Download Prognostic Significance of the Epstein-Barr Virus, p53, Bcl

Imaging, Diagnosis, Prognosis
Prognostic Significance of the Epstein-Barr Virus, p53, Bcl-2,
and Survivin in Nasopharyngeal Cancer
Kenneth W. Yip,1,3 Wei Shi,1,3 Melania Pintilie,4 Joseph D. Martin,1,2,3,7 Joseph D. Mocanu,1,3 Derek Wong,1,3
Christine MacMillan,5 Pat Gullane,6 Brian O’Sullivan,2,7 Carlo Bastianutto,1,3 and Fei-Fei Liu1,2,3,7
Abstract
Purpose: Nasopharyngeal cancer (NPC) is a malignant epithelial carcinoma which is intimately
associated with EBV. The latent presence of EBV affects the function of p53, Bcl-2, and survivin.
We thus investigated the relationship between EBV status, p53, Bcl-2, and survivin in biopsy
specimens from patients with primary NPC.
Experimental Design: Archival formalin-fixed, paraffin-embedded NPC biopsies were
evaluated in 80 patients treated with curative radiation from a single institution. The presence of
EBV was determined using EBER in situ hybridization, whereas p53, Bcl-2, and survivin were
assessed using immunohistochemistry.
Results: The majority of NPC specimens in this patient cohort were EBER-positive (64 of 78,
or 82%), which in turn, was significantly associated with ethnicity (P = 0.0007), and WHO
subtype 2A/2B (P = 0.04). EBER-positive tumors were also associated with p53 (P = 0.002),
Bcl-2 (P = 0.04), and nuclear survivin (P = 0.03) expression. Patients with EBER-positive NPC
fared better, with a 10-year overall survival of 68% versus 48% for EBER-negative patients
(P = 0.03). For nuclear survivin, patients with either low or high nuclear survivin fared worse
than patients with intermediate survivin expression (P = 0.05), suggesting that there is an optimal
proportion of survivin-expressing cells for best function and clinical outcome.
Conclusions: With an extended median follow-up time of 11.4 years, EBV status remains a strong
predictor for overall survival in NPC. EBV-positive NPC has strong molecular associations
with p53, Bcl-2, and survivin expression. Furthermore, we provide clinical data revealing the
potentially dual nature of survivin in predicting clinical outcome.
Nasopharyngeal
carcinoma (NPC) is a malignant epithelial
carcinoma of the head and neck site, with incidences of 20 to
30 per 100,000 people in areas such as Southeast Asia (a region
of >100 million people) and the Mediterranean basin (a region
of >300 million people) (1). NPC is anatomically located in
close proximity to the base of the skull, rendering it a significant
challenge for conventional and experimental treatments. In
Authors’ Affiliations: Departments of 1Medical Biophysics and 2Radiation
Oncology, University of Toronto, 3Division of Applied Molecular Oncology, Ontario
Cancer Institute, 4Clinical Study Coordination and Biostatistics, Ontario Cancer
Institute/Princess Margaret Hospital, University Health Network, 5Department of
Pathology, Mt. Sinai Hospital, and Departments of 6 Surgical Oncology and
7
Radiation Oncology, Princess Margaret Hospital, University Health Network,
Toronto, Ontario, Canada
Received 3/9/06; revised 5/25/06; accepted 7/25/06.
Grant support: Canadian Institutes of Health Research, the Ontario Cancer
Research Network, and the Elia Chair in Head & Neck Cancer Research. K. Yip is a
recipient of a Natural Sciences and Engineering Research Council of Canada
scholarship.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Fei-Fei Liu, Department of Radiation Oncology, Princess
Margaret Hospital/Ontario Cancer Institute, 610 University Avenue, Room 10-203,
Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2123; Fax: 416-946-4586;
E-mail: Fei-Fei.Liu@ rmp.uhn.on.ca.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-0571
Clin Cancer Res 2006;12(19) October 1, 2006
fact, inadequate tumor volume coverage may be partially
responsible for the modest 5-year overall survival (OS) rate
of f70% when patients are treated with conventional
radiotherapy alone (2 – 4). Although intensity-modulated radiotherapy does improve local control, the risk of distant
metastases remains high at f40% (2). A predilection for
developing distant metastases, a young median age of presentation (at f50 years), and the intimate association with
EBV are features that render NPC a unique head and neck
epithelial malignancy (5).
The latent presence of EBV is unique to malignancies such
as NPC, Burkitt’s lymphoma, Hodgkin’s lymphoma, peripheral T cell lymphomas, natural killer cell lymphomas, and
gastric carcinomas (1). Approximately 75% to 81% of patients
with NPC worldwide harbor the EBV genome, which is
present in the type II latency. This form of latency is
characterized by the expression of a limited set of viral genes,
including EBNA1, LMP1, LMP2, and EBER (1). These genes
almost certainly contribute to tumor development. EBNA1 for
example, binds and inhibits USP7/HAUSP. USP7 normally
deubiquitinates the tumor suppressor p53, thereby stabilizing
it and facilitating p53-mediated growth suppression and
apoptosis (6). Therefore, the presence of EBNA1 could
compromise the normal function of p53. LMP1 has been
shown to up-regulate Bcl-2 and antiapoptotic Bcl-2 family
members, possibly through nuclear factor nB (1). LMP1 also
induces the overexpression of survivin (7). Survivin has been
5726
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
EBV, p53, Bcl-2, and Survivin in NPC
implicated in both cell death inhibition and mitotic regulation (8), by binding to the X-linked inhibitor of apoptosis in
the cytoplasm to synergistically inhibit apoptosis (9), and by
associating with the mitotic apparatus. EBV-induced upregulation of survivin leads to increased cell proliferation
(7), whereas inhibition of survivin results in catastrophic
defects in mitosis, failed cytokinesis, and polyploidy (8). The
significance of p53, Bcl-2, and survivin in EBV-negative NPC
remains undetermined.
p53, Bcl-2, and survivin are dysregulated in other head and
neck cancers. p53, for example, is associated with poor patient
outcome in laryngeal squamous cell carcinomas and oral premalignant lesions (10). Bcl-2 is up-regulated in head and neck
squamous cell carcinomas, and inhibits chemotherapy-induced
apoptosis in these tumors (11, 12). Survivin is also up-regulated
in head and neck squamous cell carcinomas, and has been
associated with poor survival in esophageal cancer (11, 13).
Given the above reports, we evaluated a cohort of patients
with NPC who were primarily treated with radiation therapy
from a single North American institution. Our objective was to
determine the prognostic significance of key molecular factors,
specifically EBV, p53, Bcl-2, and survivin, in NPC.
Table 1. Clinical and molecular characteristics of
patients
Characteristic
Age (y)
V50
>50
Gender
Female
Male
Ethnicity
Asian
Chinese
Black
Caucasian
Stage
I
II
III
IV
WHO classification
1
2A
2B
EBER
Missing
0
1
2
3
p53 (%)
<10
z10
Bcl-2 (%)
0
>0
Nuclear survivin (%)
0 to <5
5-25
>25
Materials and Methods
Patients. Between the period of 1985 and 1992, 198 patients
with NPC were treated with curative intent at the Princess Margaret
Hospital (3). Archival formalin-fixed and paraffin-embedded tumors
were obtained from 80 of these patients prior to treatment. The
clinical characteristics of this subgroup of patients have been
previously published (14, 15). As shown in Table 1, the majority
of patients were male (60 of 80, or 75%), of Asian/Chinese ethnicity
(49 of 80, or 61%), had locally advanced stage III or IV disease
(66 of 80, or 83%), and had undifferentiated WHO type 2B
NPC (60 of 80, or 75%). All patients were treated with curative
intent. The median radiotherapy dose was 66 Gy/33 fractions/6.5
weeks delivered using a parallel pair opposed field technique. Only
four patients received additional chemotherapy, delivered in a
neoadjuvant fashion.
Tissue specimens. For each patient’s tumor block, 4-Am sections
were cut and mounted onto microscopy slides. One representative
section from each block was stained with H&E (Fig. 1A), and then
reviewed by a single pathologist (C. MacMillan) to confirm the
diagnosis and WHO classification. Keratinizing squamous cell carcinoma is classified as WHO type I, nonkeratinizing differentiated
carcinoma as type 2A, and nonkeratinizing undifferentiated carcinoma
as type 2B (also known as type 3) NPC (5). Due to the small size of
these biopsy specimens, not every patient had sufficient remaining
tumor tissues for all these variables to be evaluated.
Detection of latent EBV infection. The latent presence of EBV in NPC
biopsies was detected using EBER in situ hybridization as previously
described (14, 15). Briefly, sections were predigested with proteinase K
(1:30), probed with a fluorescein-conjugated EBV (EBER) peptide
nucleic acid probe (DAKO, Mississauga, Ontario, Canada) for 2 hours
at 55jC, and detected with an alkaline phosphatase – conjugated antifluorescein antibody (DAKO). 5-Bromo-4-chloro-3-indolylphosphate
nitroblue tetrazolium was used as a chromogen, and several slides were
counterstained with a light hematoxylin stain. Dark brown staining was
identified as the positive hybridization signal. Scoring was defined as
follows: 0 (negative), 1 (<10% positive tumor nuclei), 2 (10-50%
positive tumor nuclei), or 3 (>50% positive tumor nuclei). Tumors that
were scored 1, 2, or 3 were considered EBER-positive.
Immunohistochemistry for p53, Bcl-2, and survivin. For immunohistochemistry, microwave antigen retrieval was used in combination with
www.aacrjournals.org
N
41
39
20
60
6
43
1
30
4
10
37
29
14
6
60
2
14
16
21
27
46
34
32
48
32
37
11
the Level-2 Ultra Streptavidin (horseradish peroxidase) system (Signet
Laboratories, Dedham, MA). Detection of p53, Bcl-2, and survivin
utilized the mouse monoclonal antihuman p53 (clone PAb 1801, 1:100
dilution; Novocastra Laboratories, Newcastle upon Tyne, England),
mouse monoclonal antihuman Bcl-2 oncoprotein (clone 124, 1:50
dilution; DakoCytomation, Carpinteria, CA), and rabbit polyclonal
antisurvivin (NB 500-201, Lot 9, 1:50 dilution; Novus Biologicals,
Littleton, CO) antibodies, respectively.
In order to determine the percentage of positive tumor cells, light
microscopy was used to count at least 300 tumor cells in the three
most densely staining fields (400). For p53, a tumor was considered positive when z10% of tumor nuclei expressed p53. For
Bcl-2, a tumor was considered positive when >0% of tumor cells
expressed Bcl-2. For survivin, a tumor was considered positive for
nuclear survivin when z5% of tumor nuclei expressed the protein,
and highly positive for nuclear survivin when >25% of tumor nuclei
expressed the protein. A tumor was considered positive for cytoplasmic survivin when >0% of tumor cells expressed the cytoplasmic
protein.
Statistical analyses. OS was defined as the time of diagnosis to date
of death; disease-free survival (DFS) was defined as the time of
diagnosis to date of first failure. The OS and DFS estimates over time
were calculated using the Kaplan-Meier method (16). Associations
between EBER, p53, Bcl-2, or survivin with OS or DFS were analyzed
using the log-rank test. Correlations between EBER and p53, Bcl-2, or
survivin were analyzed using m2 test, as were correlations between the
molecular factors with ethnicity and WHO.
5727
Clin Cancer Res 2006;12(19) October 1, 2006
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
Imaging, Diagnosis, Prognosis
Fig. 1. EBER, p53, Bcl-2, and survivin expression in NPC patient biopsies. One section from each tumor biopsy was stained with H&E (A). In order to detect the latent
presence of EBV, EBER in situ hybridization was done (B). p53 (C), Bcl-2 (D), nuclear survivin (N. Survivin ; E), and cytoplasmic survivin (C. Survivin ; F) were detected using
immunohistochemistry. Bar, 10 Am; black arrow, positively staining tumor cells.
Results
Expression of molecular markers in NPC. As shown in Table 1,
the majority of these patients’ tumors are EBV-positive,
whereby EBER in situ hybridization signal intensity (Fig. 1B)
was detected in 64 of 78 (82%) of these biopsies (in two
samples, EBER could not be done due to insufficient tumor
tissues). These EBER-positive NPC’s were associated with
ethnicity, in that more Asian/Chinese patients (45 of 48, or
94%) had EBER-positive tumors than African/Caucasian
patients (19 of 30, or 63%; P = 0.0007). In addition, the
latent presence of EBV was more common in WHO type 2
(56 of 65, or 86%), than in WHO type 1 (8 of 13 or 61%)
NPC (P = 0.04).
Overexpression of p53, defined by nuclear immunostaining
for p53 in z10% of tumor nuclei, was observed in 34 of 80
(43%) of NPC specimens (Table 1; Fig. 1C). p53 overexpression was associated with EBV in that only 1 of 14 (7%)
of EBER-negative samples overexpressed p53 (Table 1; Fig. 2A).
In contrast, 33 of 64 (52%) of the EBER-positive NPC biopsies
also overexpressed p53 (Table 1; Fig. 2A), which is statistically
significant (P = 0.002). Similarly, more Asian/Chinese patients
(27 of 49, or 55%) had p53 overexpressing tumors than
African/Caucasian patients (7 of 31, or 23%; P = 0.004).
Similar to p53, Bcl-2 expression (Fig. 1D) was associated with
the presence of EBV in that 5 of 14 (36%) EBER-negative
biopsies expressed Bcl-2 (Table 1; Fig. 2B). In contrast, 42 of 64
(66%) EBER-positive tumors were also Bcl-2-positive (P = 0.04;
Table 1; Fig. 2B). Bcl-2 expression was more commonly
observed in Asian/Chinese patients (33 of 49, or 67%), as
opposed to a lower frequency in African/Caucasian patients
(15 of 31, or 48%), although the latter association was not
statistically significant (P = 0.09).
Both nuclear and cytoplasmic survivin expressions were
assessed using immunohistochemistry (Fig. 1E and F).
Expression of nuclear survivin (defined as z5% tumor nuclei
immunostaining for survivin) was associated with EBVpositivity in that only 5 of 14 (36%) of EBER-negative NPC
biopsies had nuclear survivin expression (Table 1; Fig. 2C). In
contrast, 43 of 64 (67%) EBER-positive tumors also expressed
nuclear survivin (Table 1; Fig. 2C; P = 0.03). Not surprisingly,
nuclear survivin expression correlated strongly with cytoplasmic survivin in that only 13 of 36 (36%) NPC biopsies with
no cytoplasmic survivin had nuclear survivin expression. In
contrast, 35 of 44 (80%) NPC biopsies expressed both
cytoplasmic and nuclear survivin (P < 0.0001). NPC biopsies
from Asian/Chinese patients were more commonly nuclear
Clin Cancer Res 2006;12(19) October 1, 2006
survivin-positive (35 of 49, or 71%) than biopsies from
African/Caucasian patients (13 of 31, or 42%; P = 0.009).
Prognostic value of molecular variables. The median followup time for this group of patients has now reached 11.4 years,
with 10-year OS and DFS of 63% and 50%, respectively.
Similar to our previous observations (14, 15), EBER-positive
patients continue to experience a statistically significant better
OS compared with EBER-negative patients (Fig. 3A). Specifically, the 10-year OS rates were 68% versus 48% for the
Fig. 2. EBER is associated with p53 (A), Bcl-2 (B), and nuclear survivin (C) in
NPC patient biopsies. Tumors with EBER scores of 1, 2, or 3 were considered to be
EBER-positive. For p53, Bcl-2, and nuclear survivin, tumors were scored positive
when z10%, >0%, and z5% of tumor cells were positively stained, respectively. The
number and percentage (in parentheses) of EBER-positive and EBER-negative
NPC patients that are positive for p53 (A), Bcl-2 (B), or nuclear survivin (C) are
shown.
5728
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
EBV, p53, Bcl-2, and Survivin in NPC
detailed molecular pathology analyses (14, 15). Our previous
observations noted the strong association of positive EBER
status with superior OS and DFS (14), and the negative
prognostic indicator of absent p16 expression in NPC (15).
Our current study uses a similar population of patients, but
now with an extended follow-up time of 11.4 years (versus
4.8 years [14] and 8.4 years [15] for the previous studies). In
this current study, we continue to observe that patients with
EBV-positive NPC experience better OS than EBV-negative
NPC. The difference in DFS rates is no longer observed with
the longer follow-up time (Fig. 3B). (Similar observations
were recently reported on 2,687 patients with NPC treated in
Hong Kong [17], wherein the DFS curves never plateau,
indicating that patients with EBV-positive NPC continue to
relapse, even several years after the completion of treatment).
EBV-positive tumors expressed p53, Bcl-2, and nuclear
survivin. EBV-negative tumors, on the other hand, tend not
to express p53, Bcl-2, or survivin. Similar to other reports,
EBV-positive NPC tumors are associated with both ethnicity
(Asian/Chinese versus Africans/Caucasians), and WHO subtype 2A and 2B (versus type 1 NPC; ref. 18). Most significant,
however, is the first demonstration of a polynomial prognostic factor whereby intermediate nuclear survivin expression is
associated with better OS compared to patients with a low or
high proportion of survivin immunostaining.
Our observation that WHO type 1 NPC is more commonly
EBV-negative, which in turn, is associated with reduced OS
(Fig. 3A) is corroborated by a large American population –
based study, whereby the outcome for 5,069 patients with
NPC was significantly associated with histology (19). WHO
Fig. 3. Kaplan-Meier plots for OS (A) and DFS (B) as a function of EBER status.
EBER in situ hybridization was done on NPC patient biopsies and scored. Tumors
with EBER scores of 1, 2, or 3 were considered to be EBER-positive; EBER-negative
tumor had a score of 0. A, the 5- and 10-year OS rates for EBER-positive tumors
were 80% and 68%, respectively. The 5- and 10-year OS rates for EBER-negative
tumors were 56% and 48%, respectively (P = 0.03). B, the 5- and 10-year DFS
rates for EBER-positive tumors were 62% and 54%, respectively.The 5- and 10-year
DFS rates for EBER-negative tumors were both 43% (P = 0.2).
EBER-positive and -negative groups of patients, respectively
(P = 0.03). Interestingly, the 10-year DFS rates were also
superior in the EBER-positive group (54% versus 43%), but
this relationship was no longer statistically significant (P =
0.2; Fig. 3B).
Neither p53 nor Bcl-2 expression provided any prognostic
value for either OS or DFS for this group of patients. However,
nuclear survivin had an intriguing ‘‘polynomial’’ relationship in
that the OS hazard was lowest when the percentage of tumor
nuclei expressing survivin was 10%, but this hazard increases as
the percentage decreases from 10% to 0%, or when the
percentage increases from 10% to 80% (Fig. 4A). This
relationship is reflected in the actuarial survival graph
(Fig. 4B), which shows that OS was superior when the
percentage of tumor nuclei expressing survivin was in the
intermediate group (z5% to V25%) versus the low (<5%) or
high (>25%) expressors (Table 2). For these two disparate
groups, the 5- and 10-year actuarial OS rates were 84% versus
65% and 74% versus 53%, respectively (P = 0.05).
Discussion
We have previously reported the largest North American
series of NPC samples from a single institution subjected to
www.aacrjournals.org
Fig. 4. Predicted hazard for survival (A) and OS (B) as a function of nuclear
survivin levels. Survivin immunohistochemistry was done on NPC patient biopsies.
Tumors were grouped into low (<5% of tumor nuclei), medium (5-25% of tumor
nuclei), and high (>25%) nuclear survivin groups. A, a polynomial relationship is
observed between hazard for OS, as a function of nuclear survivin wherein the
minimum risk was observed at 10% positivity. As the nuclear survivin score either
decreased to <10%, or increased to >10%, the OS hazards increased. B, the 5- and
10-year actuarial OS rates were 84% and 74%, respectively, for the intermediate
nuclear survivin group, versus 65% and 53% for the patients with low and high
nuclear survivin (P = 0.05 for comparison between intermediate versus high and
low).
5729
Clin Cancer Res 2006;12(19) October 1, 2006
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
Imaging, Diagnosis, Prognosis
Table 2. Cox regression model incorporating
survivin, age, and stage with OS
Variables
Nuclear survivin alone
Combined model
Age
Stage
Nuclear survivin
with age and stage
Hazards ratio
(confidence interval)
P value
2.04 (0.98-4.24)
0.06
1.05 (1.02-1.08)
14.1 (1.9 to >30)
2.3 (1.1-4.9)
0.0004
0.01
0.02
NOTE: Results of the Cox regression model incorporating age (at
time of diagnosis), and stage dichotomized (I/II versus III/IV),
with nuclear survivin dichotomized as either (a) <5% or >25%
versus (b) >5% to V25%.
type 1 NPC had a poorer outcome with a 5-year OS rate of
37%, as opposed to 65% for patients with either type 2A or
2B histologies (19). As we have stated previously (15), we
believe that EBER-positive and EBER-negative NPC are two
distinct clinical and biological entities, hence, EBER status
should be used as a stratification variable for future NPC
clinical trials.
These clinical data are borne out by biology, whereby the
protein expression profile of EBV-positive tumors, which
overexpress p53, Bcl-2, and nuclear survivin, are distinct from
that of EBV-negative tumors (Fig. 2). Other groups have also
reported a significant association between p53 and Bcl-2
expression in NPC (20, 21), along with an association between
EBV with both p53 and Bcl-2 overexpression in adult (22) and
pediatric (23) NPC. Thus, one would surmise that the optimal
treatment modalities or regimens for EBV-negative and EBVpositive NPC should be different, although we might require
additional insights from future gene expression microarray
studies to fully elucidate the distinct molecular profiles
between EBV-positive and EBV-negative NPC.
The association between p53 overexpression and EBV status
is reproducibly reported (24, 25), but its underlying mechanism is complex. The p53 gene is rarely mutated in primary
NPC (26), providing evidence that latent EBV genes are likely
responsible for alterations in p53 expression. The EBV protein
EBNA1 binds USP7, thereby preventing deubiquitination of
p53 (6). Ablation of USP7, however, has recently been shown
to result in p53 accumulation (27). This might be explained
by USP7 stabilizing Mdm2, thereby preventing Mdm2mediated p53 degradation (27). The mechanism of EBVinduced p53 overexpression is further complicated by other
EBV gene products such as BZLF1 and EBNA5, which can also
bind p53 (28, 29). In addition, EBNA2 can also promote p53
phosphorylation and induce p21 expression (30). Hence, it is
likely that multiple EBV players interact with p53, ultimately
resulting in its nuclear accumulation, and probable compromised function.
Bcl-2 overexpression has been reported in f80% of NPC
cases, and is also associated with EBV (31). The EBV oncogenic
protein, LMP1, can directly induce Bcl-2 expression (1). Bcl-2targeted experimental molecular therapies for NPC, such as
BimS gene therapy (32), or Bcl-2 antisense therapy (33), have
been shown to be highly effective, and hence, would be
Clin Cancer Res 2006;12(19) October 1, 2006
predicted to have a greater benefit for patients with EBVpositive NPC.
One of the most intriguing observations in this study relates
to the poor clinical outcome when the proportion of nuclear
survivin – expressing tumor cells is either too low or too high.
Survivin itself seems to be a multifunctional protein, playing
important roles in both mitotic regulation and apoptosis
inhibition (8). In terminally differentiated normal tissues,
survivin expression is undetectable, but survivin overexpression
has been observed in numerous human malignancies (8). Thus,
there has been substantial interest in survivin as a therapeutic
target. Recent evidence suggests that survivin gene transcription
is linked to mitotic progression, and because cancer cells are
globally deregulated in cell cycling, survivin is overexpressed
during all phases of the cell cycle (8). Survivin can be directly
up-regulated via LMP1 (7) and the WNT-h-catenin signaling
pathway (34), which is known to be activated in NPC from
microarray expression studies (35). Of note, wild-type p53 can
transcriptionally repress survivin (8), therefore, the dysfunction
of p53 in NPC (mediated via EBV proteins) might also
contribute to survivin up-regulation because p53 itself is rarely
mutated in NPC (26).
The inhibition of survivin can lead to aberrant mitotic
progression, with supernumerary centrosomes, multipolar
mitotic spindles, failed cytokinesis, multinucleation, premature
sister-chromatid separation, dysregulation of spindle-checkpoint activation, and/or apoptosis (8). Therefore, although very
low survivin levels would normally prevent functional cell
division, the combination of low survivin with resistance to cell
death (mediated by the overexpression of antiapoptotic
proteins such as Bcl-2) in NPC may contribute to genomic
instability.
Conversely, increased survivin levels can preserve spindle
function against microtubule poisons, promoting resistance to
chemotherapy (8), and promoting cell proliferation (7).
Similarly, high cytoplasmic survivin levels (which is associated
with high nuclear survivin expression) could lead to the
inhibition of proapoptotic caspases (8, 9). Interestingly, a
recent study showed that absent caspase-3 activation predicted
for rapid fatality in patients with NPC (36).
The literature regarding the prognostic value of survivin is
currently confusing. Some articles report that increased
nuclear survivin predicts for poor outcome (37, 38), or
improved outcome (39, 40), or has no effect (41, 42). Some
of this controversy has been attributed to technical reasons
(43), but we submit that perhaps another reason for the
inconsistencies reported in the literature might be attributed
to this unusual polynomial relationship of nuclear survivin
with biological and clinical outcome. Most investigators in the
prognostic field of research think of prognostic variables as
binary, dichotomized, or even continuous variables, but in a
linear fashion. We propose a paradigm shift, offering nuclear
survivin as a variable whereby both too high and too low a
proportion of survivin-expressing tumor cells is associated
with biological aggressiveness, translating to poorer clinical
outcome. A similar phenomenon has been encountered in
bladder cancer, in which both elevated and absent pRb
expression is associated with lower survival rates (44). Other
underlying factors may be responsible for these nonlinear
observations; for example, elevated pRb is associated with loss
of p16 in bladder cancers (45).
5730
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
EBV, p53, Bcl-2, and Survivin in NPC
The dual nature of nuclear survivin is not an uncommon
biological phenomenon; many other proteins possess the
ability to function as both tumor promoters and tumor suppressors. Members of the claudin family, such as claudin-4,
may increase or decrease invasiveness depending on the
molecular circuitry of the cell (46). Nucleophosmin inactivation leads to unrestricted centrosome duplication and genomic
instability (47), but nucleophosmin overexpression leads to
IRF-1 (antioncogenic transcription factor) inhibition and can
result in malignant transformation (48). In addition, oncogenes such as Myc and nuclear factor nB may induce apoptosis,
the latter being sometimes required for p53-mediated cell death
(49, 50).
This novel observation would obviously require corroboration by other groups, and in other human malignancies. The
total number of patients in each of the three subgroups is
somewhat limited, hence, the data are not that robust. In
addition, this specific polynomial observation was not part of
our original hypothesis, but instead, was a data-driven
evaluation.
In conclusion, this study confirms the effect of EBV
status on OS, and documents that p53, Bcl-2, and nuclear
survivin are overexpressed proteins in EBV-positive NPC. The
potentially dual nature of survivin, which predicts for poor
patient outcome at either high or low proportional expression, has been observed for the first time, in a clinical
setting.
Acknowledgments
We thank Drs. Chris Richardson and Wen-Chen Yeh for insightful discussions
regarding the data.
References
1. Young LS, Rickinson AB. Epstein-Barr virus: 40 years
on. Nat Rev Cancer 2004;4:757 ^ 68.
2. Lee N, Xia P, Quivey JM, et al. Intensity-modulated
radiotherapy in the treatment of nasopharyngeal carcinoma: an update of the UCSF experience. Int J Radiat
Oncol Biol Phys 2002;53:12 ^ 22.
3. Chow E, Payne D, O’Sullivan B, et al. Radiotherapy
alone in patients with advanced nasopharyngeal cancer: comparison with an intergroup study. Is combined modality treatment really necessary? Radiother
Oncol 2002;63:269 ^ 74.
4. Waldron J, Tin MM, Keller A, et al. Limitation of conventional two dimensional radiation therapy planning
in nasopharyngeal carcinoma. Radiother Oncol 2003;
68:153 ^ 61.
5. DeVita VT, Hellman S, Rosenberg SA, editors. Cancer: principles and practice of oncology. 7th ed. New
York: Lippincott Williams & Wilkins; 2005.
6. Saridakis V, Sheng Y, Sarkari F, et al. Structure of
the p53 binding domain of HAUSP/USP7 bound
to Epstein-Barr nuclear antigen 1 implications for
EBV-mediated immortalization. Mol Cell 2005;18:
25 ^ 36.
7. FaqingT, Zhi H, LiqunY, et al. Epstein-Barr virus LMP1
initiates cell proliferation and apoptosis inhibition via
regulating expression of Survivin in nasopharyngeal
carcinoma. Exp Oncol 2005;27:96 ^ 101.
8. Altieri DC.Validating survivin as a cancer therapeutic
target. Nat Rev Cancer 2003;3:46 ^ 54.
9. Dohi T, Okada K, Xia F, et al. An IAP-IAP complex inhibits apoptosis. J Biol Chem 2004;279:
34087 ^ 90.
10. Nylander K, Dabelsteen E, Hall PA. The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck. J Oral Pathol Med 2000;
29:413 ^ 25.
11. Sharma H, Sen S, Mathur M, Bahadur S, Singh N.
Combined evaluation of expression of telomerase,
survivin, and anti-apoptotic Bcl-2 family members in
relation to loss of differentiation and apoptosis in human head and neck cancers. Head Neck 2004;26:
733 ^ 40.
12. Andrews GA, Xi S, Pomerantz RG, et al. Mutation of
p53 in head and neck squamous cell carcinoma correlates with Bcl-2 expression and increased susceptibility to cisplatin-induced apoptosis. Head Neck 2004;
26:870 ^ 7.
13. Kato J, Kuwabara Y, Mitani M, et al. Expression of
survivin in esophageal cancer: correlation with the
prognosis and response to chemotherapy. Int J Cancer 2001;95:92 ^ 5.
14. ShiW, Pataki I, MacMillan C, et al. Molecular pathology parameters in human nasopharyngeal carcinoma.
Cancer 2002;94:1997 ^ 2006.
www.aacrjournals.org
15. Makitie AA, MacMillan C, Ho J, et al. Loss of p16
expression has prognostic significance in human nasopharyngeal carcinoma. Clin Cancer Res 2003;9:
2177 ^ 84.
16. Dinse GE, Lagakos SW. Nonparametric estimation
of lifetime and disease onset distributions from incomplete observations. Biometrics 1982;38:921 ^ 32.
17. Lee AW, Sze WM, Au JS, et al. Treatment results for
nasopharyngeal carcinoma in the modern era: the
Hong Kong experience. Int J Radiat Oncol Biol Phys
2005;61:1107 ^ 16.
18. Raab-Traub N. Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol 2002;12:431 ^ 41.
19. Marks JE, Phillips JL, Menck HR. The National Cancer Data Base report on the relationship of race and
national origin to the histology of nasopharyngeal carcinoma. Cancer 1998;83:582 ^ 8.
20. Niemhom S, Kitazawa S, Murao S, Kunachak S,
Maeda S. Co-expression of p53 and bcl-2 may correlate to the presence of Epstein-Barr virus genome and
the expression of proliferating cell nuclear antigen in
nasopharyngeal carcinoma. Cancer Lett 2000;160:
199 ^ 208.
21. Sheu LF, Chen A, Meng CL, Ho KC, Lin FG, LeeWH.
Analysis of bcl-2 expression in normal, inflamed, dysplastic nasopharyngeal epithelia, and nasopharyngeal
carcinoma: association with p53 expression. Hum
Pathol 1997;28:556 ^ 62.
22. Yang HJ, Cho YJ, Kim HS, Chang MS, Sung MW,
Kim WH. Association of p53 and BCL-2 expression
with Epstein-Barr virus infection in the cancers of
head and neck. Head Neck 2001;23:629 ^ 36.
23. Preciado MV, Chabay PA, De Matteo EN, Gismondi
MI, Rey G, Zubizarreta P. Epstein Barr virus associated
pediatric nasopharyngeal carcinoma: its correlation
with p53 and bcl-2 expression. Med Pediatr Oncol
2002;38:345 ^ 8.
24. Niedobitek G, Agathanggelou A, Barber P,
Smallman LA, Jones EL, Young LS. P53 overexpression and Epstein-Barr virus infection in undifferentiated and squamous cell nasopharyngeal
carcinomas. J Pathol 1993;170:457 ^ 61.
25. Gulley ML, Burton MP, Allred DC, et al. Epstein-Barr
virus infection is associated with p53 accumulation in
nasopharyngeal carcinoma. Hum Pathol 1998;29:
252 ^ 9.
26. Effert P, McCoy R, Abdel-Hamid M, et al. Alterations of the p53 gene in nasopharyngeal carcinoma.
J Virol 1992;66:3768 ^ 75.
27. Li M, Brooks CL, Kon N, Gu W. A dynamic role of
HAUSP in the p53 ^ 2 pathway. Mol Cell 2004;13:
879 ^ 86.
28. Zhang Q, Gutsch D, Kenney S. Functional and
physical interaction between p53 and BZLF1: implica-
5731
tions for Epstein-Barr virus latency. Mol Cell Biol 1994;
14:1929 ^ 38.
29. Szekely L, Selivanova G, Magnusson KP, Klein G,
Wiman KG. EBNA-5, an Epstein-Barr virus-encoded
nuclear antigen, binds to the retinoblastoma and p53
proteins. Proc Natl Acad Sci U S A 1993;90:5455 ^ 9.
30. Lin CS, Kuo HH, Chen JY, Yang CS, Wang WB.
Epstein-Barr virus nuclear antigen 2 retards cell
growth, induces p21(WAF1) expression, and modulates p53 activity post-translationally. J Mol Biol
2000;303:7 ^ 23.
31. Lo KW,To KF, Huang DP. Focus on nasopharyngeal
carcinoma. Cancer Cell 2004;5:423 ^ 8.
32. Yip KW, Li A, Li JH, et al. Potential utility of BimS as
a novel apoptotic therapeutic molecule. Mol Ther
2004;10:533 ^ 44.
33. Yip KW, Mocanu JD, Au PY, et al. Combination bcl2 antisense and radiation therapy for nasopharyngeal
cancer. Clin Cancer Res 2005;11:8131 ^ 44.
34. Zhang T, Otevrel T, Gao Z, Ehrlich SM, Fields JZ,
Boman BM. Evidence that APC regulates survivin expression: a possible mechanism contributing to the
stem cell origin of colon cancer. Cancer Res 2001;61:
8664 ^ 7.
35. Sriuranpong V, Mutirangura A, Gillespie JW, et al.
Global gene expression profile of nasopharyngeal carcinoma by laser capture microdissection and complementary DNA microarrays. Clin Cancer Res 2004;10:
4944 ^ 58.
36. Oudejans JJ, Harijadi A, Cillessen SA, et al. Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal
outcome. Mod Pathol 2005;18:877 ^ 85.
37. Grabowski P, Kuhnel T, Muhr-Wilkenshoff F, et al.
Prognostic value of nuclear survivin expression in
oesophageal squamous cell carcinoma. Br J Cancer
2003;88:115 ^ 9.
38. Martinez A, Bellosillo B, Bosch F, et al. Nuclear survivin expression in mantle cell lymphoma is associated
with cell proliferation and survival. Am J Pathol 2004;
164:501 ^ 10.
39. Kennedy SM, O’Driscoll L, Purcell R, et al. Prognostic importance of survivin in breast cancer. Br J Cancer
2003;88:1077 ^ 83.
40. Trieb K, Lehner R, Stulnig T, Sulzbacher I, Shroyer
KR. Survivin expression in human osteosarcoma is
a marker for survival. Eur J Surg Oncol 2003;29:
379 ^ 82.
41. Agui T, McConkey DJ, Tanigawa N. Comparative
study of various biological parameters, including expression of survivin, between primary and metastatic
human colonic adenocarcinomas. Anticancer Res
2002;22:1769 ^ 76.
42. Cohen C, Lohmann CM, Cotsonis G, Lawson D,
Clin Cancer Res 2006;12(19) October 1, 2006
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
Imaging, Diagnosis, Prognosis
Santoianni R. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis.
Mod Pathol 2003;16:574 ^ 83.
43. Li F,Yang J, Ramnath N, Javle MM,Tan D. Nuclear or
cytoplasmic expression of survivin: what is the significance? Int J Cancer 2005;114:509 ^ 12.
44. Cote RJ, Dunn MD, Chatterjee SJ, et al. Elevated
and absent pRb expression is associated with bladder
cancer progression and has cooperative effects with
p53. Cancer Res 1998;58:1090 ^ 4.
45. Benedict WF, Lerner SP, Zhou J, Shen X, Tokunaga
H, Czerniak B. Level of retinoblastoma protein expression correlates with p16 (MTS-1/INK4A/CDKN2) status in bladder cancer. Oncogene 1999;18:1197 ^ 203.
46. Morin PJ. Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer
Res 2005;65:9603 ^ 6.
47. Grisendi S, Bernardi R, Rossi M, et al. Role of nucleophosmin in embryonic development and tumorigenesis. Nature 2005;437:147 ^ 53.
Clin Cancer Res 2006;12(19) October 1, 2006
5732
4 8. Kondo T, Minamino N, Nagamura-Inoue T,
Matsumoto M, Taniguchi T, Tanaka N. Identification
and characterization of nucleophosmin/B23/numatrin
which binds the anti-oncogenic transcription factor
IRF-1 and manifests oncogenic activity. Oncogene
1997;15:1275 ^ 81.
49. Dang CV, O’Donnell KA, Juopperi T. The great MYC
escape in tumorigenesis. Cancer Cell 2005;8:177 ^ 8.
50. Perkins ND. NF-nB: tumor promoter or suppressor?
Trends Cell Biol 2004;14:64 ^ 9.
www.aacrjournals.org
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.
Prognostic Significance of the Epstein-Barr Virus, p53, Bcl-2,
and Survivin in Nasopharyngeal Cancer
Kenneth W. Yip, Wei Shi, Melania Pintilie, et al.
Clin Cancer Res 2006;12:5726-5732.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/12/19/5726
This article cites 49 articles, 10 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/12/19/5726.full.html#ref-list-1
This article has been cited by 11 HighWire-hosted articles. Access the articles at:
/content/12/19/5726.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on June 12, 2017. © 2006 American Association for Cancer
Research.