Comparison of plasma Epstein-Barr virus (EBV) DNA levels and serum EBV immunoglobulin A/virus capsid antigen antibody titers in patients with nasopharyngeal carcinoma

BACKGROUND: Serologic measurement of antibodies to Epstein-Barr virus (EBV) immunoglobulin A/viral capsid antigen (IgA/VCA) and early antigen (IgA/EA) has been used widely to screen for nasopharyngeal carcinoma (NPC) in China. Recently, it was found that plasma EBV DNA concentration is an indicator for the staging and prognosis of patients with NPC. To determine whether there is a correlation between plasma EBV DNA levels and serum levels of IgA/VCA, the authors measured both in patients with NPC and in a control group. METHODS: Real-time polymerase chain reaction was used for quantitative analysis of plasma EBV DNA concentration, and enzyme-linked immunoadsorbent assay was used to measure EBV VCA/IgA in patients with primary NPC (n = 120 patients), locally recurrent NPC (n = 8 patients), and distant metastatic NPC (n = 21 patients) among 76 patients with NPC after the completion of radiotherapy, in 60 patients with NPC in clinical remission, in 38 patients with non-NPC tumors, and in 47 control individuals. RESULTS: The median plasma EBV DNA levels were 6200 copies/mL, 9200 copies/mL, and 2050 copies/mL in patients with primary, locally recurrent, and distant metastatic NPC, respectively, but declined to 0 copies/mL in patients with clinically remissive NPC, in patients who completed radiotherapy, in patients with non-NPC tumors, and in the control group. In contrast, EBV VCA/IgA titers and detection rates remained high in all NPC groups. Plasma EBV DNA levels were significantly higher in patients who had serum VCA/IgA titers > or = 1:640 (median, 83,450 copies/mL) compared with the levels in patients who had titers < or = 1:320 (median, 17,200 copies/mL). Patients with NPC who had advanced TNM stage (Stages III and IV; median, 8530 copies/mL) and T classification (T3 and T4 tumors; median, 8530 copies/mL) had significantly higher plasma EBV DNA levels compared with patients who had early TNM stage (Stages I and II; median, 930 copies/mL) and T classification (T1 and T2 tumors; median, 3700 copies). Patients who had advanced TNM stage NPC had significantly higher mean VCA/IgA titers (1:424) compared with patients who had early TNM stage NPC (1:246), but there was no correlation between IgA/VCA titer and T or N classification of NPC. CONCLUSIONS: The results suggest that plasma EBV DNA detection is a more sensitive and specific marker than the serum IgA/VCA titer for the diagnosis and monitoring of patients with NPC. These findings provide convincing evidence for the use of plasma EBV DNA measurements for the early diagnosis and staging of NPC as well as for monitoring recurrence and metastasis of this tumor.     

血浆 EB病毒游离 DNA检测对监测鼻咽癌患者预后的意义

背景与目的:有报道 , 测定血浆中的 EB病毒游离 DNA( EBV-DNA)的拷贝数可作为诊断及监测鼻咽癌患者病情变化的手段之一.本研究旨在评价血浆 EBV-DNA检测在鼻咽癌患者预后监测上的价值, 并进一步与 VCA/IgA、 EA/IgA进行比较.方法:比较鼻咽癌放疗后 30例远处转移患者、 22例局部复发患者、 24例无 瘤生存者血浆中 EBV-DNA、 VCA/IgA、 EA/IgA水平.分别应用荧光定量 PCR方法检测血浆 EBV-DNA水平,免疫酶法检测 VCA/IgA、 EA/IgA;前瞻性观察 20例初诊鼻咽癌患者放疗前、放疗剂量达 40 Gy时及放疗结束时上述指标的变化. 结果:放疗后各组不同预后患者的血浆 EBV-DNA含量的中位数有显著性差异, 远处转移组为 135 100 copies/ml(四分线区域 5 525～ 1 003 750 copies/ml) >局部复发组的 20 500(四分线区域 0～ 58 500 copies/ml) > 无瘤生存组的 0 copy/ml(四分线区域 0～ 0 copy/ml), P均 < 0.05. 远处转移组的血浆 EBV-DNA水平高者较多, 当阳性标准为 1 000 000 copies/ml时,诊断远处转移组的敏感性为 27.3%,而诊断局部复发组的敏感性为 0.0%,特异性均为 100.0%.在初诊患者放疗前、放疗剂量达 40 Gy时及放疗结束时, EBV-DNA水平逐渐降低,平均含量分别为 32 050 copies/ml(四分线区域 3 880～ 317 750 copies/ml)、 0 copy/ml(四分线区域 0～ 14 375 copies/ml)、 0 copy/ml(四分线区域 0～ 2 940 copies/ml), P均 < 0.05, 而 VCA/IgA、 EA/IgA的水平未见明显变化. 结论: 血浆 EBV-DNA检测可用于监测鼻咽癌患者预后,其价值明显优于 VCA/IgA、 EA/IgA.     

Comparison of clinical and molecular surveillance in patients with advanced nasopharyngeal carcinoma after primary therapy: the potential role of quantitative analysis of circulating Epstein-Barr virus DNA

BACKGROUND: The importance of detecting recurrence at an early stage in patients with malignant disease is well recognized. Circulating Epstein-Barr virus (EBV) DNA can be detected in patients with nasopharyngeal carcinoma (NPC). The objective of the current study was to assess the effectiveness of plasma EBV DNA monitoring in the early detection of NPC recurrence compared with conventional methods. METHODS: Patients with NPC in two prospective clinical trials who had locoregional recurrences or distant metastases were recruited into the study. Clinical data on these patients were scrutinized for evidence of recurrence. EBV DNA copy numbers in the prospectively collected plasma samples were assayed retrospectively with real-time quantitative polymerase chain reaction analysis. RESULTS: At the time of clinical recurrence, 65% of 26 patients with locoregional recurrences and all but 1 of 28 patients with distant failure had circulating EBV DNA. The difference between the time from completion of treatment to positivity for circulating EBV DNA and the time from completion of treatment to the first observed clinical abnormality was not statistically significant for patients with local recurrence (P=0.84). However, the time to the first detection of circulating EBV DNA was significantly shorter among patients with distant metastases (P<0.0001). The Kaplan-Meier estimated median time to the emergence of plasma EBV DNA was 190 days, with a 95% confidence interval (CI) of 95-300 days, and the median time to the first observed clinical abnormality was 295 days (95% CI, 276-361 days). CONCLUSIONS: Monitoring plasma EBV DNA levels surpassed traditional methods for the early detection of distant failure in patients with NPC. The role of this technique should be evaluated in prospective studies that incorporate complementary advanced imaging technology.     

鼻咽癌患者血浆游离EBV/DNA的定量检测及其临床意义

目的:探讨血浆EBV/DNA定量分析,在鼻咽癌早期诊断、临床分期、预后判断和监测放疗后转移复发中的临床意义.方法:采用荧光定量PCR方法定量检测经病理确诊为鼻咽癌的120例初治、90例放疗后随诊患者,其中包括60例放疗后持续缓解,30例远处转移和局部复发患者的血浆EBV/DNA含量.结果:初治、远处转移和局部复发的鼻咽癌患者血浆中游离的EBV/DNA检出率分别为96.0%、95.0%和100%,显著高于治疗后持续缓解鼻咽癌患者、健康对照者和非鼻咽癌的肿瘤患者;初治鼻咽癌患者各TNM分期之间血浆EBV/DNA拷贝数有显著统计学差异,晚期患者(Ⅲ Ⅳ)期血浆EBV/DNA中位拷贝数显著高于早期患者(I Ⅱ)期;初治患者治疗后已出现局部和远处转移者.治疗前血浆EBV/DNA中位数显著高于尚未出现复发转移患者:初治患者治疗前血浆EBV/DNA≥40 000拷贝/ml与＜40 000拷贝/ml两个水平,患者22个月无复发生存率分别为46.1%和92.9%,有显著统计学差异;放疗后复发、转移鼻咽癌患者血浆EBV/DNA的中位拷贝数显著高于治疗后持续缓解患者.结论:采用荧光定量PCR方法检测鼻咽癌患者血浆中游离的EBV/DNA是一种敏感可靠的方法,对于鼻咽癌早期诊断、鉴别诊断、分期、判断预后、监测治疗后复发和远处转移具有重要的临床意义,有可能成为鼻咽癌的血清肿瘤标记物.     

Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma

Using real-time quantitative PCR, cell-free EBV DNA was detectable in the plasma of 96% (55 of 57) of nasopharyngeal carcinoma (NPC) patients (median concentration, 21058 copies/ml) and 7% (3 of 43) of controls (median concentration, 0 copies/ml). Advanced-stage NPC patients had higher plasma EBV DNA levels than those with early-stage disease. At 1 month after completion of radiotherapy, plasma EBV DNA was undetectable in 7 of 15 subjects (47%) but remained high in the remaining 8 subjects (53%). Clinical examination revealed that all of the former seven subjects had complete tumor regression, whereas six of the eight latter subjects exhibited evidence of disease persistence or had developed distant metastases. These results suggest that quantitative analysis of plasma EBV DNA may be a useful clinical and research tool in the screening and monitoring of NPC patients.     

Long-term prognostic effects of plasma epstein-barr virus DNA by minor groove binder-probe real-time quantitative PCR on nasopharyngeal carcinoma patients receiving concurrent chemoradiotherapy

PURPOSE: To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). METHODS AND MATERIALS: Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U), (2) high pretreatment EBV DNA (> or =1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. RESULTS: Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. CONCLUSION: Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT.     

Prospective validation of serum CYFRA 21-1, beta-2-microglobulin, and ferritin levels as prognostic markers in patients with nonmetastatic nasopharyngeal carcinoma undergoing radiotherapy

BACKGROUND: Patients with undifferentiated nasopharyngeal carcinoma (NPC) have elevated serum levels of CYFRA 21-1 (CYFRA), ferritin, and beta-2-microglobulin (beta2M) compared with healthy control individuals. The prognostic value of these markers has never been validated prospectively. METHODS: Paired serum samples from 160 patients with newly diagnosed, nonmetastatic, undifferentiated NPC were collected before radiotherapy (RT) and at 4-6 weeks after the completion of RT. Pre-RT and post-RT levels of serum CYFRA, ferritin, and beta2M were analyzed and correlated with overall survival (OS), progression-free survival (PFS), time to locoregional recurrence, (TLR) and time to distant recurrence (TDR). The results of pre-RT and post-RT plasma Epstein-Barr virus (EBV) DNA levels available from a previous study were included in a Cox regression model together with age, tumor (T) classification, and lymph node (N) classification. RESULTS: Sixty percent of patients had International Union Against Cancer Stage III-IV disease. At a median follow-up of 116 weeks (range, 37-239 weeks), 38 patients had disease progression. On multivariate analysis, pre-RT CYFRA and post-RT EBV DNA levels were independent predictors of poor OS, post-RT EBV DNA level and N classification predicted poor PFS and TDR; and only T classification predicted TLR. Patients who had pre-RT CYFRA levels > or = 1.5 U/mL were more likely to die (hazard ratio, 1.18; 95% confidence interval, 1.10-1.26) compared with patients who had pre-RT CYFRA levels < 1.5 U/mL. There were no associations between age, post-RT CYFRA levels and pre-RT or post-RT serum ferritin and beta2M levels, and the survival and recurrence rates on multivariate analysis. CONCLUSIONS: Serum CYFRA levels taken before RT predicted reduced survival in patients with nonmetastatic, undifferentiated NPC who underwent RT.     

血浆EB病毒DNA浓度预测鼻咽癌远处转移的研究

背景与目的：鼻咽癌治疗失败的主要原因之一是远处转移,近年多项放化疗综合治疗的临床研究未显示出明显的远处转移率的降低和远期生存的获益,综合治疗适宜个体及最佳模式尚未确立。血浆EB病毒DNA（EBV DNA）浓度是一项能反映鼻咽癌分期、治疗反应、预后的灵敏、特异的分子生物学指标。我们设计此前瞻性研究。探讨通过鼻咽癌患者血浆EBV DNA浓度预测远处转移的发生．为个体化的综合治疗模式的选择提供分子学指标。方法：69例初治鼻咽癌患者分别在治疗前和治疗结束时采用荧光定量PCR方法检测血浆EBV DNA浓度。所有患者按计划随访。进行远期疗效及生存的评价。Kaplan-Meier法计算无转移生存率及总生存率,多因素分析用Cox回归模型。结果：远处转移患者治疗前血浆EBV DNA中位浓度（27 000copies／m1）及治疗后EBVDNA检出率（55．56％）均高于持续缓解者（4 000 copies／ml,5．56％）及局部复发者（3 850 copies／ml,0％）（P值分别为0．039,0．001）。以治疗前20 000 copies／ml、治疗后0 copies／ml为界值点．EBV DNA低浓度患者的无复发生存率、无转移生存率及总生存率均高于高浓度患者,差异有显著性。Cox回归分析显示治疗前EBV DNA浓度（P=0．050,RR=3．95）、治疗后EBVDNA浓度（P=0．001,RR=11．74）均是影响无转移生存的危险因素。进一步将患者治疗前后EBVDNA浓度变化综合进行生存分析,结果表明治疗后EBV DNA浓度能否降到0是影响无转移生存最重要的预后因素（P=0．000）。结论：鼻咽癌患者治疗前、后血浆EBV DNA浓度,尤其是治疗后能否降到0．能预测远处转移的发生,有望为放化综合治疗筛选高危患者,指导放化结合模式的选择。     

Prognostic value of plasma Epstein–Barr virus DNA level during posttreatment follow-up in the patients with nasopharyngeal carcinoma having undergone intensity-modulated radiotherapy

Background: The value of Epstein–Barr virus (EBV) DNA assay during posttreatment follow-up of the patients with nasopharyngeal carcinoma (NPC) presenting with different pretreatment plasma EBV DNA levels remains unclear. In the present study, we aimed to evaluate the prognostic value of plasma EBV DNA assay during posttreatment follow-up in the patients with NPC who have undergone intensity-modulated radiotherapy. Methods: The medical records of 385 NPC patients treated with intensity-modulated radiotherapy between Novem-ber 2009 and February 2012 were reviewed. All patients underwent plasma EBV DNA assays before treatment, within 3 months after treatment, and then every 3–12 months during posttreatment follow-up period. The recurrence rates for patients with different pretreatment and posttreatment follow-up plasma EBV DNA levels were analyzed. Results: Of the 385 patients, 267 (69.4％) had detectable pretreatment plasma EBV DNA (> 0 copy/mL) and 93 (24.2％) had detectable posttreatment EBV DNA during a median follow-up of 52.8 months (range 9.3–73.8 months). Detectable EBV DNA during posttreatment follow-up was found in 14.4％ (17/118) and 28.5％ (76/267) of patients with undetectable and detectable pretreatment EBV DNA, respectively, and was significantly associated with tumor recurrence in both patient groups. EBV DNA was detectable in 12.8％ (40/313) of patients who remained disease-free, 56.4％ (22/39) of patients with locoregional recurrence alone, and 93.9％ (31/33) of patients with distant metastasis as the first recurrence event (P < 0.001); 6.5％ (19/292) of patients with undetectable EBV DNA and 57.0％ (53/93) of patient with detectable EBV DNA during posttreatment follow-up experienced tumor recurrence. Compared with other cut-off values, the cut-off value of 0 copy/mL for EBV DNA during posttreatment follow-up had the highest area under the ROC curve (AUC) value (0.804, 95％ confidence interval 0.741–0.868) for predicting tumor recurrence (sensi-tivity, specificity, and accuracy: 73.6％, 87.2％, and 84.7％, respectively).Conclusion: Plasma EBV DNA level during posttreatment follow-up is a good marker for predicting distant metasta-sis but not locoregional recurrence in the patients with NPC irrespective of the pretreatment EBV DNA levels.     

Prognostic values of the integrated model incorporating the volume of metastatic regional cervical lymph node and pretreatment serum Epstein–Barr virus DNA copy number in predicting distant metastasis in patients with N1 nasopharyngeal carcinoma

Background: According to the 7th edition of the American Joint Committee on Cancer (AJCC) staging system, over 50% of patients with nasopharyngeal carcinoma (NPC) have N1 disease at initial diagnosis. However, patients with N1 NPC are relatively under-researched, and the metastasis risk of this group is not well-stratified. This study aimed to evaluate the prognostic values of gross tumor volume of metastatic regional lymph node (GTVnd) and pretreatment serum copy number of Epstein–Barr virus (EBV) DNA in predicting distant metastasis of patients with N1 NPC, and to develop an integrated prognostic model that incorporates GTVnd and EBV DNA copy number for this group of patients. Methods: The medical records of 787 newly diagnosed patients with nonmetastatic, histologically proven N1 NPC who were treated at Sun Yat-sen University Cancer Center between November 2009 and February 2012 were ana-lyzed. Computed tomography-derived GTVnd was measured using the summation-of-area technique. Blood sam-ples were collected before treatment to quantify plasma EBV DNA. The receiver operating characteristic (ROC) curve analysis was used to evaluate the cut-off point for GTVnd, and the area under the ROC curve was used to assess the predicted validity of GTVnd. The survival rates were assessed by Kaplan–Meier analysis, and the survival curves were compared using a log-rank test. Multivariate analysis was conducted using the Cox proportional hazard regression model. Results: The 5-year distant metastasis-free survival (DMFS) rates for patients with GTVnd > 18.9 vs. ≤ 18.9 mL were 82.2% vs. 93.2% (P < 0.001), and for patients with EBV DNA copy number > 4000 vs. ≤ 4000 copies/mL were 83.5% vs. 93.9% (P < 0.001). After adjusting for GTVnd, EBV DNA copy number, and T category in the Cox regression model, both GTVnd > 18.9 mL and EBV DNA copy number > 4000 copies/mL were significantly associated with poor prognosis(both P < 0.05). According to combination of GTVnd and EBV DNA copy number, all patients were divided into low-, moderate-, and high-risk groups, with the 5-year DMFS rates of 96.1, 87.4, and 73.8%, respectively (P < 0.001). Multi-variate analysis confirmed the prognostic value of this model for distant metastatic risk stratification (hazard ratio [HR], 4.17; 95% confidence interval [CI] 2.34–7.59; P < 0.001). Conclusions: GTVnd and serum EBV DNA copy number are independent prognostic factors for predicting distant metastasis in NPC patients with N1 disease. The prognostic model incorporating GTVnd and EBV DNA copy number may improve metastatic risk stratification for this group of patients.     

Phase II study of neoadjuvant carboplatin and paclitaxel followed by radiotherapy and concurrent cisplatin in patients with locoregionally advanced nasopharyngeal carcinoma: therapeutic monitoring with plasma Epstein-Barr virus DNA.

PURPOSE: To assess the efficacy of neoadjuvant paclitaxel and carboplatin (TC) followed by concurrent cisplatin and radiotherapy (RT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC) and to monitor treatment response with plasma Epstein-Barr virus (EBV) DNA. PATIENTS AND METHODS: Thirty-one patients with International Union Against Cancer stages III and IV undifferentiated NPC had two cycles of paclitaxel (70 mg/m2 on days 1, 8, and 15) and carboplatin (area under the curve 6 mg/mL/min on day 1) on a 3-weekly cycle, followed by 6 to 8 weeks of cisplatin (40 mg/m2 weekly) and RT at 66 Gy in 2-Gy fractions. Plasma EBV DNA was measured serially using the real-time quantitative polymerase chain reaction method. Results All patients completed planned treatment. Response to neoadjuvant TC was as follows: 12 patients (39%) achieved partial response (PR) and 18 achieved (58%) complete response (CR) in regional nodes; five patients (16%) achieved PR and no patients achieved CR in nasopharynx. At 6 weeks after RT, one patient (3%) achieved PR and 30 patients (97%) achieved CR in regional nodes, and 31 patients (100%) achieved CR in nasopharynx; 29 patients (93%) had EBV DNA level of less than 500 copies/mL. Neoadjuvant TC was well tolerated, and the most common acute toxicity of cisplatin plus RT was grade 3 mucositis (55%). At median follow-up of 33.7 months (range, 7 to 39.3 months), six distant and three locoregional failures occurred. Plasma EBV DNA level increased significantly in eight of nine patients who experienced treatment failure but did not increase in those who did not. The 2-year overall and progression-free survival rates were 91.8% and 78.5%, respectively. CONCLUSION This strategy was feasible and resulted in excellent local tumor control. Serial plasma EBV DNA provides a noninvasive method of monitoring response in NPC.     

Combining plasma Epstein-Barr virus DNA and nodal maximal standard uptake values of 18F-fluoro-2-deoxy-D-glucose positron emission tomography improved prognostic stratification to predict distant metastasis for locoregionally advanced nasopharyngeal carcinoma

Background

This study aimed to evaluate the value of combining the nodal maximal standard uptake values (SUVmax) of ¹⁸ F-fluoro-2-deoxy-D-glucose positron emission tomography with Epstein-Barr virus DNA(EBV DNA) levels to predict distant metastasis for nasopharyngeal carcinoma (NPC) patients

Patients and Methods

Eight hundred seventy-four patients with stage III-IVa-b NPC were evaluated for the effects of combining SUVmax and EBV DNA levels on distant metastasis-free survival (DMFS), disease-free survival (DFS) and overall survival (OS).

Results

The optimal cutoff value was 6,220 copies/mL for EBV DNA and 7.5 for SUVmax-N. Patients with lower EBV DNA levels or SUVmax-N had a significantly better 3-year DMFS, DFS, and OS. Patients were divided into four groups based on EBV DNA and SUVmax-N, as follows: low EBV DNA and low SUVmax-N (LL), low EBV DNA and high SUVmax-N (LH), high EBV DNA and low SUVmax-N (HL), and high EBV DNA and high SUVmax-N (HH). There were significant differences between the four mentioned groups in 3-year DMFS: 95.7%, 92.2%, 92.3%, and 80.1%, respectively (P^trend < 0.001). When looking at the disease stage, the 3-year DMFS in group LL, LH, HL, HH were 94.2%, 92.9%, 95.0%, and 81.1%, respectively, in stage III patients (P^trend < 0.001) and 92.7%, 87.2%, 86.3%, and 77.0% in stage IVa–b patients (P^trend = 0.026).

Conclusion

Pretreatment EBV DNA and SUVmax of neck lymph nodes were independent prognostic factors for distant metastasis in NPC patients. Combining EBV DNA and SUVmax-N led to an improved risk stratification for distant metastasis in advanced-stage disease.     

血浆EBV DNA水平测定在监测鼻咽癌放疗后复发、转移中的临床意义

目的 探讨定量分析血浆EBV DNA水平在监测鼻咽癌复发、转移中的临床意义。方法 应用荧光定量PCR技术（real-time PCR）,检测360例根治放疗后鼻咽癌患者血浆EBV DNA水平,并与临床及影像学检查结果 比较。结果 360例患者中87例血浆EBV DNA检测阳性,273例阴性。在87例检出阳性者中,25例证实为临床复发,45例证实为远处转移,故其预测肿瘤进展阳性预测值为80%（70/87）。在273例血浆EBV DNA检测阴性患者中,共有17例复发和4例转移,故其阴性预测值为92%（252/273）。总计91例肿瘤进展患者中70例血浆EBV DNA阳性,其中复发42例,25例血浆EBV DNA阳性;转移49例,45例血浆EBVDNA阳性。269例临床缓解患者中17例血浆EBV DNA阳性,故其敏感性、特异性、假阳性率和假阴性率分别为77%（70/91）、94%（252/269）、6%（17/269）、23%（21/91）、90%（322/360）。复发患者、转移患者、肿瘤进展患者和临床缓解患者EBVDNA阳性率和中位拷贝数分别为：60%,4700 copies/ml;92%,425000 copies/ml;77%,38000 copies/ml;6%,〈500copies/ml。复发患者与转移患者比较、肿瘤进展患者与临床进展患者比较,血浆EBV DNA阳性率和中位拷贝数均有显著性差异。结论 血浆EBV DNA水平的检测是监测放疗后鼻咽癌患者转移、复发的有效指标。     

鼻咽癌患者血浆EBV DNA、血清CYFRA21-1和VCA-IgA的检测及临床应用

目的：探讨血浆EB病毒DNA、血清细胞角蛋白19片段（CYFRA21-1）和EB病毒壳抗原IgA（VCA-IgA）三种肿瘤标记物对鼻咽癌诊断、疗效监测和预后判断的临床应用价值。方法：分别采用荧光定量PCR法、电化学发光法和免疫酶法检测62例鼻咽癌患者治疗前、后及62例健康对照者血浆EBV DNA、血清CY-FRA21-1和VCA-IgA含量,并进行对比分析。结果：治疗前血浆EBV DNA、血清CYFRA21-1和VCA-IgA的阳性率明显高于健康对照组（P〈0.01）;治疗后血浆EBV DNA、血清CYFRA21-1和VCA-IgA含量高者,在随访中发现肿瘤残存、复发或远处转移。结论：血浆EBV DNA、血清CYFRA21-1和VCA-IgA的检测对鼻咽癌早期诊断、高危人群筛查、以疗效监测和预后判断有重要临床应用价值,三种肿瘤标记物联合检测有互补作用。     

Disparity of sensitivities in detection of radiation-na?ve and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2.

PURPOSE: The purpose of this research was to compare the sensitivities of plasma EBV DNA in detection of postirradiation locally recurrent nasopharyngeal carcinoma (NPC), postirradiation distant metastatic NPC, and radiation-na?ve NPC. EXPERIMENTAL DESIGN: Twenty-four patients with postirradiation local recurrence of NPC were assessed for plasma EBV DNA levels by a real-time quantitative PCR system. The results were compared with those of a cohort of 140 patients with newly diagnosed NPC and with those of 25 patients with distant metastatic relapse. EBV-encoded RNA positivity was also assessed in locally recurrent tumors and newly diagnosed tumors with undetectable plasma EBV DNA levels. RESULTS: Postirradiation locally recurrent tumors were associated with a significantly lower rate of detectable plasma EBV DNA compared with radiation-na?ve tumors of comparable stage [stage I-II tumors: 5 of 12 (42%) versus 47 of 51 (92%), P = 0.0002; stage III-IV tumors: 10 of 12 (83%) versus 88 of 89 (99%), P = 0.01; Fisher's exact test], and compared with distant metastatic recurrences [15 of 24 (63%) versus 24 of 25 (96%), P < 0.02; Fisher's exact test]. The median EBV DNA level in patients with detectable EBV DNA was also significantly lower in locally recurrent tumors than in radiation-na?ve tumors. All of the tissue samples of tumors associated with undetectable EBV DNA levels, where available, were EBV-encoded RNA positive. CONCLUSIONS: The sensitivity of EBV DNA in the detection of tumors regrowing from an irradiated site is much lower than that from a radiation-na?ve site. Although plasma EBV DNA is very effective in detecting distant metastatic relapse of NPC, it cannot be relied on as the sole surveillance tool for detection of local relapse.     

Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma.

Recently, cell-free EBV DNA has been detected in the plasma and serum of patients with nasopharyngeal carcinoma (NPC). We studied the relationship between plasma/serum EBV DNA and tumor recurrence. Using real-time quantitative PCR, the median plasma EBV DNA concentration in 10 patients with tumor recurrence was determined to be 32,350 copies/ml, whereas that in 15 patients in continuous remission for a mean period of 2 years was 0 copy/ml. Longitudinal follow-up of 17 NPC patients revealed 6 individuals with tumor recurrence and 11 patients who remained in remission. Significant elevations in serum EBV DNA, sometimes up to 6 months before detectable clinical deterioration, were observed in the patients who subsequently developed tumor recurrence. Continuously low or undetectable levels of serum EBV DNA were observed in the patients who remained in remission. These results suggest that plasma/serum cell-free EBV DNA may be a valuable tool for the monitoring of NPC patients for the early detection of tumor recurrence.     

Quantitative analysis of the transrenal excretion of circulating EBV DNA in nasopharyngeal carcinoma patients

PURPOSE: The existence of transrenal clearance of circulating cell-free DNA is controversial. In this study, we used NPC as a model to investigate if circulating EBV DNA can be excreted into urine and to quantify the contribution of renal excretion to the clearance of plasma EBV DNA. EXPERIMENTAL DESIGN: Quantitative analysis of urine EBV DNA was done for 74 NPC patients using real-time PCR with two different amplicon sizes. The urine concentration of EBV DNA was expressed as copies per millimole of creatinine (copies/mmol Cr) to minimize the effects of interindividual variations in hydration status. RESULTS: EBV DNA was detectable in the urine of 56% NPC patients using a 59-bp real-time PCR assay. The median urine EBV DNA concentrations measured by the 59- and 76-bp assays were 7,040 and 290 copies/mmol Cr, respectively. Patients with detectable urine EBV DNA had significantly higher plasma concentrations, with a positive correlation between the plasma and urine concentrations of EBV DNA. The fraction of plasma EBV DNA excreted into the urine was 0.0026% of that for creatinine. CONCLUSIONS: We have shown that circulating EBV DNA can be excreted transrenally into urine in NPC patient and the fraction of excretion is negatively associated with the size of the DNA molecules. Because there is a positive correlation between plasma and urine EBV DNA concentration, urine EBV DNA analysis may potentially be applicable as an ultra-noninvasive test for the monitoring and prognostication of NPC patients.     

Significance of cell-free epstein-barr virus DNA in monitoring prognosis of nasopharyngeal carcinoma

Objective It has been reported that cell-free Epstein-Barr virus (EBV-DNA) in plasma was useful in diagnosing and monitoring nasopharyngeal carcinoma (NPC). The current study was designed to evaluate the significance of EBV-DNA in monitoring the prognosis of nasopharyngeal carcinoma and comparing its significance with that of plasma VCA/lgA and EA/lgA levels. Methods E8V -DNA, VCA/lgA, and EA/lgA levels in plasma were determined in NPC patients with different prognosis after radiotherapy, including 30 distant metastatic patients, 22 local recurrence patients and 24 individuals with remission who had been followed-up for more than 2 years after treatment. EBV-DNA was determined using a real-time quantitative PCR system, and levels of VCA/lgA and EA/lgA were measured using standard immunofluorescence. In a cohort study, the indexes were determined after different radiation periods for the 20 new cases of nasopharyngeal carcinoma. Results The median plasma EBV-DNA concentration was 135,100 copies/ ml (interquartile range: 5,525-1,003 750) in metastatic group, 20,500 copies/ ml (interquartile range: 0 -58,500) in the local recurrence group and 0 copies/ml (interquartile range: 0-0) in the continuous remission group (P< 0.05). The levels of VCA/lgA and EA/lgA showed no significant differences among the different groups. The high level of EBV-DNA concentration in the metastatic group was more than that in the local recurrence group. A level of 1,000,000 copies/ml of EBV DNA was an indication of distant metastasis of the NPC patients with a sensitivity of 27.3%. However, the sensitivity was 0 in the local recurrence group. For the 20 new patients, EBV -DNA concentration gradually decreased during the radiation period. Before radiation there were 32,050 copies/ml (interquartile range: 3,880-317,750), 0 copies/ml (interquartile range: 0-14 375) after a 40 Gy radiation dose and 0 copies/ml (interquartile range: 0-2940) after the radiation was finished (P< 0.05). However, the levels of VCA/lgA and EA/lgA showed no significant difference. Conclusion Determination of plasma cell -free EBV -DNA level is more valuable than evaluation of VCA/lgA and EA/lgA for monitoring the prognosis of NPC patients.     

Spontaneous remission of residual post-therapy plasma Epstein–Barr virus DNA and its prognostic implication in nasopharyngeal carcinoma: A large-scale,big-data intelligence platform-based analysis

Detectable post-therapy plasma Epstein–Barr virus (EBV) DNA predicts poor survival in non-metastatic nasopharyngeal carcinoma (NPC). However, some patients subsequently experience spontaneous remission of residual EBV DNA during follow-up and it was unclear whether these patients were still at high risk of disease failure. Using the NPC database from an established big-data intelligence platform, 3269 NPC patients who had the plasma EBV DNA load measured at the end of therapy (± 1 week) were identified. In total, 93.0% (3031/3269) and 7.0% (238/3269) of patients had undetectable and detectable (> 0 copy/ml) plasma EBV DNA at the end of therapy (EBV DNA_end), respectively. Detectable EBV DNA_end was a prognostic factor for poorer 3-year disease-free survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS), and loco-regional recurrence-free survival (LRRFS) in both univariate and multivariate analyses. Of 238 patients with residual EBV DNA_end, 192 underwent EBV DNA assay 3 months after and s pontaneous remission occurred in 72.4% (139/192). However, these patients still had poorer 3-year DFS (55.1% vs. 89.8%), OS (79.1% vs. 96.2%), DMFS (68.4% vs. 94.1%) and LRRFS (84.5% vs. 95.0%) than patients with undetectable EBV DNA_end (all p < 0.001). And patients with persistent detectable post-therapy EBV DNA had the worst outcomes. These results were confirmed in multivariate analysis. In conclusion, residual EBV DNA post therapy was a robust biomarker for NPC prognosis. Although residual post-therapy EBV DNA could spontaneous remit during follow-up, these patients were still at high risk of disease failure and such patients may benefit from adjuvant therapy.     

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

BackgroundTo 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).Patients and methodsOne 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.ResultsNinety-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.ConclusionsUnfavorable 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.