Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a western population.
ABSTRACT Loco-regionally advanced nasopharyngeal carcinomas can be cured by the combination of chemotherapy and radiotherapy. In Eastern countries, plasma levels of viral Epstein-Barr deoxyribonucleic acid (DNA) are accurate in predicting recurrence, but few data are available in Western populations. The aim of this prospective study was to evaluate the relationship between viral Epstein-Barr DNA copy numbers in plasma and the response rate, progression-free survival and overall survival in a cohort of Western patients with stage IIb-IVb nasopharyngeal cancer.
We evaluated plasma samples from 36 consecutive patients treated with induction chemotherapy followed by chemoradiation. EBV copy numbers were determined after DNA extraction using real-time quantitative polymerase chain reaction. Survival curves were estimated using the Kaplan-Meier method.
Circulating Epstein-Barr virus DNA levels were measured before treatment, at the end of concomitant chemo- and radiotherapy, and during the follow-up period. Pre-treatment levels significantly correlated with the initial stage and probability of relapse. Their increase was 100% specific and 71.3% sensitive in detecting loco-regional or metastatic recurrence (an overall accuracy of 94.4%). Three-year progression-free and overall survival were respectively 78.2% and 97.1%.
The results of this study confirm that patients from a Western country affected by loco-regionally advanced nasopharyngeal carcinoma have high plasma Epstein-Barr virus DNA levels at diagnosis. The monitoring of plasma levels is sensitive and highly specific in detecting disease recurrence and metastases.
- SourceAvailable from: Rong-San Jiang[show abstract] [hide abstract]
ABSTRACT: 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). 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. 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. 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.International Journal of Radiation OncologyBiologyPhysics 08/2007; 68(5):1342-8. · 4.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In a prospective study, 42 048 adults residing in Zhongshan City, Guangdong, China, were followed for 16 years, and 171 of them developed nasopharyngeal carcinoma (NPC). Although Epstein-Barr virus (EBV) antibody levels of the cohort fluctuated, the antibody levels of 93% of the patients with NPC were raised and maintained at high levels for up to 10 years prior to diagnosis. This suggests that the serologic window affords an opportunity to monitor tumor progression during the preclinical stage of NPC development, facilitating early NPC detection. We reviewed the clinical records of the 171 patients with NPC in the prospective study to assess the efficacy of early NPC detection by serologic screening and clinical examination. Of the 171 patients, 51 had Stage I tumor (44 were among the 73 patients detected by clinical examination and 7 were among the 98 patients presented to outpatient department). Initial serologic screening predicted 58 (95.1%) of the 61 patients detected within 2 years. The risk of the screened population (58/3093) raised 13 times relative to cohort (61/42 048) during this period. Clinical examination detected all the 58 predicted cases, and 35 (60.3%) of which were diagnosed with Stage I tumor. The serologic prediction rate fell to 33.6% (37/110) 2 to 16 years after screening. The proportion of cases detected by clinical examination fell to 40.5% (15/37). The proportion of Stage I tumors among the cases detected by clinical examination during both periods remained at about 60%. We concluded that early detection of NPC can be accomplished by repeated serologic screening to maintain high prediction rates and by promptly examining screened subjects to detect tumors before the symptoms develop.Chinese journal of cancer 02/2011; 30(2):120-3.
- [show abstract] [hide abstract]
ABSTRACT: Anticancer cytotoxic agents go through a process by which their antitumor activity-on the basis of the amount of tumor shrinkage they could generate-has been investigated. In the late 1970s, the International Union Against Cancer and the World Health Organization introduced specific criteria for the codification of tumor response evaluation. In 1994, several organizations involved in clinical research combined forces to tackle the review of these criteria on the basis of the experience and knowledge acquired since then. After several years of intensive discussions, a new set of guidelines is ready that will supersede the former criteria. In parallel to this initiative, one of the participating groups developed a model by which response rates could be derived from unidimensional measurement of tumor lesions instead of the usual bidimensional approach. This new concept has been largely validated by the Response Evaluation Criteria in Solid Tumors Group and integrated into the present guidelines. This special article also provides some philosophic background to clarify the various purposes of response evaluation. It proposes a model by which a combined assessment of all existing lesions, characterized by target lesions (to be measured) and nontarget lesions, is used to extrapolate an overall response to treatment. Methods of assessing tumor lesions are better codified, briefly within the guidelines and in more detail in Appendix I. All other aspects of response evaluation have been discussed, reviewed, and amended whenever appropriate.JNCI Journal of the National Cancer Institute 03/2000; 92(3):205-16. · 14.34 Impact Factor
RESEARCH ARTICLE Open Access
Role of plasma EBV DNA levels in predicting
recurrence of nasopharyngeal carcinoma in a
Daris Ferrari1*, Carla Codecà1, Cecilia Bertuzzi1, Francesca Broggio1, Francesca Crepaldi1, Andrea Luciani1,
Irene Floriani2, Mohssen Ansarin3, Fausto Chiesa3, Daniela Alterio4and Paolo Foa1
Background: Loco-regionally advanced nasopharyngeal carcinomas can be cured by the combination of
chemotherapy and radiotherapy. In Eastern countries, plasma levels of viral Epstein-Barr deoxyribonucleic acid
(DNA) are accurate in predicting recurrence, but few data are available in Western populations. The aim of this
prospective study was to evaluate the relationship between viral Epstein-Barr DNA copy numbers in plasma and the
response rate, progression-free survival and overall survival in a cohort of Western patients with stage IIb-IVb
Methods: We evaluated plasma samples from 36 consecutive patients treated with induction chemotherapy
followed by chemoradiation. EBV copy numbers were determined after DNA extraction using real-time quantitative
polymerase chain reaction. Survival curves were estimated using the Kaplan–Meier method.
Results: Circulating Epstein-Barr virus DNA levels were measured before treatment, at the end of concomitant
chemo- and radiotherapy, and during the follow-up period. Pre-treatment levels significantly correlated with the
initial stage and probability of relapse. Their increase was 100% specific and 71.3% sensitive in detecting loco-
regional or metastatic recurrence (an overall accuracy of 94.4%). Three-year progression-free and overall survival
were respectively 78.2% and 97.1%.
Conclusions: The results of this study confirm that patients from a Western country affected by loco-regionally
advanced nasopharyngeal carcinoma have high plasma Epstein-Barr virus DNA levels at diagnosis. The monitoring
of plasma levels is sensitive and highly specific in detecting disease recurrence and metastases.
Keywords: Nasopharyngeal carcinoma, Epstein-Barr virus, Chemoradiotherapy, Induction chemotherapy, Plasma EBV
DNA copy numbers
Nasopharyngeal carcinomas (NPCs) are frequent
South-east Asia, China, the Arctic regions and North
Africa, with the highest prevalence being observed in
China’s Guangdong province and in Sarawak [1-7]. How-
ever, they are rare in Europe and, in Italy, prevalence is
about 1.4 per 100,000 inhabitants . They are often diag-
nosed late because the symptoms are subtle in the early
stage of disease. In Eastern populations, most stage I-II
tumours are successfully treated by means of radiotherapy
(RT), and about 50% of the patients with loco-regionally
advanced disease (stage III-IV) are cured by means of com-
bined chemotherapy and RT [9-12]. There are very few
screening procedures for early disease , but repeated
serological screening can be helpful in endemic regions
. A definite diagnosis depends on a combination of clin-
ical suspicion, endoscopy with biopsy, and mainly magnetic
resonance (MR) and positron emission tomography (PET)
imaging techniques. The World Health Organization classi-
fies NPCs as keratinising squamous carcinomas, which are
characterized by well-differentiated keratin-producing cells
* Correspondence: firstname.lastname@example.org
1Division of Medical Oncology and Department of Medicine, Surgery and
Dentistry, San Paolo Hospital and University of Milan, Via Di Rudinì 8, Milan,
Full list of author information is available at the end of the article
© 2012 Ferrari et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Ferrari et al. BMC Cancer 2012, 12:208
(type I); non-keratinizing squamous cell carcinoma, differ-
entiated (type IIa); non-keratinizing squamous cell carcin-
omas, undifferentiated (type IIb); basaloid squamous
cell carcinomas (type III) .
Epidemiological studies have shown that the gamma–
herpes Epstein-Barr virus (EBV) plays a major role in
the pathogenesis of NPCs as they are endemic in areas
where EBV infection is also endemic [16-18]. Potentially
EBV-induced carcinogenesis depends on the expression
of a set of oncogenes: in particular, latent membrane
protein 1 (LMP1) plays a role in preventing apoptosis
and activating a number of signalling pathways such as
nuclear factor k-B (NF-kB), mitogen-activated protein
(MAP) kinases, and phosphoinositol-3-kinase (PI3K),
thus favouring cell motility and suppressing immuno-
genic responses [19,20].
Following the demonstration of EBV in histological
samples of NPCs [21,22] and the presence of high titres
of IgA antibody against a viral capside antigen (VCA)
[23,24], it has been recently demonstrated that real-time
quantitative polymerase chain reaction (PCR) can easily
detect cell-free EBV DNA in the serum of NPC patients
[25-29]. Previous studies have found different percen-
tages of patients whose plasma was positive for EBV
DNA and, sometimes, a significant proportion of
patients with a low viral load, possibly due to differences
in PCR methods . The great majority of patients in
endemic countries have high circulating levels of EBV
DNA at diagnosis , and the higher the level, the
greater the risk of recurrence. Furthermore recurrent/
metastatic disease can be heralded by an increase in
plasma EBV DNA levels during follow-up .
There are few data concerning the role of EBV DNA in
Western populations, but an Italian study has demon-
strated similar behaviour in a low-incidence region in
which EBV DNA may be of prognostic value . The
aim of this prospective study was to evaluate EBV DNA
levels before treatment and during follow-up in a cohort
of Western patients with loco-regionally advanced NPCs.
Untreated patients with pathologically confirmed stage
IIb-IVb NPC  were considered eligible for study in-
clusion. The pre-treatment evaluation included a phys-
ical examination, the assessment of performance status,
a complete blood cell count with differential, a biochem-
ical profile, the quantification of plasma EBV DNA copy
numbers, MR imaging, and the measurement of index
lesions. The baseline imaging techniques included PET
and computed tomography (CT) in the case of suspected
The patients were treated by means of induction chemo-
therapy followed by concomitant chemo-radiotherapy
(CRT). Induction chemotherapy consisted of three cycles
of cisplatin (CDDP) 100 mg/m2i.v. on day 1 and 5-
fluorouracil (5-FU) 1000 g/m2/day administered as a con-
tinuous intravenous infusion on days 1–4 (PF regimen), or
three cycles of CDDP 75 mg/m2i.v. on day 1, docetaxel
75 mg/m2i.v. on day 1, and 5-FU 750 mg/m2/day adminis-
tered as a continuous intravenous infusion on days 1–4
(TPF regimen), both repeated every three weeks. The
chemotherapy was administered via a central venous cath-
eter in an outpatient setting. During the TPF regimen,
prophylactic antibiotics were given between the cycles.
During RT, CDDP was administered as a 100 mg/m2
intravenous infusion 60 minutes before radiation on
days 1, 22 and 43. The patients were hydrated with
mannitol and/or furosemide in order to maintain a high
urine output. The intravenous anti-emetics given before
CDDP included granisetron 3 mg and dexamethasone
Three-dimensional conformal RT was delivered in sin-
gle daily fractions of 2 Gy on five days a week without
interruptions. The planned total dose to the tumour and
involved lymph nodes was 70 Gy in 35 fractions.
Tumour response was assessed by means of a clinical
examination, endoscopy and MR using the response
evaluation criteria in solid tumours (RECIST)  at the
end of chemotherapy and 8–12 weeks after the comple-
tion of CRT. The patients were followed up by means of
a clinical examination every two months during the first
year, every three months for the following two years, and
every six months thereafter. Salvage treatment after a re-
lapse that was not eligible for surgery or re-irradiation
consisted of four cycles of carboplatin AUC 5 and pacli-
taxel 175 mg/m2on day 1 q 21.
blocks were retrieved from the archives of the Path-
ology Unit. Three 10 μm thick sections from each
block were collected in a 1.5 mL tube, dewaxed with
xylene, dehydrated with ethanol, and incubated over-
night in a digestion buffer containing 50 mM Tris,
pH 8, 5 mM EDTA, pH 8, 0.5% Tween20, and 10 μL
proteinase K (20 mg/ml). After digestion, total DNA
was purified using DNA QIAamp DNA MiniKit
(Qiagen, Hilden, Germany) spin columns as indicated
by the manufacturer. The quality of the DNA was
tested by means of the multiplex amplification of four
fragments of 100, 200, 300 and 400 bp as previously
described , and was considered acceptable in the
presence of at least the 100, 200 and 300 bp ladders.
For each sample, 5 μL of total DNA was tested for
the presence of the EBV DNA IR3 region (EBNA1)
using previously described primers . The EBNA 1
PCR is a simple method to detect EBV DNA even if
in situ hybridization for EBV-encoded RNA (EBER-
Ferrari et al. BMC Cancer 2012, 12:208
Page 2 of 7
RISH) can be considered the gold standard. The PCR
was performed in a mixture containing 10 pmol of
each primer (forward: 5’-GACGAGGGGCCAGGTACA
GG-3’; reverse: 5’-GCAGCCAATGCTTCTTGGACGTT
TTTGG-3’), 0.2 mmol/L dNTPs, 1.5 mmol/L MgCl2,
and 1.25 U TaqGold polymerase (Applied Biosystems,
Foster City, California, US) in a total volume of 50 μL.
The thermocycling conditions consisted of 40 cycles at
94 °C for 10 min, 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min, followed by a final extension phase at 72 °
C for 7 min (Thermal cycler ABI 9700, Applied Biosys-
tems, Foster City, California, US). Appropriate positive
and negative controls were used in each experiment. Ten
microlitres of PCR products were run on a QIAxcel Sys-
tem (Qiagen, Hilden, Germany) and visualised using
QIAxcel BioCalculator Sofware. The expected size of the
PCR product was 241 bp.
EDTA plasma samples were collected, centrifuged at
1000 x g for 15 minutes, and then stored at −80 °C until
further processing. EBV DNA copy numbers were evalu-
ated before treatment, 1–2 weeks after CRT, and every
six months during the follow-up period or in the case of
a suspected or documented recurrence. The plasma
samples were thawed and centrifuged at 20,000 x g for
five minutes, and plasma DNA was extracted using a
Germany). About 200–400 μL of each sample per col-
umn was used for DNA extraction, and the exact
amount of extracted plasma was documented fin order
to calculate the target DNA concentration. Fifty microli-
tres of distilled water was used to elute the DNA from
the extraction column. The plasma concentrations of EBV
DNA were measured by means of a real-time quantitative
PCR assay of the Bam HI-W region of the EBV genome.
The sequences of the forward and reverse primers were re-
TCTTAGGAGCTGTCCGAGGG-3'. A dual fluorescence-
labelled oligomer, 5'-(FAM)CA CACACTACACACAC
CCACCCGTCTC(TAMRA)-3', was used as a probe. The
real-time quantitative PCR assay and reaction set-up proce-
dures have been previously described in detail. The Bam
HI-W region showed a strong correlation with the
EBNA-1 PCR . Real-time quantitative PCR was
performed using a Rotor Gene Q (Qiagen, Hilden,
Germany) analyser, and the plasma concentrations of
EBV DNA were expressed as the number of copies of
the EBV genome per millilitre of plasma. Values of
<350 copies/mL were considered normal on the basis
of the analytical sensitivity of the test. Plasma EBV
DNA levels were also evaluated in 20 control samples
taken from patients with head-and-neck cancers other
than NPC, none of which had values of >350 copies/ml.
Informed consent was obtained from all of the patients,
and the study protocol was approved by our local Ethics
The aim of the study was to explore the relationship be-
tween plasma EBV DNA concentrations and progression-
free survival (PFS). Response to treatment and overall sur-
vival (OS) were also assessed. PFS was calculated from the
first day of treatment to the date of relapse. The patients
who had not experienced a recurrence by the analysis were
censored at their last disease assessment. OS was defined as
the time from the first day of treatment to the date of death
due to any cause. At the time of analysis, the patients who
were not reported as having died were censored at the date
they were last known to be alive. Induction chemotherapy
was started within three days of the date of diagnosis in all
patients. Survival curves were estimated using the Kaplan-
Meier method. Plasma EBV DNA concentrations were ana-
lysed as both continuous and categorical variables (<350 or
>350 copies), and compared using the Mann–Whitney
rank-sum test. Given the exploratory nature of the study,
no formal sample size calculation was made. All of the stat-
istical tests were two-sided, and a p-value <0.05 was con-
sidered statistically significant. The analyses were carried
out using SAS software, version 9.1 (SAS Institute, Cary,
North Carolina, US).
Thirty-six consecutive Italian patients with pathologic-
ally confirmed stage IIb-IVb NPC referred to the Oncol-
ogy Department of San Paolo Hospital, Milan (Italy),
were enrolled between October 2005 and August 2009.
According to the American Joint Committee on Cancer
(AJCC) classification, 7th
(30.6%) were in stage IIb, 16 (44.4%) in stage III, six
(16.7%) in stage IVa, and three (8.3%) in stage IVb. Hist-
ology was type IIa in two cases (5.6%), and type IIb in the
remaining 34 (94.4%). Twenty-six patients (72.2%) were
males, and the median age was 52 years (range 29–58).
Table 1 shows the characteristics of the enrolled patients.
Before treatment, tumoral tissue from all of the
patients contained EBV DNA. All but one patient had a
plasma EBV DNA concentration of >350 copies/mL,
with a median value of 4,701 (range 349–270,000) and
an interquartile range (IQR) of 1,462-9,625. The stage
IIb patients had significantly lower EBV DNA copy
numbers (p=0.007) than those in stages III-IV patients
(median 2,420; IQR 820–3,939 vs median 7,520 and IQR
Thirty-one patients (86.1%) received the PF induction
chemotherapy regimen and five the TPF regimen. At the
end of CRT, a complete response (CR) was achieved by
30 patients (83.3%, 95% CI 67.2- 93.6%) and a partial
edition (33), 11 patients
Ferrari et al. BMC Cancer 2012, 12:208
Page 3 of 7
response (PR) by the remaining six. EBV DNA levels
had returned to below the threshold limit in all of the
complete responders and four of the partial responders;
the two partial responders with still high EBV DNA
levels underwent a biopsy that failed to reveal the pres-
ence of tumour cells. No further treatment was given to
the patients with a PR.
After a median follow-up of 36 months (IQR 24–48
months), seven patients had experienced recurrent disease
(4 loco-regional and 3 metastatic), one of whom had died.
Three-year PFS and 3-year OS were respectively 78.2% and
97.1% Figure 1.
EBV DNA load monitoring seemed to be a reliable
molecular marker for predicting recurrence as five of the
seven patients who relapsed (sensitivity 71.3%, 95% CI
29.0%-96.3%) had EBV DNA copy numbers that were
much higher than the limit of detection (900 and 2,135
copies/mL in two patients with loco-regional relapse;
and 9,185, 15,290 and 4,398,000 copies/mL respectively
in three patients with metastases). The loco-regional re-
lapse in the remaining two patients was not heralded by
an increase in EBV DNA levels (Table 2) but by appear-
ance of latero-cervical lymphadenopathy confirmed by
node biopsy in one case (12 mm nodal relapse) and both
nodal and nasopharyngeal biopsy in the second (respect-
ively 11 mm and 17 mm; loco-regional relapse). MR im-
aging confirmed the diagnoses. The patient with the
highest EBV DNA load died of disseminated disease
13 months after diagnosis.
None of the 29 patients who did not experience a re-
currence had EBV DNA levels above the cut-off value
during the follow-up, which suggests a specificity of
100% (95%CI 88.1-100%). The overall accuracy of
plasma EBV DNA levels in truly detecting recurrence
was 94.4% (34/36, 95%CI 81.3-99.3).
Median plasma EBV DNA concentrations were 270
copies/mL in the patients without recurrent disease and
2,135 copies/mL in the seven experiencing recurrence
(p=0.037). Pre-treatment EBV DNA concentrations sig-
nificantly correlated with PFS at univariate analysis
(p=0.036), but not after adjusting for stage (p=0.051).
It has been demonstrated that NPCs are associated with
EBV DNA infection as the virus infects the epithelial
cells promoting the activation of proliferation signalling
. Tumour cells release EBV DNA during treatment,
and plasma EBV DNA levels correlate with tumour vol-
ume and TNM stage [27,38-40]. Quantitative real-time
PCR can detect circulating EBV DNA, and the concen-
tration of short cell-free EBV DNA fragments can pre-
dict recurrence and survival. It has been shown that
almost all NPC patients have variable pre-treatment
EBV DNA copy numbers, and that the highest values
correlate with a poor prognosis . In the case of a CR,
pre-treatment levels become undetectable after RT or
CRT, whereas some patients with a PR, stable disease or
progression tend to maintain high EBV DNA levels. It
has been reported that patients receiving standard CRT
can be stratified into subgroups on the basis of their
pre- and post-treatment EBV DNA levels. Five-year OS
and 5-year relapse-free survival are significantly better in
the groups with low or high pre-treatment and undetect-
able post-treatment levels than in those with still detect-
able levels at the end of treatment .
In our study, 97% of the patients had pre-treatment
levels of >350 copies/mL that significantly correlated
with tumour stage (lower values in stage IIb than stages
III-IV). According to Leung et al.  high pre-
treatment concentrations during early-stage NPCs are
associated with the risk of distant metastases, and Lin
et al.  found that pre-treatment plasma EBV DNA
concentrations were lower in patients with local recur-
rences than in those with distant metastases (1,311 vs
4,253 copies/mL) even if the difference was not statisti-
cally significant (p=0.37). They also described 10
patients (10.1%) with detectable EBV DNA levels one
week after completing RT, seven of whom experienced a
distant relapse, which reflects the importance of un-
detectable plasma EBV DNA levels in maintaining a
Hou et al.  found that both pre- and post-
treatment EBV DNA concentrations were significantly
Table 1 Patients' characteristics
Number of patients36
Median age (years)52.0
Sex – n (%)
WHO histology – n (%)
IIb 34 (94.4)
AJCC stage - n (%)
IIb 11 (30.6)
IVb 3 (8.3)
WHO Performance status
0 33 (91.7)
1 3 (8.3)
Positive EBV-DNA36 (100)
WHO=World Health Organization.
AJCC=American Joint Committee on Cancer.
Ferrari et al. BMC Cancer 2012, 12:208
Page 4 of 7
higher in their patients with distant metastases than in
those with long-term disease remission or local relapse.
However, in this retrospective study, the post-treatment
level was more important than the pre-treatment level
in predicting metastases and survival. The same conclu-
sion applies to the study of Chan et al. , who found
that a post-treatment level of >500 copies/mL in
patients treated with RT significantly correlated with the
poorest outcome, suggesting that this subgroup of
patients should be treated more aggressively; on the con-
trary, levels of <500 copies/mL were associated with a
During the follow-up period in endemic countries,
loco-regional recurrence or metastatic disease may be
heralded by an increase in plasma EBV DNA levels,
which can therefore be considered a useful marker for
monitoring NPC patients. In a recent study, Wang et al.
found that 36 out of 245 NPC patients (14.7%) had ab-
normal plasma EBV DNA levels that accurately pre-
dicted all of the NPC recurrences, and no disease was
found in five patients with clinical signs of recurrence
who were negative for EBV DNA. A PET scan helped in
identifying the recurring lesions with a sensitivity, speci-
ficity and visual accuracy of respectively 81.8%, 77.1%,
and 79.2% .
In our study on a cohort of patients of Italian origin,
we observed three cases of metastatic spread that corre-
lated with very high plasma EBV DNA levels, which
must be attributed to an enormous load of tumour cells
releasing the virus in blood while replicating. We also
found that pre-treatment EBV DNA copy numbers sig-
nificantly correlate with initial stage (stage IIb vs stage
III-IV) and PFS at univariate analysis. Furthermore, the
quantification of plasma EBV DNA levels proved to be
extremely specific in detecting loco-regional or distant
recurrences as none of the disease-free patients showed
an increase. Among the relapsing patients, all three with
disseminated disease were easily detected by their high
EBV DNA loads, whereas the load increased to >350
copies/mL in only two of the four patients with loco-
regional recurrences, thus confirming that assaying EBV
DNA is less reliable in the case of a limited volume
It has recently been shown that plasma EBV DNA clear-
ance rates have predictive and prognostic value during the
first month of palliative chemotherapy in patients with me-
tastases . However, the study involved a small number
of patients (34 treated with old chemotherapeutic drugs),
and the unusually high response rate (41.2% CR) and
Table 2 Recurring patients
Initial EBV DNA atEBV DNA at the
end of treatment
EBV DNA at
1 T2N3 290,000
< 350 2,13514
< 350900 25
3 T3N2 12,745
5T2N3 87,425 7,820 4,398,00011
< 3509,185 12
Figure 1 Kaplan-Meyer curves depicting progression-free survival and overall survival.
Ferrari et al. BMC Cancer 2012, 12:208
Page 5 of 7
unusually long OS (median 28 months), which have never
reported elsewhere [45,46], suggest there may have been a
selection bias. Furthermore, the need to analyse multiple
plasma samples limits the usefulness of this method in clin-
In a larger cohort of patients receiving chemotherapy
for metastatic disease, monitoring EBV DNA levels
proved to be useful in predicting which patients would
survive longer (i.e. those with low pre-treatment and un-
detectable post-treatment levels) , which is in line
with our experience as some of our long-term survivors
maintained low EBV DNA copy numbers years after the
end of chemotherapy (data not shown).
In an era of new biomolecular marker development, the
measurement of plasma EBV DNA levels is as essential
in the West as it is in the East. Given its high degree of
accuracy, all NPC patients with loco-regionally advanced
disease should be regularly monitored in order to detect
any increase in plasma levels and diagnose recurrence as
early as possible.
The authors declare that they have no competing interests.
DF and PF conceived the study and participated in its design and
coordination. CC, CB, FB and FC participated in designing the study. AL and
IF performed the statistical analysis. AM, FC and DA participated in
coordinating the study. All of the authors read and approved the final
We thank Barbara Cassani for her contribution to the writing of the Methods
1Division of Medical Oncology and Department of Medicine, Surgery and
Dentistry, San Paolo Hospital and University of Milan, Via Di Rudinì 8, Milan,
Italy.2Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa, Milan,
Italy.3Head and Neck Surgery, European Institute of Oncology, Via
Ripamonti, Milan, Italy.4Division of Radiotherapy, European Institute of
Oncology, Via Ripamonti, Milan, Italy.
Received: 12 November 2011 Accepted: 16 May 2012
Published: 30 May 2012
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Cite this article as: Ferrari et al.: Role of plasma EBV DNA levels in
predicting recurrence of nasopharyngeal carcinoma in a western
population. BMC Cancer 2012 12:208.
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