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.     

Serologic diagnosis of nasopharyngeal carcinoma. A double-blind study of four EB virus antibodies with evaluation by sequential discrimination

It is generally known that a close relationship exists between Epstein-Barr Virus (EBV) and nasopharyngeal carcinoma (NPC). Recently, patients with early lesions of NPC have been detected in the general population by use of serologic mass survey. Using the double-blind method, we have studied the diagnostic value of the four EBV antibody titers, VCA-IgA, VCA-IgG, EA-IgA and EA-IgG, in four groups of subjects, each consisting of 50 persons: patients with nasopharyngeal carcinoma (NPC group), patients with cancers other than NPC in the head and neck regions (HNC group), patients with cancers outside of head and neck regions (OC group) and normal individuals (NS group). The results of these four antibodies were evaluated both singularly and together by multivariate sequential discrimination. Taking 1:10 as the criterion of being positive, in the NPC group, the positive rate of VCA-IgA is 88%, the VCA-IgG rate is 100%, the EA-IgA rate is 48% and the EA-IgG rate is 74%. In the non-NPC group, the positive rates of VCA-IgA are as high as 86%-92%, but those of the other antibodies are as low as 0-42%. The positive rates and the geometric mean titers of these four antibodies were all elevated as compared with those in the three non-NPC groups. These differences are statistically significant. VCA-IgG is unimportant in the diagnosis of NPC because of its low specificity. By treating the antibody titers of VCA-IgA, VCA-IgG, EA-IgA and EA-IgG with sequential discrimination, the correlation rate between the serology and pathology of NPC is 88% and the false positive rate is 7.3%.     

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.     

Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma

This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in endemic areas.     

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。     

Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma

BACKGROUND: Epstein-Barr virus (EBV) DNA can be detected and quantified in the plasma of patients with EBV-related tumors, such as nasopharyngeal carcinoma (NPC). Although NPC at early stages can be cured by radical radiotherapy, there is a high recurrence rate in patients with advanced NPC. The pretreatment level of circulating EBV DNA is a prognostic factor for NPC, but the prognostic value of post-treatment EBV DNA has not been studied. We designed a prospective study in Hong Kong, China, to investigate the value of plasma EBV DNA as a prognostic factor for NPC. METHODS: One hundred seventy NPC patients, without metastatic disease at presentation, were treated with a uniform radiotherapy protocol. Circulating EBV DNA was measured by real-time quantitative polymerase chain reaction before treatment and 6-8 weeks after radiotherapy was completed. Risk ratios (RRs) were determined with a Cox regression model, and associations of various factors with progression-free and overall survival and recurrence rates were determined with a stepwise Cox proportional hazards model. All statistical tests were two-sided. RESULTS: Ninety-nine percent of patients achieved complete clinical remission. Levels of post-treatment EBV DNA dominated the effect of levels of pretreatment EBV DNA for progression-free survival. The RR for NPC recurrence was 11.9 (95% confidence interval [CI] = 5.53 to 25.43) for patients with higher post-treatment EBV DNA and 2.5 (95% CI = 1.14 to 5.70) for patients with higher pretreatment EBV DNA. Higher levels of post-treatment EBV DNA were statistically significantly associated with overall survival (P<.001; RR for NPC recurrence = 8.6, 95% CI = 3.69 to 19.97). The positive and negative predictive values for NPC recurrence for a higher level of post-treatment EBV DNA were 87% (95% CI = 58% to 98%) and 83% (95% CI = 76% to 89%), respectively. CONCLUSION: Levels of post-treatment plasma EBV DNA in patients with NPC appear to strongly predict progression-free and overall survival and to accurately reflect the post-treatment residual tumor load.     

EB病毒感染与早期鼻型结外NK/T细胞淋巴瘤患者疗效及预后的关系

目的 探讨早期结外鼻型NK/T细胞淋巴瘤（ENKTCL）患者治疗前EBV DNA载量、治疗前血清EA-IgA及VCA-IgA抗体水平与临床特征、治疗反应及预后的关系。方法 分析78例早期结外鼻型NK/T细胞淋巴瘤患者的临床特征及影响预后的因素。结果 治疗前EBV DNA、VCA-IgA、EA-IgA阳性率分别为43.6%、20.5%、14.1%。EBV DNA与Ann Arbor分期、原发部位、PTI、治疗后未获得CR显著相关（均P<0.05）。VCA-IgA、EA-IgA滴度分别与EBV DNA、治疗后未获得CR显著相关（均P<0.05）。多因素分析发现年龄、EBV DNA、治疗后未获得CR为早期ENKTCL患者OS的独立预后因素（均P<0.05）;年龄、EBV DNA、原发鼻腔外上呼吸消化道、治疗后未获得CR（均P<0.05）则为早期ENKTCL患者PFS的独立预后因素。结论 治疗前EBV DNA阳性与较晚的Ann Arbor分期、PTI、原发鼻腔外上呼吸消化道、治疗反应差有关。EA-IgA、VCA-IgA水平升高与EBVDNA阳性、治疗反应差有关。治疗前EBV DNA可用于ENKTCL的风险分层及预后预测,而EA-IgA、VCA-IgA对于ENKTCL的预后指导作用有限。     

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.     

A comparison study of different PCR assays in measuring circulating plasma epstein-barr virus DNA levels in patients with nasopharyngeal carcinoma.

PURPOSE: To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance. EXPERIMENTAL DESIGN: Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables. RESULTS: Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002). CONCLUSIONS: The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.     

鼻咽癌HSP70表达与血清EB病毒IgA/VCA的关系及其临床意义

背景与目的:热休克蛋白(HSP)70在许多恶性肿瘤中均有表达,但在鼻咽癌组织中的表达与血清IgA/VCA滴度及预后的关系尚不清楚。本研究旨在检测鼻咽癌组织中HSP70的表达和含量水平,探讨HSP70表达与鼻咽癌患者血清EB病毒IgA/VCA滴度及预后的关系。方法:采用SP免疫组化法检测38例鼻咽癌组织中HSP70表达,ELISA法检测38例鼻咽癌组织中HSP70含量,免疫酶标法检测38例鼻咽癌患者血清IgA/VCA滴度。结果:HSP70在38例鼻咽癌组织中表达率为60.5%。在不同性别、年龄、T分期、N分期和临床分期鼻咽癌组织中HSP70表达率均无显著性差异(P>0.05)。HSP70表达和含量与血清EB病毒抗体IgA/VCA滴度呈正相关(P=0.001)。HSP70阳性组和阴性组患者的5年生存率分别为65.2%和80.0%,5年无瘤生存率分别为40.0%和78.6%(P=0.04)。结论:HSP70在临床Ⅱ、Ⅲ期鼻咽癌患者癌组织中的表达与患者的IgA/VCA滴度正相关,HSP70阳性患者常规治疗后预后较差。     

Prognostic value of pretreatment and recovery duration of cranial nerve palsy in nasopharyngeal carcinoma

ABSTRACT: BACKGROUND: The purpose of this study was to evaluate the prognostic value of cranial nerve (CN) palsy in nasopharyngeal carcinoma (NPC) patients. METHODS: A retrospective analysis was performed on CN involvement using medical records of 178 consecutive patients with histologically diagnosed, non-disseminated NPC. RESULTS: In 178 NPC patients with CN palsy, the 5-year survival rates were as follows: overall survival (OS), 61.0%; disease-specific survival (DSS), 69.6%; local relapse-free survival (LRFS), 75.2%; distant metastasis-free survival (DMFS), 73.4%; and disease-free survival (DFS), 55.3%. Significant differences were observed in the 5-year OS rates between patients with single and multiple CN palsy (69.8% vs. 54.3%; P = 0.033) and the OS rates between patients with different pretreatment durations (68.7% vs. 43.3%, P = 0.007). However, no significant differences were observed in OS, DSS, LRFS and DFS rates between patients with upper and lower CN palsy (P = 0.581, P = 0.792, P = 0.729 and P = 0.212, respectively). The results showed that recovery duration was an independent prognostic factor for OS (HR = 2.485; P < 0.001), DSS (HR = 2.065; P = 0.016), LRFS (HR = 3.051; P = 0.001) and DFS (HR = 2.440; P < 0.001). CONCLUSIONS: Recovery duration is an independent prognostic factor for NPC patients with CN palsy and is related to recurrence, which leads to poor survival. Recovery duration requires close surveillance and different treatment regimens.     

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.     

治疗前外周血血管内皮抑素浓度与局部晚期鼻咽癌治疗后远处转移的关系

背景与目的:远处转移是局部晚期鼻咽癌治疗失败的主要原因之一.有研究提示血清血管内皮抑素(endostatin)浓度与肿瘤的复发及转移等密切相关.本研究通过检测患者外周血血管内皮抑素浓度,探讨其与局部晚期鼻咽癌患者治疗后远处转移的关系.方法:用酶联免疫吸附法(ELISA)测定218例初治局部晚期('92分期Ⅲ121例、Ⅳa 97例)患者及20例健康人外周血血管内皮抑素的浓度,并分析外周血血管内皮抑素浓度与患者性别、T分期、N分期及治疗后远处转移及局部区域复发的关系.结果:局部晚期鼻咽癌的外周血血管内皮抑素浓度中位值显著高于健康对照组(P＜0.001).以187.50 μg/L为截点将218例患者分为两组,低浓度组(≤187.5μg/L)和高浓度组(＞187.5μg/L).经使用log-rank法双侧检验可见,两组2年无区域局部复发率、2年无进展生存率、2年总生存率差异无显著性(P＞0.05);两组2年无转移生存率差异有显著性(P=0.034).多因素分析显示:治疗前外周血血管内皮抑素浓度(95%可信区间,1.039～3.696,P=0.03)是局部晚期鼻咽癌治疗后远处转移的可能影响因素.结论:治疗前外周血血管内皮抑素浓度与局部晚期鼻咽癌治疗后远处转移有关,可作为局部晚期鼻咽癌治疗后远处转移的预测指标之一.     

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.     

The association between circulating tumor cells and Epstein-Barr virus activation in patients with nasopharyngeal carcinoma

Background: Circulating tumor cells (CTCs) and microemboli (CTM) are attracting increasing attention in medical biology and clinical practice. However, the clinical relevance of CTCs in nasopharyngeal carcinoma (NPC) has not yet been ascertained, and no study has focused on the influence of Epstein-Barr virus (EBV) status on CTCs in NPC patients. These issues were therefore examined. Methods: Peripheral blood samples were prospectively obtained from 33 NPC patients before treatment. CTCs and CTM were captured using the Isolation by Size of Epithelial Tumor (ISET) method. Immunohistochemistry on CK5/6 (cytokeratin5/6) and P63, as well as in situ hybridization of EBERs (EBV-encoded RNAs) were used to validate the harvested tumor cells. Results: Out of 33 NPC patients, CTCs were detected in 22 cases (66.7%), and CTM were observed in 2 cases (6.1%). CTCs were presented in all stages of NPC patients but had no association with the TNM stages (all P > 0.05). The presence of CTCs appeared to correlate with EBV activation status. Among the total NPC patients, the EBV VCA-IgA levels in CTC-positive cases were higher than that in CTC-negative cases (negative vs. positive: 3.88 vs. 4.86, P = 0.016). A similar result was observed in the patients without distant metastasis (negative vs. positive: 3.76 vs. 4.95, P = 0.009). When the number of CTCs was considered, CTC counts were found to correlate with EBV VCA-IgA (R = 0.382, P = 0.041) and EBV DNA load (R = 0.396, P = 0.033) for all NPC patients as well as NPC patients without distant metastases. Conclusions: These findings strongly suggested detectable CTCs/CTM in all stages of NPC patients and support a positive correlation between CTCs and EBV activation in NPC patients.     

内皮素受体A表达与鼻咽癌预后的关系

背景与目的:内皮素受体A(endothelin A receptor,ETAR)激活参与肿瘤的生长及转移,包括细胞增殖、细胞凋亡、血管生成与肿瘤转移等作用。本研究探讨鼻咽癌组织中ETAR的表达与临床特征及预后的关系。方法:采用ETAR单克隆抗体对153例鼻咽癌组织中ETAR的表达进行免疫组化SABC技术检测。结果:鼻咽癌ETAR的阳性率为73.9%。ETAR的表达与性别、年龄、T分期、N分期及临床TNM分期无明显相关性(P>0.05)。ETAR阴性组和阳性组的3年总生存率分别为87.5%和73.2%(P=0.029),3年无瘤生存率分别为80.0%和57.3%(P=0.009),3年无局部区域复发生存率分别为86.9%和80.2%(P=0.228),3年无远处转移生存率分别为89.9%和70.4%(P=0.012)。Cox模型多因素分析显示ETAR表达、性别、年龄、T分期和N分期是影响总生存、无瘤生存及远处转移的独立预后因素。结论:ETAR的表达与鼻咽癌患者的远处转移风险有关,其高表达提示患者预后不良。     

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.     

Titers of Epstein-Barr virus-related antibodies in nasopharyngeal carcinoma in Japan

It is thought that nonkeratinizing or undifferentiated squamous cell carcinoma in the nasopharynx (NPC) is intimately correlated with Epstein-Barr Virus (EBV). Twenty-one patients with NPC were followed in Kyoto University Hospital and 4 in Osaka Red Cross Hospital during the past 2 years from 1980 to 1981. These patients were classified histopathologically according to the WHO classification in 1978 and staged with the TNM classification in Union Internationale Contre le Cancer (UICC) in 1978. The incidence rate of NPC among the head and neck tumors was 5.6% in the authors' university from 1980 to 1981. The sex ratio of male to female was nearly equal. The mean age of NPC patients was 56.7 years. Sera from these 25 patients with nasopharyngeal carcinoma were collected at intervals of 3 to 8 months over a 2-year period, and were examined for their spectra and titers of antibodies of EBV-related antigens. They were titrated for IgG, IgA and IgM antibodies to EB viral capsid antigen (VCA), for IgG and IgA antibodies to early antigen-DR component (EA) and for antibodies to EBV-associated nuclear antigen (EBNA). All of these patients were primarily treated with radiation, while a few who did not respond to this therapy were subsequently treated with surgery or chemotherapy. EBV antibodies of VCA-IgG, -IgA, EA(DR)-IgG, and -IgA and EBNA were elevated in 73% and 90% of the nonkeratinizing and undifferentiated NPC patients, respectively. The VCA-IgM was elevated in almost none of the cases. In contrast to this, these values were all in a normal range in the NPC patients with keratinizing squamous cell carcinoma and malignant lymphoma. Also 9% and 10% of nonkeratinizing and undifferentiated carcinomas showed the normal ranges of EBV antibodies, possibly indicating a nonassociation with EBV. When NPC disappeared with radiation therapy, EBV antibodies became normal for 6-18 months. However, those whose NPC did not respond to the combined therapy with radiation, surgery and chemotherapy maintained high titers of EBV antibodies. The prognosis was the poorest in the patients with undifferentiated carcinoma, 40% of whom died within 4 years after diagnosis.     

EA-D p45-IgG as a Potential Biomarker for Nasopharyngeal Carcinoma Diagnosis

Aim: To identify new biomarkers for NPC diagnosis with an anti-EBV Western blot test kit. Methods:Serum samples from 64 NPC patients and healthy subjects with four specific VCA-IgA/EA-IgA profiles weretested with an anti-EBV Western blot test kit from EUROIMMUN AG. Proteins were quantified with scores ofintensity visually assigned to the protein bands. The markers which showed statistical differences between theNPC and non-NPC subjects were further evaluated in another 32 NPC patients and 32 controls in comparisonwith established biomarkers including VCA-IgA, EA-IgA, EBV-related protein IgG, and EBV DNA. Results:Among the markers screened, EA-D p45-IgG showed a statistically significant difference (p < 0.05) between NPCand non-NPC subjects with VCA-IgA positivy. In 32 VCA-IgA positive NPC patients and 32 control subjects,the diagnostic accuracy of EA-D p45-IgG was 78.1% with a positive predictive value of 77.8% and a negativepredictive value of 78.6%. In the verification experiment, the specificity and sensitivity of EA-D p45-IgG were75.0% and 90.6 %, respectively. Conclusions: EA-D p45-IgG might be a potential biomarker for NPC diagnosis,especially among VCA-IgA positive subjects.     

鼻咽癌放疗前CT或MRI检查分期与预后关系的比较

背景与目的:影像学检查结果是鼻咽癌临床分期和确定治疗靶区的重要依据.本研究试图分析放疗前按CT或MRI检查分期与鼻咽癌患者预后的关系.方法:445例病理确诊的无转移初治鼻咽癌患者.根据治疗前影像学检查手段的不同,分为CT组230例,MRI组215例.全组患者均行常规放疗,局部晚期患者加用化疗.用Kaplan-Meier和Log-rank法计算和比较两组生存率,用Cox模型分析影响预后的凶素.结果:CT组1、2、3年生存率分别为96.9%,90.3%,85.3%,MRI组分别为98.6%,94.3%,92.3%,两组比较差异有显著性(x2=6.305,P=0.012).CT组与MRI组的l,2,3年无瘤生存率和无复发生存率比较差异均有显著性(P<0.05),MRI组优于CT组.单因素分析结果显示临床分期、N分期、分期方法和化疗是影响鼻咽癌预后的因素,多因素分析结果显示临床分期和分期方法是影响鼻咽癌患者预后的独立因素.结论:鼻咽癌放疗前行MRI检查分期较CT检查分期来确定放疗靶区可提高鼻咽癌患者的生存率和局控率,临床分期和分期方法是影响鼻咽癌患者预后的独立因素.