鼻咽癌患者血浆EBV DNA水平的研究进展

EB病毒与鼻咽癌的发生有密切关系。近年来,随着PCR技术的发展,在鼻咽癌患者血浆中可定量检测EBVDNA水平。大量研究表明,鼻咽癌患者血浆EBVDNA的检出率及拷贝数明显高于正常人群。放疗后转移、复发患者其EBVDNA的阳性率及拷贝数高于持续缓解患者。血浆EBVDNA水平在鼻咽癌的早期诊断、临床分期、预后判断及监测治疗后转移、复发中均有重要临床意义。     

Screening nasopharyngeal carcinoma by detection of the latent membrane protein 1 (LMP-1) gene with nasopharyngeal swabs

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. The goals of the current study were to investigate whether a nasopharyngeal swab technique could provide enough DNA for polymerase chain reaction (PCR) analysis of the Epstein-Barr virus (EBV)-derived latent membrane protein 1 (LMP-1) gene and to determine the feasibility and reliability of diagnosing NPC by detection of LMP-1 in the nasopharynx. METHODS: 320 adults underwent nasopharyngoscopy and nasopharyngeal swab to obtain cells for the LMP-1 PCR assay; some patients also underwent nasopharyngeal biopsy. RESULTS: An amount of DNA that was sufficient for PCR was extracted from 96.3% of the swab samples. By detecting LMP-1 in nasopharyngeal swabs, NPC was diagnosed with a false positive rate of 12.7% (7 of 55 patients), a false negative rate of 1.6% (4 of 253 patients), sensitivity of 87.3% (48 of 55 patients), specificity of 98.4% (249 of 253 patients), a positive predictive value of 92.3% (48 of 52 patients), and a negative predictive value of 97.3% (249/256 patients). NPC was diagnosed by nasopharyngoscopy with a false positive rate of 38% (30 of 79 patients), a false negative rate of 0.4% (1 of 241 patients), sensitivity of 62% (49 of 79 patients), specificity of 99.6% (240 of 241 patients), a positive predictive value of 98% (49 of 50 patients), and a negative predictive value of 88.9% (240 of 270 patients). Only 7 (0.2%) of 256 patients with a diagnosis other than NPC had LMP-1 detected in the nasopharyngeal space. CONCLUSIONS: Detecting EBV genomic LMP-1 by nasopharyngeal swab diagnosed NPC with 87.3% sensitivity and 98.4% specificity. EBV genomic DNA usually is not detected by PCR-based methods in the nasopharyngeal space. Its incidence is estimated to be as low as 0.2% among the general population. The nasopharyngeal swab coupled with PCR-based EBV LMP-1 detection could serve as part of a screening program for high-risk populations.     

Complementary roles of MRI and endoscopic examination in the early detection of nasopharyngeal carcinoma

BackgroundEarly-stage nasopharyngeal carcinoma (NPC) evades detection when the primary tumor is hidden from view on endoscopic examination. Therefore, in a prospective study of subjects being screened for NPC using plasma Epstein–Barr virus (EBV) DNA, we conducted a study to investigate whether magnetic resonance imaging (MRI) could detect endoscopically occult NPC.Patients and methodsParticipants with persistently positive EBV DNA underwent endoscopic examination and biopsy when suspicious for NPC, followed by MRI blinded to the endoscopic findings. Participants with a negative endoscopic examination and positive MRI were recalled for biopsy or surveillance. Diagnostic performance was assessed by calculating sensitivity, specificity and accuracy, based on the histologic confirmation of NPC in the initial study or in a follow-up period of at least two years.ResultsEndoscopic examination and MRI were performed on 275 participants, 34 had NPC, 2 had other cancers and 239 without cancer were followed-up for a median of 36 months (24–60 months). Sensitivity, specificity and accuracy were 76.5%, 97.5% and 94.9%, respectively, for endoscopic examination and 91.2%, 97.5% and 96.7%, respectively, for MRI. NPC was detected only by endoscopic examination in 1/34 (2.9%) participants (a participant with stage I disease), and only by MRI in 6/34 (17.6%) participants (stage I = 4, II = 1, III = 1), two of whom had stage I disease and follow-up showing slow growth on MRI but no change on endoscopic examination for 36 months.ConclusionMRI has a complementary role to play in NPC detection and can enable the earlier detection of endoscopically occult NPC.     

Circulating EBV DNA as a tumor marker for nasopharyngeal carcinoma

The demonstration of Epstein-Barr virus (EBV) DNA in the plasma/serum of patients suffering from nasopharyngeal carcinoma (NPC) has provided us with a new tool for NPC detection and monitoring. The sensitivity and specificity of using circulating EBV DNA for the detection of NPC, with real-time polymerase chain reaction analysis, is 96 and 93%, respectively. EBV DNA level has been shown to be more powerful than existing staging system in predicting outcomes and it could also identify patients with emergent clinical relapse. It is, therefore, expected that this promising molecular tumor marker would soon be incorporated into routine clinical use.     

Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

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

BACKGROUND:

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

METHODS:

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

RESULTS:

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

CONCLUSIONS:

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

Detection of cell free Epstein-Barr virus DNA in sera from patients with nasopharyngeal carcinoma

BACKGROUND: The detection of tumor-derived DNA within the circulation of patients with malignant disease using polymerase chain reaction (PCR)-based strategies has opened a new avenue for the diagnosis and monitoring of these patients. Because of the universal association of Epstein-Barr virus (EBV) with the nonsquamous type of nasopharyngeal carcinoma (NPC; World Health Organization types II and III), the detection of cell free EBV DNA in sera from patients with NPC may be a valuable tool for monitoring the progress of tumors or to provide advanced warning of tumor recurrence. METHODS: Serum samples were obtained from different patients, and cell free EBV DNA was detected with a conventional PCR approach. A total of 134 patients were sampled, including 36 patients with primary NPC, 28 control patients, 18 patients suffering from locoregional recurrence, 7 patients with distant metastasis, and 45 patients with NPC in clinical remission. A conventional PCR approach employing standard 35-cycle and 50-cycle reactions was used to detect cell free EBV genomes. Results from the two PCR cycles were compared to provide a semiquantitative picture of the relative quantity of EBV genome in each serum sample. RESULTS: The EBV DNA detection rates, i.e., the rates of positive detection, for 35-cycle and 50-cycle PCR analyses, respectively, were 38.9% and 75% for patients with primary NPC, 3.5% and 10.7% for control patients, 27.8% and 88.9% for patients with locoregional disease recurrence, 71.4% and 100% for patients with distant metastasis, and 7.1% and 36.5% for patients with disease in clinical remission. The rates of positive detection among patients with active disease all appeared to be significantly greater compared with the rates among patients with disease in clinical remission. Longitudinal data for six patients with recurrent tumors revealed a close correlation between the relative quantity of circulating cell free EBV genomes and the disease course of these patients. The sensitivity, specificity, positive predictive value, and negative predictive value of the 50-cycle PCR analysis for detecting recurrent disease were 92%, 63.5%, 42.6%, and 96.4%, respectively. CONCLUSIONS: This study demonstrated that, by using a 50-cycle PCR-based approach, high sensitivity and high negative predictive value for detecting recurrent disease can be obtained from the detection of the cell free EBV genome in sera from patients with NPC. The 50-cycle PCR analysis, therefore, may provide a simple, clinically useful adjuvant method for monitoring patients with NPC.     

Plasma Epstein-Barr virus DNA level strongly predicts survival in metastatic/recurrent nasopharyngeal carcinoma treated with palliative chemotherapy

BACKGROUND:

Plasma Epstein‐Barr virus (EBV) DNA is widely used in screening, monitoring, and prediction of relapse in nonmetastatic nasopharyngeal carcinoma (NPC). However, data regarding utility of plasma EBV DNA in metastatic NPC are rare. The current study was to test the prognostic implication of plasma EBV DNA level in metastatic/recurrent NPC patients treated with palliative chemotherapy.

METHODS:

Plasma EBV DNA level was measured at baseline and thereafter at the start of each treatment cycle in 127 histologically proven metastatic/recurrent NPC patients treated with palliative chemotherapy. Correlations of pre‐treatment and post‐treatment plasma EBV DNA levels to survival and response were analyzed.

RESULTS:

Patients with a low pre‐treatment plasma EBV DNA level ( CONCLUSIONS: Plasma EBV DNA is of predictive value for prognosis in metastatic/recurrent NPC patients undergoing palliative chemotherapy. The pre‐treatment plasma EBV DNA level as well as the early decrease of plasma EBV DNA after chemotherapy enabled easy and early discrimination between patients who will and those who will not benefit from continued treatment. Cancer 2011. © 2011 American Cancer Society.     

MRI联合CT诊断鼻咽癌颅底转移

目的:探讨MRI联合CT诊断鼻咽癌颅底转移的价值。方法:选取2006年8月至2013年8月经病理证实为鼻咽癌的患者50例,全部患者治疗前均行CT及MRI检查,分析鼻咽癌颅底转移的CT及MRI影像学表现,评价联合应用CT/MRI对鼻咽癌颅底转移诊断的价值。结果:CT示颅底骨质侵犯30例,MRI扫描示颅底侵犯39例,其中5例MRI扫描发现斜坡、蝶骨翼和蝶骨体早期骨髓浸润,而CT检查表现正常。另外1例MRI见肿瘤沿三叉神经向颅内侵犯,而CT未见明确的卵圆孔破坏。两种影像学方法的检出率存在显著性差异（P<0.05）。结论:CT与MRI均可显示鼻咽癌颅底转移,MRI对颅底组织侵犯更加敏感,二者联合有助于提高诊断的准确性。     

Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma

We studied the kinetics of circulating EBV DNA in the plasma of nasopharyngeal carcinoma (NPC) patients. Serial weekly sampling of 10 NPC patients revealed a rapid decline in plasma EBV DNA concentration after treatment. In two subjects, an initial rise in the circulating EBV DNA level was observed immediately after treatment initiation. Plasma EBV DNA levels were monitored daily during the first treatment week in a second cohort of five patients, and the results indicated that an initial rise in plasma EBV DNA concentration could be observed in all subjects during the first treatment week. This observation is consistent with the liberation of EBV DNA after therapy-induced cancer cell death. After this initial rise, plasma EBV DNA concentration was found to decay with a median half-life of 3.8 days (interquartile range, 2.4-4.4 days). Kinetic analysis of circulating tumor-derived DNA during treatment may be a powerful tool for evaluating the in vivo response of NPC and other tumors to antineoplastic treatment and may improve our understanding of the biology of plasma nucleic acids.     

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。     

Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important tumor in many countries. Ethnic and regional factors strongly influence disease risk. NPC is usually diagnosed late in disease development, and 10-year survival rates are as low as 10%. Epstein-Barr virus (EBV), a possibly causative agent, is present in all cells of essentially all undifferentiated NPCs. We wished to determine the following: 1) whether an ambulatory nasopharyngeal brush biopsy could provide sufficient tumor cell DNA for the detection of EBV and 2) whether the detection of EBV in this locale reflects the presence of tumor cells or simply EBV carrier status. METHODS: We collected nasopharyngeal tissue via ambulatory brush biopsies from 21 patients with newly diagnosed NPC and from 157 subjects with other otolaryngologic complaints. The majority of study subjects were from high-risk populations. Sample DNA was analyzed for the presence of EBV genomic sequences by use of the polymerase chain reaction (PCR). RESULTS: Ninety-six percent of samples yielded sufficient DNA for PCR amplification. Nineteen of 21 patients with NPC brushed positive for EBV DNA, while all but two (1.3%) of 149 informative control subjects were negative for EBV (two-sided P<.0001). One of the EBV-positive control subjects had an EBV-positive inverted sinonasal papilloma; the other EBV-positive control subject exhibited no overt clinical disease. CONCLUSION: Demonstration of EBV DNA in nasopharyngeal brush biopsy specimens detects NPC with a sensitivity of at least 90% (95% confidence interval = 89.63%-91.32%) and a specificity of approximately 99% (95% confidence interval = 98.64%-98.68%). This technique merits further testing as a possible ambulatory screening strategy in high-risk populations.     

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 Epstein-Barr virus DNA analysis and detection of gene promoter hypermethylation in nasopharyngeal (NP) brushing samples from patients with NP carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China and characterized by a strong association with EBV. We aimed to detect EBV DNA and cancer-related gene promoter hypermethylation in nasopharyngeal (NP) brushing samples and provide a novel noninvasive approach for NPC detection. EXPERIMENTAL DESIGN: Twenty-eight NPC cases and 26 noncancerous subjects were prospectively recruited. NP brushing samples were subjected to quantitative real-time PCR analysis of EBV DNA and methylation-specific PCR analysis of the DAP-kinase, RASSF1A, and p16 genes. RESULTS: EBV DNA quantity in NP brushing samples from NPC patients (median, 8.94 copies/actin) was significantly higher than that of controls (median, 0 copies/actin; P < 0.0001). Twenty-seven of 28 NPC patients had detectable EBV DNA in NP brushes, whereas 25 of 26 controls had undetectable or very low levels of EBV DNA. Elevated EBV DNA level in brushing samples as a tumor marker had a sensitivity of 96.4% and a specificity of 96.2% for NPC detection. Moreover, T(1) disease had a significantly lower EBV DNA level as compared with locally more advanced disease (P = 0.037). In brushing samples of NPC patients, the frequencies of DAP-kinase, RASSF1A, and p16 promoter hypermethylation were 50.0%, 39.3%, and 46.4%, respectively. Seventy-eight percent of cases showed methylation of at least one gene. No aberrant hypermethylation was detected in control samples. CONCLUSIONS: Our study demonstrated the feasibility of detecting multiple molecular tumor markers in NP brushing samples with a high sensitivity and specificity for NPC detection. It offers a powerful yet noninvasive approach for the diagnosis of NPC in high-risk populations.     

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

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

Rapid clearance of plasma Epstein-Barr virus DNA after surgical treatment of nasopharyngeal carcinoma.

PURPOSE: Circulating EBV DNA analysis has been shown to be valuable in the detection, prognostication, and monitoring of nasopharyngeal carcinoma (NPC) patients. A previous study has shown that, after radiotherapy, plasma EBV DNA levels of NPC patients would decline exponentially with a median half-life of 3.8 days. We postulate that this decline in plasma EBV DNA reflects the decrease in cancer cell population and, therefore, the rate of decline reflects the radiosensitivity of the tumor. However, this postulation would hold true only if EBV DNA is rapidly eliminated from the circulation. In this study, we determined the in vivo elimination rate of plasma EBV DNA in NPC patients. EXPERIMENTAL DESIGN: We monitored the level of plasma EBV DNA in NPC patients during and after surgical resection of NPC. The half-life of plasma EBV DNA was then calculated by plotting the natural logarithm of EBV DNA concentrations against time. RESULTS: The median half-life of plasma EBV DNA after surgical resection of NPC was 139 min. After a median follow-up of 6.7 days, EBV DNA was undetectable in 8 of 11 patients. One of 8 patients with undectable EBV DNA and all of the patients with detectable EBV DNA developed clinical relapse. CONCLUSIONS: The in vivo elimination of EBV DNA is very rapid after surgical resection of NPC. The failure of complete and rapid elimination of EBV DNA from the circulation predicts disease recurrence.     

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.     

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.     

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。     

Epigenetic markers for noninvasive early detection of nasopharyngeal carcinoma by methylation‐sensitive high resolution melting

Nasopharyngeal carcinoma (NPC) is a human malignancy that is closely associated with Epstein‐Barr Virus (EBV). Early diagnosis of NPC will greatly improve the overall survival. However, current EBV DNA marker detection still lacks the predictive value to perform well in high‐risk populations for early detection of NPC. Since aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is widely considered to be an important epigenetic change in early carcinogenesis, this study identified a panel of methylation markers for early detection of NPC and also assessed the clinical usefulness of these markers with noninvasive plasma specimens instead of biopsies. MS‐HRM assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1 and RARβ2) in biopsies, NP brushings and cell‐free plasma from NPC patients. High‐risk and cancer‐free groups were used as controls. DNA methylation panel showed higher sensitivity and specificity than EBV DNA marker in cell‐free plasma from NPC patients at early Stages (I and II) and in addition to the EBV DNA marker, MS‐HRM test for plasma and NP brushing DNA methylation significantly increased the detection rate at all NPC stages as well as local recurrence, using this selected four‐gene panel (p < 0.05). MS‐HRM assay on a selected gene panel has great potential to become a noninvasive and complementary test for NPC early and recurrent detection in combination with the EBV DNA test to increase the sensitivity for NPC detection at an early stage.