Plasma Epstein-Barr virus DNA as a biomarker for nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is common in southern China and Southeast Asia. Epstein-Barr virus (EBV) infection is an important etiology for NPC, and EBV genome can be detected in almost all tumor ti...     

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.     

鼻咽癌患者血浆游离EBV-DNA的研究进展

近年来,研究人员发现血浆游离EB病毒DNA对鼻咽癌的诊断、分期、疗效监测及判断预后方面都具有重要作用,并且优于目前较为普遍的抗体的检测,是新一代的有效检测指标。     

鼻咽癌患者血浆游离EBV-DNA的研究进展

近年来,研究人员发现血浆游离EB病毒DNA对鼻咽癌的诊断、分期、疗效监测及判断预后方面都具有重要作用,并且优于目前较为普遍的抗体的检测,是新一代的有效检测指标。     

鼻咽癌高危人群血浆EBV-DNA定量分析

目的：探讨鼻咽癌高危人群E病毒DNA（Epstein-Barr virusDNA,EBV—DNA）的存在情况及其在鼻咽癌早期诊断中的应用价值。方法：2009-08-13—2010-07-27在广东省中山市小榄镇开展鼻咽癌筛查,ELISA法检测16712名EBV抗体,确定鼻咽癌高危人群386名,同时收集鼻咽癌低危人群273名。收集同期中山市人民医院初诊鼻咽癌患者62例。荧光定量PCR方法检测高危人群血浆EBV-DNA,并随访1年,比较分析高危人群EBv_DNA定量检测的应用价值。结果：初筛鼻咽癌高危人群EBV—DNA阳性率为12．2％（47／386）,高于鼻咽癌低危人群的3．3％（9／273）,而低于初诊鼻咽癌患者的91．9％（57／62）,3组差异有统计学意义,P〈0．001。随访复查时,血清学持续高危人群EBV—DNA阳性率为9．7％（18／186）,而血清学转变为非高危人群的EBV-DNA阳性率为3．1％（3／97）,差异有统计学意义,P=0．045。筛查人群中血清学诊断鼻咽癌的阳性预测值为5．6％（32／572）,而在鼻咽癌高危人群中增加EBV—DNA检测,其阳性预测值提高到44．6％（29／65）。结论：鼻咽癌高危人群血浆EBV-DNA定量检测能对血清学EBV抗体检测进行有效的补充,可大大提高筛查准确性,对鼻咽癌高危人群的监测具有重要作用。     

鼻咽癌患者血浆 EBV DNA 水平和 VCA-IgA 检测的临床意义

目的:探讨鼻咽癌患者血浆EBV DNA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EBV DNA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,EBV DNA阳性率95.59,中位拷贝数93×104copies/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,EBV DVA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EBV DNA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EBV DNA和VCA-IgA抗体阳性率显著高于对照组。结论:进一步证实EBV与鼻咽癌有密切关系;EBV DNA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。     

Combination of RERG and ZNF671 methylation rates in circulating cell‐free DNA: A novel biomarker for screening of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome‐wide and targeted analyses based on next‐generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real‐time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell‐free DNA (ccfDNA) from nonhemolytic plasma, with methylation‐sensitive and/or methylation‐dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC.     

Trajectories of EBV DNA and identifying the potential long-term survivors in metastatic nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is highly incident in southern China. Distant metastasis is the leading cause of death in NPC patients. However, the phenotypical feature of this patient population is largely undefined. The current study aimed to categorize metastatic NPC patients into novel subgroups based on their EBV DNA trajectories. In this retrospective study, 446 eligible patients with metastatic NPC treated at Sun Yat-Sen University Cancer Center between 2012 and 2016 were analyzed. Using a mixture model analysis, we identified distinct trajectories based on longitudinal EBV DNA measurements. We evaluated their associations with metastatic NPC mortality using Cox regression analysis. The two-class trajectory model provided the best fit, in which 272 patients were classified as non-sustained EBV DNA class and 174 patients as sustained EBV DNA class. After a median follow-up of 60.8 months, the median OS was 61.7 months in the sustained EBV DNA clearance class versus 20.0 months in the non-sustained EBV DNA clearance class (P<0.001). Compared with patients in the non-sustained EBV DNA clearance class, patients in the sustained EBV DNA clearance class demonstrated superior PFS (HR, 3.238; 95% CI, 2.601-4.032; P<0.001). Forty-three patients experienced disease-free for longer than 36 months, defined as long-term survivors (LTS). Notably, 41 patients were presented in the sustained EBV DNA clearance class (95.3%), along with only 2 patients in the non-sustained EBV DNA clearance class. Collectively, we identified two EBV DNA trajectory sub-phenotypes of patients with metastatic NPC, providing more reliable survival information for physicians and patients during their informed decision-making process.     

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.     

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。     

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.     

Predictive value of miR-9 as a potential biomarker for nasopharyngeal carcinoma metastasis

Background:

Nasopharyngeal carcinoma (NPC) has a distinctive geographic distribution and is characterised by its strong tendency of metastasis. We aimed to examine the microRNA (miRNA) expression profiles in plasma samples of NPC patients to explore their clinical significance in disease development and progression.

Methods:

This study was divided into four steps: (1) confirmation of differentially expressed miRNAs using microarray analysis and quantitative PCR validation; (2) comparison of plasma miR-9 levels during NPC progression; (3) evaluation of the predictive performance of plasma miR-9 as a biomarker for NPC metastasis; and (4) comparison of plasma miR-9 levels between pre- and post-treatment samples.

Results:

Plasma microarray profiling identified 33 differentially expressed miRNAs between NPC patients and healthy volunteers. The significantly declined level of miR-9 in NPC patients was confirmed through two-stage validation. The low level of plasma miR-9 was significantly correlated with worse lymphatic invasion and advanced TNM stage. The plasma miR-9 could distinguish locoregional from metastatic NPC cases with a high sensitivity and specificity. Furthermore, the plasma miR-9 level was significantly elevated in post-treatment plasma compared with those pre-treatment samples.

Conclusion:

Our study reports that plasma miR-9 may serve as a useful biomarker to predict NPC metastasis and to monitor tumour dynamics.     

Screening for family members of patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is well known for its peculiarly skewed distribution with highest incidence in Southern Chinese population. Familial aggregation is evident, hence screening for early detection is offered by oncology centers in Hong Kong to first-degree relatives of patients with NPC. During the period 1994-2001, 929 family members were screened in our center. The screenees were advised to attend an annual examination that includes serological test against Epstein Barr Virus (EBV), physical examination to exclude cervical lymphadenopathy and cranial nerve palsy, and endoscopic examination of the nasopharyngeal region. Two different methods were used for the serology test: indirect immuno-fluorescent (IF) test for IgA against viral capsid antigen; and starting in 1997 enzyme-linked immunosorbent assay (ELIZA) against nuclear antigen and viral capsid antigen. Twelve cases of nasopharyngeal carcinoma were diagnosed, giving a detection rate of 5/1,155 (433/100,000) person-year for male and 7/1,404 (499/100,000) person-year for female participants observed. The corresponding average annual incidence in Hong Kong during this period was 24.1 and 9.6 per 100,000, respectively. Forty-one percent of these detected cases had Stage I disease, whereas only 2% of patients referred to the department for primary treatment presented with such early disease. Six cases were detected at first visit, and all were EBV-positive. Another 78 screenees with positive serology at first visit were followed up for 204 person years, and thus far NPC was detected in 3 after an interval of 6-32 months. Of the 845 initially EBV-negative screenees followed up for 2,337 person-years, NPC was detected in 3 after an interval of 12-45 months. One showed sero-conversion at the time of diagnosis. We conclude that family members of known patients do show a substantially higher risk of developing NPC, and regular screening by current method improves the chance of early detection.     

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.     

Systemic treatment strategies and therapeutic monitoring for advanced nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is endemic in Asia and is etiologically associated with the Epstein–Barr virus. Although radiotherapy can cure most patients with early-stage disease, those with advanced disease often develop recurrences following radiotherapy. The past decade has witnessed significant advances in the systemic treatment of advanced NPC. This article reviews the latest literature regarding: combined modality therapy for locoregionally advanced NPC; clinical trials of novel biologic and cytotoxic therapies for metastatic NPC; and disease monitoring with the plasma Epstein–Barr virus DNA assay.     

Syncope as the initial presentation of nasopharyngeal carcinoma

Syncope can be the first manifestation of nasopharyngeal carcinoma (NPC). Recently, we have encountered one such case and was able to eradiate this symptom by combined chemotherapy and radiotherapy.     

Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein‐Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV‐negative NPC cell lines, NPC‐TW01 (TW01) and HONE‐1, were treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and sodium n‐butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double‐strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double‐strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy‐number alterations, is proportional to the frequency of TPA/SB‐induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV‐negative TW01 cells with mock or TPA/SB treatment, and were few in mock‐treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC. © 2008 Wiley‐Liss, Inc.     

Post-radiation circulating tumor DNA as a prognostic factor in locally advanced esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly malignant and deadly tumor. Radiation therapy is one of the primary treatments for locally advanced ESCC. However, the biomarkers for prognosis of definitive radiation remain undefined. Peripheral blood circulating tumor (ct)DNA provides information of tumor genetic alterations and has been confirmed as a potential non-invasive biomarker for several types of cancer. The present study investigated the clinical implications of ctDNA detection in patients with ESCC and receiving definitive radiation therapy. Patients with locally advanced ESCC were retrospectively recruited. Plasma samples were collected before, during and following radiation therapy. Next-generation sequencing was performed to identify somatic mutations in 180 genes. A total of 69 baseline and post-radiation plasma samples were collected from 25 patients. A total of 59 non-silent single nucleotide variants were present in 33 genes. All pre-radiation and 58.3% (14/24) of post-radiation samples had at least one mutation. Patients with lymph node metastases (LNM) exhibited a higher number of pre-radiation mutations compared with those without LNM. The variables, progression-free survival (PFS) and overall survival (OS) of the patients with one baseline mutation were not significantly different compared with that in patients with more than one baseline mutation. Patients with initial ctDNA-positive post-radiation samples exhibited significantly reduced PFS (P=0.047) and OS (P=0.005) compared with that in patients with ctDNA-negative samples. The post-radiation plasma ctDNA status was an independent prognostic factor from univariate and multivariate analyses. Dynamic monitoring of ctDNA during follow-up was examined. The results indicated that ctDNA was a predictive and prognostic marker in patients with ESCC and receiving definitive radiation therapy, which may guide subsequent treatment.