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.     

鼻咽癌患者血浆EBVDNA水平和VCA—IgA检测的临床意义

目的：探讨鼻咽癌患者血浆EBVDNA水平和VCA—IgA联合检测对鼻咽癌早期诊断的临床价值。方法：用荧光定量PCR方法检测血浆EBVDNA水平，常规免疫酶法检测VCA—IgA抗体滴度。结果：68例鼻咽癌患者中，EBVDNA阳性率95．59％，中位拷贝数93× 10^4 copies／ml，VCA—IgA抗体阳性率92．65％，抗体滴度≥1：20；其他头颈肿瘤36例中，EBVDVA阳性率5．56％，中位拷贝数为0copies／ml，VCA—IgA抗体阳率36．11％，阳性滴度均≤1：20；健康对照组EBVDNA阳性率为35．56％，其滴度除3例≥1：40外，余均≤1：20。鼻咽癌患者中EBVDNA和VCA—IgA抗体阳性率显著高于对照组。结论：进-步证实EBV与鼻咽癌有密切关系；EBVDNA水平和VCA—IgA抗体滴度联合检测，有助于鼻咽癌的早期辅助诊断。     

The reliability of IgA antibody to Epstein-Barr virus (EBV) capsid antigen as a test for the diagnosis of nasopharyngeal carcinoma (NPC)

Since patients with nasopharyngeal carcinoma were first reported to have elevated levels of IgA antibody to Epstein-Barr virus (EBV) in their sera, workers in a number of countries have studied the possibility that this assay could be used in the diagnosis and monitoring of patients with this disease. In the United States, a collaborative project involving seven centers has been established to investigate the potential value of IgA antibody to EBV viral capsid antigen (VCA) as a clinical tool. In this report, we will summarize the results obtained from three studies: a comparison of EBV serology in three laboratories; a retrospective study of 37 nasopharyngeal carcinoma (NPC) patients and controls, and a prospective study of 126 NPC patients and 683 controls, including 149 patients with other malignancies involving the head and neck. The study of testing comparability in three laboratories demonstrated the feasibility of using this assay in a number of laboratories. The retrospective study confirmed the difference in IgA antibody titers between NPC patients and matched controls. The prospective study showed a relationship between IgA antibody titers and histopathology but not disease stage. IgA antibody titers were elevated more frequently in patients with nonkeratinizing or poorly differentiated types of NPC than for the well-differentiated squamous cell carcinomas. While IgA antibodies to EBV VCA appear to be of value in the early detection and diagnosis of NPC, it is possible that additional serologic tests for immunity to EBV, such as IgG antibody to VCA or early antigen (EA), will improve even further the clinical value of EBV serology in the management of NPC.     

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.     

Human nasopharyngeal carcinomas positive for Epstein-Barr virus DNA in North America

Nasopharyngeal carcinomas (NPC) from 2 black patients and 1 Caucasian patient were positive for Epstein-Barr virus (EBV) DNA. Of the tumors, 2 were lymphoepitheliomas (undifferentiated NPC) and 1 was a moderately differentiated NPC. All 3 patients had high IgG titers against EBV early antigen and high IgG and IgA titers against virus capsid antigen (VCA). In one patient, the levels of anti-VCA IgA were different than those of anti-VCA IgG over the course of the disease. Our data support the association of EBV and NPC in North America.     

Pretherapy quantitative measurement of circulating Epstein-Barr virus DNA is predictive of posttherapy distant failure in patients with early-stage nasopharyngeal carcinoma of undifferentiated type

BACKGROUND: Patients with International Union Against Cancer (UICC) Stage I-II nasopharyngeal carcinoma (NPC) appear to have a relatively favorable prognosis and generally are excluded from trials of combined modality treatment. More recently, plasma/serum cell-free Epstein-Barr virus (EBV) DNA has been shown to be measurable in the majority of NPC patients at the time of diagnosis, and appears to have prognostic significance. However, within Stage I-II disease, in which failure events are infrequent, the prognostic impact of the pretreatment EBV DNA level has not been addressed to our knowledge. This issue has management implications because different therapeutic strategies currently are employed for patients with good-risk and those with poor-risk NPC. METHODS: A cohort of 90 patients with UICC Stage I-II NPC (World Health Organization Grade 2/3 histology) had their pretherapy plasma/serum EBV DNA levels determined by a quantitative polymerase chain reaction assay and correlated with the probability of posttherapy failure. All patients received radiation therapy only, except for three patients who also received concurrent chemotherapy. Kaplan-Meier plots of the probability of locoregional failure, distant failure, and cancer-specific survival were compared with reference to clinical stage and EBV DNA levels. RESULTS: With a median follow-up time of 45 months, 12 patients and 7 patients, respectively, had developed locoregional and distant failures, including 2 patients with both local and distant failures. Patients with distant failure had significantly higher pretherapy EBV DNA levels than those without failure (a median of 13,219 copies/mL [interquatile-range, 274,635 copies/mL] vs. a median of 423 copies/mL [interquatile-range, 2753 copies/mL]). The probability of distant failure was significantly higher in patients with high (>4000 copies/mL plasma) compared with low EBV DNA levels (P=0.0001, log-rank test) and for Stage IIB disease compared with Stage I and Stage IIA disease combined (P=0.0149, log-rank test), but was not significantly different between patients with Stage II and those with Stage I disease. The risks of locoregional failure were not significantly different between patients with high and those with low EBV DNA levels, and also was not significantly different between clinical substages. Approximately 35% of patients with Stage IIB disease were in the at-risk group for distant failure, as identified by high EBV DNA levels. CONCLUSIONS: Within a group of patients with UICC Stage I-II NPC, the pretherapy plasma EBV DNA level was found to identify a poor-risk group with a probability of distant failure similar to that of patients with advanced stage disease. This group of patients may warrant management considerations currently applicable only to cases of Stage III-IV disease. The prognostic significance of designating Stage IIB disease as per the 1997 UICC staging was confirmed, although the pretherapy EBV DNA level appears to be a more powerful prognostic discriminator in patients with early-stage NPC.     

Laboratory markers of tumor burden in nasopharyngeal carcinoma: a comparison of viral load and serologic tests for Epstein-Barr virus

Epstein-Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real-time or end-point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non-NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end-point and real-time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.     

A comparison of EBV serology and serum cell-free DNA as screening tools for nasopharyngeal cancer: Results of the Singapore NPC screening cohort

We aimed to evaluate the effectiveness of nasopharyngeal cancer (NPC) screening by comprehensive clinical follow-up and adjunctive Epstein–Barr virus (EBV) testing. In a prospective cohort study, 524 individuals with a first-degree family history of NPC were recruited at a university clinical center in Singapore. The cohort was evaluated at baseline and at 6 monthly intervals, with a complete head and neck examination including nasopharyngeal endoscopy. Blood was taken at baseline and at yearly intervals for EBV Viral Capsid Antigen (VCA) IgA, EBV Early Antigen (EA) IgA serology and serum cell-free EBV DNA. Nasopharyngeal biopsy was performed when any irregularity in the nasopharynx was observed, or when EBV markers were elevated. The mean duration of follow-up was 57.7 months, with an average of 8.6 clinical visits per participant. Five participants (0.96%) were identified to have NPC, giving a prevalence of 199 per 100,000 person-years of screening. Four of the five NPC cases identified had asymptomatic T1 disease, at an earlier stage compared to NPC patients diagnosed in the clinic during the same time period (p = 0.0297). All NPC cases identified had elevated EBV-EA IgA titers ≥1:10, with a specificity of 94.6% and a positive predictive value of 15.2%, outperforming EBV-VCA IgA and serum EBV DNA. Two NPC cases were biopsied only because of elevated EBV serology titers, with increasing EBV-EA IgA titers preceding the diagnosis of NPC. In conclusion, screening for NPC is effective in identifying early-stage disease. Adjunctive EBV-EA IgA testing improved the effectiveness of screening.     

EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平在鼻咽癌高发区不同人群中的分布

目的 观察分析鼻咽癌高发区中的鼻咽癌患者、非鼻咽癌头颈部相似疾病患者和健康体检人群中EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA的抗体水平分布情况。方法 收集211例未经治疗的鼻咽癌患者、203例头颈部相似症状患者和210例健康体检者的血清,采用免疫酶法检测VCA/IgA,采用酶联免疫吸附法（ELISA）检测Rta/IgG和EBNA1/IgA。应用秩和检验、受试者工作特征(ROC)曲线、多分类logistic回归模型等方法对结果进行分析评价。结果 鼻咽癌组的VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平均显著高于头颈部相似疾病组和健康对照组(P<0.001)。头颈部相似疾病组的Rta/IgG及VCA/IgA抗体水平也明显高于健康对照组(P<0.001)。以头颈部相似疾病组和健康体检组为分析人群,分别作相关抗体的ROC曲线,VCA/IgA 的ROC曲线下面积为0.565,Rta/IgG抗体的ROC曲线下面积为0.604,具有统计学意义(P<0.05)。综合年龄、性别和3种EB病毒抗体等因素的多分类logistic回归分析显示,鼻咽癌、头颈部相似疾病和健康体检者的预测准确率分别为95.3％、70.9％和55.2％。结论 在鼻咽癌高发区EB病毒VCA/IgA及Rta/IgG抗体水平在头颈部相似疾病人群和健康人群中存在一定差异,在鼻咽癌的人群筛查和临床诊断中可根据具体情况设定不同的抗体阳性临界值。     

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.     

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.     

EBV 抗体和EBV-DNA 在鼻咽癌诊断及分期的研究*

目的：评估EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA对不同分期鼻咽癌的诊断效能,探讨各指标阳性率与鼻咽癌分期的关系。方法：收集2010年3 月至2015年9 月中山大学附属中山医院收治的初诊鼻咽癌患者152 例,健康体检者675 例。采用酶联免疫吸附法（ELISA）检测血清EBNA1/IgA 、Zta/IgA 和VCA/IgA 抗体ROD 值,荧光定量PCR （fluorescence quantitative PCR,FQ-PCR ）检测血浆EBV-DNA水平。比较单独和联合应用EBV 标记物对各期鼻咽癌的诊断效能,同时分析各指标阳性率与鼻咽癌分期的关系。结果：鼻咽癌患者EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA阳性率显著高于健康体检者（P < 0.01）。 EBNA1/IgA 在早期鼻咽癌表达相对较高,灵敏度为77.8% ,而EBV-DNA在晚期鼻咽癌的灵敏度最高为88.8% ,两者特异度均在96% 以上。联合检测中EBNA1/IgA 并联EBV-DNA检测的灵敏度为92.1%（早期为82.5% 、晚期为98.9%）,特异度为96.9% 。EBV-DNA阳性率与鼻咽癌临床分期和N 分期呈正相关,Zta/IgA 阳性率与N 分期呈正相关（P < 0.01）。 结论：在无症状人群中进行鼻咽癌筛查,单项指标首选EBNA1/IgA 。晚期患者的辅助诊断则推荐EBV-DNA。两者并联检测可进一步提高鼻咽癌诊断效能。EBV-DNA是鼻咽癌分期和病情监测的重要指标,Zta/IgA 可间接反映淋巴结转移情况,有望对患者病情评估起到参考作用。     

Significance of Plasma IgA and IgG Antibodies to Epstein-Barr Virus Early and Viral Capsid Antigens in Thai Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) is an important causal factor of human nasopharyngeal carcinoma (NPC). High levels ‍of serum IgA and IgG antibodies to EBV early and viral capsid antigens (IgA/EA, IgA/VCA, IgG/EA and IgG/VCA) ‍have been reported in NPC patients. Since specific serum IgA/EA, IgA/VCA and IgG/EA are claimed to be useful ‍serological markers for NPC. In order to evaluate whether plasma IgA/EA, IgA/VCA, IgG/EA and IgG/VCA antibody ‍levels are useful markers for diagnosis and prognosis of Thai NPC, we examined the prevalence of these antibodies ‍in 79 NPC patients, and 127 age-matched controls (47 healthy subjects (HS), 32 cases of other disease (OD) and 48 ‍cases of other cancer (OC)) by using an indirect immunofluorescence assay. The prevalence of plasma IgA/EA, IgA/ ‍VCA, and IgG/EA in NPC patients (55.7, 68.4 and 68.4%) was significantly higher than in the HS (0.0, 0.0 and ‍20.5%,), OD (0.0, 0.0 and 3.1%) and OC (0.0, 0.0 and 20.8%) groups (p<0.05). The prevalence of plasma IgG/VCA ‍in NPC patients (93.7%) was significantly different from those for the OD and OC groups (71.9 and 43.8%) but not ‍for the HS group (89.4%). In NPC patients, the geometric mean titers (GMT) of plasma IgA/EA, IgA/VCA and IgG/ ‍EA were increased with an advanced clinical stage of disease but not IgG/VCA. In contrast, GMT of IgG/VCA was ‍increased with aggressive type of disease (histological type) but not IgA/EA, IgA/VCA, and IgG/VCA. The results of ‍our study suggest that plasma IgA/EA, IgA/VCA and IgG/EA antibodies may be useful markers for diagnosis and ‍assessing prognosis of Thai NPC. ‍     

鼻咽癌患者血浆EB病毒DNA水平的动态变化与临床疗效的关系

背景与目的：鼻咽癌是一种与EB病毒相关的肿瘤。最近,一些学者报道了鼻咽癌患者血浆或血清中可检测到EB病毒DNA,本研究观察晚期鼻咽癌患者同期放化疗中血浆EB病毒DNA水平的动态变化以及与临床疗效的关系。方法：20例初治患者在同期放化疗期间每周抽血一次,共7次,每一次抽血时间为治疗前,所有的标本均采用荧光定量PCR的方法,在PE7700型检测仪上定量检测血浆标本中EB病毒DNA的含量。结果：90%（18/20）患者治疗前的血浆中可检测到EB病毒DNA,中位浓度为150000copies/ml,2例患者自治疗开始至结束血浆中均未检测到EB病毒DNA,18例阳性患者中,3例患者在治疗期间可检测到持续高水平的EB病毒DNA,治疗后临床结果显示三者均有肿瘤残留,7例患者EB病毒DNA水平在治疗开始后迅速下降至检测不到;8例患者血浆EB病毒DNA水平下降缓慢,但在治疗结束时,EB病毒DNA均检测不到。临床结果显示,此15例患者均达到完全缓解。结论：血浆EB病毒DNA水平的变化与鼻咽癌患者临床疗效关系密切,值得进一步研究。     

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.     

EBV DNA定量分析在监测鼻咽癌转移和复发中的临床意义

背景与目的:EB病毒(Epstein-Barrvirus,EBV)感染与鼻咽癌关系密切,近年来,有报道鼻咽癌患者血浆/血清中可检测到游离EBVDNA,但血浆EBVDNA水平对判断放疗后鼻咽癌患者转移、复发的临床意义尚缺少大宗研究报道。本研究定量检测鼻咽癌放疗后随诊患者血浆EBVDNA含量,探讨其在监测鼻咽癌转移、复发中的临床意义。方法:选择在中山大学肿瘤防治中心门诊随诊的放疗后鼻咽癌患者90例,用荧光定量PCR方法检测血浆EBVDNA含量,比较转移、复发与持续缓解患者血浆EBVDNA拷贝数。结果:放疗后转移或复发患者血浆EBVDNA的检出率为96.7%(29/30),中位拷贝数为2650copies/ml(0～5900000copies/ml);而持续缓解组患者血浆EBVDNA检出率12%(7/60),中位拷贝数为0copy/ml(0～71000copies/ml),差异均有统计学意义(P<0.01)。3例临床持续缓解但有血浆EBVDNA升高患者,在随后的3～4个月随访中,证实有肿瘤转移或复发。结论:血浆EBVDNA李宇红,等.EBVDNA定量分析在646定量检测可能成为监测放疗后鼻咽癌患者肿瘤转移、复发的敏感肿瘤标记物。     

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.     

Diagnostic Significance of Combined Detection of Epstein-Barr Virus Antibodies,VCA/IgA,EA/IgA,Rta/IgG and EBNA1/IgA for Nasopharyngeal Carcinoma

The objective of this study was to investigate the diagnostic significance of EBV antibody combined detectionfor nasopharyngeal carcinoma (NPC) in a high incidence region of southern China. Two hundred and elevenuntreated NPC patients, 203 non-NPC ENT patients, and 210 healthy controls were recruited for the study. Thetiters of VCA/IgA and EA/IgA were assessed by immunoenzyme assay, and the levels of Rta/IgG and EBNA1/IgAwere determined by enzyme-linked immunosorbent assay. The levels of VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA demonstrated no association with gender or age (p>0.05). The receiver operating characteristic curve andthe area under the curve were used to evaluate the diagnostic value. The sensitivity of VCA/IgA (98.1%) andthe specificity of EA/IgA (98.5%) were the highest. When a logistic regression model was used to combine theresults from multiple antibodies to increase the accuracy, the combination of VCA/IgA+Rta/IgG, whose areaunder the curve was 0.99, had the highest diagnostic efficiency, and its sensitivity, specificity and Youden indexwere 94.8%, 98.0% and 0.93 respectively. The data suggest that the combination of VCA/IgA+Rta/IgG may bemost suitable for NPC serodiagnosis.