Content uploaded by I-Wei Chang
Author content
All content in this area was uploaded by I-Wei Chang on Aug 16, 2018
Content may be subject to copyright.
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
304
J
Jo
ou
ur
rn
na
al
l
o
of
f
C
Ca
an
nc
ce
er
r
2016; 7(3): 304-313. doi: 10.7150/jca.13638
Research Paper
Necdin Overexpression Predicts Poor Prognosis in Pa-
tients with Urothelial Carcinomas of the Upper Urinary
Tract and Urinary Bladder
I-Wei Chang1,2, Yu-Hui Wang3,4, Wen-Jeng Wu5,6,7,8,9, Peir-In Liang10, Wei-Ming Li5,6,7,8,9, Bi-Wen Yeh5,6,7,8,9,
Ting-Feng Wu11, Hong-Lin He1, Steven Kuan-Hua Huang12,, Chien-Feng Li4,11,13,14,
1. Department of Pathology, E-DA Hospital, I-Shou University, Kaohsiung, Taiwan;
2. School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan;
3. Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan;
4. Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan;
5. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan;
6. Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan;
7. Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;
8. Department of Urology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan;
9. Center for Stem Cell Research, Kaohsiung Medical University, Kaohsiung, Taiwan;.
10. Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan;
11. Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan;
12. Department of Urology, Chi Mei Medical Center;
13. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan;
14. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
Corresponding authors: Chien-Feng Li, M.D., PhD., Department of Pathology, Chi Mei Medical Center, Tainan 701, Taiwan., E-mail: angelo.p@yahoo.com.tw,
or Steven Kuan-Hua Huang., M.D., Ph.D., Department of Urology, Chi Mei Medical Center, Tainan 701, Taiwan., E-mail: skhsteven@gmail.com
© Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See
http://ivyspring.com/terms for terms and conditions.
Received: 2015.08.23; Accepted: 2015.10.28; Published: 2016.01.10
Abstract
Background and Aims: Oncogenesis is a multistep process, resulting from the accumulations of
multiple mutations. Of these mutations, self-sufficiency in growth signals, i.e., disruption of cell growth
regulation, is the first episode. Nonetheless, the genes associated with cell growth dysregulation have
seldom been systematically evaluated in either urothelial carcinomas of upper urinary tract (UTUC) or
urothelial carcinomas of urinary baldder (UBUC). By data mining a published transcriptomic dataset of
UBUCs (GSE31684), we identified the NDN gene as one of the most significant of those associated with
the regulation of cell growth and found this gene is associated with advanced tumor status and meta-
static disease (GO:0001558). Accordingly, we analyzed NDN transcript and protein expression with
their clinicopathological significance.
Materials and Methods: We used real time RT-PCR to detect NDN transcript levels in 27 UTUCs
and 27 UBUCs, respectively. Immunohistochemical study was performed to determine NDN protein
(a.k.a. Necdin) expression evaluated by H-score method in 340 UTUCs and 295 UBUCs. NDN ex-
pression was further correlated with clinicopathological features and disease-specific survival (DSS) and
metastasis-free survival (MeFS).
Results: NDN transcriptional level was significantly higher in UCs of both sites with stepwise more
advanced pT statuses. Through immunohistochemistry, we found NDN protein expression was sig-
nificantly associated with adverse clinicopathological parameters, e.g., advanced pT status, nodal me-
tastasis, high grade histological patterns, and frequent mitotses (all P<0.05). In univariate analysis, NDN
overexpression not only predicted worse DSS and MeFS in both the UTUC and UBUC groups, it also
served as an independent prognostic factor for DSS and MeFS in multivariate analysis (all P<0.05).
Conclusions: NDN may play an important role in tumor progression in UC and could serve as a
prognostic biomarker and a potential novel therapeutic target in UC.
Key words: NDN gene, Necdin, Urothelial carcinoma, Prognosis.
Ivyspring
International Publisher
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
305
Introduction
Urothelial carcinoma (UC) is the most prevalent
histological type of malignancy throughout the uri-
nary tract, from the upper urinary tract (UT) to the
urinary bladder (UB) [1]. The former consists of the
renal pelvis and the ureter. In contrast to urothelial
carcinoma of the urinary bladder (UBUC), which is
the seventh most common cancer in the United States
[2], urothelial carcinoma of the upper urinary tract
(UTUC) is much uncommon and accounts for only 5%
to 10% of all UCs [3]. Nevertheless, the incidence of
UTUC in Taiwan is unusually high, especially in
southern Taiwan and areas of endemic “Black-foot
disease” [4-6]. Arsenic-contaminated drinking water
may contribute to the prevalence of UTUC in Taiwan
[6]. Etiologically, all UCs, regardless of anatomical
location, are attributed to identical carcinogens, such
as cigarette smoking, and aromatic amines, e.g. ben-
zidine, β-naphtylanine [7-9]. However, certain popu-
lations are particularly predisposed to UTUC. For
instance, patients with analgesic nephropathy [10],
Chinese herb nephropathy [11, 12], and Balkans
nephropathy [11, 13] are more susceptible to UTUC
than UBUC. In spite of the fact that UTUCs usually
have higher stage and grade than UBUCs, their clini-
cal behavior is similar after balancing the stages and
grades [14]. In addition, the previous study revealed
that gene expression profiles of both UBUCs and
UTUCs are much alike [15]. These findings indicate
that carcinogensis of UCs from both anatomical loca-
tions may participate in a common molecular path-
way.
Cancer is essentially a disease of regulation of
cell growth; genes that regulate cell growth must be
altered in order to transform normal cells into cancer
cells [16]. Oncogenesis is a multistep process, result-
ing from the accumulations of multiple mutations. Of
these mutations, self-sufficiency in growth signals, i.e.,
disruption of cell growth regulation is the first epi-
sode [17]. Nonetheless, the genes associated with cell
growth dysregulation have seldom been systemati-
cally evaluated in either UTUC or UBUC. By data
mining documented transcript expression profiles
(GSE31684) in the Gene Expression Omnibus (GEO,
National Center for Biotechnology Information
(NCBI), Bethesda, MD, USA) with a special focus on
growth regulation (GO:0001558), we found that tran-
scription of the NDN gene was significantly upregu-
lated from early tumor development and associated
stepwise with tumor progression. This evidence sug-
gests that the NDN gene plays an important role in
tumorigenesis and its progression.
The NDN gene encodes Necdin protein, a
member of the melanoma-associated antigen gene
(MAGE) family [18] that was first identified in neu-
rally differentiated mouse stem cells of P19 embryonal
carcinoma cell lines treated with retinoic acid [19].
Necdin was originally recognized as a suppressor of
cell proliferation in postmitotic neurons, leading the
differentiated neurons into permanent withdrawal
from the cell cycle due to constitutive and lifelong
expression of Necdin [20]. Moreover, NDN transcripts
and their encoded proteins showed downregulation
or low expression in previous UBUC cell lines and
human UBUC tissue studies [21]. Hence, we con-
ducted this study to elucidate the expression of Nec-
din protein and NDN mRNA in UCs from both ana-
tomical sites, as well as their association with clini-
copathological parameters and clinical outcomes.
Materials and Methods
Data mining the GEO to identify the most al-
tered transcripts in UCs
We performed data mining of the GEO of NCBI,
identifying dataset GSE31684 (http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE31684) in order
to analyze radical cystectomy specimens from 93 pa-
tients with UBUC using the Affymetrix GeneChip
Human Genome U133 Plus 2.0 Array. To analyze all
probe sets, we used Nexus Expression 3 statistical
software (BioDiscovery, El Segundo, CA, USA)
without preselection or filtering. Under supervision,
our comparative analysis examined the statistical sig-
nificance of differentially expressed transcripts on the
basis of primary tumor status (pT) and the develop-
ment of metastatic events. We performed functional
profiling using transcriptomes of high-stage UCs
(pT2-pT4) with metastases and low-stage UCs
(pTa-pT1) devoid of metastasis, focusing on those
related to the regulation of cell growth (GO:0001558).
We further analyzed survival patterns by dichoto-
mizing all cases into high-expression and
low-expression clusters for computing the prognostic
significance of the selected genes.
Patients and tumor specimens
The Institutional Review Board of Chi Mei
Medical Center approved this study (IRB971006). For
immunohistochemical study and survival analysis,
we enrolled 635 consecutive cases diagnosed as con-
ventional UC between 1996 and 2004, from the ar-
chives of the Department of Pathology, Chi-Mei
Medical Center. Of these cases, 340 tumors originated
from the UT and 295 arose from the UB. Other histo-
logical classifications as well as variants of UC were
excluded. The criteria for clinicopathological evalua-
tion were essentially identical to those in our previous
work [22]. Detailed information is provided in the
online supplementary material.
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
306
Transcriptional levels of the NDN gene
The materials for genetic examination were
gathered from macrodissection of fresh tumor tissue.
For quantification of NDN mRNA expression, we ex-
tracted total RNAs, quantified them, and submitted
them for reverse-transcription from 27 UTUCs and 27
UBUCs (both were composed of 9 cases of pTa; 9 pT1;
9 pT2-4), respectively. Using pre-designed TaqMan
assay reagents (Applied Biosystems, Waltham, MA,
USA), we measured mRNA abundance of NDN
(Hs00267349_s1) with the ABI StepOnePlus™ System,
as previously described [23]. We calculated the fold
expression of NDN relative to normal urothelium by
comparative Ct method, after normalization to
POLR2A (Hs01108291_m1) as the internal control.
Immunohistochemical staining and scoring of
NDN
Tissue sections underwent standard procedures
for deparaffinization, rehydration and antigen re-
trieval. Afterwards, the sections were incubated with
a primary antibody targeting NDN (TA506975, 1:150;
OriGene, Rockville, MD, USA) for an hour. Normal
brain tissue with or without incubation of primary
antibody were run in parallel as positive and negative
control, respectively. We scored NDN immunoreac-
tivity based on the combination of the percentage and
intensity of positivity of the immunostain in tumoral
nuclei in order to generate an H-score, which was
calculated using the following equation:
H-score=ΣPi(i +1), where i is the intensity of stained
tumor cells (0-3+), and Pi is the percentage of stained
tumor cells for each intensity varying from 0% to
100%. This formula produced a score range from
100-400, where 100 indicated that 100% of tumor cells
were negative and 400 indicated 100% of tumor cells
were strongly stained (3+) [24, 25].
Statistical analysis
We performed statistics using SPSS V.14.0 soft-
ware (SPSS Inc., Chicago, IL, USA). The median
H-score of NDN immunoreactivity was used as the
cut-off to dichotomize the study cohort, separating
cases into high expression and low expression groups.
We used Pearson’s χ2 test to compare NDN expres-
sion status and various categorical clinicopathological
parameters. Mann-Whitney U test was used to com-
pare NDN expression and numerical ones, i.e., mitotic
rate. The end points analyzed were disease-specific
survival (DSS) and metastasis-free survival (MeFS),
calculated from the date of tumor resection to the date
the event developed. Patients lost to follow-up were
censored on the latest follow-up date. We performed
univariate survival analyses using Kaplan-Meier plots
and compared them by log-rank test. Those parame-
ters with univariate P<0.1 were enrolled into multi-
variate tests using Cox proportional hazards regres-
sion. For all analyses, we used two-sided tests of sig-
nificance, with P<0.05 considered statistically signifi-
cant.
Results
NDN is a significant differentially upregulated
transcript linked with regulation of cell growth
in UBUCs
From the transcriptomic profiling of 93 UBUC
cases (GSE31684), which contains 78 high-stage
(T2-T4) and 15 low-stage (Ta-T1) cases, 28 with meta-
static disease and 49 without metastasis, we identified
six probes covering two transcripts associated with
regulation of cell growth (GO:0001558) (Figure 1). As
shown in Table 1, both transcripts, i.e., IGFBP5 and
NDN, were significantly upregulated with a log2 ratio
of 0.337 to 2.6399-fold and 0.7821-fold upregulation,
respectively, comparing high-stage to low-stage (all
P<0.005). IGFBP5 and NDN upregulation also showed
statistically significant association with metastatic
disease in all probes (all P≦0.0001). Comparing
high-expression (n=37) to low-expression (n=56)
clusters, high expression levels of NDN transcripts
significantly predicted worse disease-specific survival
(Figure S1, P=0.0059). We had investigated IGFBP5 in
a previous study [26]; however, NDN has not previ-
ously been systematically studied in UCs. Hence, we
further characterized the endogenous expression lev-
els and clinical significance of both NDN transcript
and its protein in UC.
Higher NDN mRNA transcriptional levels in
UBUCs and UTUCs with advanced pT stage
In the examined 27 UBUCs and 27 UTUCs, NDN
transcripts were significantly more abundant in tu-
mors with stepwise more advanced pT status, from
pTa to pT1 to pT2-4 (all P<0.05), suggesting it plays a
role in tumor progression (Figure 2).
Clinicopathological findings on UTUCs and
UBUCs
Clinicopathological features of the patients with
UTUCs and UBUCs are listed in Table 2. In the UTUC
group, no obvious sex predilection was noted despite
there being slightly more women (n=182, 53.5%). In
contrast, the majority of patients with UBUC were
male (n=216, 73.2%). For both types of UC, most pa-
tients’ age at diagnosis was older than 65 years
(n=202, 59.4% for UTUC and n=174, 59.0% for UBUC).
In the UTUC group, synchronous multifocal tumors
occurred in 62 patients (18.2%); both the renal pelvis
and ureter were involved in 49 (14.4%) of these. In
both UC groups, most tumors were classified as high
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
307
histological grade (n=284, 83.5% for UTUC and n=239,
81% for UBUC). Advanced pT stages (pT2-T4) were
seen in 46.8% (n=159) and 41.7% (n=123) of patients
with UTUCs and UBUCs, respectively. Nodal metas-
tasis was detected in 8.2% (n=28) and 9.8% (n=29) of
UTUC and UBUC patients, respectively. Vascular
invasion was noted in 31.2% of UTUC (n=106) and
16.6% of UBUC cases (n=49); while perineurial inva-
sion was observed in 5.6% of UTUC (n=19) and 6.8%
of UBUC cases (n=20). In addition, about half of the
tumors showed ten or more mitoses per ten high
power fields (n=167, 49.1% for UTUC and n=156,
52.9% for UBUC).
Correlations between immunoreactivity of
NDN and clinicopathological parameters in
UTUCs and UBUCs
NDN showed variable nuclear expression in
UCs from both sites. The median H-score for each
group was 105 and 370 for the UTUCs and UBUCs,
respectively. As Table 2 demonstrates, after dichoto-
mizing tumors into low and high NDN expression,
increased NDN expression in UC of both anatomical
sites was significantly associated with stepwise in-
creases of pT status (Figure 3, UTUC, P=0.003; UBUC,
P<0.001), lymph node metastasis (UTUC, P =0.002;
UBUC, P=0.012), high grade histological patterns
(UTUC, P=0.019; UBUC, P=0.003) and higher mitotic
rate (UTUC, P=0.004; UBUC, P=0.005). Increased
NDN expression was significantly associated with
vascular invasion and perineural invasion only in the
UBUC group (P=0.008 and 0.001, respectively).
Survival analysis for UTUC and UBUC pa-
tients
The univariate and multivariate analyses of rela-
tionships between clinical outcomes and miscellane-
ous clinicopathological parameters in both UTUC and
UBUC patients are illustrated in Tables 3-4. In mul-
tivariate analysis, multifocality (P=0.007), nodal me-
tastasis (P<0.001), high histological grade (P=0.007)
and perineurial invasion (P=0.001) are independent
prognostic factors for poor DSS in the UTUC group.
Similarly, inferior MeFS was significantly associated
with multifocality (P=0.002), nodal metastasis
(P=0.001), high histological grade (P=0.030), vascular
invasion (P<0.001) and perineurial invasion (P=0.014)
in patients with UTUCs. In UTUCs, advanced pT
status was significantly associated with worse DSS
and MeFS in univariate (P<0.0001) but not in multi-
variate analyses. Tumor location and vascular inva-
sion in UTUC group correlated with poorer patient
DSS in univariate analysis only (P=0.0079 and
P<0.0001, respectively).
Figure 1. Analysis of gene expression in urothelial carcinoma using a published
transcriptomic dataset (GSE31684). Clustering analysis of genes focused on the
regulation of cell growth (GO:0001558) revealed NDN is one of the most significantly
upregulated genes associated with increments of pT status and metastatic disease.
Tissue specimens from tumors with different pT statuses are indicated on top of the
heatmap, and expression levels of upregulated and downregulated genes are repre-
sented as a spectrum of brightness or red and green, respectively. Those with
unaltered mRNA transcriptional levels are coded black.
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
308
In the UBUC group, multivariate analysis re-
vealed advanced pT status and higher mitotic rate
were significantly associated with both dismal DDS
and MeFS (all P<0.05). Lymph node metastasis was
significantly associated with poor MeFS (P=0.037) in
multivariate analysis, as well. In UBUCs, high histo-
logical grade, and vascular and perineural invasion
were significantly associated with both adverse DSS
and MeFS in univariate analysis (all P<0.005), but not
in multivariate analysis.
Prognostic significance of NDN expression in
UC
As shown in Tables 3-4 and Figure 4, in uni-
variate analysis, either the UTUC or UBUC group
with NDN overexpression had significantly worse
DSS and MeFS (P<0.005 for all). Notably, in multi-
variate analysis, high NDN nuclear expression was
still an independent prognosticator predicting more
dismal DSS and MeFS for all UCs (P<0.05 for all).
Table 1. Summary of differentially expressed genes associated with regulation of cell growth (GO:0001558) and showed positive as-
sociations to cancer invasiveness and metastasis in the transcriptome of urothelial carcinoma of urinary bladder (GSE31684).
Probe
Comparing T2-4 to
Ta-T1
Comparing Meta. to
Non-Meta.
#
Gene Symbol
Biological Process
Molecular Function
log ratio
p-value
log ratio
p-value
1554741_s_at
0.4313
<0.0001
0.3919
<0.0001
IGFBP5
regulation of cell growth, signal transduction
growth factor binding, insu-
lin-like growth factor binding
201666_at
1.1435
0.0001
1.0825
<0.0001
IGFBP5
regulation of cell growth, signal transduction
growth factor binding, insu-
lin-like growth factor binding
205131_x_at
0.337
0.0008
0.2968
0.0001
IGFBP5
regulation of cell growth, signal transduction
growth factor binding, insu-
lin-like growth factor binding
205168_at
0.7821
0.0044
1.0228
<0.0001
NDN
axon extension involved in development, axonal
fasciculation, axonogenesis, central nervous system
development, glial cell migration, negative regula-
tion of cell proliferation, nerve growth factor re-
ceptor signaling pathway, nervous system devel-
opment, neuron development, neuron migration,
regulation of cell growth, regulation of progression
through cell cycle, regulation of transcription;
DNA-dependent, respiratory gaseous exchange,
sensory perception of pain, transcription
DNA binding, gamma-tubulin
binding, protein binding
205782_at
0.8325
0.0003
0.8815
<0.0001
IGFBP5
regulation of cell growth, signal transduction
growth factor binding, insu-
lin-like growth factor binding
207426_s_at
2.6399
<0.0001
1.4678
<0.0001
IGFBP5
regulation of cell growth, signal transduction
growth factor binding, insu-
lin-like growth factor binding
#, Meta., distal metastasis developed during follow-up; Non-Meta.: no metastatic event developed.
Table 2. Correlations between NDN expression and other important clinicopathological parameters in urothelial carcinomas.
Parameter
Category
Upper Urinary Tract Urothelial Carcinoma
Urinary Bladder Urothelial Carcinoma
Case No. (%)
NDN Expression
p-value
Case No. (%)
NDN Expression
p-value
Low (%)
High (%)
Low (%)
High (%)
Gender
Male
158 (46.5)
86 (54.4)
72 (45.6)
0.128
216 (73.2)
109 (50.5)
107 (49.5)
0.719
Female
182 (53.5)
84 (46.2)
98 (53.8)
79 (26.8)
38 (48.1)
41 (51.9)
Age (years)
< 65
138 (40.6)
70 (50.7)
68 (49.3)
0.825
121 (41.0)
59 (48.8)
62 (51.2)
0.759
≥ 65
202 (59.4)
100 (49.5)
102 (50.5)
174 (59.0)
88 (50.6)
86 (49.4)
Tumor location
Renal pelvis
141 (41.5)
71 (50.4)
70 (49.6)
0.685
-
-
-
-
Ureter
150 (44.1)
72 (48.0)
78 (52.0)
-
-
-
-
Renal pelvis & ureter
49 (14.4)
27 (55.1)
22 (44.9)
-
-
-
-
Multifocality
Single
278 (81.8)
138 (49.6)
140 (50.4)
0.779
-
-
-
-
Multifocal
62 (18.2)
32 (51.6)
30 (48.4)
-
-
-
-
Primary tumor (pT)
pTa
89 (26.2)
56 (62.9)
33 (37.1)
0.003*
84 (28.5)
62 (73.8)
22 (26.2)
<0.001*
pT1
92 (27.0)
49 (53.3)
43 (46.7)
88 (29.8)
48 (54.5)
40 (45.5)
pT2-pT4
159 (46.8)
65 (40.9)
94 (59.1)
123 (41.7)
37 (30.0)
86 (70.0)
Nodal metastasis
Negative (pN0)
312 (91.8)
164 (52.6)
148 (47.4)
0.002*
266 (90.2)
139 (52.3)
127 (47.7)
0.012*
Positive (pN1-pN3)
28 (8.2)
6 (21.4)
22 (78.6)
29 (9.8)
8 (27.6)
21 (72.4)
Histological grade
Low grade
56 (16.5)
36 (64.3)
20 (35.7)
0.019*
56 (19.0)
38 (67.9)
18 (32.1)
0.003*
High grade
284 (83.5)
134 (47.2)
150 (52.8)
239 (81.0)
109 (45.6)
130 (54.4)
Vascular invasion
Absent
234 (68.9)
122 (52.1)
112 (47.9)
0.242
246 (83.4)
131 (53.3)
115 (46.7)
0.008*
Present
106 (31.1)
48 (45.3)
58 (54.7)
49 (16.6)
16 (32.7)
33 (67.3)
Perineural invasion
Absent
321 (94.4)
164 (51.1)
157 (48.9)
0.098
275 (93.2)
144 (52.4)
131 (47.6)
0.001*
Present
19 (5.6)
6 (31.6)
13 (68.4)
20 (6.8)
3 (15.0)
17 (85.0)
Mitotic rate (per 10
high power fields)
#
340
9.66+/-7.315
14.96+/-15.324
0.004*
295
11.41+/-10.286
17.38+/-16.469
0.005*
#, Mann-Whitney U test; *, Statistically significant.
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
309
Table 3. Univariate log-rank and multivariate analyses for Disease-specific and Metastasis-free Survivals in upper urinary tract urothelial
carcinoma.
Parameter
Category
Case No.
(%)
Disease-specific Survival
Metastasis-free Survival
Univariate analysis
Multivariate analysis
Univariate analysis
Multivariate analysis
No. of event
(%)
p-value
R.R.
95% C.I.
p-value
No. of event
(%)
p-value
R.R.
95% C.I.
p-value
Gender
Male
158 (46.5)
28 (17.7)
0.8286
-
-
-
32 (20.3)
0.7904
-
-
-
Female
182 (53.5)
33 (18.1)
-
-
-
38 (20.9)
-
-
-
Age (years)
< 65
138 (40.6)
26 (18.8)
0.9943
-
-
-
30 (21.7)
0.8470
-
-
-
≥ 65
202 (59.4)
35 (17.3)
-
-
-
40 (19.8)
-
-
-
Tumor side
Right
177 (52.1)
34 (19.2)
0.7366
-
-
-
38 (21.5)
0.3074
-
-
-
Left
154 (45.3)
26 (16.9)
-
-
-
32 (20.8)
-
-
-
Bilateral
9 (2.6)
1 (11.1)
-
-
-
0 (0)
-
-
-
Tumor loca-
tion
Renal pelvis
141 (41.5)
24 (17.0)
0.0079*
1
-
0.840
31 (22.0)
0.0659
-
-
-
Ureter
150 (44.1)
22 (14.7)
0.876
0.472-1.625
25 (16.7)
-
-
-
Renal pelvis &
ureter
49 (14.4)
15 (30.6)
1.286
0.607-7.438
14 (28.6)
-
-
-
Multifocality
Single
278 (81.8)
48 (17.3)
0.0026*
1
-
0.007*
52 (18.7)
0.0127*
1
-
0.002*
Multifocal
62 (18.2)
18 (29.0)
2.821
1.321-6.024
18 (29.0)
2.402
1.381-4.4
178
Primary
tumor (pT)
pTa
89 (26.2)
2 (2.2)
<0.0001*
1
-
0.071
4 (4.5)
<0.0001*
1
-
0.227
pT1
92 (27.0)
9 (9.8)
3.042
0.646-14.324
15 (16.3)
2.344
0.756-7.2
67
pT2-pT4
159 (46.8)
50 (31.4)
5.159
1.150-23.153
51 (32.1)
2.497
0.795-7.8
46
Nodal me-
tastasis
Negative
(pN0)
312 (91.8)
42 (13.5)
<0.0001*
1
-
<0.001*
55 (17.6)
<0.0001*
1
-
0.001*
Positive
(pN1-pN3)
28 (8.2)
19 (67.9)
5.071
2.753-9.340
15 (53.6)
2.888
1.552-5.3
72
Histological
grade
Low grade
56 (16.5)
4 (7.1)
0.0215*
1
-
0.007*
3 (5.4)
0.0027*
1
-
0.030*
High grade
284 (83.5)
57 (20.0)
3.853
1.441-10.298
67 (23.6)
3.818
1.139-12.
792
Vascular
invasion
Absent
234 (68.9)
24 (10.3)
<0.0001*
1
-
0.059
26 (11.1)
<0.0001*
1
-
<0.001*
Present
106 (31.1)
37 (34.9)
1.774
0.978-3.218
44 (41.5)
2.960
1.625-5.3
92
Perineural
invasion
Absent
321 (94.4)
50 (15.6)
<0.0001*
1
-
0.001*
61 (19.0)
<0.0001*
1
-
0.014*
Present
19 (5.6)
11 (57.9)
3.463
1.661-7.222
9 (47.4)
2.562
1.208-5.4
34
Mitotic rate
(per 10 high
power fields)
< 10
173 (50.9)
27 (15.6)
0.167
-
-
30 (17.3)
0.0823
-
-
>= 10
167 (49.1)
34 (20.4)
-
-
40 (24.0)
-
-
NDN ex-
pression
Low
170 (50.0)
17 (10.0)
0.0002*
1
-
0.032*
24 (14.1)
0.0027*
1
-
0.033*
High
170 (50.0)
44 (25.9)
1.893
1.058-3.385
46 (27.1)
1.731
1.044-2.8
69
* Statistically significant.
Table 4. Univariate log-rank and multivariate analyses for Disease-specific and Metastasis-free Survivals in urinary bladder urothelial
carcinoma.
Parameter
Category
Case No.
(%)
Disease-specific Survival
Metastasis-free Survival
Univariate analysis
Multivariate analysis
Univariate analysis
Multivariate analysis
No. of
event (%)
p-value
R.R.
95% C.I.
p-value
No. of
event (%)
p-value
R.R.
95% C.I.
p-value
Gender
Male
216 (73.2)
41 (19.0)
0.4446
-
-
-
60 (27.8)
0.2720
-
-
-
Female
79 (26.8)
11 (13.9)
-
-
-
16 (20.3)
-
-
-
Age (years)
< 65
121 (41.0)
17 (14.0)
0.1136
-
-
-
31 (25.6)
0.6875
-
-
-
≥ 65
174 (59.0)
35 (20.1)
-
-
-
45 (25.9)
-
-
-
Primary tumor
(pT)
pTa
84 (28.5)
1 (1.4)
<0.0001*
1
-
<0.0001*
4 (4.8)
<0.0001*
1
-
0.011*
pT1
88 (29.8)
9 (10.2)
3.817
1.748-8.333
23 (26.1)
4.631
1.311-16.364
pT2-pT4
123 (41.7)
42 (34.1)
21.277
2.823-200.00
49 (39.8)
6.688
1.889-23.677
Nodal metastasis
Negative (pN0)
266 (90.2)
41 (15.4)
0.0002*
1
-
0.387
61 (22.9)
<0.0001*
1
-
0.037*
Positive
(pN1-pN3)
29 (9.8)
11 (37.9)
1.364
0.675-2.758
15 (51.7)
1.939
1.042-3.607
Histological grade
Low grade
56 (19.0)
2 (3.6)
0.0013*
1
-
0.889
5 (8.9)
0.0007*
1
-
0.711
High grade
239 (81.0)
50 (20.9)
0.895
0.188-4.266
71 (29.7)
0.809
0.264-2.481
Vascular invasion
Absent
246 (83.4)
37 (15.0)
0.0024*
1
-
0.185
54 (22.0)
0.0001*
1
-
0.835
Present
49 (16.6)
15 (30.6)
0.629
0.317-1.249
22 (44.9)
1.067
0.580-1.964
Perineural invasion
Absent
275 (93.2)
44 (16.0)
0.0001*
1
-
0.131
66 (24.0)
0.0007*
1
-
0.347
Present
20 (6.8)
8 (40.0)
1.908
0.824-4.420
10 (50.0)
1.429
0.679-3.009
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
310
Parameter
Category
Case No.
(%)
Disease-specific Survival
Metastasis-free Survival
Univariate analysis
Multivariate analysis
Univariate analysis
Multivariate analysis
No. of
event (%)
p-value
R.R.
95% C.I.
p-value
No. of
event (%)
p-value
R.R.
95% C.I.
p-value
Mitotic rate (per 10
high power fields)
< 10
139 (47.1)
12 (8.6)
<0.0001*
1
-
0.011*
23 (16.5)
<0.0001*
1
-
0.019*
>= 10
156 (52.9)
40 (25.6)
2.409
1.219-4.761
53 (34.0)
1.871
1.109-3.156
NDN expression
Low
147 (49.8)
10 (6.8)
<0.0001*
1
-
0.012*
21 (14.3)
<0.0001*
1
-
0.021*
High
148 (50.2)
42 (28.4)
2.477
1.220-5.028
55 (37.2)
1.852
1.098-3.123
* Statistically significant.
Figure 2. Quantitative real-time PCR (qPCR) analysis showed that NDN mRNA expression was significantly increased stepwise in both urothelial carcinomas of the
upper urinary tract (left panel) and urinary bladder (right panel) along with more advanced primary pT status, from pTa to pT1 and pT1 to pT2-4 (all P<0.05).
Figure 3. NDN immunostaining on representative sections revealed stepwise increase of NDN expression from non-tumoral urothelium (A), non-invasive (B), superficially
infiltrating (pT1) (C) and deeply infiltrating (pT2-4) urothelial carcinoma (D).
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
311
Figure 4. Kaplan-Meier plots disclosed the predictive value of NDN immunoreactivity for disease-specific survival (DSS) and metastasis-free survival (MeFS) in both groups
of UTUC (A and B for DSS and MeFS, respecitvely) and UBUC (C and D for DSS and MeFS, respectively) (all P<0.005).
Discussion
Urothelial carcinoma is prevalent worldwide
and has high recurrence rates [27]. Pathological stag-
ing is still the most decisive prognosticator of out-
comes for bladder cancer, where five-year survival
rates are approximately 60% to 85% for T2 bladder
cancers after cystectomy, but decrease rapidly to 40%
to 60% for T3 tumors [28]. In spite of the introduction
of cisplatin-based combination chemotherapy, the
prognosis is still poor for advanced stage bladder
cancers; so far bladder cancer is ranked as the thir-
teenth most common cause of cancer death in the
world (GLOBOCAN 2012 http://globocan.iarc.fr).
Therefore, developing more potent therapeutic regi-
mens is of crucial importance for high-risk patients.
Every cancer has ten hallmarks: sustaining pro-
liferative signaling, evading growth suppressors,
avoiding immune destruction, enabling replicative
immortality, tumor-promoting inflammation, acti-
vating invasion and metastasis, inducing angiogene-
sis, genome instability and mutation, resisting cell
death, and deregulating cellular energetics [29].
Among these characteristics, sustaining proliferative
signaling, that is, cell growth dysregulation, serves as
the first step of oncogenesis.
The maternally imprinted gene, NDN, is associ-
ated with Prader-Willi syndrome [30]. Necdin, en-
coded by the NDN gene, is a 325-amino acid protein
belonging to the MAGE family [18, 19]. The function
of Necdin protein is similar to Rb, which has been
shown to interact with viral oncogenes such as simian
virus 40 (SV40) large T antigen and adenovirus E1A,
as well as E2F1 and p53 [31, 32]. Nevertheless, Necdin
also bears oncogenic properties. Necdin’s inhibitory
effect on p53-mediated growth arrest and suppressive
impact on p53-dependent apoptosis have also been
demonstrated to be oncogenic functions [33, 34]. In
line with this, there have been divergent results for
Necdin expression in different cancers of each study.
The scarcity of Necdin expression in brain tumor cell
lines [35], as well as its decreased expression in mel-
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
312
anomas [36] and bladder cancer cell lines and tumors
[21] suggest NDN may be a tumor suppressor gene.
Conversely, upregulation and loss of imprinting of
Necdin in pancreatic cancers have been demonstrated
by more recent studies [37, 38]. The paradoxical re-
sults of experiments designed to recognize the role of
Necdin in oncogenesis indicate that it has complex
functions, which may be due to different microenvi-
ronments and cellular contexts [34]. Our current in-
vestigation proves the possible oncogenic role of NDN
in UCs, which is not consistent with a previous study
that found NDN was downregulated in bladder can-
cer cell lines and tumors [21]. A study by Chapman, et
al. [21] first observed that NDN was induced to be
downregulated in normal human urothelial cells
transducted by hTERT, the catalytic subunit of te-
lomerase. After that, the authors found NDN tran-
scripts were downregulated in 26 of 28 (92.9%) blad-
der cancer cell lines and 35 of 58 (60%) bladder cancer
tumors. However, the researchers neither correlated
the NDN transcription levels with clinicopathological
significance nor analyzed their association with sur-
vival. In addition, to the best of our knowledge, the
current investigation is the first to inspect the rela-
tionship between NDN expression of both mRNA and
protein levels, along with clinical outcomes in UTUC.
Conclusion
In sum, our work demonstrates that NDN gene
and NDN protein (a.k.a. Necdin) plays an influential
role in tumor progression of UCs. High expression of
Necdin in both UTUC and UBUC is associated with
worse clinicopathological parameters. Overexpres-
sion of Necdin is also an independent prognosticator
of inferior DSS and MeFS in both groups. Further re-
search to elucidate the details of the biological role of
the NDN gene and Necdin in carcinogenesis of UC is
essential for exploring the potential of NDN-targeted
therapy for UCs, as we illustrated the hopeful targets
for new strategies for UC therapy lately [39-41].
Supplementary Material
Supplementary methods and Figure S1.
http://www.jcancer.org/v07p0304s1.pdf
Acknowledgements
The authors are grateful to the BioBank at Chi
Mei Medical Center for providing tumor samples.
Grant Support
This work was sponsored by E-DA Hospital
(EDAHP104014 to I-W Chang), Taiwan Ministry of
Science and Technology (NSC99-2320-B-384-001-MY2
and NSC101-2320-B-384 -001 -MY3), Ministry of
Health and Welfare (DOH102-TD-M-111-102001 and
MOHW104- TDU-B-212–124-003, from Health and
Welfare surcharge on tobacco products, to C-F Li and
W-J Wu) and Kaohsiung Medical University
(KMU-TP103G01, KMU-TP103G00, KMU-TP103G04,
KMU-TP103G05) This study was also supported by
grants from Kaohsiung Medical University “Aim for
the Top Universities (KMU-TP103E19).
Competing Interests
The authors have declared that no competing
interest exists.
References
1. Eble JN, Sauter G, Epstein JI, Sesterhenn IA. World Health Organization
Classification of Tumours. Pathology and Genetics of Tumours of the Urinary
System and Male Genital Organs. Lyon, Fance: International Agency for
Research on Cancer (IARC) press; 2004.
2. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin.
2014;64:9-29.
3. Raman JD, Messer J, Sielatycki JA, Hollenbeak CS. Incidence and survival of
patients with carcinoma of the ureter and renal pelvis in the USA, 1973-2005.
BJU Int. 2011;107:1059-64.
4. Chou YH, Huang CH. Unusual clinical presentation of upper urothelial
carcinoma in Taiwan. Cancer. 1999;85:1342-4.
5. Yang MH, Chen KK, Yen CC, et al. Unusually high incidence of upper urinary
tract urothelial carcinoma in Taiwan. Urology. 2002;59:681-7.
6. Tan LB, Chen KT, Guo HR. Clinical and epidemiological features of patients
with genitourinary tract tumour in a blackfoot disease endemic area of
Taiwan. BJU Int. 2008;102:48-54.
7. McLaughlin JK, Silverman DT, Hsing AW, et al. Cigarette smoking and
cancers of the renal pelvis and ureter. Cancer Res. 1992;52:254-7.
8. Pommer W, Bronder E, Klimpel A, Helmert U, Greiser E, Molzahn M.
Urothelial cancer at different tumour sites: role of smoking and habitual intake
of analgesics and laxatives. Results of the Berlin Urothelial Cancer Study.
Nephrol Dial Transplant. 1999;14:2892-7.
9. Shinka T, Miyai M, Sawada Y, Inagaki T, Okawa T. Factors affecting the
occurrence of urothelial tumors in dye workers exposed to aromatic amines.
Int J Urol. 1995;2:243-8.
10. Stewart JH, Hobbs JB, McCredie MR. Morphologic evidence that
analgesic-induced kidney pathology contributes to the progression of tumors
of the renal pelvis. Cancer. 1999;86:1576-82.
11. Debelle FD, Vanherweghem JL, Nortier JL. Aristolochic acid nephropathy: a
worldwide problem. Kidney Int. 2008;74:158-69.
12. Laing C, Hamour S, Sheaff M, Miller R, Woolfson R. Chinese herbal uropathy
and nephropathy. Lancet. 2006;368:338.
13. Dragicevic D, Djokic M, Pekmezovic T, et al. Survival of patients with
transitional cell carcinoma of the ureter and renal pelvis in Balkan endemic
nephropathy and non-endemic areas of Serbia. BJU Int. 2007;99:1357-62.
14. Catto JW, Yates DR, Rehman I, et al. Behavior of urothelial carcinoma with
respect to anatomical location. J Urol. 2007;177:1715-20.
15. Zhang Z, Furge KA, Yang XJ, Teh BT, Hansel DE. Comparative gene
expression profiling analysis of urothelial carcinoma of the renal pelvis and
bladder. BMC Med Genomics. 2010;3:58.
16. Croce CM. Oncogenes and cancer. N Engl J Med. 2008;358:502-11.
17. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57-70.
18. Barker PA, Salehi A. The MAGE proteins: emerging roles in cell cycle
progression, apoptosis, and neurogenetic disease. J Neurosci Res.
2002;67:705-12.
19. Maruyama K, Usami M, Aizawa T, Yoshikawa K. A novel brain-specific
mRNA encoding nuclear protein (necdin) expressed in neurally differentiated
embryonal carcinoma cells. Biochem Biophys Res Commun. 1991;178:291-6.
20. Hayashi Y, Matsuyama K, Takagi K, Sugiura H, Yoshikawa K. Arrest of cell
growth by necdin, a nuclear protein expressed in postmitotic neurons.
Biochem Biophys Res Commun. 1995;213:317-24.
21. Chapman EJ, Kelly G, Knowles MA. Genes involved in differentiation, stem
cell renewal, and tumorigenesis are modulated in telomerase-immortalized
human urothelial cells. Mol Cancer Res. 2008;6:1154-68.
22. Huang WW, Huang HY, Liao AC, et al. Primary urothelial carcinoma of the
upper tract: important clinicopathological factors predicting bladder
recurrence after surgical resection. Pathol Int. 2009;59:642-9.
23. Wu LC, Chen LT, Tsai YJ, et al. Alpha-methylacyl coenzyme A racemase
overexpression in gallbladder carcinoma confers an independent prognostic
indicator. J Clin Pathol. 2012;65:309-14.
24. Budwit-Novotny DA, McCarty KS, Cox EB, et al. Immunohistochemical
analyses of estrogen receptor in endometrial adenocarcinoma using a
monoclonal antibody. Cancer Res. 1986;46:5419-25.
Journal of Cancer 2016, Vol. 7
http://www.jcancer.org
313
25. McClelland RA, Finlay P, Walker KJ, et al. Automated quantitation of
immunocytochemically localized estrogen receptors in human breast cancer.
Cancer Res. 1990;50:3545-50.
26. Liang PI, Wang YH, Wu TF, et al. IGFBP-5 overexpression as a poor prognostic
factor in patients with urothelial carcinomas of upper urinary tracts and
urinary bladder. J Clin Pathol. 2013;66:573-82.
27. Rodriguez-Alonso A, Pita-Fernandez S, Gonzalez-Carrero J, Nogueira-March
JL. Multivariate analysis of survival, recurrence, progression and developmen t
of mestastasis in T1 and T2a transitional cell bladder carcinoma. Cancer.
2002;94:1677-84.
28. Townsend CM JR, Evers BM, Mattox KL. Sabiston Textbook of Surgery: The
Biological Basis of Modern Surgical Practice. 16th ed. Philadelphia, USA:
Saunders; 2002.
29. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell.
2011;144:646-74.
30. Jay P, Rougeulle C, Massacrier A, et al. The human necdin gene, NDN, is
maternally imprinted and located in the Prader-Willi syndrome chromosomal
region. Nat Genet. 1997;17:357-61.
31. Taniura H, Matsumoto K, Yoshikawa K. Physical and functional interactions
of neuronal growth suppressor necdin with p53. J Biol Chem.
1999;274:16242-8.
32. Taniura H, Taniguchi N, Hara M, Yoshikawa K. Necdin, a postmitotic
neuron-specific growth suppressor, interacts with viral transforming proteins
and cellular transcription factor E2F1. J Biol Chem. 1998;273:720-8.
33. Hasegawa K, Yoshikawa K. Necdin regulates p53 acetylation via Sirtuin1 to
modulate DNA damage response in cortical neurons. J Neurosci.
2008;28:8772-84.
34. Lafontaine J, Rodier F, Ouellet V, Mes-Masson AM. Necdin, a p53-target gene,
is an inhibitor of p53-mediated growth arrest. PLoS One. 2012;7:e31916.
35. Aizawa T, Maruyama K, Kondo H, Yoshikawa K. Expression of necdin, an
embryonal carcinoma-derived nuclear protein, in developing mouse brain.
Brain Res Dev Brain Res. 1992;68:265-74.
36. Hoek K, Rimm DL, Williams KR, et al. Expression profiling reveals novel
pathways in the transformation of melanocytes to melanomas. Cancer Res.
2004;64:5270-82.
37. Tan AC, Jimeno A, Lin SH, et al. Characterizing DNA methylation patterns in
pancreatic cancer genome. Mol Oncol. 2009;3:425-38.
38. Wang L, Liu HL, Li Y, Yuan P. Proteomic analysis of pancreatic intraepithelial
neoplasia and pancreatic carcinoma in rat models. World J Gastroenterol.
2011;17:1434-41.
39. Chang IW, Liang PI, Li CC, et al. HAS3 underexpression as an indicator of
poor prognosis in patients with urothelial carcinoma of the upper urinary tract
and urinary bladder. Tumour Biol. 2015;36:5441-50.
40. Chang IW, Lin VC, He HL, et al. CDCA5 overexpression is an indicator of
poor prognosis in patients with urothelial carcinomas of the upper urinary
tract and urinary bladder. Am J Transl Res. 2015;7:710-22.
41. Li CF, Wu WJ, Wu WR et al. The cAMP responsive element binding protein 1
transactivates epithelial membrane protein 2, a potential tumor suppressor in
the urinary bladder urothelial carcinoma. Oncotarget 2015;6:9220-39.
