Survivin and laryngeal carcinoma prognosis: nuclear localization

Histopathology 2012 DOI: 10.1111/j.1365-2559.2012.04217.x
Survivin and laryngeal carcinoma prognosis: nuclear
localization and expression of splice variants
Gino Marioni,1 Marco Agostini,2,3,4 Chiara Bedin,2,4 Stella Blandamura,5 Edoardo Stellini,6
Giovanni Favero,6 Marco Lionello,1 Luciano Giacomelli,5 Silvia Burti,2,3,4 Edoardo D’Angelo,2,4
Donato Nitti,2 Alberto Staffieri1 & Cosimo De Filippis1
1
Department of Neurosciences, Otolaryngology Section, University of Padova, Padova, Italy, 2Department of Surgical,
Oncological and Gastroenterological Sciences, 2nd Surgical Clinic, University of Padova, Padova, Italy, 3Department of
Nanomedicine, The Methodist Hospital Research Institute, Houston, TX, USA, 4Instituto di Ricerca Pediatrica-Città della
Speranza, Padova, Italy, 5Department of Medicine, Anatomic Pathology Section, University of Padova, Padova, Italy, and
6
Department of Neurosciences, Odontostomatology Institute, University of Padova, Padova, Italy
Date of submission 15 November 2011
Accepted for publication 23 December 2011
Marioni G, Agostini M, Bedin C, Blandamura S, Stellini E, Favero G, Lionello M, Giacomelli L, Burti S, D’Angelo E,
Nitti D, Staffieri A & De Filippis C
(2012) Histopathology
Survivin and laryngeal carcinoma prognosis: nuclear localization and expression of splice variants
Aims: Aberrant survivin expression in cancer cells
has been associated with tumour progression, radiation ⁄ drug resistance and shorter patient survival. The
aim of the present study was to investigate survivin
expression in laryngeal carcinoma (LSCC) tissue and –
for the first time at this site – the expression of survivin
splice variants. P53 was also studied.
Methods and results: Survivin and p53 expression was
determined immunohistochemically in 86 consecutive
patients operated for LSCC. Survivin mRNA expression
was assessed by quantitative real-time polymerase
chain reaction (PCR). Hot-spot mutations in exons
5, 6, 7 and 8 of the TP53 gene were studied by
sequencing analysis. A nuclear localization for survivin
predominated. There was a significant association
between a higher nuclear survivin expression and
LSCC recurrence (P = 0.046). Disease-free survival
(DFS) for LSCC patients with a nuclear survivin
expression >7.0% was shorter than in cases whose
expression was £7.0% (P = 0.05). Wild-type survivin
correlated significantly with nuclear survivin expression (P = 0.02). p53 expression was associated with
the co-expression of wild-type survivin and survivin-2B
(P = 0.01).
Conclusions: Nuclear expression of survivin appears to
influence LSCC aggressiveness, a higher nuclear survivin expression correlating with a higher recurrence
rate and a shorter DFS. Wild-type survivin was the
most frequently detected splice variant in LSCC tissues.
Keywords: isoforms, laryngeal carcinoma, p53, prognosis, survivin
Abbreviations: BIR, baculoviral IAP repeat; DFS, disease-free survival; IA, image analysis; IAP, inhibitors
of apoptosis proteins; LSCC, laryngeal squamous cell carcinoma; OS, overall survival; PCR, polymerase chain
reaction, ROC, receiver operating curve
Introduction
Survivin is the smallest mammalian member of the
family of inhibitors of apoptosis proteins (IAP), the ‘old’
caspase inhibitors.1 Survivin is structurally unique
among the IAPs, being a 142-amino acid, 16.5-kDa
protein encoded by a single gene located on the human
17q25 chromosome (3% of the distance from the
Address for correspondence: G Marioni MD, Department of Neurosciences, Otolaryngology Section, Padova University, Italy.
e-mail: [email protected]
2012 Blackwell Publishing Limited.
2
G Marioni et al.
telomere), consisting of three introns and four exons,
and existing physiologically as a functional homodimer.2 Survivin contains a single baculoviral IAP repeat
(BIR) domain, shared by other IAPs, which contributes
to its function in inhibiting apoptosis. However, instead
of a carboxyl terminal ring finger, as shared by the
others, survivin contains an extended carboxyl terminal alpha-helical coiled coil thought to be important for
its interaction with microtubules, hence its role in cell
division. Survivin is expressed in the G2 ⁄ M phase of
the cell cycle to support the rapidly dividing cell
machinery;3 some investigators have suggested that
the primary function of survivin lies in controlling cell
division, rather than in inhibiting apoptosis.2 The more
probable scenario is that tumours globally exploit the
multifaceted biology of survivin to the greatest advantage in cell proliferation, survival and adaptation.
Consistent with this model, survivin dysregulation
profoundly affects mitotic transitions in tumour cells,
maintaining aneuploid cell viability, bypassing cellcycle checkpoints, promoting resistance to microtubule-targeting agents and cooperating with oncogenes
for disease progression.1 Alternative splicing of the
survivin gene transcript produces a number of different
splice variant mRNAs, thus encoding different proteins.
Five splice variants of human survivin have been well
described: wild-type survivin, survivin-2A, survivin-2b,
survivin-3b and survivin-DEx3. Recently found new
variants, which have yet to be characterized, suggest
that the survivin gene is functionally even more
diversified and strictly regulated.4
Survivin is expressed ubiquitously in fetal tissues, but
is restricted during development and seems to be
negligible in the majority of terminally differentiated
adult tissues, with the exception of thymocytes and
CD34 stem cells.5 Conversely, survivin is overexpressed
in a great variety of malignancies, including cancers of
the lung, oesophagus, breast, pancreas, ovaries, colon
and rectum, liver, stomach and bladder, malignant
melanoma and acute myeloid and acute lymphocytic
leukaemia.2 Survivin overexpression correlates with
advanced disease, shorter times to recurrence, a poorer
survival and resistance to treatment.6 Survivin expression and its prognostic significance in head and neck
carcinoma have also been investigated in recent years.5
Judging from recent studies, the therapeutic modulation of survivin is regulated critically by interaction
with prominent cell-signalling pathways [hypoxiainducible factor 1a (HIF-1a), heat shock protein 90
(HSP90), phosphoinositide-3-kinase (PI3K) ⁄ protein
kinase B (AKT), mammalian target of rapamycin
(mTOR), extracellular-regulated kinase (ERK), tumour
suppressor genes (p53, phosphatase and tensin homo-
logue – PTEN), oncogenes (Bcl-2, Ras, Dj-1)] and a
number of growth factors [epidermal growth factor
receptor (EGFR), vascular endothelial growth factor
(VEGF), among others].7,8 In particular, pioneering
studies by Hoffman et al.9 and Mirza et al.10 have
identified survivin as one of the relatively few known
genes to be repressed actively by wild-type p53. These
studies have been confirmed amply by others, and
discrete p53-responsive elements have been identified
in the proximal survivin promoter. Monitoring survivin
levels is now a routinely used functional assay to test
for wild-type p53 activity in cells.11
The biological functions and contributions to
cancer progression of different subcellular survivin
localizations and splice variants are much debated.
The aim of the present study was to investigate
survivin expression using immunohistochemistry, and
the expression of the wild-type survivin, survivin-2B
and survivin-DEx3 splice variants by reverse
transcription and quantitative real-time polymerase
chain reaction (PCR) (for the first time at this
anatomical site) in 86 consecutive laryngeal squamous cell carcinomas (LSCCs). p53 expression and
hot-spot mutations in exons 5, 6, 7 and 8 of the
TP53 gene were studied by immunohistochemistry
and sequencing analysis, respectively, to establish the
biological relationship between survivin and p53 in
LSCC.
Materials and methods
patients
The study was conducted on 86 consecutive cases of
LSCC (mean age of 62.8 ± 8.4 years). All patients
underwent microlaryngoscopy with laryngeal biopsy,
upper aerodigestive tract endoscopy, oesophagoscopy,
neck ultrasonography (with or without fine needle
aspiration cytology), head and neck contrast-enhanced computerized tomography and ⁄ or magnetic
resonance imaging, chest X-rays and liver ultrasonography.
Sixty-six patients had a primary partial laryngectomy
and 20 a primary total laryngectomy, all performed at
the Otolaryngology Section of Padova University. Seventy-two patients also had unilateral or bilateral cervical
lymph node dissection. The indications for neck dissection were based on currently accepted protocols.12
Postoperative radiotherapy (RT) was administered in
15 cases. Postoperative RT was indicated in cases with
one or more of the following adverse features: close or
positive surgical margins; pT4 disease; perineural or
lymphatic or vascular invasion; multiple positive nodes
2012 Blackwell Publishing Ltd, Histopathology
Survivin and laryngeal SCC prognosis
(three or more metastatic lymph nodes); extracapsular
spread or perineural involvement; N3 nodes; subglottic
extension of primary carcinoma.12 Patients’ clinicopathological characteristics (also based on the 7th
edition of the TNM Classification of Malignant Tumors13)
are reported in detail in Table 1. No distant metastases
(M) were detected at diagnosis. The mean follow-up was
61.3 ± 38.4 months. The use of biological samples was
approved by the local Ethics Committee (protocol number 448P).
Table 1. Clinicopathological characteristics of the 86 considered patients with laryngeal carcinoma (LSCC)
No. of cases
Sex
Male
Female
76
10
pT
pT1
23
pT2
32
pT3
22
pT4a
9
N
N0 (cN0+pN0)
65 (14+51)
N+*
21
3
i mm u no h i sto c h e mi s t r y
Immunohistochemical staining on formalin-fixed, paraffin-embedded tissue sections was performed with a
fully automated system (Bond-maX; Leica, Newcastle
Upon Tyne, UK). In brief, one 4-lm-thick section was cut
from each paraffin-embedded block. The sections were
deparaffinized in Bond Dewax solution (Leica, Microsystems, Newcastle Upon Tyne, UK) at 72C, rinsed in
ethanol and rehydrated in distilled water. Antigen
retrieval was performed by heating sections for 30 min
at 99C in Bond Epitope retrieval solution 1 (Leica).
Endogenous peroxidase was blocked by 3% hydrogen
peroxide before 30 min of incubation with mouse
monoclonal anti-survivin (clone D-8; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1:50) and
mouse monoclonal antihuman p53 (clone DO-7; Dako,
Glostrup, Denmark; ready-to-use). Specimens were then
washed with phosphate-buffered saline (pH 7.0) and
incubated with Bond Polymer Refine Detection Kit
(Leica Microsystems), according to the manufacturer’s
protocols. The staining was visualized with 3,3¢-diaminobenzidine (DAB) and the slides were counterstained
with Mayer’s haematoxylin. The sections were then
dehydrated, cleared and mounted. Samples from formalin-fixed, paraffin-embedded human breast tumour were
used as positive controls, and serum without the primary
antibody as a negative control.
i ma g e an al y si s ( ia )
Recurrence
Without locoregional recurrence
67
With locoregional recurrence
19
All evaluations were performed on an image analysis
workstation consisting of a conventional Zeiss Axioskop
light microscope (Zeiss, Jena, Germany) with a colour
digital, Peltier-cooled videocamera (MicroPublisher 5.0
RTV) connected to a personal computer with the ImagePro Plus version 7 for Windows image analysis
program (Media Cybernetics Inc., Bethesda, MD, USA).
In all cases, 1378 · 954 lm2 areas of tumour tissue were
evaluated comprehensively with a 495-point sampling
grid superimposed by the program on the image acquired
with a ·50 field of view. The three areas with the relatively
highest survivin expression were chosen, irrespective
of their position in the considered carcinoma tissue. The
points intercepting the positive and negative subareas
were counted for survivin (nuclear and cytoplasmic)
and p53; the proportion of the positive subarea (area
fraction) was calculated and reported as a percentage.
68
nucleic a cid e xt raction
Stage grouping
Stage I
23
Stage II
22
Stage III
18
Stage IV
23
Grading
G1
21
G2
44
G3
21
Final status
Alive without evidence of disease
Dead
18
*Pathologically positive (N1 in 6, and N2 in 15).
2012 Blackwell Publishing Ltd, Histopathology
For each sample, we used five to seven sections
about 10 lm thick from the same formalin-fixed and
4
G Marioni et al.
paraffin-embedded tissue block. The sections were
distributed between two tubes, allowing two to three
sections for DNA and four to five sections for RNA
extraction.
Prior to nucleic acid purification, the paraffin had to
be removed to enable the sample to be exposed to
proteinase K, so the paraffin was first dissolved in
xylene and, after sample precipitation and removal of
the supernatant, the residual xylene was removed by
washing with ethanol.
Total RNA and DNA were extracted using the
miRNeasy FFPE kit and the QIAmp DNA minikit
(Qiagen, Hilden, Germany), respectively, according to
the manufacturer’s protocol. The concentration and
purity of all samples were assessed using the NanoDrop
spectrophotometer (Thermo Scientific, Waltham, MA,
USA).
r e v e r s e t ra n s c r i p t i o n a n d q u a n t i t a t i v e
real-time p cr
cDNA was synthesized from 0.7 lg of total RNA using
the high-capacity cDNA reverse transcription kit
(Applied Biosystems, Foster City, CA, USA), according
to the manufacturer’s protocol.
qPCR was performed using the 7300 real-time PCR
system (Applied Biosystems) with glyceraldehyde 3phosphate dehydrogenase (GAPDH) as an endogenous
control. PCR was conducted in a final volume of 10 ll
using 1 ll of cDNA, TaqMan Universal PCR Master
Mix · 1 (Applied Biosystems) and specific TaqMan
Assay · 1 (TaqMan Gene Expression Assays; Applied
Biosystems): wild-type survivin Hs00153353_m1;
survivin 2B Hs0304574_m1; survivin DEx3
Hs0304576_m1; GAPDH 432617E. The thermal
cycling conditions included one cycle at 95C for
10 min followed by 50 cycles at 95C for 15 s and at
60C for 1 min.
Each measurement was performed in duplicate and
the average result was used for the calculations. The
results from each sample were compared with sample
RNA as a calibrator, using the 2)DDCt calculation
method.
sequencing o f tp 5 3 gene
Sequencing analysis was used to identify hot-spot
mutations in exons 5, 6, 7 and 8 of the TP53 gene.
Thirty ng of DNA were amplified in a final volume of
25 ll by using the following: PCR buffer · 1 (Promega,
Madison, WI, USA), 0.2 mm of dNTPs mix (GE Healthcare, Fairfield, CT, USA), 0.4 lm of each primer and
0.625 U of GoTaq polymerase (Promega). PCR was
conducted in a 9700 GeneAmp PCR system (Applied
Biosystems) thermal cycler. The primer and the protocol for amplification analysis were as described previously by Ganci et al.14
After PCR, samples were purified with Exo-SapIT (GE
Healthcare, Fairfield, CT, USA) and submitted to cycle
sequencing with the BigDye Terminator version
1.1 · 1 (Applied Biosystems), sequencing buffer · 1
(Applied Biosystems) and 0.16 lm of universal primers
M13 in a final volume of 20 ll. After purification with
ethylenediamine tetraacetic acid (EDTA) 125 mm and
ethanol precipitation, samples were analysed with the
3130xl Genetic Analyzer (Applied Biosystems).
statistical a nalysis
The statistical tests applied were Student’s t-test,
Fisher’s exact test, the Mann–Whitney U-test, the
non-parametric test for trend (Kruskal–Wallis modification) and Spearman’s rank correlation test, as
appropriate. The Kaplan–Meier product limit estimator,
the log-rank test and Cox’s proportional hazard regression model were also used to compare disease-free
survival times and overall survival (OS) stratified by the
different parameters analysed.
The receiver operating curve (ROC) approach (failure
versus parameter) was used to set the analytically bestfitting cut-off to binarize the continuous variable
survivin (nuclear or cytoplasmic) expression according
to the highest level of the positive likelihood ratio. A Pvalue <0.05 was considered significant. The STATA
8.1 (Stata Corp., College Station, TX, USA) statistical
package was used for all analyses.
Results
clinical outcome
Sixty-seven of the 86 cases of LSCC experienced no
disease relapse after treatment, while 19 developed
locoregional recurrences. Fisher’s exact test identified
significant differences in the distributions for lymph
node status (N0 ⁄ N+) (P = 0.002) and stage grouping
(P = 0.006), but not for pT (P = 0.54) or grade
(P = 0.20), in the two subgroups of patients with and
without locoregional carcinoma recurrences. The logrank test showed a significant difference in disease-free
survival (DFS) (in months) when patients were stratified by N and stage grouping (P = 0.003 and 0.001,
respectively). Statistical analysis failed to disclose any
significant difference in DFS when patients were
stratified by pT (log-rank test, P = 0.55) or grade
(log-rank test, P = 0.16). The final status of patients
2012 Blackwell Publishing Ltd, Histopathology
Survivin and laryngeal SCC prognosis
at the end of considered follow-up was reported by
Table 1. Mean overall survival in months was 63.1 ±
38.7 (median value 54.5 months).
s u r v i v i n an d p 5 3 immunohistochemical
e x p r e s s i o n : cl i n i c o p a t h o l o g i c a l f e a t u r e s
Epithelial cells from normal laryngeal mucosa showed
weak immunohistochemical staining for survivin in
scattered groups of cells in basal and parabasal layers.
A nuclear reaction predominated in most of the
primary LSCC specimens (Figure 1A,B). None of the
cases showed exclusively cytoplasmic survivin staining.
The immunohistochemical expression of nuclear survivin ranged from 1.0% to 35.0% (mean 9.4% ±
9.5%). Only 16 cases revealed a cytoplasmic survivin
expression, in the range of 2–70% (mean 3.5% ±
11.7%) (Table 2).
The Mann–Whitney U-test confirmed a significantly higher nuclear survivin expression in N+
than in N0 cases (P = 0.037). Statistical analysis
5
failed to disclose any significant differences in
nuclear survivin expression when these patients were
distributed by pT (non-parametric test for
trend, P = 0.62), stage grouping (non-parametric test
for trend, P = 0.19) or grade (test for trend, P =
0.29).
Statistical analysis ruled out any significant differences in cytoplasmic survivin expression when patients
were stratified by pT (non-parametric test for trend,
P = 0.82), N (Mann–Whitney U-test, P = 0.09), stage
grouping (non-parametric test for trend, P = 0.46) or
grade (test for trend, P = 0.39).
The presence of cells with clear, unequivocal nuclear
staining identified p53-positive cells. There was no
nuclear p53 expression in the squamous epithelial cells
from the normal mucosa. Nuclear p53 protein expression was found in 62 cases of LSCC (Figure 1C), and
ranged from 2.0% to 90.0% (mean 31.8% ± 34.2%).
Statistical analysis failed to disclose any significant
differences in p53 expression when the patients
considered were distributed by pT (non-parametric test
Table 2. Immunohistochemical expression of survivin and p53 according to classical clinicopathological and prognostic
parameters
Mean nuclear
survivin expression
(%) ± SD (%)
Mean cytoplasmic
survivin expression
(%) ± SD (%)
Mean p53
expression
(%) ± SD (%)
pT1
9.2 ± 9.2
4.0 ± 14.7
37.5 ± 37.9
pT2
9.4 ± 9.5
3.5 ± 13.0
30.0 ± 33.6
pT3
9.7 ± 11.1
4.6 ± 8.5
29.2 ± 32.6
pT4a
9.6 ± 7.3
0.0 ± 0.0
30.2 ± 36.5
N0 (cN0+pN0)
7.7 ± 8.4
3.4 ± 12.7
32.4 ± 35.0
14.8 ± 10.8
4.0 ± 8.0
30.0 ± 33.2
Stage I
9.2 ± 9.2
4.0 ± 14.7
37.5 ± 37.9
Stage II
8.2 ± 9.5
4.0 ± 15.3
30.9 ± 32.6
Stage III
6.8 ± 9.6
2.8 ± 6.7
26.7 ± 35.2
Stage IV
12.9 ± 9.4
3.2 ± 7.6
30.9 ± 33.2
N+
G1
8.8 ± 10.1
1.6 ± 4.7
32.4 ± 36.1
G2
8.7 ± 9.3
3.5 ± 11.9
28.7 ± 32.9
G3
11.4 ± 9.6
5.6 ± 15.6
37.7 ± 36.5
7.4 ± 7.3
4.1 ± 13.1
28.7 ± 33.7
16.3 ± 12.7
1.4 ± 3.2
42.6 ± 35.6
Without locoregional recurrence
With locoregional recurrence
SD, Standard deviation.
2012 Blackwell Publishing Ltd, Histopathology
G Marioni et al.
6
s u r vi v i n a n d p 5 3 immunohistochemical
e x p r es s io n : pr o gn o si s i n l sc c
A
500 µm
B
200 µm
C
The ROC approach was used to find the analytically
best-fitting nuclear survivin expression cut-off for
prognostic purposes, and the value calculated was
7.0% [area under the curve (AUC) = 0.70, 95%
confidence interval (CI) 0.55–0.85]. The locoregional
recurrence rate was significantly higher among LSCC
patients with a nuclear survivin expression >7.0%
(Fisher’s exact test, P = 0.046), and their DFS was
shorter than for those with a nuclear survivin expression £7.0% (log-rank test, P = 0.05) (Figure 2). The
log-rank test ruled out a significant association
between nuclear survivin expression (cut-off value
7.0%) and OS (P = 0.20).
Cytoplasmic survivin expression did not correlate
with recurrence rate (Mann–Whitney U-test, P = 0.96)
or DFS (Cox’s proportional hazard regression,
P = 0.43). The log-rank test ruled out a significant
association between cytoplasmic survivin expression
(cut-off value 50.0% calculated by ROC approach, AUC
= 0.58, 95% CI 0.48–0.65) and OS (P = 0.90).
Furthermore, statistical analysis ruled out any significant relationship between p53 expression and recurrence rate (Mann–Whitney U-test, P = 0.21) or DFS
(Cox’s proportional hazard regression, P = 0.16).
Spearman’s rank correlation test showed that p53
expression was associated significantly with cytoplasmic survivin expression (P = 0.03), but not with
nuclear survivin expression (P = 0.93).
e x p r es s io n o f m r n a s u r vi v i n t r a n s c ri p t
v a ri a n t s in l s c c ti ss u es
1.00
Among the 84 tumour samples, wild-type survivin
expression was detected in 48 samples (57.14%),
survivin-2B expression in eight samples (9.52%) and
0.75
200 µm
Survivin nuclear expression ≤7.0%
p=0.05
for trend, P = 0.37), N (Mann–Whitney U-test,
P = 0.38), stage grouping (non-parametric test for
trend, P = 0.12) or grade (test for trend, P = 0.68).
0.25
0.00
Figure 1. A, Survivin expression in normal laryngeal mucosa
(bottom left) and poorly differentiated laryngeal carcinoma (LSCC)
cells; B, same patient: nuclear survivin expression in LSCC cells; C,
p53 expression in LSCC cells.
0.50
Survivin nuclear expression >7.0%
0
50
100
150
Figure 2. Disease-free survival in laryngeal carcinoma (LSCC)
patients estimated from nuclear survivin expression; time (abscissa)
calculated in months.
2012 Blackwell Publishing Ltd, Histopathology
Survivin and laryngeal SCC prognosis
survivin-DEx3 in none. Seven samples were positive for
the expression of both wild-type survivin and survivin2B (8.33%). The median relative expression (2)DDCt) of
wild-type survivin and survivin-2B was 1.684 (range
0.025–69.889) and 3.837 (range 0.183–10.218),
respectively.
No association emerged between the expression of
survivin isoforms and clinicopathological parameters
(pT, N status, stage grouping, grading, recurrence rate
and DFS). When the association between the survivin
transcript variants and immunohistochemical expression of survivin and p53 was examined (Table 3), wildtype survivin was found to correlate significantly with
nuclear survivin expression (Spearman’s rank correlation test, P = 0.02), while the coexpression of wild-type
survivin and survivin-2B correlated significantly with
p53 expression (Mann–Whitney U-test, P = 0.01).
tp 5 3 g en e m u t at io n a na ly s is
p53 mutational status was explored at exons 5–8 in 61
tumour samples (71.8%), revealing an E198K missense mutation responsible for the substitution of a
negative- with a positive-charged amino acid, already
described in head and neck carcinoma,15 and an I255I
non-sense mutation.16
Discussion
The diagnosis and management of LSCC have improved
significantly in recent decades, but long-term survival
rates have not. Locoregional relapse after therapy is a
major cause of death in advanced LSCC despite modern
therapeutic strategies combining radio- and chemotherapy with sophisticated surgical approaches. Using
conventional clinicopathological criteria, it is often
difficult to establish a reliable prognosis and the
response to treatment of advanced LSCC.17It is therefore crucial to search for potential biomarkers applicable to LSCC that can not only reflect the biological
7
characteristics of the tumour, but also help clinicians to
arrive at a prognosis and personalize therapy for
patients.18
Because of its up-regulation in malignancies, and its
functional involvement in apoptosis as well as proliferation, survivin is currently attracting considerable
interest as a potential cancer biomarker. Survivin
expression and its prognostic role in LSCC have been
studied in only a few publications in the English
language literature.5 Dong et al.19 used immunohistochemistry to examine survivin expression in 102 cases
of LSCC: 66% of tumours were positive for mostly
cytoplasmic survivin, and its expression was associated
significantly with tumour site, poor differentiation,
tumour size, lymph node metastases, advanced stage
and a shorter disease-free and overall survival. In
2004, Pizem et al.20 again used immunohistochemistry
to investigate survivin and p53 expression in 68
archival biopsy specimens of LSCC. Survivin was
identified in the nucleus and ⁄ or cytoplasm of carcinoma cells and a strong survivin expression emerged as
an independent adverse prognostic factor. The number
of survivin-positive cells was higher in the p53-positive
group. In 2006, Marioni et al.21 studied survivin
expression in 37 LSCCs and 12 cervical lymph node
metastases. A nuclear reaction predominated in the
LSCCs. Survivin expression was significantly higher in
pN+ than in pN0 LSCCs, and in LSCCs that recurred
locoregionally than in those that did not. Survivin
expression was also significantly higher in cervical
lymph node metastases than in the corresponding
primary LSCCs, and the authors hypothesized that
survivin expression might facilitate the survival of
carcinoma cells at distant sites. Zhao et al.22 studied
survivin and CD44v6 expression immunohistochemically in 112 specimens of LSCC, finding survivin
expression in surgical margins associated with a higher
incidence of tumour progression and a worse DFS. Very
recently, Garcı́a-Fernández et al.23 determined survivin, Aurora B, Aurora A and p53 expressions in a large
Table 3. Immunohistochemical expression of survivin and p53 in patients stratified by detected survivin transcript variants (49
cases with at least one variant detected)
mRNA survivin
transcript variants
No. of
cases
Mean nuclear
survivin expression
± SD
Mean cytoplasmic
survivin expression
± SD
Mean p53
expression
± SD
Wild-type survivin
48
10.5 ± 9.9
4.4 ± 14.4
25.6 ± 31.3
Survivin-2B
8
10.8 ± 13.9
3.8 ± 7.4
6.3 ± 10.6
Coexpression of wild-type survivin and survivin-2B
7
12.3 ± 14.3
4.3 ± 7.9
5.7 ± 11.3
SD, Standard deviation.
2012 Blackwell Publishing Ltd, Histopathology
8
G Marioni et al.
series of 259 LSCCs. Survivin expression was assessable
in 234 cases: 100% of the cases showed cytoplasmic
survivin expression and 45% a nuclear-cytoplasmic
pattern. Survivin expression was associated significantly neither with considered clinicopathological
parameters nor with patients’ survival.
In the present study, we considered only surgical
specimens (not biopsies) of LSCC, and only from larynxes
treated primarily and consecutively by the same surgical
team, with a view to reducing the risk of a significant
bias deriving from the heterogeneity typical of retrospective series of head and neck carcinomas. In addition,
a computer-based IA system was used to measure
survivin expression to ensure an accurate, precise and
reproducible immunostained slide analysis. In the present cohort of consecutive patients with LSCC, N+
classification correlated strongly with a poor prognosis,
in terms of disease recurrence rate and DFS. A nuclear
survivin reaction predominated in most of the specimens
of primary LSCC considered. Nuclear survivin expression was significantly higher in N+ than in N0 primary
LSCCs. Statistical analysis identified a significant association between a higher nuclear survivin expression
and disease recurrence, and the DFS was shorter in
patients whose nuclear survivin expression was >7.0%
than in cases in which it was £7.0%. Nuclear survivin
expression in LSCC can thus be considered a potentially
useful prognostic marker for identifying patients at
higher risk of recurrent disease after treatment. Possibly
because of the limited number of considered patients, we
could not demonstrate a significant association between
nuclear survivin expression and OS.
The exact molecular mechanisms behind the predominantly nuclear localization of survivin, seen in our
experience with LSCC and as in some other tumours,
remain to be explained. Mutations in survivin’s nuclear
export signal (NES), an inhibited nuclear transport
machinery or enhanced binding to overexpressed
nuclear survivin interaction partners may contribute
to the pronounced nuclear localization of survivin.24
The biological functions and contribution to cancer
progression of different subcellular survivin localizations and splice variants are still controversial, and
very little information is available on the expression of
different survivin splice variants in head and neck
carcinoma tissue.25 In particular, the expression and
role of survivin splice variants in LSCC have yet to be
investigated. The significant relationship emerging
from our results between the relative expression of
the transcription of wild-type survivin and the protein
expression of survivin presumably indicates that its first
localization is mainly in the nucleus. The nuclear
survivin pool is suspected of controlling cell division.
Survivin functions as a subunit of the chromosomal
passenger complex (CPC) and interacts with the other
CPC subunits such as Aurora B and inner centromere
protein (INCENP) in regulating cell division. Survivin’s
dynamics increase at centromeres during G2 ⁄ M phase
transition and are regulated by microtubule attachment. Qi et al.26 found that: (i) nuclear survivin
expression correlated well with Aurora B expression
in head and neck carcinoma; (ii) head and neck
carcinoma cases strongly expressing both nuclear
survivin and Aurora B had higher numbers of mitoses;
and (iii) survivin knockdown resulted in a cell division
defect, cell growth inhibition and tumour sphere
formation largely overlapping the situation seen on
Aurora B knockdown in head and neck carcinoma
cells. Conversely, in our experience, the protein expression of p53 was associated significantly with the
coexpression of wild-type survivin and survivin-2B;
indeed, p53 proteins can increase apoptotic survivin
variant expression, reducing cell survival.27 Being
located on mitochondria, survivin-2B colocalizes and
interacts with c-tubulin in the microtubule organization centre (MTOC), as well as blocking tubulin
polymerization, and inducing mitochondria-dependent,
caspase 8-independent apoptosis.28
Survivin possesses many attributes that make it
attractive as a potential new target for treating cancer:
(i) it is minimally expressed in most normal cells and
up-regulated in malignant tissue; (ii) it is a nodal
protein, i.e. it is involved in multiple signaling mechanisms controlling tumour maintenance; (iii) targeting
it may block angiogenesis as well as tumour cell
growth; and (iv) it is responsible for resistance to
numerous types of cancer therapy.29 As survivin is not
a cell surface protein and has no intrinsic enzymatic
activity, targeting it however for therapeutic purposes
might prove difficult. Nonetheless, numerous strategies
have been used to target survivin expression ⁄ function,
by binding to the survivin promoter (e.g. YM155),
inhibiting protein translation (e.g. antisense oligonucleotides and small interfering RNA), or interfering
with survivin function (e.g. dominant-negative mutants).29,30 Survivin-based vaccines (vaccination strategies to generate an antigen-specific immune response
against survivin-bearing tumour cells31) administered
to experimental animals have been found to induce
tumour regression in several types of malignancy, such
as lung, pancreatic and prostate cancers, lymphoma
and neuroblastomas.29 Several classes of survivintargeted cancer therapeutics are currently being
evaluated in Phase I ⁄ II clinical trials (in cases of acute
myeloid leukaemia, non-small-cell lung cancer, prostate cancer, breast cancer, renal cell carcinoma,
2012 Blackwell Publishing Ltd, Histopathology
Survivin and laryngeal SCC prognosis
malignant melanoma, etc.), and include antisense
oligonucleotides (LY2181308), transcriptional repressors (terameprecol, YM155) and immunotherapy.2
Many preliminary lines of evidence indicate that
survivin inhibition by gene therapy (antisense oligonucleotides) and by small molecule inhibitors in head
and neck carcinoma cell lines increases the antitumour activity of several cytotoxic and other targeted
therapies significantly.5 Only a very limited number of
the reported experiences of survivin inhibition have
focused on LSCC cell lines, however.32–34
Our current investigation supports the notion that
nuclear survivin expression influences the aggressiveness of the LSCC phenotype, a stronger nuclear
survivin expression correlating significantly with a
higher carcinoma recurrence rate and a shorter DFS. In
our experience with quantitative real-time PCR, we first
found that wild-type survivin was the more frequently
detected splice variant in LSCC tissues; the significant
relationship between the relative expression of the
transcription of wild-type survivin and the protein
expression of survivin presumably indicates that its
primary localization in LSCC cells is in the nucleus.
Concerning efforts to deliver personalized treatments
with a view to improving the prognosis in LSCC,
further investigations are needed to establish the utility
of incorporating survivin-targeted treatments in multimodality approaches, combined with conventional
chemotherapy, or in multi-target strategies against
advanced LSCC.
Acknowledgements
This study was supported in part by a research
laboratory patronized by both the Odontostomatology
Institute (E. Stellini) and the Otolaryngology Section
(C. de Filippis) and managed by the former Department
of Medical and Surgical Specialties, University of Padova
and by grant no. 60A07-9312 ⁄ 10 (G. Marioni) from
the University of Padova. The authors thank Frances
Coburn for correcting the English version of this paper.
Conflicts of interest
The authors have no conflict of interest to declare.
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