time PCR. The salivary EBV DNA level in patients at an advanced overall stage appeared to be higher than in patients at an early stage.

Prognostication of survival from nasopharyngeal carcinoma by reduction of plasma Epstein-Barr viral DNA load at midpoint of radiotherapy course: A new paradigm of prognostication.

6015 Background: To test the hypothesis that prognostication of treatment outcome is feasible by biomarker response at mid-course of chemoradiotherapy (CRT)/ radiotherapy (RT), with respect to the plasma load of Epstein-Barr viral (EBV) DNA in nasopharyngeal carcinoma (NPC). Methods: 107 patients with stage IIB-IV NPC were prospectively studied. Plasma EBV DNA load was measured by quantitative PCR before therapy (pre-DNA), at completion of 4 weeks of CRT/RT (mid-DNA), and within 3 months of completion of therapy (post-DNA). The endpoints are post-DNA load, a recognized surrogate of survival, and clinical outcome. Results: 93% of patients had detectable EBV DNA before therapy (median load = 972 copies/ml). EBV DNA became undetectable in 55 (51%) patients at the end of week 4 of therapy. Detectable mid-DNA was associated with worse clinical outcome (median follow-up time, 6.2 years), for distant failure (HR 12.02, 95% CI 2.78-51.93; P&lt;0.0001), progression-free survival (HR 4.05, 95% CI 1.89-8.67, P&lt;0.0001), and overall survival (HR 3.29, 95% CI 1.37-7.90, P=0.0077). About three-quarters of all failures were associated with detectable mid-DNA, whereas about one-third of all failures were associated with detectable post-DNA. Patients with detectable mid-DNA which disappeared after RT continued to sustain a less favourable prognosis than those with undetectable mid-DNA. Stratification by tumor stage (II, III, IV) has no significant prognostic effect. Conclusions: Unfavorable EBV DNA response at mid-course of radiotherapy/chemoradiotherapy is an adverse prognosticator for treatment outcome, is linked to majority of all failures, and discriminates outcome better than tumor stage. The data could provide a basis for trial design that addresses alteration of therapy intensity during the latter phase of chemoradiotherapy, and adjuvant therapy.

Investigation into the Origin and Tumoral Mass Correlation of Plasma Epstein–Barr Virus DNA in Nasopharyngeal Carcinoma

Despite the increasing number of clinical applications of circulating Epstein–Barr virus (EBV) DNA analysis for the detection (1)(2), monitoring(3)(4), and prognostication(5)(6) of nasopharyngeal carcinoma (NPC), several fundamental questions concerning plasma EBV DNA, including its tissue origin and relationship with tumor mass, remain unanswered. In the first part of this study, we investigated the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA. Previous studies have shown that different EBV genotypes are harbored by latently EBV-infected lymphoid tissues and blood cells (7)(8)(9)(10) in healthy persons and patients with malignancies. On the other hand, it is well established that the EBV is monoclonal in NPC tumor tissues and other EBV-associated cancers(11)(12)(13)(14). Therefore, investigation of the genotype of plasma EBV DNA should reveal the relative contributions of tumor cells and other latently EBV-infected lymphoid tissues to the pool of circulating EBV DNA in NPC patients. For this study, 25 patients with newly diagnosed NPC, 15 patients with systemic lupus erythematosus, and 13 renal transplant recipients were recruited and gave informed consent. This study was approved by the ethics committee of the Prince of Wales Hospital, Hong Kong. Plasma samples were collected from all patients, and tumor specimens for microdissection were obtained from 14 of the 25 NPC patients. DNA extracted from the plasma samples and the microdissected tumor tissues was used for the amplification of EBV DNA, targeting a region encoding the carboxy terminus of latent membrane protein (LMP-1), using the primers 5′-ATGGTAATGCCTAGAAGTAAAGAAAGG-3′ (LMP1f) and 5′-CATAGCCCTAGCGACTCTGCTG-5′ (R2b). The PCR products were ligated to the pGEM®-T Easy Vectors (Promega), and the ligation products were transformed into competent Escherichia coli cells. Twelve bacterial clones were …

Frequent Hypermethylation of RASSF1A, TSLC1, High Viral Load of Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma, Matched Tumor-Adjacent Tissues

We examined the promoter hypermethylation of tumorsuppressor genes RASSF1A, TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), matched tumor-adjacent tissues outside 0.5 cm (P tissues), outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A, TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A, TSLC1, quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%), P tissues (75%), but less frequently in Z tissues (46%). The average quantities of EBV DNA (copies/μg DNA) in matched T, P, Z tissues were 673,000, 90,000, 7000. The differences of promoter hypermethylation of RASSF1A, EBV viral load among T, P, Z tissues were statistically significant, with more frequent methylation, higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A, high EBV load might be important events in NPC pathogenesis, they may be useful molecular diagnostic markers for this cancer.

High frequency of common deletion (4981 bp) in mitochondrial DNA in nasopharyngeal carcinoma and its correlation with patient age and clinical stages

Mitochondrial DNA (mtDNA) has a high mutation rate due at least in part to a lack of protective histones and an inefficient DNA repair system. The most frequently change in mtDNA is the so-called Common Deletion (CD), which accumulates in patients with hetero-plasmic mtDNA mutations and in normal individuals during aging. In this study, wild type mtDNA (WT-mtDNA) and mitochondrial DNA with CD (CD-mtDNA) were quantitatively analyzed in different nasopharynx lesions. A novel type of CD-mtDNA (4981 bp) was de-tected significantly higher in nasopharyngeal carcinoma (NPC) (93%, 54/58) than in naso-pharyngitis (60%, 28/47) and the paired white blood cells (WBC) (26%, 8/31). The ratio of CD-mtDNA to WT-mtDNA in NPC (0.000625, median) was 10 times that in nasopharyngitis (0.000064, median) (P = 0.003), and was significantly higher than that in paired WBC (0.000000, median) (P = 0.000). The CD/WT-mtDNA ratio was 0.000564 (quartile range, 0.000184 - 0.000919) in late stage NPC, which was nearly 3 times the ratio in early stage NPC (0.000164, quartile range, 0.000042 - 0.000353) (P = 0.015, Mann-Whitney Test). In NPC patients with ages °› 48yrs (mean age), the ratio of CD-mtDNA to WT-mtDNA was 0.000625, which was nearly 10 times that in NPC patients with ages < 48yrs (0.000064) (P = 0.005, Mann-Whitney Test). This is the first quantitative study of CD-mtDNA mutations in NPC, which provides evidences that CD-mtDNA mutation might be involved in the devel-opment and progression of NPC.

Quantitative analysis of plasma cell-free Epstein-Barr virus DNA in nasopharyngeal carcinoma after salvage nasopharyngectomy: a prospective study.

The quantity of circulating cell-free Epstein-Barr virus (EBV) DNA in patients with nasopharyngeal carcinoma (NPC) managed by radiotherapy has prognostic relevance. We measured the copy number of EBV DNA in patients with early recurrent NPC before and after salvage nasopharyngectomy. Nasopharyngectomy with the maxillary swing approach was performed for 28 patients. Serum blood samples were taken prospectively before nasopharyngectomy and on postoperative day 7. Plasma cell-free EBV DNA copies were measured with a real-time quantitative polymerase chain reaction for the BamHI-W fragment of the EBV genome. Cell-free EBV DNA was detected in 17 patients before nasopharyngectomy. Surgical resection reduced the copy number of EBV DNA significantly (p = .016). Negative surgical margins achieved during nasopharyngectomy is associated with a zero EBV DNA copy postoperatively (p = .022). Cell-free EBV DNA was detected in 61% of patients with recurrent NPC, and its quantity postoperatively reflects whether the salvage nasopharyngectomy has achieved a negative surgical margin.

Quantitative analysis of Epstein-Barr virus DNA in plasma and serum: applications to tumor detection and monitoring.

The recent interest in cell-free tumor-derived DNA in the plasma and serum of cancer patients has opened up numerous diagnostic possibilities. One type of tumor-derived DNA that has been detected in plasma/serum is viral DNA. One example of circulating viral nucleic acid is Epstein-Barr virus (EBV) DNA, which has been found in the plasma and serum of patients with nasopharyngeal carcinoma (NPC), certain lymphomas, and gastric carcinoma. Quantitative analysis of circulating EBV DNA in NPC has demonstrated a positive correlation with disease stage and a strong relationship with clinical events, as well as being of prognostic importance. For EBV-associated lymphomas, quantitative EBV DNA analysis has also been found to correlate closely with clinical progress. It is expected that plasma/serum EBV DNA analysis will soon be incorporated into the routine investigative protocol of EBV-associated malignancies.

Prognostic implication of pretreatment plasma/serum concentration of Epstein-Barr virus DNA in nasopharyngeal carcinoma

Recently, much interest has been focused on the diagnostic application and biology of tumor-derived DNA in the plasma and serum of cancer patients. Such interest has resulted in the demonstration of Epstein-Barr virus (EBV) DNA in the plasma/serum of patients with nasopharyngeal carcinoma (NPC). Using quantitative real-time polymerase chain reaction (PCR) technology, circulating EBV DNA has been found not only to correlate with disease staging, but also to provide additional prognostic information. Following treatment, circulating EBV DNA analysis has been shown to provide useful information for the monitoring for tumor recurrence. In addition to NPC, it is envisaged that circulating DNA technology will find wide applications in the detection and monitoring of many other types of malignancies.

Transforming activity of nasopharyngeal carcinoma DNA detectable in mouse JB6 cells

A JB6 mouse epidermal recipient cell line has been used to detect nasopharyngeal carcinoma (NPC) DNA-associated transforming activity that is not detectable in the NIH 3T3 focus assay. NPC DNA showed both transforming activity and activity for transferring sensitivity to tumor-promoter-induced neoplastic transformation, assayed in 2 different variants of mouse JB6 cells. Comparison of DNAs from various NPC sources that did or did not harbor EBV DNA and that varied in degree of differentiation showed similar transforming activities and similar activities for transferring promotion sensitivity. Thus both a NPC DNA-associated promotion sensitivity and an oncogenic activity function independently of concurrent EBV gene expression.